Medical Physiology: A Cellular and Molecular Approach [2 ed.] 1437717535, 9781437717532

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Me d ic a l Ph y s i o l o g y

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Medical Physiology A Cellular and Molecular Approach U P DATE D S ECO N D E D I TI O N

Walter F. Boron, MD, PhD

Professor David N. and Inez Myers/Antonio Scarpa Chairman Department of Physiology and Biophysics Case Western Reserve University Cleveland, Ohio

Emile L. Boulpaep, MD

Professor Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

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1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899 MEDICAL PHYSIOLOGY: A CELLULAR AND MOLECULAR APPROACH

ISBN: 978-1-4377-1753-2

International Edition

ISBN: 978-0-8089-2449-4

Copyright © 2012 by Saunders, an imprint of Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Rights Department: phone: (+1) 215 239 3804 (US) or (+44) 1865 843830 (UK); fax: (+44) 1865 853333; e-mail: [email protected]. You may also complete your request online via the Elsevier website at http://www.elsevier.com/permissions.

Notice Knowledge and best practice in this field are constantly changing. As new research and experience broaden our knowledge, changes in practice, treatment, and drug therapy may become necessary or appropriate. Readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of the practitioner, relying on his or her experience and knowledge of the patient, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the Authors assume any liability for any injury and/or damage to persons or property arising out of or related to any use of the material contained in this book. The Publisher Previous editions copyrighted 2003, 2005, 2009 Library of Congress Cataloging-in-Publication Data Medical physiology : a cellular and molecular approach / [edited by] Walter F. Boron, Emile L. Boulpaep. – 2nd ed. p. ; cm. Includes bibliographical references and index. ISBN 978-1-4377-1753-2 1. Human physiology—Textbooks. I. Boron, Walter F. II. Boulpaep, Emile L. [DNLM: 1. Physiology. 2. Cell Physiology. 3. Genomics. QT 104 M4894 2009] QP34.5.B65 2009 612–dc22 2008000942 Acquisitions Editor: Elyse O’Grady Developmental Editor: Andrew Hall Publishing Services Manager: Patricia Tannian Senior Project Manager: John Casey Design Manager: Steven Stave Printed in United States Last digit is the print number: 9 8 7 6 5 4 3 2 1

Working together to grow libraries in developing countries www.elsevier.com | www.bookaid.org | www.sabre.org

C O N T R I B U TO R S

Michael Apkon, MD, PhD

Michael J. Caplan, MD, PhD

Associate Clinical Professor Department of Pediatrics Yale University School of Medicine New Haven, Connecticut

Professor Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

Peter S. Aronson, MD

Barry W. Connors, PhD

Professor Section of Nephrology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Professor and Chair Department of Neuroscience Brown University Providence, Rhode Island Arthur DuBois, MD

Eugene J. Barrett, MD, PhD

Professor Department of Internal Medicine University of Virginia School of Medicine Charlottesville, Virginia

Professor Emeritus of Epidemiology and Public Health and Cellular and Molecular Physiology John B. Pierce Laboratory New Haven, Connecticut Gerhard Giebisch, MD

Paula Barrett, PhD

Professor Department of Pharmacology University of Virginia School of Medicine Charlottesville, Virginia

Professor Emeritus of Cellular and Molecular Physiology Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

Henry J. Binder, MD

Fred S. Gorelick, MD

Professor of Medicine Professor of Cellular and Molecular Physiology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Professor Section of Digestive Diseases Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Walter F. Boron, MD, PhD

Peter Igarashi, MD

Professor David N. and Inez Myers/Antonio Scarpa Chairman Department of Physiology and Biophysics Case Western Reserve University Cleveland, Ohio

Professor University of Texas Southwestern Medical Center at Dallas Dallas, Texas Ervin E. Jones, MD, PhD

Emile L. Boulpaep, MD

Professor Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut Lloyd Cantley, MD, FASN

Professor Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Department of Obstetrics and Gynecology Yale University School of Medicine New Haven, Connecticut W. Jonathan Lederer, MD, PhD

Director, Medical Biotechnology Center and Department of Physiology University of Maryland Biotechnology Institute University of Maryland School of Medicine Baltimore, Maryland

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Contributors

Christopher R. Marino, MD

George B. Richerson, MD, PhD

Professor of Medicine and Physiology University of Tennessee Health Science Center Chief, Medical Service VA Medical Center Memphis, Tennessee

Professor Department of Neurology Yale University School of Medicine New Haven, Connecticut Steven S. Segal, PhD

Edward J. Masoro, PhD

Professor Emeritus of Physiology University of Texas Health Science Center at San Antonio San Antonio, Texas

Professor Department of Medical Pharmacology and Physiology University of Missouri School of Medicine Columbia, Missouri

Edward G. Moczydlowski, PhD

Gerald I. Shulman, MD, PhD

Professor and Chair Department of Biology Clarkson University Potsdam, New York

Professor Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Kitt Falk Petersen, MD

Associate Professor Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

John T. Stitt, PhD

Professor Emeritus of Epidemiology and Public Health John B. Pierce Laboratory New Haven, Connecticut Frederick J. Suchy, MD

Bruce R. Ransom, MD, PhD

Professor and Chair Department of Neurology University of Washington Health Sciences Center Seattle, Washington

Professor and Chair Pediatrics, Hepatology Mount Sinai Medical Center New York, New York Erich E. Windhager, MD

Adrian Reuben, MBBS, FRCP, FACG

Director of Liver Studies Department of Gastroenterology and Hepatology Medical University of South Carolina Charleston, South Carolina

Professor Department of Physiology and Biophysics Weill Medical College Cornell University New York, New York

P R E FA C E TO T H E S E C O N D E D I T I O N

We are very grateful for the enthusiastic reception with which the academic community received the first edition of our book. In producing this second edition, three guiding principles have remained the same as before. First, create a modern textbook of physiology that provides the expertise of several authors but the consistency of a single pen. Second, weave an integrative story that extends from the level of DNA and proteins to the level of cells, tissues, and organs, and finally to the interaction among organ systems. Third, illustrate important physiological principles with examples from pathophysiology, thereby putting physiology in a clinical context. In addition, we have strived to improve the book along the lines suggested by our readers. Moreover, we have updated the material—reflecting new molecular insights— as well as the presentation of this material. The result is two new chapters, new authors for seven chapters, the reordering or reorganization of several chapters, and—throughout the book—countless improvements to the text. In addition, the second edition includes 65 new or redrawn figures as well as enhancements to 488 others. In Section II (The Physiology of Cells and Molecules), fresh insights into genetics led to substantial revisions in Chapter 4 (Regulation of Gene Expression). Moreover, advances in genomics and the understanding of genetic diseases led to the creation of new tables to organize families of transporter proteins in Chapters 5 (Transport of Solutes and Water) and ion channels in Chapter 6 (Electrophysiology of the Cell Membrane). In Section III (The Nervous System), new molecular developments led to major changes in Chapter 15 (Sensory Transduction). In Section IV (The Cardiovascular System), we have added new Chapter 18 on Blood. In Section V (The Respiratory System), we have shifted some pulmonary function tests into Chapter 26 (Organization of the Respiratory System). In Section VI (The Urinary System), genomic progress led to a new table on amino-acid transporters. In Section VII (The Gastrointestinal System), Chapter 45 (Nutrient Digestion and Absorption) now contains a section on nutritional requirements. In Section VIII (The Endocrine System), we have renamed Chapter 48 to Endocrine Regulation of Growth and Body Mass to reflect updated coverage of the regulation of appetite. In Section IX (The Reproductive System), we have modified figures to clarify mitosis versus meiosis in males versus meiosis in females, as well as to clarify the development of the follicle. Finally, in Section X (The Physiology of Cells and Molecules), we have largely rewritten Chapter 58 (Metabolism), with special emphasis on energy interconversion (e.g., gluconeogenesis); energy

capture after ingestion of carbohydrate, protein, or fats; and the integrative response to fasting. Moreover, we have added new Chapter 62 (The Physiology of Aging). To create the second edition, we recruited as new authors several outstanding scientist-educators: Lloyd Cantley (Chapter 3), Gerald Shulman and Kitt Petersen (Chapter 58), John Stitt (Chapter 59), Arthur DuBois (Chapter 61), and Edward Masoro (Chapter 62). In addition, two previous authors picked up additional chapters: Edward Moczydlowski (Chapter 9) and Steven Segal (Chapter 60). Online Access. The Web site www.StudentConsult.com

offers the reader access to the online edition of the textbook, with the ability to search, bookmark, post notes, download highlighted text to a handheld device, access all of the images in the book, and more. The hundreds of “mouse” icons in the text direct the reader to “webnotes” that likewise are available on the Student Consult website. These webnotes provide derivations of mathematical equations, amplification of concepts, supplementary details, additional clinical illustrations, and links that may be of interest (e.g., biographies of famous physiologists). Acknowledgments. A textbook is the culmination of successful collaborations among many individuals. First, we thank our authors. Second, we thank Philine Wangemann, who made invaluable suggestions for the Vestibular and Auditory Transduction subchapter in Chapter 15. Third, we thank our colleagues who provided advice on parts of the book: Samuel Cukierman, Sarah Garber, and Mark Shapiro (Chapters 6-8); R. John Solaro and John Walsh (Chapter 9); T. Richard Nichols (Chapter 16); Don McCrimmon and Frank Powell (Chapter 32); Franz Beck, Gerhard Burkhardt, Bruce Koeppen, Patricia Preisig, Luis Reuss, James Schafer, Jurgen Schnermann, James Wade, and Carsten Wagner (Chapters 33-40); Mark Donowitz (Chapter 44); Charles Mansbach (Chapter 45); as well as Harold Behrman and Richard Ehrenkranz (Chapters 53-57). We thank all of our readers who sent us their suggestions. At the art studio Dartmouth Publishing Inc, we thank Stephanie Davidson for developing new figures and updating others, while maintaining the textbook’s aesthetic appeal originally established by JB Woolsey and Associates. At Elsevier, we are very grateful to William R. Schmitt, Acquisitions Editor, for his trust and endurance. Andrew Hall, Developmental Editor, was the project’s communica-

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Preface to the Second Edition

tions hub, responsible for coordinating all parties working on the textbook, and for assembling the many elements that comprised the final product. His meticulous care was indispensable. We thank Sharon Lee, Project Manager, for overseeing production of the textbook. Finally, at Yale University and Case Western Reserve University we thank Charleen Bertolini, who used every ounce

of her friendly, good-humored, and tenacious personality to keep our authors—and us—on track. As we did in the First Edition, we again invite the reader to enjoy learning physiology. If you are pleased with our effort, tell others. If not, tell us.

P R E FA C E TO T H E F I R S T E D I T I O N

We were intrigued by an idea suggested to us by W.B. Saunders: write a modern textbook of physiology that combines the expertise of a multi-author book with the consistency of a single pen. Our approach has been, first, to recruit as writers mainly professors who teach medical physiology at the Yale University School of Medicine, and then to recast the professors’ manuscripts in a uniform style. After much effort, we now present our book, which we hope will bring physiology to life and at the same time be a reliable resource for students. Target Audience. We wrote Medical Physiology primarily as

an introductory text for medical students, although it should also be valuable for students in the allied health professions and for graduate students in the physiological sciences. The book should continue to be useful for the advanced medical student who is learning pathophysiology and clinical medicine. Finally, we hope that physicians in training, clinical fellows, and clinical faculty will find the book worthwhile for reviewing principles and becoming updated on new information pertinent for understanding the physiological basis of human disease. Content of the Textbook. Aside from Part I, which is a brief

introduction to the discipline of physiology, the book consists of nine major Parts. Part II (Physiology of Cells and Molecules) reflects that, increasingly, the underpinnings of modern physiology have become cellular and molecular. Chapters 2, 4, and 5 would not be present in a traditional physiology text. Chapter 2 (Functional Organization of the Cell), Chapter 4 (Signal Transduction), and Chapter 5 (Regulation of Gene Expression) provide the essentials of cell biology and molecular biology necessary for understanding cell and organ function. The other chapters in Part II cover the cellular physiology of transport, excitability, and muscle— all of which are classic topics for traditional physiology texts. In this book we have extended each of these subjects to the molecular level. The remainder of the book will frequently send the reader back to the principles introduced in Part II. Parts III to IX address individual organ systems. In each case, the first chapter provides a general introduction to the system. Part III (Cellular Physiology of the Nervous System) is untraditional in that it deliberately omits those aspects of the physiology of the central nervous system that neuroscience courses generally treat and that require extensive knowledge of neuroanatomical pathways. Rather, Part III focuses on cellular neurophysiology, including synaptic transmission in the nervous system, sensory transduction, and neural cir-

cuits. In addition, Part III also treats two subjects—the autonomic nervous system and the neuronal microenvironment— that are important for understanding other physiological systems. Finally, Part X (The Physiology of Everyday Life) is an integrated, multisystem approach to metabolism, temperature regulation, exercise, and adaptations to special environments. Emphasis of the Textbook. Some important aspects of physiology remain as fundamentally important today as when the pioneers of physiology discovered them a century or more ago. These early observations were generally phenomenological descriptions that physiologists have since been trying to understand at a mechanistic level. Where possible, a goal of this textbook is to extend this understanding all the way to the cell and molecule. Moreover, although some areas are evolving rapidly, we have tried to be as up to date as practical. To make room for the cellular and molecular bricks, we have omitted some classic experimental observations, especially when they were of a “black-box” nature. Just as each major Part of the textbook begins with an introductory chapter, each chapter generally first describes— at the level of the whole body or organ system (e.g., the kidney)—how the body performs a certain task and/or controls a certain parameter (e.g., plasma K+ concentration). As appropriate, our discussion then progresses in a reductionistic fashion from organ to tissue to cell and organelles, and ultimately to the molecules that underlie the physiology. Finally, most chapters include a discussion of how the body regulates the parameter of interest at all levels of integration, from molecules to the whole body. Creating the Textbook. The first draft of each chapter was written by authors with extensive research and/or teaching experience in that field. The editors, sitting shoulder to shoulder at a computer, then largely rewrote all chapters line by line. The goal of this exercise was for the reader to recognize, throughout the entire book, a single voice—a unity provided by consistency in style, in organization, in the sequence for presenting concepts, and in terminology and notation, as well as in consistency in the expression of standard values (e.g., a cardiac output of 5 liters/min). The editors also attempted to minimize overlap among chapters by making extensive use of cross references (by page, figure, or table number) to principles introduced elsewhere in the book. After the first round of editing, Dr. Malcolm Thaler—a practicing physician and accomplished author in his own right—improved the readability of the text and sometimes

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Preface to the First Edition

added clinical examples. Afterwards, the editors again went through the entire text line by line to decide on the material to be included in specific illustrations, and to match the main text of the book with the content of each figure. The editors then traveled to Philadelphia to visit the art studio of JB Woolsey and Associates. Over many visits, John Woolsey and the editors together developed the content and format for each of the approximately 760 full-color illustrations used in the textbook. These meetings were unique intellectual and pedagogical dialogues concerning the design of the figures. To a large extent, the figures owe their pedagogical style to the creativity of John Woolsey. The illustrations evolved through several iterations of figure editing, based on suggestions from both the editors and authors. This evolution, as well as text changes requested by authors, led to yet a third round of editing of the entire book, often line by line. Throughout this seemingly endless process, our goal has been to achieve the proper balance among reader friendliness, depth, and accuracy. Special Features. Compared with other major textbooks of

physiology, a much larger fraction of the space in this book is devoted to illustrations. Thus, although our textbook may appear thick, it actually has fewer text words than most other leading medical physiology books. Virtually all illustrations in our book are in full color, conceived de novo, with consistent style and pedagogy. Many of the figures feature “dialogue balloons” that tell a story. The illustrations are also available in digital format on the Evolve Web site (http://evolve.elsevier.com/productPages/s_417.html) for use in the classroom. The textbook makes considerable use of clinical boxes— highlighted on a color background—that present examples of diseases illustrating important physiological principles. The text includes over 2000 cross references that send the reader from the current page to specific pages, figures, or tables elsewhere in the book for relevant concepts or data. The text also includes hundreds of web icons, which direct the reader to our website at http://www.wbsaunders.com/ MERLIN/BandB/. These web links provide derivations of mathematical equations, amplification of concepts, material that was deleted for the sake of brevity from earlier drafts of the textbook, and clinical illustrations not included in the clinical boxes. The website will also contain several other features, including summaries for each subchapter, an expanded list

of references (sometimes with direct links to the primary literature), other links that may be of interest to the physiology student (e.g., biographies of famous physiologists), latebreaking scientific developments that occur after publication of the book, and—alas—the correction of errors. Finally, we invite the reader to visit our website to comment on our book, to point out errors, and to make other helpful suggestions. Acknowledgments. A textbook is the culmination of successful collaborations among many individuals. First, we would like to thank our authors. Second, we acknowledge the expert input of Dr. Malcolm Thaler, both in terms of style and clinical insight. We also thank Dr. Thaler for emphasizing the importance of telling a “good story.” The textbook’s aesthetic appeal is largely attributable to JB Woolsey and Associates, particularly John Woolsey and Joel Dubin. At W.B. Saunders, we are especially thankful to William R. Schmitt—Acquisitions Editor—for his trust and patience over the years that this book has been in gestation. At the times when the seas were rough, he steered a safe course. Melissa Dudlick—Developmental Editor at W.B. Saunders— was the project’s nerve center, responsible for day-to-day communication among all parties working on the textbook, and for assembling all of the many components that went into making the final product. Her good humor and careful attention to detail greatly facilitated the creation of the textbook. We thank Frank Polizzano—Publishing Services Manager at W.B. Saunders—for overseeing production of the textbook. Before this textbook was completed, the author of Part X (The Physiology of Everyday Life), Ethan Nadel, passed away. We are indebted to those who generously stepped up to carefully check the nearly finished manuscripts for the final four chapters: Dr. Gerald Shulman for Chapter 57, Dr. John Stitt for Chapter 58, the late Dr. Carl Gisolfi for Chapter 59, and Dr. Arthur DuBois for Chapter 60. In addition, Dr. George Lister provided expert advice for Chapter 56. We are also grateful to Dr. Bruce Davis for researching the sequences of the polypeptide hormones, to Mr. Duncan Wong for expert information-technology services, and to Mrs. Leisa Strohmaier for administrative assistance. We now invite the reader to enjoy the experience of learning physiology. If you are pleased with our effort, tell others. If not, tell us.

C O N T E N TS

SECTION

I

INTRODUCTION 1

Foundations of Physiology

13 Synaptic Transmission in the Nervous 1 3

Emile L. Boulpaep and Walter F. Boron

System

14 The Autonomic Nervous System

2 Functional Organization of the Cell

7 9

Michael J. Caplan

3 Signal Transduction

75

Peter Igarashi

5 Transport of Solutes and Water

106

147

179

Edward G. Moczydlowski

System

18 Blood 19 Arteries and Veins

429 448

212

Edward G. Moczydlowski

467

Emile L. Boulpaep 482

Emile L. Boulpaep

21 Cardiac Electrophysiology and the Electrocardiogram

8 Synaptic Transmission and the

504

W. Jonathan Lederer

22 The Heart as a Pump

529

Emile L. Boulpaep

9 Cellular Physiology of Skeletal, Cardiac, and Smooth Muscle

427

17 Organization of the Cardiovascular

20 The Microcirculation

7 Electrical Excitability and Action

Neuromuscular Junction

IV

THE CARDIOVASCULAR SYSTEM

Emile L. Boulpaep

Edward G. Moczydlowski Potentials

408

Barry W. Connors

Emile L. Boulpaep

Peter S. Aronson, Walter F. Boron, and Emile L. Boulpaep

6 Electrophysiology of the Cell Membrane

16 Circuits of the Central Nervous System

SECTION 48

Lloyd Cantley

4 Regulation of Gene Expression

371

Barry W. Connors

II

PHYSIOLOGY OF CELLS AND MOLECULES

351

George B. Richerson

15 Sensory Transduction SECTION

323

Barry W. Connors

237

23 Regulation of Arterial Pressure and Cardiac Output

Edward G. Moczydlowski and Michael Apkon

554

Emile L. Boulpaep SECTION

24 Special Circulations

III

THE NERVOUS SYSTEM

265

25 Integrated Control of the Cardiovascular System

10 Organization of the Nervous System

577

Steven S. Segal

267

593

Emile L. Boulpaep

Bruce R. Ransom

11 The Neuronal Microenvironment

289

12 Physiology of Neurons Barry W. Connors

SECTION

V

THE RESPIRATORY SYSTEM

Bruce R. Ransom 310

26 Organization of the Respiratory System

611 613

Walter F. Boron

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xii

Contents

27 Mechanics of Ventilation

630

Walter F. Boron

28 Acid-Base Physiology

652 SECTION

29 Transport of Oxygen and Carbon Dioxide 672 685 700

Walter F. Boron

32 Control of Ventilation

725

George B. Richerson and Walter F. Boron SECTION

33 Organization of the Urinary System

747 749

55 The Female Reproductive System

835

56 Fertilization, Pregnancy, and Lactation

851

57 Fetal and Neonatal Physiology

881 883 895

Henry J. Binder 912

Christopher R. Marino and Fred S. Gorelick 933

Henry J. Binder

45 Nutrient Digestion and Absorption Henry J. Binder and Adrian Reuben

1146 1170

Ervin E. Jones 1193

X

PHYSIOLOGY OF EVERYDAY LIFE

Henry J. Binder

44 Intestinal Fluid and Electrolyte Movement

1128

Ervin E. Jones

SECTION

41 Organization of the Gastrointestinal

43 Pancreatic and Salivary Glands

1113

Ervin E. Jones

866

VII

42 Gastric Function

1111

Ervin E. Jones

Gerhard Giebisch and Erich Windhager

System

1094

IX

THE REPRODUCTIVE SYSTEM

821

Gerhard Giebisch and Erich Windhager

THE GASTROINTESTINAL SYSTEM

52 The Parathyroid Glands and Vitamin D

54 The Male Reproductive System

Gerhard Giebisch and Erich Windhager

SECTION

1074

Eugene J. Barrett

797

Gerhard Giebisch and Erich Windhager

40 Integration of Salt and Water Balance

1057

Ervin E. Jones

Gerhard Giebisch and Erich Windhager

39 Transport of Acids and Bases

51 The Endocrine Pancreas

53 Sexual Differentiation

36 Transport of Urea, Glucose, Phosphate,

38 Urine Concentration and Dilution

1044

Eugene J. Barrett

SECTION 782

Gerhard Giebisch and Erich Windhager

37 Transport of Potassium

49 The Thyroid Gland

767

Gerhard Giebisch and Erich Windhager

Calcium, Magnesium, and Organic Solutes

1028

Eugene J. Barrett

Eugene J. Barrett and Paula Barrett

34 Glomerular Filtration and Renal 35 Transport of Sodium and Chloride

Body Mass

Eugene J. Barrett

Gerhard Giebisch and Erich Windhager Blood Flow

1011

48 Endocrine Regulation of Growth and

50 The Adrenal Gland

VI

THE URINARY SYSTEM

47 Organization of Endocrine Control

1009

Eugene J. Barrett

Walter F. Boron

31 Ventilation and Perfusion of the Lungs

VIII

THE ENDOCRINE SYSTEM

Walter F. Boron

30 Gas Exchange in the Lungs

980

Frederick J. Suchy

Walter F. Boron in the Blood

46 Hepatobiliary Function

949

58 Metabolism

1211 1213

Gerald I. Shulman and Kitt Falk Petersen

59 Regulation of Body Temperature

1237

John Stitt

60 Exercise Physiology and Sports Science

1249

Steven S. Segal

61 Environmental Physiology

1268

Arthur DuBois

62 The Physiology of Aging

1281

Edward J. Masoro Index

1293

SECTION

I

I NTRODUCTION

1

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CHAPTER

1

F O U N D AT I O N S O F P H Y S I O L O G Y Emile L. Boulpaep and Walter F. Boron

WHAT IS PHYSIOLOGY? Physiology is the dynamic study of life. Physiology describes the “vital” functions of living organisms and their organs, cells, and molecules. For centuries, the discipline of physiology has been closely intertwined with medicine. Although physiology is not primarily concerned with structure—as is the case of anatomy, histology, and structural biology— structure and function are inextricably linked because the living structures perform the functions. For some, physiology is the function of the whole person (e.g., exercise physiology). For many practicing clinicians, physiology may be the function of an individual organ system, such as the cardiovascular, respiratory, or gastrointestinal system. For still others, physiology may focus on the cellular principles that are common to the function of all organs and tissues. This last field has traditionally been called general physiology, a term that is now supplanted by “cellular and molecular physiology.” Although one can divide physiology according to varying degrees of reductionism, it is also possible to define a branch of physiology—for example, comparative physiology—that focuses on differences and similarities among different species. Indeed, comparative physiology may deal with all degrees of reductionism, from molecule to whole organism. In a similar way, medical physiology deals with how the human body functions, which depends on how the individual organ systems function, which depends on how the component cells function, which in turn depends on the interactions among subcellular organelles and countless molecules. Thus, medical physiology takes a global view of the human body; but in doing so, it requires an integrated understanding of events at the level of molecules, cells, and organs. Physiology is the mother of several biological sciences, having given birth to the disciplines of biochemistry, biophysics, and neuroscience as well as their corresponding scientific societies and journals. Thus, it should come as no surprise that the boundaries of physiology are not sharply delineated. Conversely, physiology has its unique attributes. For example, physiology has evolved over the centuries from a more qualitative to a more quantitative science. Indeed, many of the leading physiologists were—and still are—

trained as engineers.

chemists,

physicists,

mathematicians,

or

Physiological genomics is the link between the organ and the gene The life of the human body requires not only that individual organ systems do their jobs but also that these organ systems work “hand in hand” with each other. They must share information. Their actions must be interdependent. The cells within an organ or a tissue often share information, and certainly the individual cells must act in concert to perform the proper function of the organ or tissue. In fact, cells in one organ must often share information with cells in another organ and make decisions that are appropriate for the health of the individual cell as well as for the health of the whole person. In most cases, the sharing of information between organs and between cells takes place at the level of atoms or molecules. Cell-to-cell messengers or intracellular messengers may be atoms such as H+ or K+ or Ca2+. The messengers may also be more complex chemicals. A cell may release a molecule that acts on a neighboring cell or that enters the bloodstream and acts on other cells a great distance away. In other cases, a neuron may send an axon a centimeter or even a meter away and rapidly modulate, through a neurotransmitter molecule, the activity of another cell or another organ. Cells and organs must interact with one another, and the method of communication is almost always molecular. The grand organizer—the master that controls the molecules, the cells, and the organs and the way they interact—is the genome. Traditionally, the discipline of physiology has, in its reductionistic journey, always stopped at about the level of cells and certain subcellular organelles as well as their component and controlling molecules. The discipline of physiology left to molecular biology and molecular genetics the business of how the cell controls itself through its DNA. The modern discipline of physiology has become closely intertwined with molecular biology, however, because DNA encodes the proteins in which physiologists are most interested. Very often, physiologists painstakingly develop elegant

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4

Section I • Introduction

strategies for cloning of the genes relevant to physiology. Sometimes, brute force approaches, such as the Human Genome Project in the United States, hand the physiologist a candidate gene, homologous to one of known function, on a silver platter. In still other cases, molecular biologists may clone a gene with no known function. In this case, it may be up to the physiologist to determine the function of the gene product, that is, to determine its physiology. Physiological genomics (or functional genomics) is a new branch of physiology devoted to understanding of the roles that genes play in physiology. Traditionally, physiologists have moved in a reductionistic direction from organ to cell to molecule to gene. One of the most fascinating aspects of physiological genomics is that it has closed the circle and linked organ physiology directly with molecular biology. Perhaps one of the most striking examples is the knockout mouse. Knocking out the gene encoding a protein that, according to conventional wisdom, is very important will sometimes have no obvious effect or sometimes unexpected effects. It is up to the physiologist, at least in part, to figure out why. It is perhaps rather sobering to consider that to truly understand the impact of a transgene or a knockout on the physiology of a mouse, one would have to carefully reevaluate the totality of mouse physiology. To grasp the function of a gene product, the physiologist must retrace the steps up the reductionistic road and achieve an integrated understanding of that gene’s function at the level of the cells, organs, and whole body. Physiology is unique among the basic medical sciences in that it is both broad in its scope (i.e., it deals with multiple systems) and integrative in its outlook. In some cases, important physiological parameters, such as blood pressure, may be under the control of many genes. Certain polymorphisms in several of these many genes could have a cumulative effect that produces high blood pressure. How would one identify which polymorphisms of which genes may underlie high blood pressure? This sort of complex problem does not easily lend itself to a physiologist’s controlled studies. One approach would be to study a population of people, or strains of experimental animals, and use statistical tools to determine which polymorphisms correlate with high blood pressure in a population. Indeed, epidemiologists use statistical tools to study group effects in populations. However, even after the identification of variants in various genes, each of which may make a small contribution to high blood pressure, the physiologist has an important role. First, the physiologist, performing controlled experiments, must determine whether a particular genetic variant does indeed have at least the potential to modulate blood pressure. Second, the physiologist must determine the mechanism of the effect. Cells live in a highly protected milieu intérieur In his lectures on the phenomena of life, Claude Bernard wrote in 1878 on the conditions of the constancy of life, which he considered a property of higher forms of life. According to Bernard, animals have two environments: the milieu extérieur that physically surrounds the whole organism; and the milieu intérieur, in which the tissues and cells of the organism live. This internal environment is neither the

air nor the water in which an organism lives but rather—in the case of the human body—the well-controlled liquid environment that Bernard called “the organic liquid that circulates and bathes all the anatomic elements of the tissues, the lymph or the plasma.” In short, this internal environment is what we today call the extracellular fluid. He argued that physiological functions continue in a manner indifferent to the changing environment because the milieu intérieur isolates the organs and tissues of the body from the vagaries of the physical conditions of the environment. Indeed, Bernard described the milieu intérieur as if an organism had placed itself in a greenhouse. According to Bernard’s concept of milieu intérieur, some fluids contained within the body are not really inside the body at all. For example, the contents of the gastrointestinal tract, sweat ducts, and renal tubules are all outside the body. They are all continuous with the milieu extérieur. Bernard compares a complex organism to an ensemble of anatomical elements that live together inside the milieu intérieur. Therefore, in Part II of this textbook, we examine the physiology of these cells and molecules. In Chapter 2 (“Functional Organization of the Cell”), we begin our journey through physiology with a discussion of the biology of the cells that are the individual elements of the body. Chapter 3 (“Signal Transduction”) discusses how cells communicate directly through gap junctions or indirectly by molecules released into the extracellular fluid. These released molecules can bind to receptors on the cell membrane and initiate signal transduction cascades that can modify gene transcription (a genomic response) and a wide range of other cell functions (nongenomic responses). Alternatively, these released molecules can bind to receptors in the cytoplasm or nucleus and alter the transcription of genes. In Chapter 4 (“Regulation of Gene Expression”), we examine the response of the nucleus. Chapter 5 (“Transport of Solutes and Water”) addresses how the plasma membrane separates the cell interior from Bernard’s milieu intérieur and establishes the composition of the cell interior. In the process of establishing the composition of the intracellular fluid, the plasma membrane also sets up ion and voltage gradients across itself. Excitable cells—mainly nerve and muscle cells—can exploit these gradients for the long-distance “electrical” transmission of information. The property of “excitability,” which requires both the perception of a change (a signal) and the reaction to it, is the topic of Chapters 6 to 9. In Part III, we examine how the nervous system exploits excitability to process information. Another theme developed by Bernard was that the “fixité du milieu intérieur” (the constancy of the extracellular fluid) is the condition of “free, independent life.” He explains that organ differentiation is the exclusive property of higher organisms and that each organ contributes to “compensate and equilibrate” against changes in the external environment. In that sense, each of the systems discussed in Parts IV to VIII permits the body to live within an adverse external environment because the cardiovascular system, the respiratory system, the urinary system, the gastrointestinal system, and the endocrine system create and maintain a constant internal environment. Individual cell types in various organ systems act in concert to support the constancy of the internal milieu, and the internal milieu in

Chapter 1 • Foundations of Physiology

turn provides these cells with a culture medium in which they can thrive. The discipline of physiology also deals with those characteristics that are the property of a living organism as opposed to a nonliving organism. Four fundamental properties distinguish the living body. First, only living organisms exchange matter and energy with the environment to continue their existence. Several organ systems of the body participate in these exchanges. Second, only living organisms can receive signals from their environment and react accordingly. The principles of sensory perception, processing by the nervous system, and reaction are discussed in the chapters on excitability and the nervous system. Third, what distinguishes a living organism is the life cycle of growth and reproduction, as discussed in the chapters on reproduction (Part IX). Finally, the living organism is able to adapt to changing circumstances. This is a theme that is developed throughout this textbook but especially in the chapters on everyday life (Part X). Homeostatic mechanisms—operating through sophisticated feedback control mechanisms—are responsible for maintaining the constancy of the milieu intérieur Homeostasis is the control of a vital parameter. The body carefully controls a seemingly endless list of vital parameters. Examples of tightly controlled parameters that affect nearly the whole body are arterial pressure and blood volume. At the level of the milieu intérieur, tightly regulated parameters include body core temperature and plasma levels of oxygen, glucose, potassium ions (K+), calcium ions (Ca2+), and hydrogen ions (H+). Homeostasis also occurs at the level of the single cell. Thus, cells regulate many of the same parameters that the body as a whole regulates: volume, the concentrations of many small inorganic ions (e.g., Na+, Ca2+, H+), and energy levels (e.g., ATP). One of the most common themes in physiology is the negative feedback mechanism responsible for homeostasis. Negative feedback requires at least four elements. First, the system must be able to sense the vital parameter (e.g., glucose) or something related to it. Second, the system must be able to compare the input signal with some internal reference value called a set-point, thereby forming a difference signal. Third, the system must multiply the error signal by some proportionality factor (i.e., the gain) to produce some sort of output signal (e.g., release of insulin). Fourth, the output signal must be able to activate an effector mechanism (e.g., glucose uptake and metabolism) that opposes the source of the input signal and thereby brings the vital parameter closer to the set-point (e.g., decrease of blood glucose levels to normal). Sometimes the body controls a parameter, in part, by cleverly employing positive feedback loops. A single feedback loop often does not operate in isolation but rather as part of a larger network of controls. Thus, a complex interplay may exist among feedback loops within single cells, within a tissue, within an organ or organ system, or at the level of the whole body. After studying these individual feedback loops in isolation, the physiologist may find that two feedback loops act either synergistically or

antagonistically. For example, insulin lowers blood glucose levels, whereas epinephrine and cortisol have the opposite effect. Thus, the physiologist must determine the relative weights of feedback loops in competition with one another. Finally, the physiologist must also establish hierarchy among various feedback loops. For example, the hypothalamus controls the anterior pituitary, which controls the adrenal cortex, which releases cortisol, which helps control blood glucose levels. Another theme of homeostasis is redundancy. The more vital a parameter is, the more systems that the body mobilizes to regulate it. If one system should fail, others are there to help maintain homeostasis. It is probably for this reason that genetic knockouts sometimes fail to have their expected deleterious effects. The result of many homeostatic systems controlling many vital parameters is a milieu intérieur with a stable composition. Whether at the level of the milieu intérieur or the cytoplasm of a single cell, homeostasis occurs at a price: energy. When a vital parameter (e.g., the blood glucose level) is well regulated, that parameter is not in equilibrium. Equilibrium is a state that does not involve energy consumption. Instead, a well-regulated parameter is generally in a steady state. That is, its value is constant because the body or the cell carefully matches actions that lower the parameter value with other actions that raise it. The net effect is that the vital parameter is held at a constant value. An important principle in physiology, to which we have already alluded, is that each cell plays a specialized role in the overall function of the body. In return, the body—which is the sum of all these cells—provides the milieu intérieur appropriate for the life of each cell. As part of the bargain, each cell or organ must respect the needs of the body as a whole and not run amok for its own greedy interests. For example, during exercise, the system that controls body core temperature sheds heat by elaborating sweat for evaporation. However, the production of sweat ultimately reduces blood volume. Because the body as a whole places a higher priority on the control of blood volume than on the control of body core temperature, at some point the system that controls blood volume will instruct the system that controls body core temperature to reduce the production of sweat. Unfortunately, this juggling of priorities works only if the individual stops exercising; if not, the result may be heat stroke. The adaptability of an organism depends on its ability to alter its response. Indeed, flexible feedback loops are at the root of many forms of physiological adaptation. For instance, at sea level, experimentally lowering the level of oxygen (the sensory stimulus) in the inspired air causes an increase in breathing (the response). However, after acclimatization at high altitude to low oxygen levels, the same low level of oxygen (the same sensory stimulus) causes one to breathe much faster (a greater response). Thus, the response may depend on the previous history and therefore the “state” of the system. In addition to acclimatization, genetic factors can also contribute to the ability to respond to an environmental stress. For example, certain populations of humans who have lived for generations at high altitude withstand hypoxia better than lowlanders do, even after the lowlanders have fully acclimatized.

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Section I • Introduction

Medicine is the study of “physiology gone awry” Medicine borrows its physicochemical principles from physiology. Medicine also uses physiology as a reference state: it is essential to know how organs and systems function in the healthy person to grasp which components may be malfunctioning in a patient. A large part of clinical medicine is simply dealing with the abnormal physiology brought about by a disease process. One malfunction (e.g., heart failure) can lead to a primary pathological effect (e.g., a decrease in cardiac output) that—in chain reaction style—leads to a series of secondary effects (e.g., fluid overload) that are the appropriate responses of physiological feedback loops. Indeed, as clinician-physiologists have explored the basis of disease, they have discovered a great deal about physiology. For this reason, we have tried to illustrate physiological principles with clinical examples, some of which are displayed in clinical boxes in this text. Physiologists have developed many tools and tests to examine normal function. A large number of functional tests—used in diagnosis of a disease, monitoring of the

evolution of an illness, and evaluation of the progress of therapy—are direct transfers of technology developed in the physiology laboratory. Typical examples are cardiac monitoring, pulmonary function tests, and renal clearance tests as well as the assays used to measure plasma levels of various ions, gases, and hormones. Refinements of such technology in the hospital environment, in turn, benefit the study of physiology. Thus, the exchange of information between medicine and physiology is a two-way street. The understanding of physiology summarized in this book comes from some experiments on humans but mostly from research on other mammals and even on squids and slime molds. However, our ultimate focus is on the human body. REFERENCES Bernard C: Leçons sur les phénomènes de la vie communs aux animaux et aux végétaux. Cours de physiologie générale du Museum d’Histoire Naturelle. Paris: Baillière et Fils, 1878. Cannon WB: The Wisdom of the Body. New York: Norton, 1932. Smith HW: From Fish to Philosopher. New York: Doubleday, 1961.

SECTION

II

P H YS I O LO GY O F C E L LS A N D MOLECULES Chapter 2

• Functional Organization of the Cell ...... 9

Chapter 3

• Signal Transduction ...... 48

Chapter 4 • Regulation of Gene Expression ...... 75 Chapter 5

• Transport of Solutes and Water ...... 106

Chapter 6

• Electrophysiology of the Cell Membrane ...... 147

Chapter 7

• Electrical Excitability and Action Potentials ...... 179

Chapter 8 • Synaptic Transmission and the Neuromuscular Junction ...... 212 Chapter 9 • Cellular Physiology of Skeletal, Cardiac, and Smooth Muscle ...... 237

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CHAPTER

2

F U N C T I O N A L O R G A N I Z AT I O N OF THE CELL Michael J. Caplan

In the minds of many students, the discipline of physiology is linked inextricably to images from its past. This prejudice is not surprising because many experiments from physiology’s proud history, such as those of Pavlov and his dogs, have transcended mere scientific renown and entered the realm of popular culture. Some might believe that the science of physiology devotes itself exclusively to the study of whole animals and is therefore an antique relic in this era of molecular reductionism. Nothing could be further from the truth. Physiology is and always has been the study of the homeostatic mechanisms that allow an organism to persist despite the ever-changing pressures imposed by a hostile environment. These mechanisms can be appreciated at many different levels of resolution. Certainly it would be difficult to understand how the body operates unless one appreciates the functions of its organs and the communication between these organs that allows them to influence one another’s behaviors. It would also be difficult to understand how an organ performs its particular tasks unless one is familiar with the properties of its constituent cells and molecules. The modern treatment of physiology that is presented in this textbook is as much about the interactions of molecules in cells as it is about the interactions of organs in organisms. It is necessary, therefore, at the outset to discuss the structure and characteristics of the cell. Our discussion focuses first on the architectural and dynamic features of a generic cell. We then examine how this generic cell can be adapted to serve in diverse physiological capacities. Through adaptations at the cellular level, organs acquire the machinery necessary to perform their individual metabolic tasks.

STRUCTURE OF BIOLOGICAL MEMBRANES The surface of the cell is defined by a membrane The chemical composition of the cell interior is very different from that of its surroundings. This observation applies equally to unicellular paramecia that swim freely in a fresh-

water pond and to neurons that are densely packed in the cerebral cortex of the human brain. The biochemical processes involved in cell function require the maintenance of a precisely regulated intracellular environment. The cytoplasm is an extraordinarily complex solution, the constituents of which include myriad proteins, nucleic acids, nucleotides, and sugars that the cell synthesizes or accumulates at great metabolic cost. The cell also expends tremendous energy to regulate the intracellular concentrations of numerous ions. If there were no barrier surrounding the cell to prevent exchange between the intracellular and extracellular spaces, all of the cytoplasm’s hard-won compositional uniqueness would be lost by diffusion in a few seconds. The requisite barrier is provided by the plasma membrane, which forms the cell’s outer skin. The plasma membrane is impermeable to large molecules such as proteins and nucleic acids, thus ensuring their retention within the cytosol. It is selectively permeable to small molecules such as ions and metabolites. However, the metabolic requirements of the cell demand a plasma membrane that is much more sophisticated than a simple passive barrier that allows various substances to leak through at different rates. Frequently, the concentration of a nutrient in the extracellular fluid is several orders of magnitude lower than that required inside the cell. If the cell wishes to use such a substance, therefore, it must be able to accumulate it against a concentration gradient. A simple pore in the membrane cannot concentrate anything; it can only modulate the rate at which a gradient dissipates. To accomplish the more sophisticated feat of creating a concentration gradient, the membrane must be endowed with special machinery that uses metabolic energy to drive the uphill movements of substances—active transport—into or out of the cell. In addition, it would be useful to rapidly modulate the permeability properties of the plasma membrane in response to various metabolic stimuli. Active transport and the ability to control passive permeabilities underlie a wide range of physiological processes, from the electrical excitability of neurons to the resorptive and secretory functions of the kidney. In Chapter 5, we will explore how cells actively transport solutes across the plasma membrane. The mechanisms through which the plasma membrane’s dynamic selectivity is achieved, modified, and regulated are

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Section II • Physiology of Cells and Molecules

discussed briefly later in this chapter and in greater detail in Chapter 7. The cell membrane is composed primarily of phospholipids Our understanding of biological membrane structure is based on studies of red blood cells, or erythrocytes, that were conducted in the early part of the 20th century. The erythrocyte lacks the nucleus and other complicated intracellular structures that are characteristic of most animal cells. It consists of a plasma membrane surrounding a cytoplasm that is rich in hemoglobin. It is possible to break open erythrocytes and release their cytoplasmic contents. The plasma membranes can then be recovered by centrifugation, providing a remarkably pure preparation of cell surface membrane. Biochemical analysis reveals that this membrane is composed of two principal constituents: lipid and protein. Most of the lipid associated with erythrocyte plasma membranes belongs to the molecular family of phospholipids. In general, phospholipids share a glycerol backbone, two hydroxyl groups of which are esterified to various fatty acid or acyl groups (Fig. 2-1A). These acyl groups may have different numbers of carbon atoms and also may have double bonds between carbons. For glycerol-based phospholipids, the third glycerolic hydroxyl group is esterified to a phosphate group, which is in turn esterified to a small molecule referred to as a head group. The identity of the head group determines the name as well as many of the properties of the

individual phospholipids. For instance, glycerol-based phospholipids that bear an ethanolamine molecule in the head group position are categorized as phosphatidylethanolamines (Fig. 2-1A). Phospholipids form complex structures in aqueous solution The unique structure and physical chemistry of each phospholipid (Fig. 2-1B) underlie the formation of biological membranes and explain many of their most important properties. Fatty acids are nonpolar molecules. Their long carbon chains lack the charged groups that would facilitate interactions with water, which is polar. Consequently, fatty acids dissolve poorly in water but readily in organic solvents; thus, fatty acids are hydrophobic. On the other hand, the head groups of most phospholipids are charged or polar. These head groups interact well with water and consequently are very water soluble. Thus, the head groups are hydrophilic. Because phospholipids combine hydrophilic heads with hydrophobic tails, their interaction with water is referred to as amphipathic. When mixed with water, phospholipids organize themselves into structures that prevent their hydrophobic tails from making contact with water while simultaneously permitting their hydrophilic head groups to be fully dissolved. When added to water at fairly low concentrations, phospholipids form a monolayer (Fig. 2-1C) on the water’s surface at the air-water interface. It is energetically less costly to the

B—PHOSPHOLIPID ICON

A—PHOSPHATIDYLETHANOLAMINE

This icon is used in this text to represent this and other phospholipid molecules.

+

NH3

Ethanolamine

CH2 CH2

C—MONOLAYER

O

Phosphate O

P

Hydrophobic lipid tails Hydrophilic head groups

–

O

O

Glycerol

CH2

CH

O

O

C CH2

O

C

CH2

Water

O

CH2

D—PHOSPHOLIPID BILAYER

Fatty acid

In an aqueous environment, polar hydrophilic head groups orient toward the polar water… …and nonpolar (hydrophobic) tails orient away from the water. Thus, a phospholipid bilayer is formed.

R1

Figure 2-1

Phospholipids.

R2

Chapter 2 • Functional Organization of the Cell

system for the hydrophobic tails to stick up in the air than to interact with the solvent. At higher concentrations, phospholipids assemble into micelles. The hydrophilic head groups form the surfaces of these small spheres, whereas the hydrophobic tails point toward their centers. In this geometry, the tails are protected from any contact with water and instead are able to participate in energetically favorable interactions among themselves. At still higher concentrations, phospholipids spontaneously form bilayers (Fig. 2-1D). In these structures, the phospholipid molecules arrange themselves into two parallel sheets or leaflets that face each other tail to tail. The surfaces of the bilayer are composed of hydrophilic head groups; the hydrophobic tails form the center of the sandwich. The hydrophilic surfaces insulate the hydrophobic tails from contact with the solvent, leaving the tails free to associate exclusively with one another. The physical characteristics of a lipid bilayer largely depend on the chemical composition of its constituent phospholipid molecules. For example, the width of the bilayer is determined by the length of the fatty acid side chains. Dihexadecanoic phospholipids (whose two fatty acid chains are each 16 carbons long) produce bilayers that are 2.47 nm wide; ditetradecanoic phospholipids (bearing 14-carbon fatty acids) generate 2.3-nm bilayers. Similarly, the nature of the head groups determines how densely packed adjacent phospholipid molecules are in each leaflet of the membrane. Detergents can dissolve phospholipid membranes because like the phospholipids themselves, they are amphipathic. They possess very hydrophilic head groups and hydrophobic tails and are water soluble at much higher concentrations than are the phospholipids. When mixed together in aqueous solutions, detergent and phospholipid molecules interact through their hydrophobic tails, and the resulting complexes are water soluble, either as individual dimers or in mixed micelles. Therefore, adding sufficient concentrations of detergent to phospholipid bilayer membranes disrupts the membranes and dissolves the lipids. Detergents are extremely useful tools in research into the structure and composition of lipid membranes. The diffusion of individual lipids within a leaflet of a bilayer is determined by the chemical makeup of its constituents Despite its highly organized appearance, a phospholipid bilayer is a fluid structure. An individual phospholipid molecule is free to diffuse within the entire leaflet in which it resides. The rate at which this two-dimensional diffusion occurs is extremely temperature dependent. At high temperatures, the thermal energy of any given lipid molecule is greater than the interaction energy that would tend to hold adjacent lipid molecules together. Under these conditions, lateral diffusion can proceed rapidly, and the lipid is said to be in the sol state. At lower temperatures, interaction energies exceed the thermal energies of most individual molecules. Thus, phospholipids diffuse slowly because they lack the energy to free themselves from the embraces of their neighbors. This behavior is characteristic of the gel state.

The temperature at which the bilayer membrane converts from the gel to the sol phase (and vice versa) is referred to as the transition temperature. The transition temperature is another characteristic that depends on the chemical makeup of the phospholipids in the bilayer. Phospholipids with long, saturated fatty acid chains can extensively interact with one another. Consequently, a fair amount of thermal energy is required to overcome these interactions and permit diffusion. Not surprisingly, such bilayers have relatively high transition temperatures. For example, the transition temperature for dioctadecanoic phosphatidylcholine (which has two 18-carbon fatty acid chains, fully saturated) is 55.5°C. In contrast, phospholipids that have shorter fatty acid chains or double bonds (which introduce kinks) cannot line up next to each other as well and hence do not interact as well. Considerably less energy is required to induce them to participate in diffusion. For example, if we reduce the length of the carbon chain from 18 to 14, the transition temperature falls to 23°C. If we retain 18 carbons but introduce a single, double bond (making the fatty acid chains monounsaturated), the transition temperature also falls dramatically. By mixing other types of lipid molecules into phospholipid bilayers, we can markedly alter the membrane’s fluidity properties. The glycerol-based phospholipids, the most common membrane lipids, include the phosphatidylethanolamines described earlier (Fig. 2-1A) as well as the phosphatidylinositols (Fig. 2-2A), phosphatidylserines (Fig. 2-2B), and phosphatidylcholines (Fig. 2-2C). The second major class of membrane lipids, the sphingolipids (derivatives of sphingosine), are made up of three subgroups: sphingomyelins (Fig. 2-2D), glycosphingolipids such as the galactocerebrosides (Fig. 2-2E), and gangliosides (not shown). Cholesterol (Fig. 2-2F) is another important membrane lipid. Because these other molecules are not shaped exactly like the glycerol-based phospholipids, they participate to different degrees in intermolecular interactions with phospholipid side chains. The presence of these alternative lipids changes the strength of the interactions that prevent lipid molecules from diffusing. Consequently, the membrane has a different fluidity and a different transition temperature. This behavior is especially characteristic of the cholesterol molecule, whose rigid steroid ring binds to and partially immobilizes fatty acid side chains. Therefore, at modest concentrations, cholesterol decreases fluidity. However, when it is present in high concentrations, cholesterol can substantially disrupt the ability of the phospholipids to interact among themselves, which increases fluidity and lowers the gel-sol transition temperature. This issue is significant because animal cell plasma membranes can contain substantial quantities of cholesterol. Bilayers composed of several different lipids do not undergo the transition from gel to sol at a single, well-defined temperature. Instead, they interconvert more gradually over a temperature range that is defined by the composition of the mixture. Within this transition range in such multicomponent bilayers, the membrane can become divided into compositionally distinct zones. The phospholipids with long-chain, saturated fatty acids will adhere to one another relatively tightly, which results in the formation of regions with “gel-like” properties. Phospholipids bearing

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Section II • Physiology of Cells and Molecules

A

PHOSPHATIDYLINOSITOL

B

PHOSPHATIDYLSERINE

C

PHOSPHATIDYLCHOLINE CH3

+

NH3 OH

OH

H

Serine Inositol

H3C COO

C

–

OH O

CH3

CH2

Choline

CH2

HO

N +

CH2

O

O

OH O

Phosphate Glycerol

O

CH

O

O O

C

–

O

O

P

O

CH2

C

–

O

P

O

CH2

CH2

CH

O

O O

C

O

C

–

O

P O

CH2

CH2

CH

O

O

C

O

O

C

CH2

CH2

CH2

CH2

CH2

CH2

R1

R2

R1

R2

R1

R2

CH2

O

Fatty acid

D

SPHINGOMYELIN

E

GALACTOCEREBROSIDE

CH3 CH3

Choline

+

N

Galactose

OH

O OH CH2

Sphingosine

H

CH

CH

N

CH

–

CH3

O

P O

Sphingosine

OH

OH

CH2 O

CHOLESTEROL

O

HO

CH3

CH2

O

F

CH2OH

OH

CH2 H

CH

CH

N

CH

C

O

C

O

CH3 CH3

CH

CH CH2

CH2 CH2 CH2

CH

CH2

H3C

CH CH3

Figure 2-2

Structures of some common membrane lipids.

short-chain, unsaturated fatty acids will be excluded from these regions and migrate to sol-like regions. Hence, “lakes” of lipids with markedly different physical properties can exist side-by-side in the plane of a phospholipid membrane. Thus, the same thermodynamic forces that form the elegant bilayer structure can partition distinct lipid domains within the bilayer. As discussed later, the segregation of lipid lakes in the plane of the membrane may be important for sorting membrane proteins to different parts of the cell.

Although phospholipids can diffuse in the plane of a lipid bilayer membrane, they do not diffuse between adjacent leaflets (Fig. 2-3). The rate at which phospholipids spontaneously “flip-flop” from one leaflet of a bilayer to the other is extremely low. As mentioned earlier, the center of a bilayer membrane consists of the fatty acid tails of the phospholipid molecules and is an extremely hydrophobic environment. For a phospholipid molecule to jump from one leaflet to the other, its highly hydrophilic head group would have to transit

Chapter 2 • Functional Organization of the Cell

PM

Phospholipids can move laterally, rotate, or flex. Rarely do they flip to the other leaflet.

ER

PM M

Cholesterol aids in stiffening the membrane and can flip easily.

Figure 2-3

Mobility of lipids within a bilayer.

this central hydrophobic core, which would have an extremely high energy cost. This caveat does not apply to cholesterol (Fig. 2-3), whose polar head is a single hydroxyl group. The energy cost of dragging this small polar hydroxyl group through the bilayer is relatively low, thus permitting relatively rapid cholesterol flip-flop.

E

Phospholipid bilayer membranes are impermeable to charged molecules The lipid bilayer is ideally suited to separate two aqueous compartments. Its hydrophilic head groups interact well with water at both membrane surfaces, whereas the hydrophobic center ensures that the energetic cost of crossing the membrane is prohibitive for charged atoms or molecules. Pure phospholipid bilayer membranes are extremely impermeable to almost any charged water-soluble substance. Ions such as Na+, K+, Cl−, and Ca2+ are insoluble in the hydrophobic membrane core and consequently cannot travel from the aqueous environment on one side of the membrane to the aqueous environment on the opposite side. The same is true of large water-soluble molecules, such as proteins, nucleic acids, sugars, and nucleotides. Whereas phospholipid membranes are impermeable to water-soluble molecules, small uncharged polar molecules can cross fairly freely. This is often true for O2, CO2, NH3, and, remarkably, water itself. Water molecules may, at least in part, traverse the membrane through transient cracks between the hydrophobic tails of the phospholipids, without having to surmount an enormous energetic barrier. The degree of water permeability (and perhaps that of CO2 and NH3 as well) varies extensively with lipid composition; some phospholipids (especially those with short or kinked fatty acid chains) permit a much greater rate of transbilayer water diffusion than others do. The plasma membrane is a bilayer As may be inferred from the preceding discussion, the membrane at the cell surface is, in fact, a phospholipid bilayer. The truth of this statement was established by a remarkably straightforward experiment. In 1925, Gorter and Grendel measured the surface area of the lipids they extracted from erythrocyte plasma membranes. They used a device called a Langmuir trough in which the lipids are allowed to line up

Figure 2-4 Transmission electron micrograph of a cell membrane. The photograph shows two adjacent cells of the pancreas of a frog (magnification ×43,000). The inset is a high-magnification view (×216,000) of the plasma membranes (PM) of the cells. Note that each membrane includes two dense layers with an intermediate layer of lower density. The dense layers represent the interaction of the polar head groups of the phospholipids with the OsO4 used to stain the preparation. ER, endoplasmic reticulum; M, mitochondrion. (From Porter KR, Bonneville MR: Fine Structure of Cells and Tissues, 4th ed. Philadelphia: Lea & Febiger, 1973.)

at an air-water interface (Fig. 2-1C) and are then packed together into a continuous monolayer by a sliding bar that decreases the surface available to them. The area of the monolayer that was created by the erythrocyte lipids was exactly twice the surface area of the erythrocytes from which they were derived. Therefore, the plasma membrane must be a bilayer. Confirmation of the bilayer structure of biological membranes has come from x-ray diffraction studies performed on the repetitive whorls of membrane that form the myelin sheaths surrounding neuronal axons (see Chapter 11). The membrane’s bilayer structure can be visualized directly in the high-magnification electron micrograph depicted in Figure 2-4. The osmium tetraoxide molecule (OsO4), with which the membrane is stained, binds to the head groups of phospholipids. Thus, both surfaces of a phospholipid bilayer appear black in electron micrographs, whereas the membrane’s unstained central core appears white. The phospholipid compositions of the two leaflets of the plasma membrane are not identical. Labeling studies performed on erythrocyte plasma membranes reveal that the

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Section II • Physiology of Cells and Molecules

surface that faces the cytoplasm contains phosphatidylethanolamine and phosphatidylserine, whereas the outwardfacing leaflet is composed almost exclusively of phosphatidylcholine. As is discussed later in this chapter, this asymmetry is created during the biosynthesis of the phospholipid molecules. It is not entirely clear what advantage this distribution provides to the cell. It appears likely that the interactions between certain proteins and the plasma membrane may require this segregation. The lipid asymmetry may be especially important for those phospholipids that are involved in second-messenger cascades (see Chapter 3). Finally, the phospholipids that are characteristic of animal cell plasma membranes generally have one saturated and one unsaturated fatty acid residue. Consequently, they are less likely to partition into sol-like or gel-like lipid domains than are phospholipids that bear identical fatty acid chains. Membrane proteins can be integrally or peripherally associated with the plasma membrane The demonstration that the plasma membrane’s lipid components form a bilayer leaves open the question of how the membrane’s protein constituents are organized. Membrane proteins can belong to either of two broad classes, peripheral or integral. Peripherally associated membrane proteins are neither embedded within the membrane nor attached to it by covalent bonds; instead, they adhere tightly to the cytoplasmic or extracellular surfaces of the plasma membrane (Fig. 2-5A). They can be removed from the membrane,

Peripheral protein

however, by mild treatments that disrupt ionic bonds (very high salt concentrations) or hydrogen bonds (very low salt concentrations). In contrast, integral membrane proteins are intimately associated with the lipid bilayer. They cannot be eluted from the membrane by these high- or low-salt washes. To dislodge integral membrane proteins, the membrane itself must be dissolved by adding detergents. Integral membrane proteins can be associated with the lipid bilayer in any of three ways. First, some proteins actually span the lipid bilayer once or several times (Fig. 2-5B, C) and hence are referred to as transmembrane proteins. Experiments performed on erythrocyte membranes reveal that these proteins can be labeled with protein-tagging reagents applied to either side of the bilayer. The second group of integral membrane proteins is embedded in the bilayer without actually crossing it (Fig. 25D). A third group of membrane-associated proteins is not actually embedded in the bilayer at all. Instead, these lipidanchored proteins are attached to the membrane by a covalent bond that links them either to a lipid component of the membrane or to a fatty acid derivative that intercalates into the membrane. For example, proteins can be linked to a special type of glycosylated phospholipid molecule (Fig. 25E), which is most often glycosylphosphatidylinositol (GPI), on the outer leaflet of the membrane. This family is referred to collectively as the glycophospholipid-linked proteins. Another example is a direct linkage to a fatty acid (e.g., a myristyl group) or a prenyl (e.g., farnesyl) group that intercalates into the inner leaflet of the membrane (Fig. 2-5F).

Integral proteins

Extracellular space

Some proteins are linked to membrane phospholipids via an oligosaccharide...

Most integral membrane proteins have membrane-spanning α-helical domains of about 20 amino acids.

Peripheral proteins are noncovalently bonded with integral proteins.

Some have multiple membranespanning domains.

A

B

C

E

D H N

Integral protein

P

Figure 2-5

Classes of membrane proteins. In E, protein is coupled by a GPI linkage.

O

P

F

Cytosol

C

…or are linked directly to fatty acids or prenyl groups.

Chapter 2 • Functional Organization of the Cell

The membrane-spanning portions of transmembrane proteins are usually hydrophobic a helices How can membrane-spanning proteins remain stably associated with the bilayer in a conformation that requires at least some portion of their amino acid sequence to be in continuous contact with the membrane’s hydrophobic central core? The answer to this question can be found in the special structures of those protein domains that actually span the membrane. The side chains of the eight amino acids listed in the upper portion of Table 2-1 are hydrophobic. These aromatic or uncharged aliphatic groups are almost as difficult to solvate in water as are the fatty acid side chains of the membrane phospholipids themselves. Not surprisingly, therefore, these hydrophobic side chains are quite comfortable in the hydrophobic environment of the bilayer core. Most membrane-spanning segments—that is, the short stretch of amino acids that passes through the membrane once—are composed mainly of these nonpolar amino acids, in concert with polar, uncharged amino acids. The hydrophobic, membrane-spanning segments of transmembrane proteins are specially adapted to the hydrophobic milieu in which they reside. The phospholipid molecules of the membrane bilayer actually protect these portions of transmembrane proteins from energetically unfavorable interactions with the aqueous environment. Transmembrane proteins tend to be extremely insoluble in water. If we separate the membrane-spanning segments of these proteins from the amphipathic phospholipids that surround them, these hydrophobic sequences tend to interact tightly with one another rather than with water. The resulting large protein aggregates are generally insoluble and precipitate out of solution. If, however, we disrupt the phospholipid membrane by adding detergent, the amphipathic detergent molecules can substitute for the phospholipids. The hydrophobic membrane-spanning sequences remain insulated from interactions with the aqueous solvent, and the proteins remain soluble as components of detergent micelles. This ability of detergents to remove transmembrane proteins from the lipid bilayer—while maintaining the solubility and native architectures of these proteins—has proved important for purifying individual membrane proteins. Transmembrane proteins can have a single membranespanning segment (Fig. 2-5B) or several (Fig. 2-5C). Those with a single transmembrane segment can be oriented with either their amino (N) or their carboxyl (C) termini facing the extracellular space. Multispanning membrane proteins weave through the membrane like a thread through cloth. Again, the N or C termini can be exposed to either the cytoplasmic or extracellular compartments. The pattern with which the transmembrane protein weaves across the lipid bilayer defines its membrane topology. The amino acid sequences of membrane-spanning segments tend to form α helices, with ∼3.6 amino acids per turn of the helix (Fig. 2-5B). In this conformation, the polar atoms of the peptide backbone are maximally hydrogen bonded to one another—from one turn of the helix to the next—so they do not require the solvent to contribute

hydrogen bond partners. Hence, this structure ensures the solubility of the membrane-spanning sequence in the hydrophobic environment of the membrane. Whereas most transmembrane proteins appear to traverse the membrane with α-helical spans, it is clear that an intriguing subset of membrane polypeptides makes use of a very different structure. The best studied member of this class is the porin protein, which serves as a channel in bacterial membranes. As discussed in Chapter 5, the membrane-spanning portions of porin are arranged as a β barrel. In the case of multispanning membrane proteins, their transmembrane helices probably pack together tightly (Fig. 2-5C). Molecular analysis of a number of known membranespanning sequences has helped in the development of algorithms predicting the likelihood that a given amino acid sequence can span the membrane. These algorithms are widely used to assess the likelihood that newly identified genes encode transmembrane proteins and to predict the number and location of membrane-spanning segments. Many membrane proteins form tight, noncovalent associations with other membrane proteins in the plane of the bilayer. These multimeric proteins can be composed of a single type of polypeptide or of mixtures of two or more different proteins. The side-to-side interactions that hold these complexes together can involve the membrane-spanning segments or regions of the proteins that protrude at either surface of the bilayer. By assembling into multimeric complexes, membrane proteins can increase their stability. They can also increase the variety and complexity of the functions that they are capable of performing. Some membrane proteins are mobile in the plane of the bilayer As is true for phospholipid molecules (Fig. 2-3), some transmembrane proteins can also diffuse within the surface of the membrane. In the absence of any protein-protein attachments, transmembrane proteins are free to diffuse over the entire surface of a membrane. This fact was demonstrated by Frye and Edidin in 1970 (Fig. 2-6). They labeled the surface proteins of a population of mouse lymphocytes with a lectin (a plant protein that binds strongly to certain sugar groups attached to proteins) that was linked to the fluorescent dye fluorescein. They also tagged the surface proteins of a second population of human lymphocytes with a lectin that was conjugated to a different fluorescent dye, rhodamine. Because fluorescein glows green and rhodamine glows red when excited by the light of the appropriate wavelengths, these labeling molecules can be easily distinguished from one another in a fluorescence microscope. Frye and Edidin mixed the two lymphocyte populations and treated them with a reagent that caused the cells to fuse to each other. Immediately after fusion, the labeled surface proteins of the newly joined cells remained separate; half of the fused cell surface appeared red, whereas the other half appeared green. During a period of ∼30 minutes, however, the green and red protein labels intermixed until the entire surface of the fused cell was covered with both labeling molecules. The rate at which this intermingling occurred increased with temperature, which is not surprising, given the temperature dependence of membrane fluidity.

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Section II • Physiology of Cells and Molecules

Table 2-1

Classification of the Amino Acids Based on the Chemistry of Their Side Chains

Nonpolar

Name

3-Letter Code

Single-Letter Code

Structure of the Side Chain

Hydropathy Index*

Alanine Valine Leucine Isoleucine

Ala Val Leu Ile

A V L I

—CH3 —CH(CH3)2 —CH2CH(CH3)2 CH CH2 CH3

+1.8 +4.2 +3.8 +4.5

CH3 Proline

Pro

P C H

−1.6

H2 C CH2 N CH2

Phenylalanine

Phe

F

CH2

Tryptophan

Trp

W

CH2

+2.8 −0.9

N H

Polar uncharged

Methionine

Met

M

—CH2—CH2—S—CH3

+1.9

Glycine

Gly

G

—H

−0.4

Serine

Ser

S

—CH2—OH

−0.8

Threonine

Thr

T

CH CH3

−0.7

OH +2.5

Cysteine

Cys

C

Tyrosine

Tyr

Y

CH2

Asparagine

Asn

N

CH2 C O

—CH2—SH

−1.3

OH

−3.5

NH2 Glutamine

Gln

Q

CH2

CH2 C

−3.5

O

NH2 Polar, charged, acidic

Aspartate

Asp

D

CH2 C

−3.5

O

O–

Polar, charged, basic

Glutamate

Glu

E

Lysine

Lys

K

Arginine

Arg

R

CH2 C O O–

−3.5

—CH2—CH2—CH2—CH2—NH+3

−3.9

CH2

CH2

CH2

CH2 NH C NH2

−4.5

NH+2 Histidine

His

H

CH2

N

N

−3.2

H *Kyte and Doolittle generated these values (arbitrary scale from -4.5 to +4.5) by averaging two kinds of data. The first is an index of the energy that is required to transfer the side chain from the vapor phase into water. The second indicates how likely it is to find the side chain buried in (as opposed to being on the surface of) 12 globular proteins, whose structures were solved by x-ray crystallography. A positive value indicates that the side chain is hydrophobic. Note: The portion shown in red is part of the peptide backbone. From Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157:105-132.

Chapter 2 • Functional Organization of the Cell

Rhodamine-tagged membrane proteins

Figure 2-6 Diffusion of membrane proteins within the plane of the cell membrane. The surface proteins of a human lymphocyte are tagged with a lectin conjugated to rhodamine, a fluorescent dye; the surface proteins of a mouse lymphocyte are tagged with a lectin linked to fluorescein, another fluorescent dye. Immediately after fusion of the two cells, the labeled surface proteins remained segregated. However, the membrane proteins intermingled during a period of ∼30 minutes.

Cell fusion

Immediately after cell fusion Fluorescein-tagged membrane proteins

After about 1/2 hour, tagged proteins spread throughout the membrane.

Because transmembrane proteins are large molecules, their diffusion in the plane of the membrane is much slower than that of lipids. Even the fastest proteins diffuse ∼1000 times more slowly than the average phospholipid. The diffusion of many transmembrane proteins appears to be further impeded by their attachments to the cytoskeleton, just below the surface of the membrane. Tight binding to this meshwork can render proteins essentially immobile. Other transmembrane proteins appear to travel in the plane of the membrane by directed processes that are much faster and less directionally random than diffusion is. Motor proteins that are associated with the cytoplasmic cytoskeleton (discussed later) appear to grab onto certain transmembrane proteins, dragging them in the plane of the membrane like toy boats on strings. Finally, like phospholipids, proteins can diffuse only in the plane of the bilayer. They cannot flip-flop across it. The energetic barrier to dragging a transmembrane protein’s hydrophilic cytoplasmic and extracellular domains across the bilayer’s hydrophobic core is very difficult to surmount. Thus, a membrane protein’s topology does not change over its life span.

soluble hormones such as epinephrine to influence cellular behavior, their presence in the extracellular fluid compartment must be made known to the various intracellular mechanisms whose behaviors they modulate. The interaction of a hormone with the extracellular portion of the hormone receptor, which forms a high-affinity binding site, produces conformational changes within the receptor protein that extend through the membrane-spanning domain to the intracellular domain of the receptor. As a consequence, the intracellular domain either becomes enzymatically active or can interact with cytoplasmic proteins that are involved in the generation of so-called second messengers. Either mechanism completes the transmission of the hormone signal across the membrane. The transmembrane disposition of a hormone receptor thus creates a single, continuous communication medium that is capable of conveying, through its own structural modifications, information from the environment to the cellular interior. The process of transmembrane signal transduction is discussed in Chapter 3.

FUNCTION OF MEMBRANE PROTEINS

Cells can also exploit integral membrane proteins as adhesion molecules that form physical contacts with the surrounding extracellular matrix (i.e., cell-matrix adhesion molecules) or with their cellular neighbors (i.e., cell-cell adhesion molecules). These attachments can be extremely important in regulating the shape, growth, and differentiation of cells. The nature and extent of these attachments must be communicated to the cell interior so that the cell can adapt appropriately to the physical constraints and cues that are provided by its immediate surroundings. Numerous classes of transmembrane proteins are involved in these communication processes. The integrins are examples of matrix receptors or cell matrix adhesion molecules. They comprise a large family of transmembrane proteins that link cells to components of the extracellular matrix (e.g., fibronectin, laminin) at adhesion plaques (Fig. 2-7B). These linkages produce conformational changes in the integrin molecules that are transmitted to their cytoplasmic tails. These tails, in turn, communicate the linkage events to

Integral membrane proteins can serve as receptors All communication between a cell and its environment must involve or at least pass through the plasma membrane. For the purposes of this discussion, we define communication rather broadly as the exchange of any signal between the cell and its surroundings. Except for lipid-soluble signaling molecules such as steroid hormones, essentially all communication functions served by the plasma membrane occur through membrane proteins. From an engineering perspective, membrane proteins are perfectly situated to transmit signals because they form a single, continuous link between the two compartments that are separated by the membrane. Ligand-binding receptors comprise the group of transmembrane proteins that perhaps most clearly illustrate the concept of transmembrane signaling (Fig. 2-7A). For water-

Integral membrane proteins can serve as adhesion molecules

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Section II • Physiology of Cells and Molecules

A

LIGAND-BINDING RECEPTOR

B CELL-MATRIX ADHESION MOLECULE (INTEGRIN) Matrix-binding domain

N Ligand-binding domain

Extracellular matrix 7 transmembrane segments Transmembrane segments

The cytoplasmic domains are linked to intracellular proteins.

The cytoplasmic domain interacts with the intracellular proteins. C

The helical domains form a compact unit in the membrane.

Figure 2-7 Integral membrane proteins that transmit signals from the outside to the inside of a cell. A, The ligand may be a hormone, a growth factor, a neurotransmitter, an odorant, or another local mediator. B, An integrin is an adhesion molecule that attaches the cell to the extracellular matrix.

various structural and signaling molecules that participate in formulating a cell’s response to its physical environment. In contrast to matrix receptors, which attach cells to the extracellular matrix, several enormous superfamilies of cellcell adhesion molecules attach cells to each other. These cell-cell adhesion molecules include the Ca2+-dependent cell adhesion molecules (cadherins) and Ca2+-independent neural cell adhesion molecules (N-CAMs). The cadherins are glycoproteins (i.e., proteins with sugars attached) with one membrane-spanning segment and a large extracellular domain that binds Ca2+. The N-CAMs, on the other hand, generally are members of the immunoglobulin superfamily. The two classes of cell-cell adhesion molecules mediate similar sorts of transmembrane signals that help organize the cytoplasm and control gene expression in response to intercellular contacts. Some cell-cell adhesion molecules belong to the GPI-linked class of membrane proteins. These polypeptides lack a transmembrane and cytoplasmic tail. It is not clear, therefore, how (or if) interactions mediated by this unique class of adhesion molecules are communicated to the cell interior. Adhesion molecules orchestrate processes that are as diverse as the directed migration of immune cells and the guidance of axons in the developing nervous system. Loss of cell-cell and cell-matrix adhesion is a hallmark of metastatic tumor cells.

Integral membrane proteins can carry out the transmembrane movement of water-soluble substances Earlier in this discussion, we noted that a pure phospholipid bilayer does not have the permeability properties that are normally associated with animal cell plasma membranes. Pure phospholipid bilayers also lack the ability to transport substances uphill. Transmembrane proteins endow biological membranes with these capabilities. Ions and other membrane-impermeable substances can cross the bilayer with the assistance of transmembrane proteins that serve as pores, channels, carriers, and pumps. Pores and channels serve as conduits that allow water, specific ions, or even very large proteins to flow passively through the bilayer. Carriers can either facilitate the transport of a specific molecule across the membrane or couple the transport of a molecule to that of other solutes. Pumps use the energy that is released through the hydrolysis of adenosine triphosphate (ATP) to drive the transport of substances into or out of cells against energy gradients. Each of these important classes of proteins is discussed in Chapter 5. Channels, carriers, and pumps succeed in allowing hydrophilic substances to cross the membrane by creating a hydrophilic pathway in the bilayer. Previously, we asserted that membrane-spanning segments are as hydrophobic as the fatty acids that surround them. How is it possible for these

Chapter 2 • Functional Organization of the Cell

hydrophobic membrane-spanning domains to produce the hydrophilic pathways that permit the passage of ions through the membrane? The solution to this puzzle appears to be that the α helices that make up these membrane-spanning segments are amphipathic. That is, they possesses both hydrophobic and hydrophilic domains. For each α helix, the helical turns produce alignments of amino acids that are spaced at regular intervals in the sequence. Thus, it is possible to align all the hydrophilic or hydrophobic amino acids along a single edge of the helix. In amphipathic helices, hydrophobic amino acids alternate with hydrophilic residues at regular intervals of approximately three or four amino acids (recall that there are ∼3.6 amino acids per turn of the helix). Thus, as the helices pack together, side-by-side, the resultant membrane protein has distinct hydrophilic and hydrophobic surfaces. The hydrophobic surfaces of each helix will face either the membrane lipid or the hydrophobic surfaces of neighboring helices. Similarly, the hydrophilic surfaces of each helix will face a common central pore through which water-soluble particles can move. Depending on how the protein regulates access to this pore, the protein could be a channel, a carrier, or a pump. The mix of hydrophilic amino acids that line the pore presumably determines, at least in part, the nature of the substances that the pore can accommodate. In some instances, the amphipathic helices that line the pore are contributed by several distinct proteins—or subunits—that assemble into a single multimeric complex. Figure 2-8 shows an example of a type of K+ channel that is discussed in Chapter 7. This channel is formed by the apposition of four identical subunits, each of which has six membrane-spanning segments. The pore of this channel is created by the amphipathic helices

as well as by short, hydrophilic loops (P loops) contributed by each of the four subunits. Integral membrane proteins can also be enzymes Ion pumps are actually enzymes. They catalyze the hydrolysis of ATP and use the energy released by that reaction to drive ion transport. Many other classes of proteins that are embedded in cell membranes function as enzymes as well. Membrane-bound enzymes are especially prevalent in the cells of the intestine, which participate in the final stages of nutrient digestion and absorption (see Chapter 45). These enzymes— located on the side of the intestinal cells that faces the lumen of the intestine—break down small polysaccharides into single sugars, or break down polypeptides into shorter polypeptides or amino acids, so that they can be imported into the cells. By embedding these enzymes in the plasma membrane, the cell can generate the final products of digestion close to the transport proteins that mediate the uptake of these nutrient molecules. This theme is repeated in numerous other cell types. Thus, the membrane can serve as an extremely efficient two-dimensional reaction center for multistep processes that involve enzymatic reactions or transport. Many of the GPI-linked proteins are enzymes. Several of the enzymatic activities that are classically thought of as extracellular markers of the plasma membrane, such as alkaline phosphatase and 5¢-nucleotidase, are anchored to the external leaflet of the bilayer by covalent attachment to a GPI. The biological utility of this arrangement has yet to be determined. However, the GPI linkage is itself a substrate for

K+

Ion passes through the pore that is surrounded by the subunits.

A single amphipathic helix with a hydrophilic surface along one edge and hydrophobic surfaces elsewhere. Channels are made from multiple subunits or pseudomultimeric proteins. P loop

Figure 2-8 Amphipathic α helices interacting to form a channel through the cell membrane. This is an example of a potassium channel.

For some classes of channels, each subunit has 6 transmembrane helices. Parts of the molecule facing the pore have hydrophilic surfaces.

Hydrophilic

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Section II • Physiology of Cells and Molecules

enzymatic cleavage. Phospholipase C, which is present at appreciable levels in the serum, can cleave the covalent bond between the protein and its lipid anchor, thereby releasing the protein from the membrane. The released protein subsequently behaves like a soluble polypeptide. Integral membrane proteins can participate in intracellular signaling Some integral proteins associate with the cytoplasmic surface of the plasma membrane by covalently attaching to fatty acids or prenyl groups that in turn intercalate into the lipid bilayer (Fig. 2-5F). The fatty acids or prenyl groups act as hydrophobic tails that anchor an otherwise soluble protein to the bilayer. These proteins are all located at the intracellular leaflet of the membrane bilayer and often participate in intracellular signaling and growth regulation pathways. The family of lipid-linked proteins includes the small and heterotrimeric guanosine triphosphate (GTP)–binding proteins, kinases, and oncogene products (see Chapter 3). Many of these proteins are involved in relaying the signals that are received at the cell surface to the effector machinery within the cell interior. Their association with the membrane, therefore, brings these proteins close to the cytoplasmic sides of receptors that transmit signals from the cell exterior across the bilayer. The medical relevance of this type of membrane association is beginning to be appreciated. For example,

Spectrin

Ankyrin

denying certain oncogene products their lipid modifications—and hence their membrane attachment—eliminates their ability to induce tumorigenic transformation. Peripheral membrane proteins participate in intracellular signaling and can form a submembranous cytoskeleton Peripheral membrane proteins attach loosely to the lipid bilayer but are not embedded within it. Their association with the membrane can take one of two forms. First, some proteins interact through ionic interactions with phospholipid head groups. Many of these head groups are positively or negatively charged and thus can participate in salt bridges with adherent proteins. For a second group of peripheral membrane proteins, attachment is based on the direct binding of peripheral membrane proteins to the extracellular or cytoplasmic surfaces of integral membrane proteins (Fig. 2-5A). This form of attachment is epitomized by the cytoskeleton. For instance, the cytoplasmic surface of the erythrocyte plasma membrane is in close apposition to a dense meshwork of interlocking protein strands known as the subcortical cytoskeleton. It consists of a long, fibrillar molecule called spectrin, short polymers of the cytoskeletal protein actin, and other proteins including ankyrin and band 4.1 (Fig. 2-9).

Actin p55

Protein 4.1

Protein 4.2 CD47

Rh complex

Glycophorin C

Glycophorin A

Glycophorin B

Band 3

Figure 2-9 Attachments of the cell membrane to the submembranous cytoskeleton in red blood cells. Integral membrane proteins form the bridges that link the cell membrane to the interlocking system of proteins that form the subcortical cytoskeleton.

Chapter 2 • Functional Organization of the Cell

Two closely related isoforms of spectrin (α and β) form dimers, and two of these dimers assemble head-to-head with one another to form spectrin heterotetramers. The tail regions of spectrin bind the globular protein band 4.1, which in turn can bind to actin fibrils. Each actin fibril can associate with more than one molecule of band 4.1 so that, together, spectrin, actin, and band 4.1 assemble into an extensive interlocking matrix. The protein known as ankyrin binds to spectrin as well as to the cytoplasmic domain of band 3, the integral membrane protein responsible for transporting Cl− and HCO3− ions across the erythrocyte membrane. Thus, ankyrin is a peripheral membrane protein that anchors the spectrin-actin meshwork directly to an integral membrane protein of the erythrocyte. The subcortical cytoskeleton provides the erythrocyte plasma membrane with strength and resilience. People who carry mutations in genes encoding their components have erythrocytes that do not have the characteristic biconcave disk shape. These erythrocytes are extremely fragile and are easily torn apart by the shear stresses (see Chapter 17) associated with circulation through capillaries. It would appear, therefore, that the subcortical cytoskeleton forms a scaffolding of peripheral membrane proteins whose direct attachment to transmembrane proteins enhances the bilayer’s structural integrity. The subcortical cytoskeleton is not unique to erythrocytes. Numerous cell types, including neurons and epithelial cells, have submembranous meshworks that consist of proteins very similar to those first described in the erythrocyte. In addition to band 3, transmembrane proteins found in a wide variety of cells (including ion pumps, ion channels, and cell adhesion molecules) bind ankyrin and can thus serve as focal points of cytoskeletal attachment. In polarized cells (e.g., neurons and epithelial cells), the subcortical cytoskeleton appears to play a critically important role in organizing the plasma membrane into morphologically and functionally distinct domains.

CELLULAR ORGANELLES AND THE CYTOSKELETON The cell is composed of discrete organelles that subserve distinct functions When a eukaryotic cell is viewed through a light microscope, a handful of recognizable intracellular structures can be discerned. The intracellular matrix, or cytoplasm, appears grainy, suggesting the presence of components that are too small to be discriminated by this technique. With the much higher magnifications available with an electron microscope, the graininess gives way to clarity that reveals the cell interior to be remarkably complex. Even the simplest nucleated animal cell possesses a wide variety of intricate structures with specific shapes and sizes. These structures are the membrane-enclosed organelles, the functional building blocks of cells. Figure 2-10 illustrates the interior of a typical cell. The largest organelle in this picture is the nucleus, which houses the cell’s complement of genetic information. This structure, which is visible in the light microscope, is usually round or

oblong, although in some cells it displays a complex, lobulated shape. Depending on the cell type, the nucleus can range in diameter from 2 to 20 μm. With some exceptions, including skeletal muscle and certain specialized cells of the immune system, each animal cell has a single nucleus. Surrounding the nucleus is a web of tubules or saccules known as the endoplasmic reticulum (ER). This organelle can exist in either of two forms, rough or smooth. The surfaces of the rough ER tubules are studded with ribosomes, the major sites of protein synthesis. Ribosomes can also exist free in the cytosol. The surfaces of the smooth ER, which participates in lipid synthesis, are not similarly endowed. The ER also serves as a major reservoir for calcium ions. The ER membrane is endowed with a Ca2+ pump that uses the energy released through ATP hydrolysis to drive the transport of Ca2+ from the cytoplasm into the ER lumen (see Chapter 5). This Ca2+ can be rapidly released in response to messenger molecules and plays a major role in intracellular signaling (see Chapter 3). The Golgi complex resembles a stack of pancakes. Each pancake in the stack represents a discrete, flat saccule. The number and size of the saccules in the Golgi stack vary among cell types. The Golgi complex is a processing station that participates in protein maturation and targets newly synthesized proteins to their appropriate subcellular destinations. Perhaps the most intriguing morphological appearance belongs to the mitochondrion, which is essentially a balloon within a balloon. The outer membrane and inner membrane define two distinct internal compartments: the intermembrane space and the matrix space. The surface of the inner membrane is thrown into dramatic folds called cristae. This organelle is ∼0.2 μm in diameter, placing it at the limit of resolution of the light microscope. The mitochondrion is the power plant of the cell, a critical manufacturer of ATP. Many cellular reactions are also catalyzed within the mitochondrion. The cell’s digestive organelle is the lysosome. This large structure frequently contains several smaller round vesicles called exosomes within its internal space. The cytoplasm contains numerous other organelles whose shapes are not quite as distinguishing, including endosomes, peroxisomes, and transport vesicles. Despite their diversity, all cellular organelles are constructed from the same building blocks. Each is composed of a membrane that forms the entire extent of its surface. The membranes of the subcellular organelles are what can be visualized in electron micrographs. The biochemical and physical properties of an organelle’s limiting membrane dictate many of its functional properties. The nucleus stores, replicates, and reads the cell’s genetic information The nucleus serves as a cell’s repository for its complement of chromosomal DNA. To conceive of the nucleus as simply a hermetically sealed vault for genetic information, however, is a gross oversimplification. All of the machinery necessary to maintain, to copy, and to transcribe DNA is in the nucleus, which is the focus of all of the cellular pathways that regulate gene expression and cell division. Transcriptional control is

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Section II • Physiology of Cells and Molecules

ANIMAL CELL

NUCLEUS

Centrioles

Ribosomes Nucleolus

Golgi apparatus Smooth endoplasmic reticulum

Chromatin

Lysosomes: (endosomes, peroxisomes, and transport vesicles)

Rough endoplasmic reticulum

Nuclear lamina

Basket Transporter subunit Scaffold

Nucleoplasmic ring subunit Inner nuclear membrane

MITOCHONDRION

Proteins

Intermembrane space

Cytoplasmic ring subunit

Outer nuclear membrane

Thick filament NUCLEAR PORE COMPLEX Matrix space Outer membrane

Figure 2-10

Inner membrane

Ultrastructure of a typical animal cell.

discussed in Chapter 4. The focus of this section is nuclear structure. The nucleus is surrounded by a double membrane (Fig. 2-10). The outer membrane is studded with ribosomes and is continuous with the membranes of the rough ER. The inner membrane is smooth and faces the intranuclear space, or nucleoplasm. The space between these concentric membranes is continuous with the lumen of the rough ER. The inner and outer nuclear membranes meet at specialized structures known as nuclear pores, which penetrate the nuclear envelope and provide a transport pathway between the cytoplasm and the nuclear interior (see Chapter 5). All RNA transcripts that are produced in the nucleus must pass through nuclear pores to be translated in the cytoplasm. Similarly, all the signaling molecules that influence nuclear function as well as all proteins of the nuclear interior (which are synthesized in the cytoplasm) enter the nucleus through nuclear pores.

Nuclear pores are selective in choosing the molecules that they allow to pass. Cytoplasmic proteins destined for the nuclear interior must be endowed with a nuclear localization sequence to gain entry. Several nuclear localization sequences have been characterized, and all seem to share common structural elements. For example, they all have short stretches of four to eight basic amino acids that can be located anywhere in the protein’s sequence. Evidence implies that the ability of these signals to mediate nuclear localization can be modulated by phosphorylation, which suggests that the entry of proteins into the nucleus may be under the control of the cell’s second-messenger systems. The selectivity of the nuclear pore is surprising, considering its size. The outer diameter of the entire nuclear pore is ∼100 nm, considerably larger than the proteins whose passage it controls. The nuclear pore’s specificity is provided by the nuclear pore complex (NPC), an intricate matrix of protein that is distributed in a highly organized octagonal array. In

Chapter 2 • Functional Organization of the Cell

its resting state, the NPC forms an aqueous channel that is ∼9 nm in diameter, restricting the movement of any protein larger than 60 kDa. However, when it is confronted with a protein bearing a nuclear localization signal or a messenger RNA (mRNA) transcript, the pore complex can dilate to many times this size. The mechanisms by which the pore’s permeability is regulated remain unknown. The NPC has a barrier that prevents the diffusion of intrinsic membrane proteins between the outer and inner membranes of the nuclear envelope. Thus, although the inner and outer nuclear membranes are continuous with one another at nuclear pores, their protein contents remain distinct. Between mitoses, the chromosomal DNA is present in the nucleus as densely packed heterochromatin and more loosely arrayed euchromatin. Chromatin is a complex between DNA and numerous DNA-binding proteins, which organize the chromosome into a chain of tightly folded DNA-protein assemblies called nucleosomes (see Chapter 4). Interspersed within the nucleoplasm are round, dense nucleoli, where the transcription of ribosomal RNA and the assembly of ribosomal subunits appear to occur. The interior surface of the inner nuclear membrane is apposed to a fibrillar protein skeleton referred to as the nuclear lamina. This meshwork, composed of proteins known as lamins, is presumably involved in providing structural support to the nuclear envelope. The nuclear lamina may also play a role in orchestrating nuclear reassembly. During mitosis, the nuclear envelope breaks down into small vesicles, and the contents of the nucleoplasm mix with the cytoplasm. After mitosis, these vesicles fuse with one another to regenerate the double-walled nuclear membrane. The means by which these vesicles find one another and assemble correctly is the subject of intense study. Similarly, the mechanisms involved in maintaining the compositional discreteness of the inner and outer membranes during vesiculation and reassembly have yet to be determined. After reconstitution of the nuclear envelope, the proteins of the nucleoplasm are re-imported from the cytoplasm through the nuclear pores by virtue of their nuclear localization sequences. Lysosomes digest material that is derived from the interior and exterior of the cell In the course of normal daily living, cells accumulate waste. Organelles become damaged and dysfunctional. Proteins denature and aggregate. New materials are constantly being brought into the cells from the extracellular environment through the process of endocytosis (discussed later). In specialized cells of the immune system, such as macrophages, the collection of foreign materials (in the form of pathogens) from the extracellular milieu is the cellular raison d’être. If this material were allowed to accumulate indefinitely, it would ultimately fill the cell and essentially choke it to death. Clearly, cells must have mechanisms for disposing of this waste material. The lysosome is the cell’s trash incinerator. It is filled with a broad assortment of degradative enzymes that can break down most forms of cellular debris. Proton pumps embedded within the lysosome’s limiting membrane ensure that this space is an extremely acidic environment, which aids in protein hydrolysis. A rare group of inherited disorders, called

lysosomal storage diseases (see the box on page 43 about this topic), result from the deficiency of lysosomal enzymes that are involved in the degradation of a variety of substances. The lysosomal membrane is specially adapted to resist digestion by the enzymes and the acid that it encapsulates, thus ensuring that the harsh conditions necessary for efficient degradation are effectively contained. Loss of lysosomal membrane integrity may underlie some clinically important inflammatory conditions, such as gout. Material that has been internalized from the cell exterior by endocytosis is surrounded by the membrane of an endocytic vesicle. To deliver this material to the lysosome, the membranes of the endocytic vesicles fuse with the lysosomal membrane and discharge their cargo into the lysosomal milieu. Intracellular structures that are destined for degradation, such as fragments of organelles, are engulfed by the lysosome in a process called autophagy. Autophagy results in the formation of membrane-enclosed structures within the lysosomal lumen; hence, the lysosome is often referred to as a multivesicular body. The mitochondrion is the site of oxidative energy production Oxygen-dependent ATP production—or oxidative phosphorylation—occurs in the mitochondrion. Like the nucleus, the mitochondrion (Fig. 2-10) is a double-membrane structure. The inner mitochondrial membrane contains the proteins that constitute the electron transport chain, which generates pH and voltage gradients across this membrane. According to the “chemiosmotic” model (see Chapter 5), the inner membrane uses the energy in these gradients to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. The mitochondrion maintains and replicates its own genome. This circular DNA strand encodes mitochondrial transfer RNAs (tRNAs) and (in humans) 13 mitochondrial proteins. Several copies of the mitochondrial genome are located in the inner mitochondrial matrix, which also has all of the machinery necessary to transcribe and to translate this DNA, including ribosomes. Whereas the proteins encoded in mitochondrial DNA contribute to the structure and function of the mitochondrion, they account for a relatively small fraction of total mitochondrial protein. Most mitochondrial proteins are specified by nuclear DNA and are synthesized on cytoplasmic ribosomes. The two mitochondrial membranes enclose two distinct compartments: the intermembrane space and the inner mitochondrial matrix space. The intermembrane space lies between the two membranes; the inner mitochondrial matrix space is completely enclosed by the inner mitochondrial membrane. These compartments have completely different complements of soluble proteins, and the two membranes themselves have extremely different proteins. In addition to its role in energy metabolism, the mitochondrion also serves as a reservoir for intracellular Ca2+. It is not clear whether—under physiological conditions—the mitochondrion releases Ca2+ from this reservoir. The mitochondrial Ca2+ stores are released as a consequence of energy starvation, which leads to cell injury and death. Finally, the

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Section II • Physiology of Cells and Molecules

mitochondrion plays a central role in the process called apoptosis, or programmed cell death (see Chapter 62). Certain external or internal signals can induce the cell to initiate a signaling cascade that leads ultimately to the activation of enzymes that bring about the cell’s demise. One of the pathways that initiates this highly ordered form of cellular suicide depends on the participation of the mitochondrion. Apoptosis plays an extremely important role during tissue development and is also involved in the body’s mechanisms for identifying and destroying cancer cells. The cytoplasm is not amorphous but is organized by the cytoskeleton Our discussion thus far has focused almost exclusively on the cell’s membranous elements. We have treated the cytoplasm as if it were a homogeneous solution in which the organelles and vesicles carry out their functions while floating about unimpeded and at random. Rather, the cytoplasm is enormously complex with an intricate local structure and the capacity for locomotion. The cytoplasmic cytoskeleton is composed of protein filaments that radiate throughout the cell, serving as the beams, struts, and stays that determine cell shape and resilience. On the basis of their appearance in the electron microscope, these filaments were initially divided into several classes (Table 2-2): thick, thin, and intermediate filaments as well as microtubules. Subsequent biochemical analysis has revealed that each of these varieties is composed of distinct polypeptides and differs with respect to its formation, stability, and biological function. Intermediate filaments provide cells with structural support Intermediate filaments are so named because their 8- to 10nm diameters, as measured in the electron microscope, are intermediate between those of the actin thin filaments and

Table 2-2

Components of the Cytoskeleton

Subunits

Diameter (nm)

Intermediate filaments

Tetramer of two coiled dimers

8-10

Microtubules

Heterodimers of α and β tubulin form long protofilaments, 5 nm in diameter

25

Thin filaments

Globular or G-actin, 5 nm in diameter, arranged in a double helix to form fibrous or F-actin

5-8

Thick filaments

Assembly of myosin molecules

10

the myosin thick filaments. As with all of the cytoskeletal filaments that we will discuss, intermediate filaments are polymers that are assembled from individual protein subunits. There is a very large variety of biochemically distinct subunit proteins that are all structurally related to one another and that derive from a single gene family. The expression of these subunit polypeptides can be cell type specific or restricted to specific regions within a cell. Thus, vimentin is found in cells that are derived from mesenchyme, and the closely related glial fibrillary acidic protein is expressed exclusively in glial cells (see Chapter 11). Neurofilament proteins are present in neuronal processes. The keratins are present in epithelial cells as well as in certain epithelially derived structures. The nuclear lamins that form the structural scaffolding of the nuclear envelope are also members of the intermediate filament family. Intermediate filament monomers are themselves fibrillar in structure. They assemble to form long, intercoiled dimers that in turn assemble side-to-side to form the tetrameric subunits. Finally, these tetrameric subunits pack together, end-to-end and side-to-side, to form intermediate filaments. Filament assembly can be regulated by the cell and in some cases appears to be governed by phosphorylation of the subunit polypeptides. Intermediate filaments appear to radiate from and to reinforce areas of a cell that are subject to tensile stress. They emanate from the adhesion plaques that attach cells to their substrata. In epithelial cells, they insert at the desmosomal junctions that attach neighboring cells to one another. The toughness and resilience of the meshworks formed by these filaments is perhaps best illustrated by the keratins, the primary constituents of nails, hair, and the outer layers of skin. Microtubules provide structural support and provide the basis for several types of subcellular motility Microtubules are polymers formed from heterodimers of the proteins α and β tubulin (Fig. 2-11A). These heterodimers assemble head to tail, creating a circumferential wall of a microtubule, which surrounds an empty lumen. Because the tubulin heterodimers assemble with a specific orientation, microtubules are polar structures, and their ends manifest distinct biochemical properties. At one tip of the tubule, designated the plus end, tubulin heterodimers can be added to the growing polymer at three times the rate that this process occurs at the opposite minus end. The relative rates of microtubule growth and depolymerization are controlled in part by an enzymatic activity that is inherent in the tubulin dimer. Tubulin dimers bind to GTP, and in this GTP-bound state they associate more tightly with the growing ends of microtubules. Once a tubulin dimer becomes part of the microtubule, it hydrolyzes the GTP to guanosine diphosphate (GDP), which lowers the binding affinity of the dimer for the tubule and helps hasten disassembly. Consequently, the microtubules can undergo rapid rounds of growth and shrinkage, a behavior termed dynamic instability. Various cytosolic proteins can bind to the ends of microtubules and serve as caps that prevent assembly and disassembly and thus stabilize the structures of the microtubules. A large and diverse family of microtubule-associated proteins appears

Chapter 2 • Functional Organization of the Cell

A MICROTUBULE AND ITS MOLECULAR MOTORS Kinesin Tail

Dynein Light and intermediate chains

Base

Coiled-coil

Head domain β Tubulin α Tubulin

5 nm

Cross section 13 protofilaments form a hollow microtubule.

+

+

+

+

GTP

Longitudinal view

– end

B MICROTUBULE-ORGANIZING CENTER Interphase cell

Head domain

C

Tubulin dimers (heterodimers)

β α

β α

25 nm

Light chain

+ end

GDP

MOTILE CILIUM Nine outer microtubule doublets

Two central microtubules

Tubulin dimers

Dynein arms A

B

13

11

Cell membrane Radial spokes

+

+

+

+

Microtubule organizing centers Astral (centrosomes) microtubules

Microtubule Axenome Dynein arms

+

Kinetochore microtubules

+

+

+ +

Metaphase cell

+

+

Cilium

+ + +

+

+

+

+

+

+

+

+

+

ATP

Figure 2-11 Microtubules. A, Heterodimers of α and β tubulin form long protofilaments, 13 of which surround the hollow core of a microtubule. The microtubule grows more rapidly at its plus end. The molecular motor dynein moves along the microtubule in the plus-to-minus direction, whereas the molecular motor kinesin moves in the opposite direction. ATP is the fuel for each of these motors. B, The microtubules originate from a microtubule-organizing center or centrosome, which generally consists of two centrioles (green cylinders). C, A motile cilium can actively bend as its microtubules slide past each other. The molecular motor dynein produces this motion, fueled by ATP.

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Section II • Physiology of Cells and Molecules

to modulate not only the stability of the tubules but also their capacity to interact with other intracellular components. In most cells, all of the microtubules originate from the microtubule-organizing center or centrosome. This structure generally consists of two centrioles, each of which is a small (∼0.5 μm) assembly of nine triplet microtubules that are arranged obliquely along the wall of a cylinder (see upper portion of Fig. 2-11B). The two centrioles in a centrosome are oriented at right angles to one another. The minus ends of all of a cell’s microtubules are associated with proteins that surround the centrosome, whereas the rapidly growing plus ends radiate throughout the cytoplasm in a star-like arrangement (“astral” microtubules). Microtubules participate in a multitude of cellular functions and structures. For example, microtubules project down the axon of neurons. Microtubules also provide the framework for the lacy membranes of the ER and Golgi complex. Disruption of microtubules causes these organelles to undergo dramatic morphological rearrangements and vesicularization. Microtubules also play a central role in cell division. Early in mitosis, the centrioles that make up the centrosomes replicate, forming two centrosomes at opposite poles of the dividing nucleus. Emanating from these centrosomes are the microtubules that form the spindle fibers, which in turn align the chromosomes (see lower portion of Fig. 2-11B). Their coordinated growth and dissolution at either side of the chromosomes may provide the force for separating the genetic material during the anaphase of mitosis. A pair of centrioles remains with each daughter cell. The architectural and mechanical capacities of microtubules are perhaps best illustrated by their role in motility. An electron microscopic cross section of a cilium demonstrates the elegance, symmetry, and intricacy of this structure (Fig. 2-11C). Every cilium arises out of its own basal body, which is essentially a centriole that is situated at the ciliary root. Cilia are found on the surfaces of many types of epithelial cells, including those that line the larger pulmonary airways (see Chapter 26). Their oar-like beating motions help propel foreign bodies and pathogens toward their ultimate expulsion at the pharynx. At the center of a cilium is a structure called the axoneme, which is composed of a precisely defined 9 + 2 array of microtubules. Each of the 9 (which are also called outer tubules) consists of a complete microtubule with 13 tubulin monomers in cross section (the A tubule) to which is fused an incomplete microtubule with 11 tubulin monomers in cross section (the B tubule). Each of the 2, which lie at the core of the cilium, is a complete microtubule. This entire 9 + 2 structure runs the entire length of the cilium. The same array forms the core of a flagellum, the serpentine motions of which propel sperm cells (see Chapter 56). Radial spokes connect the outer tubules to the central pair, and outer tubules attach to their neighbors by two types of linkages. One is composed of the protein dynein, which acts as a molecular motor to power ciliary and flagellar motions. Dynein is an ATPase that converts the energy released through ATP hydrolysis into a conformational change that produces a bending motion. Because dynein attached to one outer tubule interacts with a neighboring

outer tubule, this bending of the dynein molecule causes the adjacent outer tubules to slide past one another. It is this sliding-filament motion that gives rise to the coordinated movements of the entire structure. To some extent, this coordination is accomplished through the action of the second linkage protein, called nexin. The nexin arms restrict the extent to which neighboring outer tubules can move with respect to each other and thus prevent the dynein motor from driving the dissolution of the entire complex. The utility of the dynein motor protein is not restricted to its function in cilia and flagella. Cytoplasmic dynein, which is a close relative of the motor molecule found in cilia, and a second motor protein called kinesin provide the force necessary to move membrane-bound organelles through the cytoplasm along microtubular tracks (Fig. 2-11A). The ability of vesicular organelles to move rapidly along microtubules was first noted in neurons, in which vesicles carrying newly synthesized proteins must be transported over extremely long distances from the cell body to the axon tip. Rather than trust this critical process to the vagaries of slow, nondirected diffusion, the neuron makes use of the kinesin motor, which links a vesicle to a microtubule. Kinesin hydrolyzes ATP and, like dynein, converts this energy into mechanical transitions that cause it to “walk” along the microtubule. Kinesin will move only along microtubules and thereby transport vesicles in the minus-to-plus direction. Thus, in neurons, kinesin-bound vesicles move from the microtubular minus ends, originating at the centrosome in the cell body, toward the plus ends in the axons. This direction of motion is referred to as anterograde fast axonal transport. Cytoplasmic dynein moves in the opposite plus-to-minus (or retrograde) direction. The motor-driven movement of cellular organelles along microtubular tracks is not unique to neurons. This process, involving both kinesin and cytoplasmic dynein, appears to occur in almost every cell and may control the majority of subcellular vesicular traffic. Thin filaments (actin) and thick filaments (myosin) are present in almost every cell type Thin filaments, also called microfilaments, are 5 to 8 nm in diameter. They are helical polymers composed of a single polypeptide called globular actin or G-actin. Thin filaments are functionally similar to microtubules in two respects: (1) the actin polymers are polar and grow at different rates at their two ends, and (2) actin binds and then hydrolyzes a nucleotide. However, whereas tubulin binds GTP and then hydrolyzes it to GDP, actin binds ATP and then hydrolyzes it to ADP. After G-actin binds ATP, it may interact with another ATP-bound monomer to form an unstable dimer (Fig. 2-12A). Adding a third ATP-bound monomer, however, yields a stable trimer that serves as a nucleus for assembly of the polymer of fibrous actin or F-actin. Once it is part of Factin, the actin monomer hydrolyzes its bound ATP, retaining the ADP and releasing the inorganic phosphate. The ADP-bound actin monomer is more likely to disengage itself from its neighbors, just as GDP-bound tubulin dimers are more likely to disassemble from tubulin. Even though the length of the F-actin filament may remain more or less constant, the polymer may continually grow at its plus end but

Chapter 2 • Functional Organization of the Cell

A

FORMATION OF F-ACTIN

ATP

ATP-bound G-actin

Activation G-actin molecule B

Unstable actin dimer

F-actin filament

Assembly

Nucleus formation Stable actin oligomer

Pi

TREADMILLING ATP actin ATP cap

+ This end + end growing Figure 2-12 Thin filaments. A, Single molecules of G-actin form F-actin filaments. B, F-actin can grow at the plus end while shrinking at the minus end, with no change in length.

+

end

disassemble at its minus end (Fig. 2-12B). This “treadmilling” requires the continuous input of energy (i.e., hydrolysis of ATP) and illustrates the unique dynamic properties of actin filament elongation and disassembly. Thick filaments are composed of dimers of a remarkable force-generating protein called myosin. All myosin molecules have helical tails and globular head groups that hydrolyze ATP and act as motors to move along an actin filament. The energy liberated by ATP hydrolysis is invested in bending the myosin molecule around a pivot point called the hinge region, which marks the junction between the globular and tail regions. By means of this bending, myosin, like the dynein and kinesin that interact with microtubules, acts as a molecular motor that converts chemical into mechanical energy. In muscle, the myosin molecules are in the myosin II subfamily and exist as dimers with their long tails intertwined (Fig. 2-13A). In muscle, each of the two myosin II heads binds two additional protein subunits that are referred to as myosin light chains. Non-muscle cells, in addition to myosin II, may have a variety of other, smaller myosin molecules. These other myosins, the most widely studied of which is myosin I, have shorter tails and, at least in some cases, act as molecular motors that move vesicles along actin filaments. In muscle, the myosin II dimers stack as antiparallel arrays to form a bipolar structure with a bare central region that contains only tails (Fig. 2-13A). The ends of the thick filament contain the heads that bend toward the filament’s central region. The pivoting action of the myosin head groups drags the neighboring thin filament (Fig. 2-13B), which includes other molecules besides actin. This sliding-

ADP actin

This end – shrinking end

Pi

–

end

filament phenomenon underlies muscle contraction and force generation (Fig. 2-13C). Actin as well as an ever-growing list of myosin isoforms is present in essentially every cell type. The functions of these proteins are easy to imagine in some cases and are less obvious in many others. Many cells, including all of the fibroblast-like cells, possess actin filaments that are arranged in stress fibers. These linear arrays of fibers interconnect adhesion plaques to one another and to interior structures in the cell. They orient themselves along lines of tension and can, in turn, exert contractile force on the substratum that underlies the cell. Stress fiber contractions may be involved in the macroscopic contractions that are associated with wound healing. Frequently, actin filaments in non-muscle cells are held together in bundles by cross-linking proteins. Numerous classes of cross-linking proteins have been identified, several of which can respond to physiological changes by either stabilizing or severing filaments and filament bundles. In motile cells, such as fibroblasts and macrophages, arrays of actin-myosin filaments are responsible for cell locomotion. A Ca2+-stimulated myosin light chain kinase regulates the assembly of myosin and actin filaments and thus governs the generation of contractile force. The precise mechanism by which these fibers cooperate in causing the cell to crawl along a substrate remains poorly understood. In contrast to fibroblasts and circulating cells of the immune system, cells such as neurons and epithelial cells generally do not move much after their differentiation is complete. Despite this lack of movement, however, these cells are equipped with remarkably intricate actin and myosin filament networks. In some cases, these cytoskeletal elements

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Section II • Physiology of Cells and Molecules

A MYOSIN Hinge region

Dense plaque material

Short tail Myosin I

Plasma membrane

Head

S1

Heavy meromyosin S2

Light meromyosin

Light chains

Actin filaments

Villin Fimbrin

Myosin II

Myosin I The myosin that forms the contractile apparatus in muscle.

Antiparallel pairs of myosinII dimers assemble…

Terminal web

Fodrin

… to form a myosin thick filament.

B

MYOFILAMENTS Tropomyosin Actin (thin filament)

Intermediate filaments

Figure 2-14 Actin filaments at the brush border of an epithelial cell.

Troponin Myosin (thick filament) C

SARCOMERE A band H band M band

Z line

Myosin Actin

Nebulin

Titin

The striated appearance of skeletal muscle is due to the overlapping of thick and thin filaments.

Figure 2-13 Thick filaments. A, Myosin I is one of a large number of widely distributed myosins that have short tails. Myosin II is the myosin that participates in muscle contraction. B, The pivoting action of the myosin head, fueled by ATP, moves the thick filament past the thin filament. C, In skeletal and cardiac muscle, the sarcomere is the fundamental contractile unit.

permit the cell to extend processes to distant locations. This is the case in neurons, in which the growth and migration of axons during development or regeneration of the nervous system bear a striking morphological resemblance to the crawling of free-living amoebae. The tip of a growing axon, known as a growth cone, is richly endowed with contractile fibers and is capable of the same types of crawling motions that characterize motile cells. In epithelial cells, the role of the actin-myosin cytoskeleton is somewhat less obvious but still important to normal physiological function. The microvilli at the apical surfaces of many epithelial cell types (e.g., those that line the renal proximal tubule and the small intestine) are supported by an intricate scaffolding of actin filaments that form their cores (Fig. 2-14). This bundle of actin fibers is held together and anchored to the overlying plasma membrane by a variety of cross-linking proteins, including various myosin isoforms. The roots of the microvillar actin filament bundles emerge from the bases of the microvilli into a dense meshwork of cytoskeletal filaments known as the terminal web. Included among the components of the terminal web network are fodrin (the nonerythroid homologue of spectrin) and myosin. It remains unclear whether the myosin in the terminal web is present simply to interconnect the actin filaments of neighboring microvilli or if this actin-myosin complex is capable of generating contractile movements.

Chapter 2 • Functional Organization of the Cell

Actin and myosin filaments also form an adhesion belt that encircles the cytoplasmic surface of the epithelial plasma membrane at the level of the tight junctions that interconnect neighboring cells. These adhesion belts are apparently capable of contraction and thus cause epithelial cells that normally form a continuous sheet to pull away from one another, temporarily loosening tight junctions and creating direct passages that connect the luminal space to the extracellular fluid compartment. Actin and myosin also participate in processes common to most if not all cell types. The process of cytokinesis, in which the cytoplasm of a dividing cell physically separates into two daughter cells, is driven by actin and myosin filaments. Beneath the cleavage furrow that forms in the membrane of the dividing cell is a contractile ring of actin and myosin filaments. Contraction of this ring deepens the cleavage furrow; this invagination ultimately severs the cell and produces the two progeny.

SYNTHESIS AND RECYCLING OF MEMBRANE PROTEINS Secretory and membrane proteins are synthesized in association with the rough endoplasmic reticulum Transmembrane proteins are composed of hydrophobic domains that are embedded within the phospholipid bilayer and hydrophilic domains that are exposed at the intracellular and extracellular surfaces. These proteins do not “flip” through the membrane. How, then, do intrinsic membrane proteins overcome the enormous energetic barriers that should logically prevent them from getting inserted into the membrane in the first place? The cell has developed several schemes to address this problem. Mammalian cells have at least three different membrane insertion pathways, each associated with specific organelles. The first two are mechanisms for inserting membrane proteins into peroxisomes and mitochondria. The third mechanism inserts membrane proteins destined for delivery to the plasma membrane and to the membranes of organelles (the endomembranous system) other than the peroxisome and mitochondrion. This same mechanism is involved in the biogenesis of essentially all proteins that mammalian cells secrete and is the focus of the following discussion. The critical work in this field centered on studies of the rough ER. The membrane of the rough ER is notable for the presence of numerous ribosomes that are bound to its cytosol-facing surface. Whereas all nucleated mammalian cells have at least some rough ER, cells that produce large quantities of secretory proteins—such as the exocrine cells of the pancreas, which function as factories for digestive enzymes (see Chapter 43)—are endowed with an abundance of rough ER. Roughly half of the cytoplasmic space in an exocrine pancreatic acinar cell is occupied by rough ER. In early experiments exploring cell fractionation techniques, membranes that were derived from the rough ER were separated from the other membranous and cytoplasmic components of pancreatic acinar cells. The mRNAs associ-

ated with rough ER membranes were isolated and the proteins they encoded were synthesized by in vitro translation. Analysis of the resultant polypeptides revealed that they included the cell’s entire repertoire of secretory proteins. It is now appreciated that the mRNA associated with the ER also encodes the cell’s entire repertoire of membrane proteins, with the exception of those destined for either the peroxisome or the mitochondrion. When the same experiment was performed with mRNAs isolated from ribosomes that are freely distributed throughout the cytoplasm, the products were not secretory proteins but rather the soluble cytosolic proteins. Later work showed that the ribosomes bound to the ER are biochemically identical to and in equilibrium with those that are free in the cytosol. Therefore, a ribosome’s subcellular localization—that is, whether it is free in the cytosol or bound to the rough ER—is somehow dictated by the mRNA that the ribosome is currently translating. A ribosome that is involved in assembling a secretory or membrane protein will associate with the membrane of the rough ER, whereas the same ribosome will be free in the cytosol when it is producing cytosolic proteins. Clearly, some localization signal that resides in the mRNA or in the protein that is being synthesized must tell the ribosome what kind of protein is being produced and where in the cell that production should occur. The nature of this signal was discovered in 1972 during studies of the biosynthesis of immunoglobulin light chains. Light chains synthesized in vitro, in the absence of rough ER membranes, have a 15–amino acid extension at their amino terminus that is absent from the same light chains synthesized and secreted in vivo by B lymphocytes. Similar amino-terminal extensions are present on most secretory or membrane proteins but never with the soluble proteins of the cytosol. Although they vary in length and composition, these extensions are present on most acids that are interspersed with occasional basic residues. These signal sequences, as they have come to be known, serve as the localization devices discussed earlier. As it emerges from a ribosome and is freely floating in the cytosol, the signal sequence of a nascent protein (Fig. 2-15, stage 1) targets the ribosome-mRNA complex to the surface of the rough ER where the protein’s biogenesis will be completed. Ribosome-mRNA complexes that lack a signal sequence complete the translation of the mRNA—which encodes neither secretory nor membrane proteins—without attaching to the rough ER. For his work on signal sequences, Günter Blobel received the 1999 Nobel Prize for Physiology or Medicine. Why does the cell bother to segregate the synthesis of different protein populations to different cellular locales? Proteins that are destined either to reside in a membrane or to be secreted are inserted into or across the membrane of the rough ER at the same time that they are translated; this is called cotranslational translocation. As the nascent polypeptide chain emerges from the ribosome, it traverses the rough ER membrane and ultimately appears at the ER’s luminal face. There, an enzyme cleaves the amino-terminal signal sequence while the protein is still being translocated. This is why proteins that are synthesized in vitro in the absence of membranes are longer than the same proteins that are produced by intact cells.

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Section II • Physiology of Cells and Molecules

1 Protein synthesis begins.

mRNA 5´

2 Protein synthesis is inhibited.

3 Protein synthesis resumes.

4 The signal sequence is cleaved.

6 The ribosome dissociates.

5 Protein synthesis continues to completion.

3´

Ribosome Cleaved signal sequence

Signal sequence N

Nascent protein

Signal recognition particle

N

Carbohydrate

SRP receptor Translocon

Figure 2-15

Signal peptidase

C

N

Completed protein

Rough endoplasmic reticulum

Synthesis and translocation of a secretory protein.

Simultaneous protein synthesis and translocation through the rough endoplasmic reticulum membrane requires signal recognition and protein translocation machinery The information embodied within a signal sequence explains how a nascent protein can direct a cell to complete that protein’s translation at the time of translocation in the rough ER. However, the signal sequence by itself is not sufficient. Two critical pieces of targeting machinery are also necessary to direct the ribosome and its attached nascent peptide to the ER. The first is a ribonucleoprotein complex called the signal recognition particle (SRP), which binds to the signal sequence on the nascent peptide (Fig. 2-15, stage 2). The SRP is composed of seven distinct polypeptides and a short strand of RNA. When the SRP binds to a nascent chain, it also binds a GTP molecule. The second vital piece of targeting machinery is a transmembrane component of the rough ER, the SRP receptor, also called the docking protein. Interaction between a signal sequence and the SRP, and subsequently between the SRP–nascent peptide–ribosome complex and the docking protein, directs the nascent chain to the rough ER’s translocation apparatus. Because the membrane of the rough ER has a finite number of docking sites, the cell must coordinate the synthesis of secretory and membrane proteins with the availability of docking sites. If all docking sites were occupied, and if the synthesis of nascent secretory and membrane proteins were allowed to continue unabated, these nascent peptides would be synthesized entirely in the cytoplasm on free ribosomes. As a consequence, these newly synthesized proteins would never arrive at their proper destination. The SRP serves as a regulatory system that matches the rate of secretory and membrane protein syntheses to the number of unoccupied translocation sites. By associating with a nascent

signal sequence, the SRP causes the ribosome to halt further protein synthesis (Fig. 2-15, stage 2). This state of translation arrest persists until the SRP–nascent peptide–ribosome complex finds an unoccupied docking protein with which to interact. Thus, SRP prevents secretory and membrane proteins from being translated until their cotranslational translocation can be ensured. Because SRP interacts only with nascent chains that bear signal sequences, ribosomes that synthesize proteins destined for release into the cytosol never associate with SRP, and their translation is never arrested. Thus, SRP serves as a highly specific spatial and temporal sorting machine, guaranteeing the accurate and efficient targeting of secretory and membrane proteins. How does the cell terminate the translation arrest of the SRP–nascent peptide–ribosome complex? When this complex interacts with a docking protein (Fig. 2-15, stage 3), one of the SRP’s subunits hydrolyzes the previously bound GTP, thereby releasing the SRP from a successfully targeted nascent peptide–ribosome complex. In this way, the docking protein informs the SRP that its mission has been accomplished and it can return to the cytosol to find another ribosome with a signal peptide. A second GTP hydrolysis step transfers the nascent peptide from the docking protein to the actual translocation tunnel complex. GTP hydrolysis is a common event and is involved in the transmission of numerous cellular messages (see Chapter 3). In this case, the two separate instances of GTP hydrolysis serve a quality-control function because the activation of the GTPase activity depends on the delivery of the nascent peptide to the appropriate component in the translocation apparatus. Adjacent to the docking protein in the membrane of the rough ER is a protein translocator termed a translocon (Fig. 2-15, stage 3), which contains a tunnel through which the nascent protein will pass across the rough ER membrane. It appears that delivery of a nascent chain to the translocon

Chapter 2 • Functional Organization of the Cell

causes the entrance of the translocator’s tunnel, which is normally closed, to open. This opening of the translocon also allows the flow of small ions. The electrical current carried by these ions can be measured by the patch-clamp technique (see Chapter 6). By “gating” the translocon so that it opens only when it is occupied by a nascent protein, the cell keeps the tunnel’s entrance closed when it is not in use. This gating prevents the Ca2+ stored in the ER from leaking into the cytoplasm. Because the tunnel of the translocon is an aqueous pore, the nascent secretory or membrane protein does not come into contact with the hydrophobic core of the ER membrane’s lipid bilayer during cotranslational translocation. Thus, this tunnel allows hydrophilic proteins to cross the membrane. As translation and translocation continue and the nascent protein enters the lumen of the rough ER, an enzyme called signal peptidase cleaves the signal peptide, which remains in the membrane of the rough ER (Fig. 2-15, stage 4). Meanwhile, translation and translocation of the protein continue (Fig. 2-15, stage 5). In the case of secretory proteins (i.e., not membrane proteins), the peptide translocates completely through the membrane. The ribosome releases the complete protein into the lumen of the rough ER and then dissociates from the rough ER (Fig. 2-15, stage 6). Proper insertion of membrane proteins requires start-transfer and stop-transfer sequences Unlike soluble proteins, nascent membrane proteins do not translocate completely through the membrane of the rough ER (Fig. 2-16A, stage 1). The current concept is that the hydrophobic amino acid residues that will ultimately become the transmembrane segment of a membrane protein also function as a stop-transfer sequence (Fig. 2-16A, stage 2). When a stop-transfer sequence emerges from a ribosome, it causes the translocon to disassemble, releasing the hydrophobic membrane-spanning segment into the hospitable environment of the rough ER membrane’s hydrophobic core (Fig. 2-16A, stage 3). In the meantime, the ribosomal machinery continues to translate the rest of the nascent protein. If the signal peptidase cleaves the amino terminus at this time, the end result is a protein with a single transmembrane segment, with the amino terminus in the lumen of the rough ER and the carboxyl terminus in the cytoplasm (Fig. 2-16A, stage 4). There is another way of generating a protein with a single transmembrane segment. In this case, the protein lacks a signal sequence at the N terminus but instead has—somewhere in the middle of the nascent peptide—a bifunctional sequence that serves both as a signal sequence that binds SRP and as a hydrophobic membrane-spanning segment. This special sequence is called an internal start-transfer sequence. The SRP binds to the internal start-transfer sequence and brings the nascent protein to the rough ER, where the internal start-transfer sequence binds to the translocon in such a way that the more positively charged residues that flank the start-transfer sequence face the cytosol. Because these positively charged flanking residues can either precede or follow the hydrophobic residues of the internal start-transfer sequence, either the carboxyl (C) terminus or the N termi-

nus can end up in the cytosol. If the more positively charged flanking residues are at the carboxyl-terminal end of the internal start-transfer sequence (Fig. 2-16B), the protein will be oriented with its carboxyl terminus in the cytosol. If the more positively charged flanking residues are at the aminoterminal end of the internal start-transfer sequence (Fig. 216C), the protein will be oriented with its amino terminus in the cytosol. By alternating both stop-transfer sequences (Fig. 2-16A) and internal start-transfer sequences (Fig. 2-16B, C), the cell can fabricate membrane proteins that span the membrane more than once. Figure 2-16 shows how the cell could synthesize a multispanning protein with its N terminus in the cytosol. The process starts just as in Figure 2-16C, as the translation machinery binds to the rough ER (Fig. 2-16D, stage 1) and the protein’s first internal start-transfer sequence inserts into the translocon (Fig. 2-16D, stage 2). However, when the first stop-transfer sequence reaches the translocon (Fig. 2-16D, stage 3), the translocon disassembles, releasing the protein’s first two membrane-spanning segments into the membrane of the rough ER. Note that the first membrane-spanning segment is the internal start-transfer sequence and the second is the stop-transfer sequence. In the meantime, an SRP binds to the second internal start-transfer sequence (Fig. 2-16D, stage 4) and directs it to the rough ER (Fig. 2-16D, stage 5) so that cotranslational translocation can once again continue (Fig. 2-16D, stage 6). If there are no further stop-transfer sequences, we will end up with a protein with three membrane-spanning segments. Several points from the preceding discussion deserve special emphasis. First, translocation through the ER membrane can occur only cotranslationally. If a secretory or membrane protein were synthesized completely on a cytoplasmic ribosome, it would be unable to interact with the translocation machinery and consequently would not be inserted across or into the bilayer. As discussed later, this is not true for the insertion of either peroxisomal or mitochondrial proteins. Second, once a signal sequence emerges from a ribosome, there is only a brief period during which it is competent to mediate the ribosome’s association with the ER and to initiate translocation. This time constraint is presumably due to the tendency of nascent polypeptide chains to begin to fold and acquire tertiary structure very soon after exiting the ribosome. This folding quickly buries hydrophobic residues of a signal sequence so that they cannot be recognized by the translocation machinery. Third, because the translocation channel appears to be fairly narrow, the nascent protein cannot begin to acquire tertiary structure until after it has exited at the ER’s luminal face. Thus, the peptide must enter the translocation tunnel as a thin thread immediately after emerging from the ribosome. These facts explain why translocation is cotranslational. In systems in which posttranslational translocation occurs (e.g., peroxisomes and mitochondria), special adaptations keep the newly synthesized protein in an unfolded state until its translocation can be consummated. Finally, because the protein cannot flip once it is in the membrane, the scheme just outlined results in proteins that are inserted into the rough ER membrane in their final or “mature” topology. The number and location of a membrane protein’s transmembrane segments, as well as its cytoplasmic

31

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Section II • Physiology of Cells and Molecules

A

SINGLE MEMBRANE-SPANNING SEGMENT, CYTOPLASMIC C TERMINUS

Cytosol

1

2

3

4 3´

3´

5´

3´

5´

C

5´

Dissociation of translocon

Signal sequence Stop-transfer sequence

N

N

Signal peptidase Endoplasmic reticulum lumen B

SINGLE MEMBRANE-SPANNING SEGMENT, CYTOPLASMIC C TERMINUS (ALTERNATE MECHANISM)

SRP

C

SINGLE MEMBRANE-SPANNING SEGMENT, CYTOPLASMIC N TERMINUS

N

N

C

N

N

N

N

SRP receptor

Internal start-transfer sequence

N

Internal start-transfer sequence

N

D MULTIPLE MEMBRANE-SPANNING SEGMENTS 1

2

3

N

N

N

6

SRP

N

Second signal sequence

N

Stop-transfer sequence

Internal start-transfer sequence

5

4

Reinsertion into membrane

N

C

33

Chapter 2 • Functional Organization of the Cell

Figure 2-16 Synthesis of integral membrane proteins. A, Like a secreted protein, the membrane protein can have a cleavable signal sequence. In addition, it has a stop-transfer sequence that remains in the membrane as a membrane-spanning segment. B, The emerging protein lacks a signal sequence but instead has an internal start-transfer sequence, which is a bifunctional sequence that serves both as a signal sequence that binds signal recognition particles and as a hydrophobic membrane-spanning segment. In this example, the positively charged region flanking the internal start-transfer sequence is on the carboxyl-terminal end of the internal start-transfer sequence. Therefore, the C-terminal end is in the cytoplasm. C, The example is similar to that in B except that the positively charged region flanking the internal start-transfer sequence is on the amino-terminal end of the internal start-transfer sequence. D, The emerging peptide has alternating internal start-transfer and stop-transfer sequences.

and extracytoplasmic loops, are entirely determined during the course of its cotranslational insertion into the ER membrane. The order in which signal, internal start-transfer, and stop-transfer sequences appear in a membrane protein’s primary structure completely determines how that protein will be arrayed across whatever membrane it ultimately comes to occupy. Newly synthesized secretory and membrane proteins undergo post-translational modification and folding in the lumen of the rough endoplasmic reticulum As a newly synthesized secretory or membrane protein exits the tunnel of the translocon and enters the lumen of the rough ER, it may undergo a series of post-translational modifications that will help it to acquire its mature conformation. The first alteration, as discussed earlier, is cleavage of the signal sequence (if present) and is accomplished very soon after the signal sequence has completed its translocation. Other covalent modifications that occur as translocation continues include glycosylation and formation of intramolecular disulfide bonds. Glycosylation refers here to the enzymatic, en bloc coupling of preassembled, branched oligosaccharide chains that contain 14 sugar molecules (Fig. 2-17A) to asparagine (Asn) residues that appear in the sequence Asn-X-Ser or Asn-X-Thr (X can be any amino acid A

N-LINKED GLYCOSYLATION

except proline). These N-linked sugars (N is the single-letter amino acid code for asparagine) will go on to be extensively modified as the protein passes through other organellar compartments. The addition of sugar groups to proteins can serve numerous functions, which include increasing the protein’s stability and endowing it with specific antigenic, adhesive, or receptor properties. Disulfide bond formation is catalyzed by protein disulfide isomerase, an enzyme that is retained in the ER lumen through noncovalent interactions with ER membrane proteins. Because the cytoplasm is a reducing environment, disulfide bonds can form only between proteins or protein domains that have been removed from the cytosolic compartment through translocation to the ER’s interior. Other, more specialized modifications also take place in the lumen of the rough ER. For example, the ER contains the enzymes responsible for the hydroxylation of the proline residues that are present in newly synthesized collagen chains. The ER also catalyzes the formation of GPI linkages to membrane proteins (Fig. 2-17B). GPI-linked proteins are synthesized as transmembrane polypeptides, with a typical membrane-spanning region. Shortly after their translation, however, their lumen-facing domains are cleaved from the membrane-spanning segments and covalently transferred to the GPI phospholipid. They retain this structure and orientation throughout the remainder of their journey to the cell surface. A defect in the synthesis of GPI-linked proteins B

FORMATION OF A GPI LINKAGE

Cytosol

Ribosome

Cytosol

5´

C

Glycosyl phosphatidylinositol

Cleaved C-terminal peptide

C

3´

mRNA N

Rough ER

N

Lipid bilayer ER lumen Asn

P P

Asn

H N H

P P

P P

C

Growing polypeptide chain N

ER lumen

P

O

N

N

N H

P

Protein bound covalently to lipid anchor in membrane

Figure 2-17 Post-translational modifications of integral membrane proteins. A, An enzyme in the ER lumen attaches a preassembled, branched, oligosaccharide chain to an asparagine (Asn or N) residue on the nascent protein. B, An enzyme in the ER lumen cleaves the protein and couples the protein’s new terminal carboxyl group to the terminal amino group on the GPI molecule.

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Section II • Physiology of Cells and Molecules

underlies the human disease paroxysmal nocturnal hematuria (see the box on this topic). Perhaps the most important maturational process for a nascent chain emerging into the ER lumen is the acquisition of tertiary structure. The folding of a secretory or membrane protein is determined during and immediately after its cotranslational translocation. The progress of protein folding influences—and is influenced by—the addition of sugar residues and the formation of disulfide bridges. Proteins fold into conformations that minimize their overall free energies. Their extramembranous surfaces are composed of hydrophilic residues that interact easily with the aqueous solvent. Hydrophobic residues are hidden in internal globular domains where they can be effectively isolated from contact with water or charged molecules. Left to its own devices, a linear strand of denatured protein will spontaneously fold to form a structure that reflects these thermodynamic considerations. Thus, protein folding requires no catalysis and can occur without help from any cellular machinery. However, the cell is not content to allow protein folding to follow a random course and instead orchestrates the process through the actions of molecular chaperones.

The chaperones constitute a large class of ATP-hydrolyzing proteins that appear to participate in a wide variety of polypeptide-folding phenomena, including the initial folding of newly synthesized proteins as well as the refolding of proteins whose tertiary structures have been damaged by exposure to high temperature (i.e., heat shock) or other denaturing conditions. Chaperones bind to unfolded protein chains and stabilize them in an unfolded conformation, thus preventing them from spontaneously folding into what might be an energetically favorable but biologically useless arrangement. Using energy that is provided through ATP hydrolysis, the chaperones sequentially release domains of unfolded proteins and thus allow them to fold in an ordered fashion. Distinct subclasses of chaperones are present in several cell compartments, including the cytoplasm, the mitochondrion, and the lumen of the rough ER. Newly synthesized secretory and membrane proteins appear to interact with ER chaperones as they exit from the tunnel of the translocon and subsequently disengage from the chaperones to assume their mature tertiary structure. The acquisition of tertiary structure is followed quickly by the acquisition of quaternary structure. As noted earlier

Paroxysmal Nocturnal Hematuria

T

he list of proteins embedded in the plasma membrane through a GPI linkage is remarkably long and ever-growing. In red blood cells, the inventory of GPI-linked proteins includes a pair of polypeptides, decay-accelerating factor (DAF) and CD59, which help protect the erythrocytes from being accidentally injured by constituents of the immune system. One of the mechanisms that the immune system uses to rid the body of invading bacteria involves the activation of the complement cascade. Complement is a complex collection of proteins that circulate in the blood plasma. The complement system recognizes antibodies that are bound to the surface of a bacterium or polysaccharides in the bacterial membrane. This recognition initiates a cascade of enzymatic cleavages that results in the assembly of a subset of complement proteins to form the membrane attack complex, which inserts itself into the membrane of the target organism and forms a large pore that allows water to rush in (see Chapter 5). The target bacterium swells and undergoes osmotic lysis. Unfortunately, the complement system’s lethal efficiency is not matched by its capacity to discriminate between genuine targets and normal host cells. Consequently, almost every cell type in the body is equipped with surface proteins that guard against a misdirected complement attack. DAF and CD59 are two such proteins that interfere with distinct steps in the complement activation pathway. Because GPI linkages couple both proteins to the membrane, any dysfunction of the enzymes that participate in the transfer of GPIlinked proteins from their transmembrane precursors to their GPI tails in the ER would interfere with the delivery of DAF and CD59 to their sites of functional residence at the cell surface. One of the proteins that participates in the synthesis of the GPI anchor is a sugar transferase encoded by the phosphatidylinositol glycan class A (PIG-A) gene. This gene is located on the

X chromosome. Because every cell has only one working copy of the X chromosome (although female cells are genetically XX, one of the two X chromosomes is inactivated in every cell), if a spontaneous mutation occurs in the PIG-A gene in a particular cell, that cell and all of its progeny will lose the ability to synthesize GPI-linked proteins. In paroxysmal nocturnal hemoglobinuria (i.e., hemoglobin appearing in the urine at night, with a sharp onset), a spontaneous mutation occurs in the PIG-A gene in just one of the many precursor cells that give rise to erythrocytes. All of the erythrocytes that arise from this particular precursor, therefore, are deficient in GPI-linked protein synthesis. Consequently, these cells lack DAF and CD59 expression and are susceptible to complement attack and lysis. For reasons that are largely unknown, the complement system is somewhat more active during sleep, so the hemolysis (lysis of erythrocytes) occurs more frequently at night in these patients. Some of the hemoglobin released by this lysis is excreted in the urine. Because the PIG-A gene product is required for the synthesis of all GPI-linked proteins, the plasma membranes of affected red blood cells in patients with paroxysmal nocturnal hemoglobinuria are missing a number of different proteins that are found in the surface membranes of their normal counterparts. It is the lack of DAF and CD59, however, that renders the cells vulnerable to complement-mediated killing and that creates the symptoms of the disease. Paroxysmal nocturnal hemoglobinuria is an uncommon disease. Because it is the result of an acquired mutation, it is much more likely to occur in people of middle age rather than in children. Patients with paroxysmal nocturnal hemoglobinuria are likely to become anemic and can suffer life-threatening disorders of clotting and bone marrow function. It is a chronic condition, however, and more than half of patients survive at least 15 years after diagnosis.

Chapter 2 • Functional Organization of the Cell

in this chapter, many membrane proteins assemble into oligomeric complexes in which several identical or distinct polypeptides interact with one another to form a macromolecular structure. Assembly of these multimers occurs in the ER. It is unknown whether the oligomeric assembly process occurs entirely spontaneously or if, like folding, it is orchestrated by specialized cellular mechanisms. Cells clearly go to great trouble to ensure that proteins inserted into or across their ER membranes are appropriately folded and oligomerized before allowing them to continue with their postsynthetic processing. As discussed later, proteins destined for secretion from the cell or for residence in the cell membrane or other organellar membranes depart the ER for further processing in the membranous stacks of the Golgi complex. This departure is entirely contingent on successful completion of the protein folding and assembly operations. Misfolded or unassembled proteins are retained in the ER and ultimately degraded. The ER chaperone proteins play a critical role both in identifying proteins with incorrect tertiary or quaternary structures and in actively preventing their egress to the Golgi complex. Proteins that have not folded or assembled correctly are destroyed through a process known as ERAD (endoplasmic reticulum–associated degradation). The sequential, covalent addition of ubiquitin monomers results in the formation of a branched-chain ubiquitin polymer that marks these proteins for destruction. Ubiquitin is a small protein of 76 amino acid residues. The process known as retrotranslocation removes ubiquitin-tagged proteins from the ER membrane, and a large cytoplasmic complex of proteolytic enzymes—the proteosome—degrades the ubiquitinated proteins. Secretory and membrane proteins follow the secretory pathway through the cell The rough ER is the common point of origin for the cell’s secretory and membrane proteins. Most of these proteins are

not retained in the rough ER but depart for distribution to their sites of ultimate functional residence throughout the cell. As is true for their arrival in the rough ER, the departure of these proteins is a highly organized and regimented affair. In fact, the rough ER is the first station along the secretory pathway, which is the route followed (at least in part) by all secretory and membrane proteins as they undergo their post-translational modifications (Fig. 2-18). The elucidation of the secretory pathway occurred in the 1960s, mainly in the laboratory of George Palade. For his contribution, Palade was awarded the 1975 Nobel Prize in Physiology or Medicine. This work also exploited the unique properties of pancreatic acinar cells to illuminate the central themes of secretory protein biogenesis. Because ∼95% of the protein that is synthesized by pancreatic acinar cells are digestive enzymes destined for secretion (see Chapter 43), when these cells are fed radioactively labeled amino acids, the majority of these tracer molecules are incorporated into secretory polypeptides. Within a few minutes after the tracer is added, most of the label is associated with a specialized subregion of the rough ER. Known as transitional zones, these membranous saccules are ribosome studded on one surface and smooth at the opposite face (Fig. 2-18). The smooth side is directly apposed to one pole of the pancakelike membrane stacks (or cisternae) of the Golgi complex. Smooth-surfaced carrier vesicles crowd the narrow moat of cytoplasm that separates the transitional zone from the Golgi. These vesicles “pinch off ” from the transitional zone and fuse with a Golgi stack. From this first or cis-Golgi stack, carrier vesicles ferry the newly synthesized proteins sequentially and vectorially through each Golgi stack, ultimately delivering them to the trans-most saccule of the Golgi. Finally, the newly synthesized secretory proteins appear in secretory vesicles (also called secretory granules in many tissues). The journey from the rough ER to the secretory vesicle takes ∼45 minutes in pancreatic acinar cells and requires the

Rough ER

Transitional zone

cis

Carrier vesicles Medial

Golgi

trans

Figure 2-18 The secretory pathway. After their synthesis in the rough ER, secretory and membrane proteins destined for the plasma membrane move through the Golgi stacks and secretory vesicles. In the constitutive pathway, vesicles fuse spontaneously with the plasma membrane. In the regulated pathway, the vesicles fuse only when triggered by a signal such as a hormone.

Constitutive secretory vesicle

Blood

Regulated secretory vesicle

Integral membrane protein Secretory proteins Exocytosis Constitutive pathway: secretion is continuous and unregulated

Regulated pathway: secretion is directed by hormonal or neural signal.

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Section II • Physiology of Cells and Molecules

expenditure of metabolic energy. Each nucleated eukaryotic cell possesses a secretory pathway that shares this same general outline, although the specific features reflect the cell’s particular function. The secretory pathway of the pancreatic acinar cell, for example, is specially adapted to accommodate the controlled secretion of protein by the so-called regulated pathway. Instead of being released from the cell continuously as they are produced, newly synthesized secretory proteins are held in specialized secretory vesicles that serve as an intracellular storage depot. This type of storage occurs in several cells, including those of endocrine and exocrine secretory tissues, and neurons. When the cells receive the requisite message, the storage vesicles fuse with the plasma membrane, sometimes at a specialized structure called a porosome, in a process known as exocytosis. The vesicles then dump their contents into the extracellular space. In the case of the pancreatic acinar cells, the enzymes are secreted into the pancreatic ductules and then make their way to the site of digestion in the duodenum (see Chapter 43). Most cell types, however, deliver newly synthesized secretory and membrane proteins to the cell surface in a continuous and unregulated fashion, which is referred to as the constitutive pathway. Specialized cells that have the capacity for regulated delivery also send an important subset of their secretory and membrane protein synthetic products to the cell surface constitutively. The regulated and constitutive secretory pathways are identical except for the final station of the Golgi complex. At this point, the “regulated” proteins divert to the specialized secretory vesicles described in the previous paragraph. The “constitutive” proteins, at the transmost cisterna of the Golgi complex, sort into other secretory vesicles, which move directly to the cell surface. There, the constitutive membrane proteins are delivered to the plasma membrane, and the constitutive secretory proteins are immediately exocytosed. This section has provided a broad overview of the secretory pathway. In the following sections, we examine the details of how newly synthesized proteins move between organellar compartments of the secretory pathway, how the proteins are processed during this transit, and how they are sorted to their final destination. Carrier vesicles control the traffic between the organelles of the secretory pathway As the preceding discussion suggests, the secretory pathway is not a single, smooth, continuous highway but rather a series of saltatory translocations from one discrete organellar compartment to the next. Each of these steps requires some orchestration to ensure that the newly synthesized proteins arrive at their next terminus. The cell solves the problem of moving newly synthesized proteins between membranous organelles by using membrane-enclosed carrier vesicles (or vesicular carriers). Each time proteins are to be moved from one compartment to the next, they are gathered together within or beneath specialized regions of membrane that subsequently evaginate or pinch off to produce a carrier vesicle (Fig. 2-18). Secretory proteins reside within the lumen of the carrier vesicle, whereas membrane proteins span the vesicle’s own encapsulating bilayer. On arrival at the appropriate destination, the

carrier vesicle fuses with the membrane of the acceptor organelle, thus delivering its contents of soluble proteins to the organelle’s lumen and its cargo of membrane proteins to the organelle’s own membrane. Carrier vesicles mediate the transport of secretory and membrane proteins across the space between the ER’s transition zone and the cis-Golgi stack and also between the rims of the Golgi stacks themselves. The movement between one vesicular compartment and the next is mediated by the cytoskeleton and molecular motors that were discussed earlier. A few critical facts deserve emphasis. First, throughout the formation, transit, and fusion of a carrier vesicle, no mixing occurs between the vesicle lumen and cytosol. The same principle applies to the carrier vesicle’s membrane protein passengers, which were inserted into the membrane of the rough ER with a particular topology. Those domains of a membrane protein that are exposed to the cytosol in the rough ER remain exposed to the cytosol as the protein completes its journey through the secretory pathway. Second, the flow of vesicular membranes is not unidirectional. The rate of synthesis of new membrane lipid and protein in the ER is less than the rate at which carrier vesicles bud off of the ER that is bound for the Golgi. Because the sizes of the ER and Golgi are relatively constant, the membrane that moves to the Golgi by carrier vesicles must return to the ER. This return is again accomplished by vesicular carriers. Each discrete step of the secretory pathway must maintain vesicle-mediated backflow of membrane from the acceptor to the donor compartment so that each compartment can retain a constant size. Finally, we have already noted that each organelle along the secretory pathway is endowed with a specific set of “resident” membrane proteins that determines the properties of the organelle. Despite the rapid forward and backward flow of carrier vesicles between successive stations of the secretory pathway, the resident membrane proteins do not get swept along in the flow. They are either actively retained in their home organelles’ membranes or actively retrieved by the returning “retrograde” carrier vesicles. Thus, not only the size but also the composition of each organelle of the secretory pathway remains essentially constant despite the rapid flux of newly synthesized proteins that it constantly handles. Specialized protein complexes, such as clathrin and coatamers, mediate the formation and fusion of vesicles in the secretory pathway The formation of a vesicle through evagination appears to be geometrically indistinguishable from its fusion with a target membrane. In both cases, a cross-sectional view in the electron microscope reveals an “omega” profile, which is so named because the vesicle maintains a narrow opening to the organellar lumen that resembles the shape of the Greek letter omega (Ω). However, different problems are confronted during the formation and fusion of membrane vesicles. Vesicle Formation in the Secretory Pathway

To form a spherical vesicle from a planar membrane, the mechanism that pulls the vesicle off from the larger membrane must grab onto the membrane over the entire surface of the

Chapter 2 • Functional Organization of the Cell

nascent vesicle. The mechanism that achieves this makes use of a scaffolding that is composed of coat proteins. The cell has at least two and probably more varieties of coat proteins. The best characterized of these is clathrin, which mediates the formation of secretory vesicles from the trans Golgi. Clathrin also mediates the internalization of membrane from the cell surface during the process of endocytosis, which is the reverse of exocytosis. Another major protein coat, which is involved in nonselective trafficking of vesicles between the ER and Golgi and between the stacks of the Golgi, is a protein complex known as coatamer. Both clathrin and coatamer proteins form the borders of a cage-like lattice. In the case of clathrin, the coat proteins preassemble in the cytoplasm to form three-armed “triskelions” (Fig. 2-19A). A triskelion is not planar but resembles the three adjoining edges of a tetrahedron. As triskelions attach to one another, they produce a three-dimensional structure resembling a geodesic dome with a roughly spherical shape. A triskelion constitutes each vertex in the lattice of hexagons and pentagons that form the cage. The triskelions of clathrin attach indirectly to the surface of the membrane that is to be excised by binding to the

A

cytosolic tails of membrane proteins. Mediating this binding are adapter proteins, called adaptins, that link the membrane protein tails to the triskelion scaffold. The specificity for particular membrane proteins is apparently conferred by specialized adaptins. Triskelions assemble spontaneously to form a complete cage that attaches to the underlying membrane and pulls it up into a spherical configuration. Completion of the cage occurs simultaneously with the pinching off of the evaginated membrane from the planar surface, forming a closed sphere. The pinching off, or fission, process appears to involve the action of a GTP-binding protein called dynamin, which forms a collar around the neck of the forming vesicle and may sever it. The fission process must include an intermediate that resembles the structure depicted in Figure 2-19A. According to the prevalent view, each of the lumen-facing leaflets of membrane lipids fuse, leaving only the cytoplasmic leaflets to form a continuous bridge from the vesicle to the donor membrane. This bridge then breaks, and fission is complete. Once formed, the clathrin-coated vesicle cannot fuse with its target membrane until it loses its cage, which prevents the two membranes from achieving the close contact required

FORMATION OF CLATHRIN-COATED VESICLES Golgi lumen

trans-Golgi membrane

The triskelions and adaptins from uncoated vesicles recycle.

Adaptin Receptors

The vesicles go on to fuse with other membranous structures such as the endosome and the plasma membrane.

Triskelion

Uncoated vesicle

B

Figure 2-19 Vesicle formation and fusion. A, Clathrin mediates the formation of secretory vesicles that bud off from the trans Golgi as well as the internalization of membrane from the cell surface during the process of endocytosis. B, A complex of proteins forms a bridge between the vesicle and the target membranes. ATP provides the fuel for fusion. The Rab appears to be a molecular switch. NSF, N-ethylmaleimide–sensitive factor; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor.

ATP

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VESICLE DOCKING AND FUSION

+

FUSION Rab

DOCKING

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NSF

+ P

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Rab-GTP NSF t-SNARE

SNAPs

Omega profile

37

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Section II • Physiology of Cells and Molecules

to permit fusion. Because formation of the clathrin cage is spontaneous and energetically favorable, dissolution of the cage requires energy. Uncoating is accomplished by a special class of cytoplasmic enzymes that hydrolyze ATP and use the energy thus liberated to disassemble the scaffold (Fig. 2-19A). The function of coatamers is similar to that of clathrin in that coatamer forms a cage around the budding membrane. However, coatamer coats differ from clathrin in several respects. First, coatamer coats are composed of several coatamer proteins, one of which is related to the adaptins. Second, unlike the spontaneous assembly of the clathrin triskelions, assembly of the coatamer coat around the budding vesicle requires ATP. Third, a coatamer-coated vesicle retains its coat until it docks with its target membrane. Vesicle Fusion in the Secretory Pathway Membrane fusion occurs when the hydrophobic cores of two bilayers come into contact with one another, a process that requires the two membranes to be closely apposed. Because the cytoplasmic leaflets of most cellular membranes are predominantly composed of negatively charged phospholipids, electrostatic repulsion prevents this close apposition from occurring spontaneously. To overcome this charge barrier and perhaps to assist in targeting as well, a multicomponent protein complex forms and acts as a bridge, linking vesicular membrane proteins to membrane proteins in the target bilayer (Fig. 2-19B). Investigators have established the components of this complex by use of three approaches: studies of the membrane fusion steps involved in vesicular transport between successive Golgi stacks, genetic analysis of protein secretion in yeast, and molecular dissection of the protein constituents of the synaptic vesicles of nerve terminals. In each case, the same proteins are instrumental in attaching the donor and target membranes to one another. The central components of the bridge are proteins known as SNAREs (so named because they act as receptors for the SNAPs discussed in the next paragraph). There are SNAREs in both the vesicular membrane (v-SNAREs) and the membrane of the target organelle (t-SNAREs). The best studied SNARE family members are those that participate in the fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane of the axons in neurons (see Chapter 8). In that setting, the v-SNARE is known as synaptobrevin, and proteins known as syntaxin and SNAP-25 together act as t-SNAREs. The t-SNAREs and v-SNAREs bind to each other extremely tightly, pulling the vesicular and target membranes close together. This proximity alone may be sufficient to initiate fusion, although this point remains controversial. In cells that employ rapid and tightly regulated membrane fusion, such as neurons, increases in the cytoplasmic concentration of Ca2+, sensed by the SNARE fusion complex, trigger fusion (see Chapter 8). Although the nature of the fusion event itself remains unclear, clues have emerged about its regulation. Fusion requires the participation of a class of small GTP-binding proteins called Rabs that are important for signaling. Rabs appear to act as molecular switches that assemble with the SNARE fusion complex when they are binding GTP but dissociate from the complex after they hydrolyze the GTP to GDP. Rab-GTP must associ-

ate with the fusion complex for fusion to occur. Numerous Rab isoforms exist, each isoform associated with a different vesicular compartment and a distinct membrane-to-membrane translocation step. Once fusion occurs, the former vesicle generally loses its spherical shape rapidly as it becomes incorporated into the target membrane. This “flattening out” is the result of surface tension, inasmuch as the narrow radius of curvature demanded by a small spherical vesicle is energetically unfavorable. After fusion, it is also necessary to disassemble the v-SNARE/t-SNARE complex so that its components can be reused in subsequent fusion events. The dissociation step involves the activity of two additional components that participate in the SNARE complex. The first is an ATP-hydrolyzing enzyme; because it is inhibited by the alkylating agent N-ethylmaleimide (NEM), it was named NEM-sensitive factor (NSF). Soluble NSF attachment proteins (the SNAPs mentioned before), which target NSF to the SNARE complex, are the second. Hydrolysis of ATP by NSF causes dissociation of the SNARE complex, thus regenerating the fusion machinery. Homologues of the neuronal t-SNARE and v-SNARE proteins are found in almost every cell type in the body and are thought to participate in most if not all membrane fusion events. Newly synthesized secretory and membrane proteins are processed during their passage through the secretory pathway While in the rough ER, newly synthesized secretory and membrane proteins undergo the first in a series of posttranslational modifications. As discussed earlier, this first group includes glycosylation, disulfide bond formation, and the acquisition of tertiary structure. On delivery to the cis stack of the Golgi complex, these proteins begin a new phase in their postsynthetic maturation. For many proteins, the most visible byproduct of this second phase is the complete remodeling of their N-linked sugar chains, originally attached in the rough ER. Of the 14 sugar residues transferred en bloc to newly synthesized proteins during N-linked glycosylation, nine are mannose and three are glucose (Fig. 2-20A). Enzymes called glucosidases and one called a mannosidase are associated with the luminal face of the ER; these enzymes remove the three glucose residues and one mannose. As proteins arrive from the ER, mannosidases in the cis Golgi attack the N-linked sugar trees, thereby shearing off all except two N-acetylglucosamine and five mannose residues. As the proteins pass from the cis-Golgi cisterna to the medial cisterna and ultimately to the trans-Golgi cisterna, another mannosidase removes two additional mannose residues, and other enzymes add sugars to the stump of the original sugar tree in a process referred to as complex glycosylation. The addition of new sugars occurs one residue at a time and is accomplished by enzymes called sugar transferases that face the lumens of the Golgi stacks. Each sugar is transported from the cytoplasm to the Golgi lumen by a carrier protein that spans the Golgi membrane. Throughout the maturation process, the N-linked sugar chains are always exposed only to the luminal face of the Golgi.

Chapter 2 • Functional Organization of the Cell

A

REMODELING OF N-LINKED SUGARS

Cytoplasm

Rough ER

cis face

cis-Golgi network

In rough ER

Protein Ribosome

cis cisternae

trans-Golgi network

Medial cisternae trans cisternae

Golgi apparatus

Secretory granule

trans Face Secretion

B

ER membrane N-linked sugars

Nucleus

Asn

Protein In cis Golgi

Asn

In medial Golgi

Asn

Lysosome Plasma membrane N-Acetylglucosamine

PROTEOGLYCANS

Mannose Galactose Glycosaminoglycan

Glucose n

Asn

Sialic acid Xylose

Link trisaccharide In trans Golgi O

linkage

O

Asn

Ser residue Protein main chain

Vesicle

Figure 2-20 Modification and assembly of the sugar chains on proteins in the Golgi. A, Remodeling of Nlinked sugars. B, Proteoglycans. A trisaccharide links glycosaminoglycan chains to the protein by the -OH group of a serine residue. The glycosaminoglycan is made up of n repeating disaccharide units, one of which is always an amino sugar.

Each cisterna of the Golgi is characterized by a different set of sugar transferases and sugar transporters. Thus, each Golgi compartment catalyzes a distinct step in the maturation of the N-linked chains. Complex glycosylation, therefore, proceeds like an assembly line from one modification station to the next. Because proteins have different shapes and sizes, however, the degree to which a sugar chain of any given polypeptide has access to each transferase can vary quite extensively. Thus, each protein emerges from the assembly line with its own particular pattern of complex glycosylation. The Golgi’s trans-most cisterna houses the enzymes responsible for adding the terminal sugars, which cap the N-linked chain. The final residue of these terminal sugars is frequently N-acetylneuraminic acid, also known as sialic acid. At neutral pH, sialic acid is negatively charged. This acidic sugar residue therefore is responsible for the net negative electrostatic charge that is frequently carried by glycoproteins.

The Golgi’s function is not limited to creating N-linked sugar tree topiaries. It oversees a number of other post-translational modifications, including the assembly of O-linked sugars. Many proteins possess O-linked sugar chains, which attach not to asparagine residues but to the hydroxyl groups (hence, O) of serine and threonine residues. The O-linked sugars are not preassembled for en bloc transfer the way that the original 14-sugar tree is added in the rough ER in the case of their N-linked counterparts. Instead, the O-linked sugars are added one residue at a time by sugar transferases such as those that participate in the remodeling of complex N-linked glycosylation. O-linked chains frequently carry a great deal of negatively charged sialic acid. Proteoglycans contain a very large number of a special class of O-linked sugar chains that are extremely long (Fig. 2-20B). Unlike other O-linked sugars that attach to the protein core by an N-acetylglucosamine, the sugar chain in a proteoglycan attaches by a xylose-containing three-sugar

39

Chapter 3 • Signal Transduction

of a second messenger or the activation of a catalytic cascade. Step 3: Transmission of the second messenger’s signal to the appropriate effector. These effectors represent a diverse array of molecules, such as enzymes, ion channels, and transcription factors. Step 4: Modulation of the effector. These events often result in the activation of protein kinases (which put phosphate groups on proteins) and phosphatases (which take them off), thereby altering the activity of other enzymes and proteins. Step 5: Response of the cell to the initial stimulus. This collection of actions represents the summation and integration of input from multiple signaling pathways. Step 6: Termination of the response by feedback mechanisms at any or all levels of the signaling pathway. Cells can also communicate by direct interactions Gap Junctions Neighboring cells can be electrically and metabolically coupled by means of gap junctions formed between apposing cell membranes. These water-filled channels facilitate the passage of inorganic ions and small molecules, such as Ca2+ and 3′,5′-cyclic adenosine monophosphate (cAMP), from the cytoplasm of one cell into the cytoplasm of an adjacent cell. Mammalian gap junctions permit the passage of molecules that are less than ∼1200 Da but restrict the movement of molecules that are greater than ∼2000 Da. Gap junctions are also excellent pathways for the flow of electrical current between adjacent cells, playing a critical role in cardiac and smooth muscle. The permeability of gap junctions can be rapidly regulated by changes in cytosolic concentrations of Ca2+, cAMP, and H+ as well as by the voltage across the cell membrane or membrane potential (Vm) (see Chapter 5). This type of modulation is physiologically important for cell-to-cell communication. For example, if a cell’s plasma membrane is damaged, Ca2+ passively moves into the cell and raises [Ca2+]i to toxic levels. Elevated intracellular [Ca2+] in the damaged cell triggers closure of the gap junctions, thus preventing the flow of excessive amounts of Ca2+ into the adjacent cell. Adhering and Tight Junctions

Adhering junctions form as the result of the Ca2+-dependent interactions of the extracellular domains of transmembrane proteins called cadherins (see Chapter 2). The clustering of cadherins at the site of interaction with an adjacent cell causes secondary clustering of intracellular proteins known as catenins, which in turn serve as sites of attachment for the intracellular actin cytoskeleton. Thus, adhering junctions provide important clues for the maintenance of normal cell architecture as well as the organization of groups of cells into tissues. In addition to a homeostatic role, adhering junctions can serve a signaling role during organ development and remodeling. In a cell that is stably associated with its neighbors, a catenin known as β-catenin is mainly sequestered at the adhering junctions, minimizing concentration of free βcatenin. However, disruption of adhering junctions by certain growth factors, for example, causes β-catenin to disassociate from cadherin. The resulting rise in free β-catenin

levels promotes the translocation of β-catenin to the nucleus. There, β-catenin regulates the transcription of multiple genes, including ones that promote cell proliferation and migration. Similar to adhering junctions, tight junctions (see Chapter 2) comprise transmembrane proteins that link with their counterparts on adjacent cells as well as intracellular proteins that stabilize the complex and also have a signaling role. The transmembrane proteins—including claudins, occludin, and junctional adhesion molecule—and their extracellular domains create the diffusion barrier of the tight junction. One of the integral cytoplasmic proteins in tight junctions, zonula occludin 1 (ZO-1), colocalizes with a serine/threonine kinase known as WNK1, which is found in certain renal tubule epithelial cells that reabsorb Na+ and Cl− from the tubule lumen. Because WNK1 is important for determining the permeability of the tight junctions to Cl−, mutations in WNK1 can increase the movement of Cl− through the tight junctions (see Chapter 35) and thereby lead to hypertension. Membrane-Associated Ligands

Another mechanism by which cells can directly communicate is by the interaction of a receptor in the plasma membrane with a ligand that is itself a membrane protein on an adjacent cell. Such membrane-associated ligands can provide spatial clues in migrating cells. For example, an ephrin ligand expressed on the surface of one cell can interact with an Eph receptor on a nearby cell. The resulting activation of the Eph receptor can in turn provide signals for regulating such developmental events as axonal guidance in the nervous system and endothelial cell guidance in the vasculature. Second-messenger systems amplify signals and integrate responses among cell types

Once a signal has been received at the cell surface, it is typically amplified and transmitted to specific sites within the cells through second messengers. For a molecule to function as a second messenger, its concentration, or window of activity, must be finely regulated. The cell achieves this control by rapidly producing or activating the second messenger and then inactivating or degrading it. To ensure that the system returns to a resting state when the stimulus is removed, counterbalancing activities function at each step of the cascade. The involvement of second messengers in catalytic cascades provides numerous opportunities to amplify a signal. For example, the binding of a ligand to its receptor can generate hundreds of second-messenger molecules, which can in turn alter the activity of thousands of downstream effectors. This modulation usually involves the conversion of an inactive species into an active molecule or vice versa. An example of such a cascade is the increased intracellular concentration of the second messenger cAMP. Receptor occupancy activates a G protein, which in turn stimulates a membrane-bound enzyme, adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP from adenosine triphosphate (ATP), and a 5-fold increase in the intracellular concentration of cAMP is achieved in ∼5 seconds. This sudden rise in cAMP levels is rapidly counteracted by its

51

Chapter 2 • Functional Organization of the Cell

brane. Carrier vesicles incorporate these clusters into their own bilayers. Proteins bound for different destinations cocluster in different subdomains of the TGN. Secretory and membrane proteins that are earmarked for the same destination can cluster in the same subdomain of the TGN and can be incorporated into the same carrier vesicle. Therefore, the TGN appears to function as a cellular transportation terminal that is able to direct groups of passengers who are carrying the same tickets to a common waiting area and ultimately to load them onto a common shuttle. Ticket agents herd passengers bearing different tickets into different waiting lounges. A mannose 6-phosphate recognition marker is required to target newly synthesized hydrolytic enzymes to lysosomes The most thoroughly established sorting paradigm is the pathway for newly synthesized lysosomal enzymes. Like secretory proteins, lysosomal enzymes carry amino-terminal signal sequences that direct their cotranslational translocation across the membrane of the rough ER. Their N-linked glycosylation and folding proceed in the usual fashion, after which they join all of the other simultaneously synthesized proteins in the Golgi complex (Fig. 2-21, stage 1). A special sugar transferase in the cis-Golgi cisterna recognizes newly synthesized lysosomal enzymes and adds a unique sugar. This enzyme adds N-acetylglucosamine phosphate to the mannose residues at the termini of the lysosomal enzymes’ N-linked sugar trees. This enzyme differs from the usual sugar transferases in that it adds a phosphosugar group to the mannose residue, rather than just a sugar. This enzyme is also unique in recognizing specific amino acid sequences that are exclusively in these lysosomal enzymes. A second cis-Golgi enzyme removes the additional N-acetylglucosamine sugar, leaving its phosphate group behind. As a result, the sugar trees of the lysosomal enzymes terminate in mannose 6-phosphate residues (Fig. 2-21, stage 2). A special class of mannose 6-phosphate receptors, localized predominantly in the elements of the trans Golgi, recognize proteins that carry mannose 6-phosphate groups (Fig. 2-21, stage 3). This recognition step constitutes the first stage of the cosegregation and clustering process discussed earlier. The mannose 6-phosphate receptors are transmembrane proteins. Their luminal portions bind to the newly synthesized lysosomal enzymes, whereas their cytoplasmically facing tails possess a particular signal that allows them to interact with adaptins and hence to be incorporated into clathrin-coated vesicles. The assembly of the clathrin lattice causes the mannose 6-phosphate receptors to cluster, along with their associated lysosomal enzymes, in the plane of the TGN membrane. Completion of the clathrin cage results in the formation of a vesicle whose membrane contains the mannose 6-phosphate receptors that bind their cargo of lysosomal enzymes. After departing the TGN, these transport vesicles lose their clathrin coats (Fig. 2-21, stage 4) and fuse with structures referred to as late endosomes or prelysosomal endosomes. Proton pumps in the membranes of these organelles ensure that their luminal pH is acidic (Fig. 2-21, stage 5).

When exposed to this acidic environment, the mannose 6phosphate receptors undergo a conformational change that releases the mannose 6-phosphate–bearing lysosomal enzymes (Fig. 2-21, stage 6). Consequently, the newly synthesized enzymes are dumped into the lumen of the prelysosomal endosome, which will go on to fuse with or mature into a lysosome. The empty mannose 6-phosphate receptors join vesicles that bud off from the lysosome (Fig. 2-21, stage 7) and return to the TGN (Fig. 2-21, stage 8). The luminal environment of the TGN allows the receptors to recover their affinity for mannose 6-phosphate, thus allowing them to participate in subsequent rounds of sorting. Disruption of lysosomal sorting can be produced in several ways. For example, a drug called tunicamycin blocks the addition of N-linked sugars to newly synthesized proteins and thereby prevents attachment of the mannose 6phosphate recognition marker. Compounds that elevate the luminal pH of the prelysosomal endosomes prevent newly synthesized enzymes from dissociating from the mannose 6-phosphate receptors and consequently block recycling of the receptor pool back to the TGN. The resulting shortage of unoccupied receptors allows mannose 6-phosphate– bearing proteins to pass through the TGN unrecognized (see the box titled Lysosomal Storage Diseases). Thus, instead of diverting to the lysosomes, these lysosomal enzymes continue along the secretory pathway and are ultimately released from the cell by constitutive secretion. Cells internalize extracellular material through the process of endocytosis The same fundamental mechanisms in the secretory pathway that produce vesicles by evaginating regions of Golgi membrane can also move material in the opposite direction by inducing vesicle formation through the invagination of regions of the plasma membrane. Vesicles created in this fashion are delimited by membrane that had formerly been part of the cell surface, and their luminal contents derive from the extracellular compartment. This internalization process, referred to as endocytosis, serves the cell in at least four ways. First, certain nutrients are too large to be imported from the extracellular fluid into the cytoplasm by transmembrane carrier proteins; they are instead carried into the cell by endocytosis. Second, endocytosis of hormone-receptor complexes can terminate the signaling processes that are initiated by numerous hormones. Third, endocytosis is the first step in remodeling or degrading of portions of the plasma membrane. Membrane that is delivered to the surface during exocytosis must be retrieved and ultimately returned to the TGN. Fourth, proteins or pathogens that need to be cleared from the extracellular compartment are brought into the cell by endocytosis and subsequently condemned to degradation in the lysosomes. Because endocytosed material can pursue a number of different destinies, there must be sorting mechanisms in the endocytic pathway, just as in the secretory pathway, that allow the cell to direct the endocytosed material to its appropriate destination. Fluid-phase endocytosis is the uptake of the materials that are dissolved in the extracellular fluid (Fig. 2-22, stage 1) and not specifically bound to receptors on the cell surface.

41

42

Section II • Physiology of Cells and Molecules

3 A clathrin-coated endocytotic vesicle carries the endocytosed material. 2B

4 Acidification of the endosome dissociates Nucleus the ligand and its receptors.

Rough endoplasmic reticulum Lysosome

The ligand then attaches to receptors.

Caveolin-coated endocytotic vesicle Golgi

2A Particles not bound to receptors are endocytosed.

Uncoated endocytotic vesicle

Clathrincoated pit FLUID PHASE ENDOCYTOSIS RECEPTOR-MEDIATED ENDOCYTOSIS 1 The molecule begins in solution.

Figure 2-22

GPI-linked receptor molecules

H+

5 The receptors are recycled to the plasma membrane.

Caveolin-coated pit Caveolin 1,2,3

CAVEOLAE ENDOCYTOSIS

Small indentations in the plasma membrane called caveolae can mediate clathrin-independent endocytosis.

Endocytosis.

This process begins when a clathrin cage starts to assemble on the cytoplasmic surface of the plasma membrane. Earlier we discussed the physiology of clathrin-coated vesicles in the secretory pathway (Fig. 2-19). The clathrin attaches to the membrane through interactions with adaptin proteins, which in turn adhere to the cytoplasmic tail domains of certain transmembrane polypeptides. Construction of the cage causes its adherent underlying membrane to invaginate and to form a coated pit (Fig. 2-22, stage 2A). Completion of the cage creates a closed vesicle, which detaches from the cell surface through the process of membrane fission (Fig. 2-22, stage 3). The resultant vesicle quickly loses its clathrin coat through the action of the uncoating ATPase and fuses with an organelle called an endosome. Receptor-mediated endocytosis is responsible for internalizing specific proteins Most of the proteins that a cell seeks to import by endocytosis are present in the extracellular fluid in extremely low concentrations. Furthermore, the volume of extracellular fluid that is internalized by an individual coated vesicle is very small. Consequently, the probability that any particular target molecule will enter the cell during a given round of fluid-phase endocytosis is low. To improve the efficiency of endocytosis and to ensure that the desired extracellular components are gathered in every endocytic cycle, the cell has devised a method for concentrating specific proteins at the site of endocytosis before initiating their uptake. This concentration is achieved in a process known as receptor-mediated endocytosis, in which molecules to be

internalized (Fig. 2-22, stage 1) bind to cell surface receptors with high affinity (Fig. 2-22, stage 2B). Through this interaction, the substrates for endocytosis become physically associated with the plasma membrane, thus greatly enhancing the probability that they will be successfully internalized. Cells increase this probability even further by ensuring that the receptors themselves cluster in regions of the membrane destined to be endocytosed. The cytoplasmic tails of these receptors are endowed with recognition sequences that allow them to serve as binding sites for adaptins. Consequently, these receptors congregate in regions of the cell membrane where clathrin cages are assembling and are incorporated into coated pits as they are forming. The affinity of these receptors for the endocytic machinery ensures that their ligands are internalized with maximum efficiency. Most endocytic receptors are constitutively associated with coated pits and are endocytosed whether or not they have bound their specific ligands. The cytoplasmic tails of certain receptors, however, interact with adaptins only when the receptor is in the bound state. For example, in the absence of epidermal growth factor (EGF), the EGF receptor is excluded from regions of the membrane in which coated pits are assembling. Modifications induced by ligand binding alter these receptors’ tails, which allows them to participate in coated vesicle formation and hence in endocytosis. After the clathrin-coated vesicle forms (Fig. 2-22, stage 3), it quickly loses its clathrin coat, as described earlier for fluidphase endocytosis, and fuses with an endosome. Although endosomes can be wildly pleomorphic, they frequently have a frying pan–like appearance in which a round vesicular body is attached to a long tubular “handle” (Fig. 2-22, stage

Chapter 2 • Functional Organization of the Cell

Lysosomal Storage Diseases

T

he experimental elucidation of lysosomal enzyme sorting was achieved only because of the existence of a remarkable, naturally occurring human disease that was traced to a genetic defect in the sorting machinery. In lysosomal storage diseases, the absence of a particular hydrolase—or group of hydrolases—from the lysosome prevents the lysosomes from degrading certain substances, resulting in the formation of overstuffed lysosomes that crowd the cytoplasm and impede cell function. In I-cell disease, most hydrolases are missing from the lysosomes of many cell types. As a result, lysosomes become engorged with massive quantities of undigested substrates. The enormously swollen lysosomes that characterize this disease were named inclusion bodies, and the cells that possess them were designated inclusion cells, or I cells for short. Whereas I cells lack most lysosomal enzymes, the genes that encode all of the hydrolases are completely normal. The mutation responsible for I-cell disease resides in the gene for the phosphosugar transferase that creates the mannose 6-phosphate recognition marker (Fig. 2-21). Without this enzyme, the cell cannot sort any of the hydrolases to the lysosomes. Instead, the hydrolases pass through the trans-Golgi network unnoticed by the mannose 6-phosphate receptors and are secreted constitutively from the affected cells. Certain cell types from I-cell individuals can sort newly synthesized hydrolases normally, suggesting that alternative, as yet unelucidated pathways for the targeting of lysosomal enzymes must also exist. In some other lysosomal storage diseases, specific hydrolases are not missorted but rather are genetically defective. For example, children who suffer from Tay-Sachs disease carry a homozygous mutation in the gene that encodes the lysosomal enzyme hexosaminidase A (HEX A). Consequently, their lysosomes are unable to degrade substances that contain certain specific sugar linkages. Because they cannot be broken down, these substances accumulate in lysosomes. Over time, these substances fill the lysosomes, which swell and crowd the cytoplasm. The resulting derangements of cellular function are toxic to a number of cell types and ultimately underlie this disease’s uniform fatality within the first few years of life. Carriers of the Tay-Sachs trait can be detected either by HEX A enzyme testing or by DNA analysis of the HEX A gene. Among the Ashkenazi Jewish population, in which 1 in 27 individuals is a carrier, three distinct HEX A mutations account for 98% of all carrier mutations.

4). The cytoplasmic surfaces of the handles are often decorated with forming clathrin lattices and are the sites of vesicular budding. Endocytosed proteins can be targeted to lysosomes or recycled to the cell surface In many cell types, endocytosis is so rapid that each hour, the cell internalizes a quantity of membrane that is equivalent in area to the entire cell surface. To persist in the face of this tremendous flux of membrane, the cell must retrieve

most of the endocytosed membrane components and return them to the plasmalemma. However, substances that a cell wishes to degrade must be routed to lysosomes and prevented from escaping back to the surface. The sophisticated sorting operation required to satisfy both of these conditions takes place in the endosome. Proton pumps embedded in its membrane ensure that like the lysosome, the endosome maintains an acidic luminal pH (Fig. 2-22, stage 4). This acidic environment initiates the separation of material that is destined for lysosomal destruction from those proteins that are to be recycled. Most endocytic receptors bind their ligands tightly at neutral pH but release them rapidly at pH values below 6.0. Therefore, as soon as a surface-derived vesicle fuses with an endosome, proteins that are bound to receptors fall off and enter the endosomal lumen. The receptor proteins segregate in the membranes of the handles of the frying pan–shaped endosomes and are ultimately removed from the endosome in vesicles that shuttle them back to the cell surface (Fig. 2-22, stage 5). The soluble proteins of the endosome lumen, which include the receptors’ former ligands, are ultimately delivered to the lysosome. This sorting scheme allows the receptors to avoid the fate of their cargo and ensures that the receptors are used in many rounds of endocytosis. The low-density lipoprotein (LDL) receptor follows this regimen precisely. On arrival of the LDL-laden receptor at the endosome, the acidic environment of the endosome induces the LDL to dissociate from its receptor, which then promptly recycles to the cell surface. The LDL travels on to the lysosome, where enzymes destroy the LDL and liberate its bound cholesterol. A variation on this paradigm is responsible for the cellular uptake of iron. Iron circulates in the plasma bound to a protein called transferrin. At the mildly alkaline pH of extracellular fluid, the iron-transferrin complex binds with high affinity to a transferrin receptor in the plasma membranes of almost every cell type. Bound transferrin is internalized by endocytosis and delivered to endosomes. Instead of inducing transferrin to fall off its receptor, the acid environment of the endosome lumen causes iron to fall off transferrin. Apotransferrin (i.e., transferrin without bound iron) remains tightly bound to the transferrin receptor at an acidic pH. The released iron is transported across the endosomal membrane for use in the cytosol. The complex of apotransferrin and the transferrin receptor recycles to the cell surface, where it is again exposed to the extracellular fluid. The mildly alkaline extracellular pH causes the transferrin receptor to lose its affinity for apotransferrin and promptly releases it. Thus, the cell uses the pH-dependent sorting trick twice to ensure that both the transferrin receptor and apotransferrin recycle for subsequent rounds of iron uptake. Certain molecules are internalized through an alternative process that involves caveolae Clathrin-coated pits are not the only cellular structures involved in receptor-mediated internalization. Electron microscopic examination of vascular endothelial cells that line blood vessels long ago revealed the presence of clusters of small vesicles that display a characteristic appearance, in close association with the plasma membrane. These caveolae

43

44

Section II • Physiology of Cells and Molecules

were thought to be involved in the transfer of large molecules across the endothelial cells, from the blood space to the tissue compartment. Actually, caveolae are present in most cell types. The caveolae are rich in cholesterol and sphingomyelin. Rather than having a clathrin lattice, they contain intrinsic membrane proteins called caveolins, which face the cytosol (Fig. 2-22). In addition, caveolae appear to be rich in membrane-associated polypeptides that participate in intracellular signaling, such as the Ras-like proteins as well as heterotrimeric GTP-binding proteins (see Chapter 5). They are also enriched in the receptor for folate, a vitamin required by several metabolic pathways (see Chapter 45). Unlike the receptors in the plasma membrane discussed earlier, the folate receptor has no cytoplasmic tail that might allow it to associate with coated pits. Instead, it belongs to the GPIlinked class of proteins that are anchored to the membrane through covalent attachment to phospholipid molecules. It appears that caveolae mediate the internalization of folate. In fact, a large number and variety of GPI-linked proteins are embedded in the outer leaflet of the caveolar membrane that faces its lumen. The role of caveolae in the uptake of other substances, the significance of the large inventory of GPI-linked proteins in caveolae, and the functions served by their cache of signaling molecules remain to be determined. It is clear, however, that the caveolae represent a novel endocytic structure that participates in pathways distinct from those involving coated vesicles and endosomes.

SPECIALIZED CELL TYPES All cells are constructed of the same basic elements and share the same basic metabolic and biosynthetic machinery. What distinguishes one cell type from another? Certainly, cells have different shapes and molecular structures. In addition, out of an extensive repertory of molecules that cells are capable of making, each cell type chooses which molecules to express, how to organize these molecules, and how to regulate them. It is this combination of choices that endows them with specific physiological functions. These specializations are the product of cell differentiation. Each of these cell types arises from a stem cell. Stem cells are mitotically active and can give rise to multiple, distinct cellular lineages; thus, they are referred to as pluripotent. Clearly, the zygote is the ultimate stem cell because through its divisions, it gives rise to every cell lineage present in the complete organism. Specific cell types arise from stem cells by activating a differentiation-specific program of gene expression. The interplay of environmental signals, temporal cues, and transcription factors that control the processes of cellular differentiation constitutes one of the great unraveling mysteries of modern biology. Epithelial cells form a barrier between the internal and external milieu How can an organism tightly regulate its internal fluid environment (i.e., internal milieu) without allowing this environment to come into direct and disastrous contact with the external world (i.e., external milieu)? The body has solved

these problems by arranging a sheet of cells—an epithelium—between two disparate solutions. Because of their unique subcellular designs and intercellular relationships, epithelial cells form a dynamic barrier that can import or expel substances, sometimes against steep concentration gradients. Two structural features of epithelia permit them to function as useful barriers between two very different solutions (Fig. 2-23). First, epithelial cells connect to one another by tight junctions, which constrain the free diffusion of solutes and fluids around the epithelial cells, between the internal and external compartments. Second, the tight junctions define a boundary between an apical and a basolateral domain of the plasma membrane. Each of these two domains is endowed with distinct protein and lipid components, and each subserves a distinct function. Thus, the surface membranes of epithelial cells are polarized. In Chapter 5, we discuss the mechanisms by which polarized epithelial cells exploit their unique geometry to transport salts and water from one solution to the other. However, it is worth touching on a few of the cellular specializations that characterize polarized epithelia and permit them to perform their critical roles. The apical membranes of the epithelial cells (Fig. 2-23) face the lumen of a compartment that is often topologically continuous with the outside world. For example, in the stomach and intestine, apical membranes form the inner surface of the organs that come into contact with ingested matter. The apical membranes of many epithelial cells, including those lining kidney tubules, are endowed with a single nonmotile cilium. Known as the central cilium, this structure may sense the mechanical deformation associated with fluid flow. Mutations that disrupt individual components of the central cilium are associated with cystic disease of the kidney, in which the normal architecture of the kidney is replaced by a collection of large fluid-filled cysts. The basolateral membranes of epithelial cells face the extracellular fluid compartment—which indirectly makes contact with the blood—and rest on a basement membrane. The basement membrane is composed of extracellular matrix proteins that the epithelial cells themselves secrete and include collagens, laminin, and proteoglycans. The basement membrane provides the epithelium with structural support and, most important, serves as an organizing foundation that helps the epithelial cells to establish their remarkable architecture. Each epithelial cell is interconnected to its neighbors by a variety of junctional complexes (Fig. 2-23). The lateral surfaces of epithelial cells participate in numerous types of cell-cell contacts, including tight junctions, adhering junctions, gap junctions, and desmosomes. Tight Junctions A tight junction (or zonula occludens) is a complex structure that impedes the passage of molecules and ions between the cells of the epithelial monolayer. This pathway between the cells is termed the paracellular pathway. Although the complete molecular structure of the tight junction has yet to be elucidated, it is clear that its functional properties are related to its intriguing architecture (Fig. 2-23). Viewed by transmission electron microscopy, tight junctions include regions of apparent fusion between

Chapter 2 • Functional Organization of the Cell

TIGHT JUNCTION

ADHERING JUNCTION Groove Ridge

Cadherins

Extracellular space

Actin filaments

Strands of transmembrane proteins (e.g., claudins)

Microvilli

Apical membrane

Extracellular space

Epithelial cells Basolateral membrane Cadherins

Cytosol brane

t mem

en Basem Connexons Adjacent plasma membranes GAP JUNCTION

Intermediate filaments

Plaque DESMOSOME

Figure 2-23 Epithelial cells. In an epithelial cell, the tight junction separates the cell membrane into apical and basolateral domains that have very different functional properties.

the outer leaflets of the lipid bilayer membranes of neighboring epithelial cells. Freeze-fracture electron microscopy reveals that the tight junction comprises parallel strands of closely packed particles, which presumably represent the transmembrane proteins participating in the junction’s formation. The degree of an epithelium’s impermeability—or “tightness”—is roughly proportional to the number of these parallel strands. The claudins, a large family of proteins, are the principal structural elements of the tight junction. Interactions between the claudins present in the apposing membranes of neighboring cells form the permeability barrier (see Chapter 5). Tight junctions play several roles. First, they are barriers in that they separate one compartment from another. In some epithelial cells, such as those of the renal thick ascending limb, the tight junctions form an essentially impenetrable boundary that completely blocks the flow of ions and water between cells. In contrast, the tight junctions of the renal proximal tubule are leaky, permitting significant transepithelial movement of fluid and solutes. Second, tight junctions can act as selective gates in that they permit certain solutes to flow more easily than others. Examples are the leaky tight junctions of tissues such as the proximal tubule. As discussed in Chapter 5, the permeability and selectivity of an epithelium’s tight junctions are critical

variables for determining that epithelium’s transport characteristics. Moreover, the permeability properties of the gate function of tight junctions can be modulated in response to various physiological stimuli. The inventory of claudins expressed by an epithelium appears to determine in large measure the permeability properties of the tight junctions. Third, tight junctions act as fences that separate the polarized surfaces of the epithelial plasma membrane into apical and basolateral domains. The presence of distinct populations of proteins and lipids in each plasma membrane domain is absolutely essential for an epithelium to mediate transepithelial fluid and solute transport (see Chapter 5). Adhering Junction An adhering junction (or zonula adherens) is a belt that encircles an entire epithelial cell just below the level of the tight junction. Epithelial cells need two pieces of information to build themselves into a coherent epithelium. First, the cells must know which end is up. The extracellular matrix (see earlier) provides this information by defining which side will be basolateral. Second, the cells must know that there are like neighbors with which to establish cell-cell contacts. Adhering junctions provide epithelial cells with clues about the nature and proximity of their neighbors. These cell-cell contacts are mediated by the

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extracellular domains of members of the cadherin family, transmembrane proteins discussed earlier. Epithelial cells will organize themselves into a properly polarized epithelium—with differentiated apical and basolateral plasma membranes—only if the cadherins of neighboring cells have come into close enough apposition to form an adhering junction. Formation of these junctions initiates the assembly of a subcortical cytoskeleton, in which anchor proteins (e.g., vinculin, catenins, α-actinin) link the cytosolic domains of cadherins to a network of actin filaments that is associated with the cytosolic surfaces of the lateral membranes. Conversely, the disruption of adhering junctions can lead to a loss of epithelial organization. In epithelial tumors, for example, loss of expression of the adhering junction cadherins tends to correlate with the tumor cell’s loss of controlled growth and its ability to metastasize, that is, to leave the epithelial monolayer and form a new tumor at a distant site in the body. Gap Junctions

Gap junctions, which are discussed in Chapter 6, are channels that interconnect the cytosols of neighboring cells. They allow small molecules (less than ∼1000 in molecular weight) to diffuse freely between cells. In some organs, epithelial cells are interconnected by an enormous number of gap junctions, which organize into paracrystalline hexagonal arrays. Because ions can flow through gap junctions, cells that communicate through gap junctions are electrically coupled. The permeability of gap junctions, and hence the extent to which the cytoplasmic compartments of neighboring cells are coupled, can be regulated in response to a variety of physiological stimuli. Desmosome

A desmosome (or macula adherens) holds adjacent cells together tightly at a single, round spot. Desmosomes are easily recognized in thin-section electron micrographs by the characteristic dense plaques of intermediate filaments. The extracellular domains of transmembrane proteins in the cadherin family mediate the interaction of adjacent cells. Anchor proteins link the cytosolic domains of the cadherins to intermediate filaments that radiate into the cytoplasm from the point of intercellular contact (Fig. 2-23). These filaments interact with and organize the cytoplasmic intermediate filaments, thus coupling the structurally stabilizing elements of neighboring cells to one another. Epithelial cells are often coupled to adjacent cells by numerous desmosomes, especially in regions where the epithelium is subject to physical stress. Epithelial cells are polarized In many epithelia, the apical surface area is amplified by the presence of a brush border that is composed of hundreds of finger-like, microvillar projections (Fig. 2-23). In the case of the small intestine and the renal proximal tubule, the membrane covering each microvillus is richly endowed with enzymes that digest sugars and proteins as well as with transporters that carry the products of these digestions into the cells. The presence of a microvillar brush border can amplify the apical surface area of a polarized epithelial cell by as much as 20-fold, thus greatly enhancing its capacity to inter-

act with, to modify, and to transport substances present in the luminal fluid. The basolateral surface area of certain epithelial cells is amplified by the presence of lateral interdigitations and basal infoldings (Fig. 2-23). Although they are not as elegantly constructed as microvilli, these structures can greatly increase the basolateral surface area. In epithelial cells that are involved in large volumes of transport—or in transport against steep gradients—amplifying the basolateral membrane can greatly increase the number of basolateral Na-K pumps that a single cell can place at its basolateral membrane. Although the morphological differences between apical and basolateral membranes can be dramatic, the most important distinction between these surfaces is their protein composition. As noted earlier, the “fence” function of the tight junction separates completely different rosters of membrane proteins between the apical and basolateral membranes. For example, the Na-K pump is restricted to the basolateral membrane in almost all epithelial cells, and the membrane-bound enzymes that hydrolyze complex sugars and peptides are restricted to apical membranes in intestinal epithelial cells. The polarized distribution of transport proteins is absolutely necessary for the directed movement of solutes and water across epithelia. Furthermore, the restriction of certain enzymes to the apical domain limits their actions to the lumen of the epithelium and therefore offers the advantage of not wasting energy putting enzymes where they are not needed. The polarity of epithelial membrane proteins also plays a critical role in detecting antigens present in the external milieu and in transmitting signals between the external and internal compartments. The maintenance of epithelial polarity involves complex intermolecular interactions that are only beginning to be understood. When tight junctions are disrupted, diffusion in the plane of the membrane leads to intermingling of apical and basolateral membrane components and thus a loss of polarity. The subcortical cytoskeleton beneath the basolateral surface may play a similar role by physically restraining a subset of membrane proteins at the basolateral surface. However, such mechanisms for stabilizing the polarized distributions of membrane proteins do not explain how newly synthesized proteins come to be distributed at the appropriate plasma membrane domain. We give two examples of mechanisms that cells can use to direct membrane proteins to either the basolateral or apical membrane. The first example focuses on protein-protein interactions. As noted during our discussion of the secretory protein pathway, the sorting operation that separates apically from basolaterally directed proteins apparently occurs in the TGN. Some proteins destined for the basolateral membrane have special amino acid motifs that act as sorting signals. Some of these motifs are similar to those that allow membrane proteins to participate in endocytosis. Members of the adaptin family may recognize these motifs during the formation of clathrin-coated vesicles at the TGN and segregate the basolateral proteins into a vesicle destined for the basolateral membrane. Another example of mechanisms that cells use to generate a polarized distribution of membrane proteins focuses on lipid-lipid interactions. In many epithelia, GPI-linked pro-

Chapter 2 • Functional Organization of the Cell

teins are concentrated exclusively at the apical surface. It appears that the phospholipid components of GPI-linked proteins are unusual in that they cluster into complexes of fairly immobile gel-phase lipids during their passage through the Golgi apparatus. We saw earlier how lakes of phospholipids with different physical properties may segregate within a membrane. The “glycolipid rafts” of GPI-linked proteins incorporate into apically directed vesicles so that sorting can occur through lipid-lipid interactions in the plane of the membrane rather than through protein-protein interactions at the cytoplasmic surface of the Golgi membrane. From these two examples, it should be clear that a number of different mechanisms may contribute to protein sorting and the maintenance of epithelial polarity. REFERENCES Books and Reviews Goldstein JL, Brown MS, Anderson RGW, et al: Receptor-mediated endocytosis: Concepts emerging from the LDL receptor system. Annu Rev Cell Dev Biol 1985; 1:1-39. Mellman I: Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 1996; 12:575-625.

Palade GE: Intracellular aspects of the process of protein synthesis. Science 1985; 189:347-358. Rodriguez-Boulan E, Powell SK: Polarity of epithelial and neuronal cells. Annu Rev Cell Dev Biol 1992; 8:395-427. Rothman JE: The protein machinery of vesicle budding and fusion. Protein Sci 1995; 5:185-194. Sheetz MP: Microtubule motor complexes moving membranous organelles. Cell Struct Funct 1996; 21:369-373. Journal Articles Frye LD, Edidin M: The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. J Cell Sci 1970; 7:319-335. Griffiths G, Hoflack B, Simons K, et al: The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell 1988; 52:329-341. Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157:105-132. Walter P, Ibrahimi I, Blobel G: Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in vitro assembled polysomes synthesizing secretory protein. J Cell Biol 1981; 91:545-550.

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CHAPTER

3 SIGNAL TRANSDUCTION Lloyd Cantley

The evolution of multicellular organisms necessitated the development of mechanisms to tightly coordinate the activities among cells. Such communication is fundamental to all biological processes, ranging from the induction of embryonic development to the integration of physiological responses in the face of environmental challenges. As our understanding of cellular and molecular physiology has increased, it has become evident that all cells can receive and process information. External signals such as odorants, chemicals that reflect metabolic status, ions, hormones, growth factors, and neurotransmitters can all serve as chemical messengers linking neighboring or distant cells. Even external signals that are not considered chemical in nature (e.g., light and mechanical or thermal stimuli) may ultimately be transduced into a chemical messenger. Most chemical messengers interact with specific cell surface receptors and trigger a cascade of secondary events, including the mobilization of diffusible intracellular second-messenger systems that mediate the cell’s response to that stimulus. However, hydrophobic messengers, such as steroid hormones and some vitamins, can diffuse across the plasma membrane and interact with cytosolic or nuclear receptors. It is now clear that cells use a number of different, often intersecting intracellular signaling pathways to ensure that the cell’s response to a stimulus is tightly controlled.

MECHANISMS OF CELLULAR COMMUNICATION

48

However, many other cells and tissues not classically thought of as endocrine in nature also produce hormones. For example, the kidney produces 1,25-dihydroxyvitamin D3, and the salivary gland synthesizes nerve growth factor. It is now recognized that intercellular communication can involve the production of a “hormone” or chemical signal by one cell type that acts in any (or all) of three ways, as illustrated in Figure 3-1: on distant tissues (endocrine), on a neighboring cell in the same tissue (paracrine), or on the same cell that released the signaling molecule (autocrine). For paracrine and autocrine signals to be delivered to their proper targets, their diffusion must be limited. This restriction can be accomplished by rapid endocytosis of the chemical signal by neighboring cells, its destruction by extracellular enzymes, or its immobilization by the extracellular matrix. The events that take place at the neuromuscular junction are excellent examples of paracrine signaling. When an electrical impulse travels down an axon and reaches the nerve terminal (Fig. 3-2), it stimulates release of the neurotransmitter acetylcholine (ACh). In turn, ACh transiently activates a ligand-gated cation channel on the muscle cell membrane. The resultant transient influx of Na+ causes a localized positive shift of Vm (i.e., depolarization), initiating events that result in propagation of an action potential along the muscle cell. The ACh signal is rapidly terminated by the action of acetylcholinesterase, which is present in the synaptic cleft. This enzyme degrades the ACh that is released by the neuron.

Cells can communicate with one another by chemical signals

Soluble chemical signals interact with target cells by binding to surface or intracellular receptors

Early insight into signal transduction pathways was obtained from studies of the endocrine system. The classic definition of a hormone is a substance that is produced in one tissue or organ and released into the blood and carried to other organs (targets), where it acts to produce a specific response. The idea of endocrine or ductless glands developed from the recognition that certain organs—such as the pituitary, adrenal, and thyroid gland—can synthesize and release specific chemical messengers in response to particular physiological states.

Four types of chemicals can serve as extracellular signaling molecules: amines, such as epinephrine; peptides and proteins, such as angiotensin II and insulin; steroids, including aldosterone, estrogens, and retinoic acid; and other small molecules, such as amino acids, nucleotides, ions (e.g., Ca2+), and gases (e.g., nitric oxide). For a molecule to act as a signal, it must bind to a receptor. A receptor is a protein (or in some cases a lipoprotein) on the cell surface or within the cell that specifically binds a

Chapter 3 • Signal Transduction

A

B

ENDOCRINE Cell of endocrine tissue

PARACRINE

C AUTOCRINE Hormones

Nucleus

Blood vessel Hormones

Signaling molecules

Hormone receptor

Nucleus

Target receptors Nucleus Non-target cells

Figure 3-1

Target receptor

Target cells

Modes of cell communication.

are initiated by the binding of any one ligand to its receptor. Receptors can be divided into four categories on the basis of their associated mechanisms of signal transduction (Table 3-1).

Axon Electrical stimulus Nerve terminal

ACh +

Na Muscle cell

Acetylcholine receptor

Arrival of an electrical stimulus triggers release of acetylcholine, which binds to the acetylcholine receptor on the muscle cell…

…activating the entry of sodium, which causes a local membrane depolarization.

Acetylcholinesterase degrades the transmitter, terminating the signal.

Figure 3-2 Example of paracrine signaling. The release of ACh at the neuromuscular junction is a form of paracrine signaling because the nerve terminal releases a chemical (i.e., ACh) that acts on a neighboring cell (i.e., the muscle).

signaling molecule (the ligand). In some cases, the receptor is itself an ion channel, and ligand binding produces a change in Vm. Thus, the cell can transduce a signal with no machinery other than the receptor. In most cases, however, interaction of the ligand with one or more specific receptors results in an association of the receptor with an effector molecule that initiates a cellular response. Effectors include enzymes, channels, transport proteins, contractile elements, and transcription factors. The ability of a cell or tissue to respond to a specific signal is dictated by the complement of receptors it possesses and by the chain of intracellular reactions that

1. Ligand-gated ion channels. Integral membrane proteins, these hybrid receptor/channels are involved in signaling between electrically excitable cells. The binding of a neurotransmitter such as ACh to its receptor—which in fact is merely part of the channel—results in transient opening of the channel, thus altering the ion permeability of the cell. 2. G protein–coupled receptors. These integral plasma membrane proteins work indirectly—through an intermediary—to activate or to inactivate a separate membrane-associated enzyme or channel. The intermediary is a heterotrimeric guanosine triphosphate (GTP)–binding complex called a G protein. 3. Catalytic receptors. When activated by a ligand, these integral plasma membrane proteins are either enzymes themselves or part of an enzymatic complex. 4. Nuclear receptors. These proteins, located in the cytosol or nucleus, are ligand-activated transcription factors. These receptors link extracellular signals to gene transcription. In addition to these four classes of membrane signaling molecules, some other transmembrane proteins act as messengers even though they do not fit the classic definition of a receptor. In response to certain physiological changes, they undergo regulated intramembrane proteolysis within the plane of the membrane, liberating cytosolic fragments that enter the nucleus to modulate gene expression. We discuss this process later in the chapter. Signaling events initiated by plasma membrane receptors can generally be divided into six steps: Step 1: Recognition of the signal by its receptor. The same signaling molecule can sometimes bind to more than one

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Table 3-1

Classification of Receptors and Associated Signal Transduction Pathways

Class of Receptor

Subunit Composition of Receptor

Ligand

Signal Transduction Pathway Downstream from Receptor

Ligand-gated ion channels (ionotropic receptors)

Heteromeric or homomeric oligomers

Extracellular GABA Glycine ACh: muscle ACh: nerve 5-HT Glutamate: non-NMDA Glutamate: NMDA ATP (opening) Intracellular cGMP (vision) cAMP (olfaction) ATP (closes channel) IP3 Ca2+ or ryanodine

Ion Current Cl− > HCO3− Cl− > HCO3− Na+, K+, Ca2+ Na+, K+, Ca2+ Na+, K+ Na+, K+, Ca2+ Na+, K+, Ca2+ Ca2+, Na+, Mg2+ Na+, K+ Na+, K+ K+ Ca2+ Ca2+

Receptors coupled to heterotrimeric (αβγ) G proteins

Single polypeptide that crosses the membrane seven times

Small transmitter molecules ACh Norepinephrine Peptides Oxytocin Parathyroid hormone Neuropeptide Y Gastrin Cholecystokinin Odorants Certain cytokines, lipids, and related molecules

bg Directly activates downstream effector: Muscarinic ACh receptor activates atrial K+ channel α Activates an enzyme: Cyclases that make cyclic nucleotides (cAMP, cGMP) Phospholipases that generate IP3 and diacylglycerols Phospholipases that generate arachidonic acid and its metabolites

Catalytic receptors

Single polypeptide that crosses the membrane once May be dimeric or may dimerize after activation

ANP TGF-β

Receptor guanylyl cyclase Receptor serine/threonine kinases Receptor tyrosine kinase Tyrosine kinase–associated receptor Receptor tyrosine phosphatase

Intracellular (or nuclear) receptors

Homodimers of polypeptides, each with multiple functional domains

Heterodimers of polypeptides, each with multiple functional domains

NGF, EGF, PDGF, FGF, insulin, IGF-1 IL-3, IL-5, IL-6, EPO, LIF, CNTF, GH, IFN-α, IFN-β, IFN-γ, GM-CSF CD45 Steroid hormones Mineralocorticoids Glucocorticoids Androgens Estrogens Progestins Others Thyroid hormones Retinoic acid Vitamin D Prostaglandin

kind of receptor. For example, ACh can bind to both ligand-gated channels and G protein–coupled receptors. Binding of a ligand to its receptor involves the same three types of weak, noncovalent interactions that characterize substrate-enzyme interactions. Ionic bonds are formed between groups of opposite charge. In van der Waals interactions, a transient dipole in one atom generates the opposite dipole in an adjacent atom, thereby creating an

Bind to regulatory DNA sequences and directly or indirectly increase or decrease the transcription of specific genes

electrostatic interaction. Hydrophobic interactions occur between nonpolar groups. Step 2: Transduction of the extracellular message into an intracellular signal or second messenger. Ligand binding causes a conformational change in the receptor that triggers the catalytic activities intrinsic to the receptor or causes the receptor to interact with membrane or cytoplasmic enzymes. The final consequence is the generation

Chapter 3 • Signal Transduction

of a second messenger or the activation of a catalytic cascade. Step 3: Transmission of the second messenger’s signal to the appropriate effector. These effectors represent a diverse array of molecules, such as enzymes, ion channels, and transcription factors. Step 4: Modulation of the effector. These events often result in the activation of protein kinases (which put phosphate groups on proteins) and phosphatases (which take them off), thereby altering the activity of other enzymes and proteins. Step 5: Response of the cell to the initial stimulus. This collection of actions represents the summation and integration of input from multiple signaling pathways. Step 6: Termination of the response by feedback mechanisms at any or all levels of the signaling pathway. Cells can also communicate by direct interactions gap Junctions Neighboring cells can be electrically and metabolically coupled by means of gap junctions formed between apposing cell membranes. These water-filled channels facilitate the passage of inorganic ions and small molecules, such as Ca2+ and 3′,5′-cyclic adenosine monophosphate (cAMP), from the cytoplasm of one cell into the cytoplasm of an adjacent cell. Mammalian gap junctions permit the passage of molecules that are less than ∼1200 Da but restrict the movement of molecules that are greater than ∼2000 Da. Gap junctions are also excellent pathways for the flow of electrical current between adjacent cells, playing a critical role in cardiac and smooth muscle. The permeability of gap junctions can be rapidly regulated by changes in cytosolic concentrations of Ca2+, cAMP, and H+ as well as by the voltage across the cell membrane or membrane potential (Vm) (see Chapter 5). This type of modulation is physiologically important for cell-to-cell communication. For example, if a cell’s plasma membrane is damaged, Ca2+ passively moves into the cell and raises [Ca2+]i to toxic levels. Elevated intracellular [Ca2+] in the damaged cell triggers closure of the gap junctions, thus preventing the flow of excessive amounts of Ca2+ into the adjacent cell. Adhering and Tight Junctions

Adhering junctions form as the result of the Ca2+-dependent interactions of the extracellular domains of transmembrane proteins called cadherins (see Chapter 2). The clustering of cadherins at the site of interaction with an adjacent cell causes secondary clustering of intracellular proteins known as catenins, which in turn serve as sites of attachment for the intracellular actin cytoskeleton. Thus, adhering junctions provide important clues for the maintenance of normal cell architecture as well as the organization of groups of cells into tissues. In addition to a homeostatic role, adhering junctions can serve a signaling role during organ development and remodeling. In a cell that is stably associated with its neighbors, a catenin known as β-catenin is mainly sequestered at the adhering junctions, minimizing concentration of free βcatenin. However, disruption of adhering junctions by certain growth factors, for example, causes β-catenin to disassociate from cadherin. The resulting rise in free β-catenin

levels promotes the translocation of β-catenin to the nucleus. There, β-catenin regulates the transcription of multiple genes, including ones that promote cell proliferation and migration. Similar to adhering junctions, tight junctions (see Chapter 2) comprise transmembrane proteins that link with their counterparts on adjacent cells as well as intracellular proteins that stabilize the complex and also have a signaling role. The transmembrane proteins—including claudins, occludin, and junctional adhesion molecule—and their extracellular domains create the diffusion barrier of the tight junction. One of the integral cytoplasmic proteins in tight junctions, zonula occludin 1 (ZO-1), colocalizes with a serine/threonine kinase known as WNK1, which is found in certain renal tubule epithelial cells that reabsorb Na+ and Cl− from the tubule lumen. Because WNK1 is important for determining the permeability of the tight junctions to Cl−, mutations in WNK1 can increase the movement of Cl− through the tight junctions (see Chapter 35) and thereby lead to hypertension. Membrane-Associated Ligands

Another mechanism by which cells can directly communicate is by the interaction of a receptor in the plasma membrane with a ligand that is itself a membrane protein on an adjacent cell. Such membrane-associated ligands can provide spatial clues in migrating cells. For example, an ephrin ligand expressed on the surface of one cell can interact with an Eph receptor on a nearby cell. The resulting activation of the Eph receptor can in turn provide signals for regulating such developmental events as axonal guidance in the nervous system and endothelial cell guidance in the vasculature. Second-messenger systems amplify signals and integrate responses among cell types

Once a signal has been received at the cell surface, it is typically amplified and transmitted to specific sites within the cells through second messengers. For a molecule to function as a second messenger, its concentration, or window of activity, must be finely regulated. The cell achieves this control by rapidly producing or activating the second messenger and then inactivating or degrading it. To ensure that the system returns to a resting state when the stimulus is removed, counterbalancing activities function at each step of the cascade. The involvement of second messengers in catalytic cascades provides numerous opportunities to amplify a signal. For example, the binding of a ligand to its receptor can generate hundreds of second-messenger molecules, which can in turn alter the activity of thousands of downstream effectors. This modulation usually involves the conversion of an inactive species into an active molecule or vice versa. An example of such a cascade is the increased intracellular concentration of the second messenger cAMP. Receptor occupancy activates a G protein, which in turn stimulates a membrane-bound enzyme, adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP from adenosine triphosphate (ATP), and a 5-fold increase in the intracellular concentration of cAMP is achieved in ∼5 seconds. This sudden rise in cAMP levels is rapidly counteracted by its

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breakdown to adenosine 5′-monophosphate by cAMP phosphodiesterase. Second-messenger systems also allow specificity and diversity. Ligands that activate the same signaling pathways in cells usually produce the same effect. For example, epinephrine, adrenocorticotropic hormone (ACTH), glucagon, and thyroid-stimulating hormone induce triglyceride breakdown through the cAMP messenger system. However, the same signaling molecule can produce distinct responses in different cells, depending on the complement of receptors and signal transduction pathways that are available in the cell as well as the specialized function that the cell carries out in the organism. For example, ACh induces contraction of skeletal muscle cells but inhibits contraction of heart muscle. It also facilitates the exocytosis of secretory granules in pancreatic acinar cells. This signaling molecule achieves these different endpoints by interacting with distinct receptors. The diversity and specialization of second-messenger systems are important to a multicellular organism, as can be seen in the coordinated response of an organism to a stressful situation. Under these conditions, the adrenal gland releases epinephrine. Different organ systems respond to epinephrine in a distinct manner, such as activation of glycogen breakdown in the liver, constriction of the blood vessels of the skin, dilation of the blood vessels in skeletal muscle, and increased rate and force of heart contraction. The overall effect is an integrated response that readies the organism for attack, defense, or escape. In contrast, complex cell behaviors, such as proliferation and differentiation, are generally stimulated by combinations of signals rather than by a single signal. Integration of these stimuli requires crosstalk among the various signaling cascades. As discussed later, most signal transduction pathways use elaborate cascades of signaling proteins to relay information from the cell surface to effectors in the cell membrane, the cytoplasm, or the nucleus. In Chapter 4, we discuss how signal transduction pathways that lead to the nucleus can affect the cell by modulating gene transcription. These are genomic effects. Signal transduction systems that project to the cell membrane or to the cytoplasm produce nongenomic effects, the focus of this chapter.

stoichiometry of 2 : 1 : 1 : 1. This receptor is called nicotinic because the nicotine contained in tobacco can activate or open the channel and thereby alter Vm. Note that the nicotinic AChR is very different from the muscarinic AChR discussed later, which is not a ligand-gated channel. Additional examples of ligand-gated channels are the IP3 receptor and the Ca2+ release channel (also known as the ryanodine receptor). Both receptors are tetrameric Ca2+ channels located in the membranes of intracellular organelles.

RECEPTORS COUPLED TO G PROTEINS G protein–coupled receptors (GPCRs) constitute the largest family of receptors on the cell surface, with more than 1000 members. GPCRs mediate cellular responses to a diverse array of signaling molecules, such as hormones, neurotransmitters, vasoactive peptides, odorants, tastants, and other local mediators. Despite the chemical diversity of their ligands, most receptors of this class have a similar structure (Fig. 3-3). They consist of a single polypeptide chain with seven membrane-spanning α-helical segments, an extracellular N terminus that is glycosylated, a large cytoplasmic loop that is composed mainly of hydrophilic amino acids between helices 5 and 6, and a hydrophilic domain at the cytoplasmic C terminus. Most small ligands (e.g., epinephrine) bind in the plane of the membrane at a site that involves several membrane-spanning segments. In the case of larger protein ligands, a portion of the extracellular N terminus also participates in ligand binding. The 5,6-cytoplasmic loop appears to be the major site of interaction with the intracellular G protein, although the 3,4-cytoplasmic loop and the cytoplasmic C terminus also contribute to binding in some cases. Binding of the GPCR to its extracellular ligand regulates this interaction between the receptor and the G proteins, thus transmitting a signal to downstream effectors. In the next four sections of this subchapter, we discuss the general principles of how G proteins function; three major

Extracellular space

RECEPTORS THAT ARE ION CHANNELS N

Ligand-gated ion channels transduce a chemical signal into an electrical signal The property that defines this class of multisubunit membrane-spanning receptors is that the signaling molecule itself controls the opening and closing of an ion channel by binding to a site on the receptor. Thus, these receptors are also called ionotropic receptors to distinguish them from the metabotropic receptors, which act through “metabolic” pathways. One superfamily of ligand-gated channels includes the ionotropic receptors for ACh, serotonin, γ-aminobutyric acid (GABA), and glycine. Most structural and functional information for ionotropic receptors comes from the nicotinic ACh receptor (AChR) present in skeletal muscle (Fig. 3-2). The nicotinic AChR is a cation channel that consists of four membrane-spanning subunits, α, β, γ, and δ, in a

C G protein binding Cytosol

Figure 3-3

Receptor coupled to a G protein.

Chapter 3 • Signal Transduction

second-messenger systems that are triggered by G proteins are then considered.

GENERAL PROPERTIES OF G PROTEINS G proteins are heterotrimers that exist in many combinations of different α, b, and g subunits G proteins are members of a superfamily of GTP-binding proteins. This superfamily includes the classic heterotrimeric G proteins that bind to GPCRs as well as the so-called small GTP-binding proteins, such as Ras. Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between an active GTP-bound state and an inactive guanosine diphosphate (GDP)–bound state. Heterotrimeric G proteins are composed of three subunits, α, β, and γ. At least 16 different α subunits (∼42 to

Table 3-2

50 kDa), 5 β subunits (∼33 to 35 kDa), and 11 γ subunits (∼8 to 10 kDa) are present in mammalian tissue. The α subunit binds and hydrolyzes GTP and also interacts with “downstream” effector proteins such as adenylyl cyclase. Historically, the α subunits were thought to provide the principal specificity to each type of G protein, with the βγ complex functioning to anchor the trimeric complex to the membrane. However, it is now clear that the βγ complex also functions in signal transduction by interacting with certain effector molecules. Moreover, both the α and γ subunits are involved in anchoring the complex to the membrane. The α subunit is held to the membrane by either a myristyl or a palmitoyl group; the γ subunit is held by a prenyl group. The multiple α, β, and γ subunits demonstrate distinct tissue distributions and interact with different receptors and effectors (Table 3-2). Because of the potential for several hundred combinations of the known α, β, and γ subunits, G proteins are ideally suited to link a diversity of receptors to a diversity of effectors. The many classes of G proteins, in

Families of G Proteins

Family/Subunit

% Identity

Toxin

Distribution

Receptor

Effector/Role

αs αs(s) αs(l)

100

CTX

Ubiquitous

β-adrenergic, TSH, glucagon

↑ Adenylyl cyclase ↑ Ca2+ channel ↑ Na+ channel

αolf

88

CTX

Olfactory epithelium

Odorant

↑ Adenylyl cyclase Open K+ channel

Gi αi1 αi2 αi3

100 88

PTX PTX PTX

∼Ubiquitous Ubiquitous ∼Ubiquitous

M2, α2-adrenergic, others

↑ IP3, DAG, Ca2+, and AA release ↓ Adenylyl cyclase

αO1A αO1B

73 73

PTX PTX

Brain, others Brain, others

Met-enkephalin, α2adrenergic, others

αt1 αt2

68 68

PTX, CTX PTX, CTX

Retinal rods Retinal cones

Rhodopsin Cone opsin

↑ cGMP-phosphodiesterase

αg αz

67 60

PTX, CTX (?)

Taste buds Brain, adrenal, platelet

Taste (?) M2 (?), others (?)

? ↓ Adenylyl cyclase

Gq αq α11 α14 α15 α16

100 88 79 57 58

∼Ubiquitous ∼Ubiquitous Lung, kidney, liver B cell, myeloid T cell, myeloid

M1, α1-adrenergic, others

↑ PLCβ1, β2, β3

Several receptors

↑ PLCβ1, β2, β3

G12 α12 α13

100 67

Ubiquitous Ubiquitous

CTX, cholera toxin; M1 and M2, muscarinic cholinergic receptors; PTX, pertussis toxin; TSH, thyrotropin (thyroid-stimulating hormone).

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conjunction with the presence of several receptor types for a single ligand, provide a mechanism whereby a common signal can elicit the appropriate physiological changes in different tissues. For example, when epinephrine binds β1adrenergic receptors in the heart, it stimulates adenylyl cyclase, which increases heart rate and the force of contraction. However, in the periphery, epinephrine acts on α2adrenergic receptors coupled to a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular resistance and consequently increasing venous return and blood pressure. Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase. The heterotrimeric G protein known as Gs was so named because it stimulates adenylyl cyclase. A separate class of G proteins was given the name Gi because it is responsible for the hormone-dependent inhibition of adenylyl cyclase. Identification of these classes of G proteins was greatly facilitated by the observation that the α subunits of individual G proteins are substrates for adenosine diphosphate (ADP) ribosylation catalyzed by bacterial toxins. The toxin from Vibrio cholerae activates Gs, whereas the toxin from Bordetella pertussis inactivates the cyclase-inhibiting Gi (see the box titled Action of Toxins on Heterotrimeric G Proteins). For their work in identifying G proteins and elucidating the physiological role of these proteins, Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine. G protein activation follows a cycle In their inactive state, heterotrimeric G proteins are a complex of α, β, and γ subunits in which GDP occupies the guanine nucleotide–binding site of the α subunit. After ligand binding to the GPCR (Fig. 3-4, step 1), the activated receptor interacts with the αβγ heterotrimer to promote a conformational change that facilitates the release of bound GDP and simultaneous binding of GTP (step 2). This GDP-GTP exchange stimulates dissociation of the complex from the receptor (step 3) and causes disassembly of the trimer into a free α subunit and βγ complex (step 4). The free, active GTP-bound α subunit can now interact in the plane of the membrane with downstream effectors such as adenylyl cyclase and phospholipases (step 5). Similarly, the βγ subunit can now activate ion channels or other effectors. The α subunit terminates the signaling events that are mediated by the α and βγ subunits by hydrolyzing GTP to GDP and inorganic phosphate (Pi). The result is an inactive α-GDP complex that dissociates from its downstream effector and reassociates with a βγ subunit (Fig. 3-4, step 6), thus completing the cycle (step 1). The βγ subunit stabilizes αGDP and thereby substantially slows the rate of GDP-GTP exchange (step 2) and dampens signal transmission in the resting state. The RGS (for “regulation of G protein signaling”) family of proteins appears to enhance the intrinsic guanosine triphosphatase (GTPase) activity of some but not all α subunits. Investigators have identified at least 15 mammalian RGS proteins and shown that they interact with specific α subunits. RGS proteins bind the complex Gα/GDP/AlF4−, which is the structural analogue of the GTPase transition

state. By stabilizing the transition state, RGS proteins may promote GTP hydrolysis and thus the termination of signaling. As noted earlier, α subunits can be anchored to the cell membrane by myristyl or palmitoyl groups. Activation can result in the removal of these groups and the release of the α subunit into the cytosol. Loss of the α subunit from the membrane may decrease the interaction of G proteins with receptors and downstream effectors (e.g., adenylyl cyclase). Activated α subunits couple to a variety of downstream effectors, including enzymes, ion channels, and membrane trafficking machinery Activated α subunits can couple to a variety of enzymes. A major enzyme that acts as an effector downstream of activated α subunits is adenylyl cyclase (Fig. 3-5A). This enzyme can be either activated or inhibited by G protein signaling, depending on whether it associates with the GTP-bound form of Gαs (stimulatory) or Gαi (inhibitory). Thus, different hormones—acting through different G protein complexes—can have opposing effects on the same intracellular messenger. G proteins can also activate enzymes that break down cyclic nucleotides. For example, the G protein called transducin, which plays a key role in phototransduction (see Chapter 15), activates the cyclic guanosine monophosphate (cGMP) phosphodiesterase, which catalyzes the breakdown of cGMP to GMP (Fig. 3-5B). Thus, in retinal cells expressing transducin, light leads to a decrease in [cGMP]i. G proteins can also couple to phospholipases. These enzymes catabolize phospholipids, as discussed in detail later in the section on G protein second messengers. This superfamily of phospholipases can be grouped into phospholipases A2, C, or D on the basis of the site at which the enzyme cleaves the phospholipid. The G protein αq subunit activates phospholipase C, which breaks phosphatidylinositol bisphosphate (PIP2) into two intracellular messengers, membraneassociated diacylglycerol and cytosolic IP3 (Fig. 3-5C). Diacylglycerol stimulates protein kinase C, whereas IP3 binds to a receptor on the endoplasmic reticulum membrane and triggers the release of Ca2+ from intracellular stores. Some G proteins interact with ion channels. Agonists that bind to the β-adrenergic receptor activate the L-type Ca2+ channel in the heart and skeletal muscle (see Chapter 7). The G protein Gs directly stimulates this channel as the α subunit of Gs binds to the channel, and Gs also indirectly stimulates this channel through a signal transduction cascade that involves cAMP-dependent protein kinase. A clue that G proteins serve additional functions in membrane trafficking (see Chapter 2) in the cell comes from the observation that many cells contain intracellular pools of heterotrimeric G proteins, some bound to internal membranes and some free in the cytosol. Experiments involving toxins, inhibitors, and cell lines harboring mutations in G protein subunits have demonstrated that these intracellular G proteins are involved in vesicular transport. G proteins have been implicated in the budding of secretory vesicles from the trans-Golgi network, fusion of endosomes, recruitment of non–clathrin coat proteins, and transcytosis and apical secretion in polarized epithelial cells. The receptors

Chapter 3 • Signal Transduction

N Receptor (R) consists of seven membranespanning segments.

C

2 Receptor interacts with the G protein to promote a conformational change and the exchange of GDP for GTP.

Extracellular space

1 Ligand binds, receptor activates. E1

E1 E1

γ

R R

β

α

E2

γ

β

R

α

E2

E3 Cytosol

G protein

3 G protein dissociates from the receptor.

4

α-GTP and βγ subunits dissociate.

E1

R R

γ

β

α

E2

γ

R R

E1

α

β

E2

6 5 Both α-GTP and βγ can now interact with their appropriate effectors (E1, E2).

α-catalyzed hydrolysis of GTP to GDP inactivates α and promotes reassembly of the trimer.

E1

R R

α

E1

γ

β

E2

R

γ

α

β

Pi

Figure 3-4

Enzymatic cycle of heterotrimeric G proteins.

RGS

E2

Members of the RGS family of G-protein regulators stimulate GTP hydrolysis with some but not all α subunits.

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A

G PROTEINS ACTING VIA ADENYLYL CYCLASE Extracellular space

Adenylyl cyclase

γ

αs

β

αs

αi

AC

G protein complex (stimulatory)

cAMP

β

γ

G protein complex (inhibitory) NH2

Cyclic AMP activates protein kinase A.

Cytosol B

PKA

Adenine N

N

N

N CH2 O

G PROTEIN ACTING VIA A PHOSPHODIESTERASE Light

H

O

H

H

H

OH O

Extracellular space

P

O –

O

Phosphodiesterase

Cyclic AMP γ

Cytosol

αt

αt

β

G protein complex (transducin)

PDE

cGMP

GMP O

The breakdown of cGMP leads to the closure of cGMP-dependent channels. O O–

H2N

P O

N

H

H

H

H

GMP

Phospholipase C

β

N CH2 O

OH OH

G PROTEIN ACTING VIA A PHOSPHOLIPASE

γ

O

–

Extracellular space C

N

N

cGMP

Guanine

C

αq

αq

DAG activates the enzyme protein kinase C.

PIP2

PKC

PLC

PKC

Ca

G protein complex

DAG

2+

IP3

IP3 signals the release of Ca2+ from the ER. ER

Figure 3-5 Downstream effects of activated G protein α subunits. A, When a ligand binds to a receptor coupled to αs, adenylyl cyclase (AC) is activated, whereas when a ligand binds to a receptor coupled to αi, the enzyme is inhibited. The activated enzyme converts ATP to cAMP, which then can activate protein kinase A (PKA). B, In phototransduction, a photon interacts with the receptor and activates the G protein transducin. The αt activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP and lowers the intracellular concentrations of cGMP and therefore closes the cGMP-activated channels. C, In this example, the ligand binds to a receptor that is coupled to αq, which activates phospholipase C (PLC). This enzyme converts PIP2 to IP3 and diacylglycerol (DAG). The IP3 leads to the release of Ca2+ from intracellular stores, whereas the diacylglycerol activates protein kinase C (PKC). ER, endoplasmic reticulum.

Chapter 3 • Signal Transduction

Action of Toxins on Heterotrimeric G Proteins

I

nfectious diarrheal disease has a multitude of causes. Cholera toxin, a secretory product of the bacterium Vibrio cholerae, is responsible in part for the devastating characteristics of cholera. The toxin is an oligomeric protein composed of one A subunit and five B subunits (AB5). After cholera toxin enters intestinal epithelial cells, the A subunit separates from the B subunits and becomes activated by proteolytic cleavage. The resulting active A1 fragment catalyzes the ADP ribosylation of Gαs. This ribosylation, which involves transfer of the ADP-ribose moiety from the oxidized form of nicotinamide adenine dinucleotide (NAD+) to the α subunit, inhibits the GTPase activity of Gαs. As a result of this modification, Gαs remains in its activated, GTP-bound form and can activate adenylyl cyclase. In intestinal epithelial cells, the constitutively activated Gαs elevates levels of cAMP, which causes an increase in Cl− conductance and water flow and thereby contributes to the large fluid loss characteristic of this disease. A related bacterial product is pertussis toxin, which is also an AB5 protein. It is produced by Bordetella pertussis, the causative agent of whooping cough. Pertussis toxin ADPribosylates Gαi. This ADP-ribosylated Gαi cannot exchange its bound GDP (inactive state) for GTP. Thus, αi remains in its GDP-bound inactive state. As a result, receptor occupancy can no longer release the active αi-GTP, so adenylyl cyclase cannot be inhibited. Thus, both cholera toxin and pertussis toxin increase the generation of cAMP.

and effectors that interact with these intracellular G proteins have not been determined. The bg subunits of G proteins can also activate downstream effectors Considerable evidence now indicates that the βγ subunits can also interact with downstream effectors. The neurotransmitter ACh released from the vagus nerve reduces the rate and strength of heart contraction. This action in the atria of the heart is mediated by muscarinic M2 AChRs (see Chapter 14). These receptors can be activated by muscarine, an alkaloid found in certain poisonous mushrooms. Muscarinic AChRs are very different from the nicotinic AChRs discussed earlier, which are ligand-gated channels. Binding of ACh to the muscarinic M2 receptor in the atria activates a heterotrimeric G protein, resulting in the generation of both activated Gαi as well as a free βγ subunit complex. The βγ complex then interacts with a particular class of K+ channels, increasing their permeability. This increase in K+ permeability keeps the membrane potential relatively negative and thus renders the cell more resistant to excitation. The βγ subunit complex also modulates the activity of adenylyl cyclase and phospholipase C and stimulates phospholipase A2. Such effects of βγ can be independent of, synergize with, or antagonize the action of the α subunit. For example, studies using

various isoforms of adenylyl cyclase have demonstrated that purified βγ stimulates some isoforms, inhibits others, and has no effect on still others. Different combinations of βγ isoforms may have different activities. For example, β1γ1 is one tenth as efficient at stimulating type II adenylyl cyclase as is β1γ2. An interesting action of some βγ complexes is that they bind to a special protein kinase called the β-adrenergic receptor kinase (βARK). As a result of this interaction, βARK translocates to the plasma membrane, where it phosphorylates the ligand-receptor complex (but not the unbound receptor). This phosphorylation results in the recruitment of β-arrestin to the GPCR, which in turn mediates disassociation of the receptor-ligand complex and thus attenuates the activity of the same β-adrenergic receptors that gave rise to the βγ complex in the first place. This action is an example of receptor desensitization. These phosphorylated receptors eventually undergo endocytosis, which transiently reduces the number of receptors that are available on the cell surface. This endocytosis is an important step in resensitization of the receptor system. Small GTP-binding proteins are involved in a vast number of cellular processes A distinct group of proteins that are structurally related to the α subunit of the heterotrimeric G proteins are the small GTP-binding proteins. More than 100 of these have been identified to date, and they have been divided into five groups including the Ras, Rho, Rab, Arf, and Ran families. These 21-kDa proteins can be membrane associated (e.g., Ras) or may translocate between the membrane and the cytosol (e.g., Rho). The three isoforms of Ras (N, Ha, and Ki) relay signals from the plasma membrane to the nucleus through an elaborate kinase cascade (see Chapter 4), thereby regulating gene transcription. In some tumors, mutation of the genes encoding Ras proteins results in constitutively active Ras. These mutated genes are called oncogenes because the altered Ras gene product promotes the malignant transformation of a cell and can contribute to the development of cancer (oncogenesis). In contrast, Rho family members are primarily involved in rearrangement of the actin cytoskeleton; Rab and Arf proteins regulate vesicle trafficking. Similar to the α subunit of heterotrimeric G proteins, the small GTP-binding proteins switch between an inactive GDP-bound form and an active GTP-bound form. Two classes of regulatory proteins modulate the activity of these small GTP-binding proteins. The first of these includes the GTPase-activating proteins (GAPs) and neurofibromin (a product of the neurofibromatosis type 1 gene). GAPs increase the rate at which small GTP-binding proteins hydrolyze bound GTP and thus result in more rapid inactivation. Counteracting the activity of GAPs are guanine nucleotide exchange proteins (GEFs) such as “son of sevenless” or SOS, which promote the conversion of inactive RasGDP to active Ras-GTP. Interestingly, cAMP directly activates several GEFs, such as Epac (exchange protein activated by cAMP), demonstrating crosstalk between a classical heterotrimeric G protein signaling pathway and the small Ras-like G proteins.

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G PROTEIN SECOND MESSENGERS: CYCLIC NUCLEOTIDES

cAMP-dependent kinase (PKA) is composed of two regulatory (R) and 2 catalytic (C) subunits. Binding of cAMP to the regulatory subunits induces a conformational change that reduces their affinity for the catalytic subunits.

cAMP usually exerts its effect by increasing the activity of protein kinase A Activation of Gs-coupled receptors results in the stimulation of adenylyl cyclase and a rise in intracellular concentrations of cAMP (Fig. 3-5A). The downstream effects of this increase in [cAMP]i depend on the specialized functions that the responding cell carries out in the organism. For example, in the adrenal cortex, ACTH stimulation of cAMP production results in the secretion of aldosterone and cortisol; in the kidney, vasopressin-induced changes in cAMP levels facilitate water reabsorption (see Chapters 38 and 50). Excess cAMP is also responsible for certain pathologic conditions. One is cholera (see the box on page 57, titled Action of Toxins on Heterotrimeric G Proteins). Another pathologic process associated with excess cAMP is McCune-Albright syndrome, characterized by a triad of (1) variable hyperfunction of multiple endocrine glands, including precocious puberty in girls, (2) bone lesions, and (3) pigmented skin lesions (café au lait spots). This disorder is caused by a somatic mutation that constitutively activates the G protein αs subunit in a mosaic pattern. cAMP exerts many of its effects through cAMP-dependent protein kinase A (PKA). This enzyme catalyzes transfer of the terminal phosphate of ATP to certain serine or threonine residues within selected proteins. PKA phosphorylation sites are present in a multitude of intracellular proteins, including ion channels, receptors, and signaling pathway proteins. Phosphorylation of these sites can influence either the localization or the activity of the substrate. For example, phosphorylation of the β2-adrenergic receptor causes receptor desensitization in neurons, whereas phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) increases its Cl− channel activity. To enhance regulation of phosphorylation events, the cell tightly controls the activity of PKA so that the enzyme can respond to subtle—and local—variations in cAMP levels. One important control mechanism is the use of regulatory subunits that constitutively inhibit PKA. In the absence of cAMP, two catalytic subunits of PKA associate with two of these regulatory subunits, resulting in a heterotetrameric protein complex that has a low level of catalytic activity (Fig. 3-6). Binding of cAMP to the regulatory subunits induces a conformational change that diminishes their affinity for the catalytic subunits, and the subsequent dissociation of the complex results in activation of kinase activity. In addition to the short-term effects of PKA activation noted before, the free catalytic subunit of PKA can also enter the nucleus, where substrate phosphorylation can activate the transcription of specific PKA-dependent genes (see Chapter 4). Although most cells use the same catalytic subunit, different regulatory subunits are found in different cell types. Another mechanism that contributes to regulation of PKA is the targeting of the enzyme to specific subcellular locations. Such targeting promotes the preferential phosphorylation of substrates that are confined to precise locations within the cell. PKA targeting is achieved by the

cAMP cAMP

R

C cAMP

cAMP

C

R

R C

R cAMP

cAMP cAMP

PKA

C

cAMP

The complex dissociates and the catalytic subunits are free to catalyze the phosphorylation of protein substrates.

Figure 3-6

Activation of protein kinase A by cAMP.

association of a PKA regulatory subunit with an A kinase anchoring protein (AKAP), which in turn binds to cytoskeletal elements or to components of cellular subcompartments. More than 35 AKAPs are known. The specificity of PKA targeting is highlighted by the observation that in neurons, PKA is localized to postsynaptic densities through its association with AKAP79. This anchoring protein also targets calcineurin—a protein phosphatase—to the same site. This targeting of both PKA and calcineurin to the same postsynaptic site makes it possible for the cell to tightly regulate the phosphorylation state of important neuronal substrates. The cAMP generated by adenylyl cyclase does not interact only with PKA. For example, olfactory receptors (see Chapter 15) interact with a member of the Gs family called Golf. The rise in [cAMP]i that results from activation of the olfactory receptor activates a cation channel, a member of the family of cyclic nucleotide–gated (CNG) ion channels. Na+ influx through this channel leads to membrane depolarization and the initiation of a nerve impulse. For his work in elucidating the role played by cAMP as a second messenger in regulating glycogen metabolism, Earl Sutherland received the 1971 Nobel Prize in Physiology or Medicine. In 1992, Edmond Fischer and Edwin Krebs shared the prize for their part in demonstrating the role of protein phosphorylation in the signal transduction process. This coordinated set of phosphorylation and dephosphorylation reactions has several physiological advantages. First, it allows a single molecule (e.g., cAMP) to regulate a range of enzymatic reactions. Second, it affords a large amplification to a small signal. The concentration of epinephrine needed to stimulate glycogenolysis in muscle is ∼10−10 M. This subnanomolar level of hormone can raise [cAMP]i to ∼10−6 M. Thus, the catalytic cascades amplify the hormone signal 10,000-fold, resulting in the liberation of enough glucose to raise blood glucose levels from ∼5 to ∼8 mM. Although the effects of cAMP on the synthesis and degradation of glycogen are confined to muscle and liver, a

Chapter 3 • Signal Transduction

wide variety of cells use cAMP-mediated activation cascades in the response to a wide variety of hormones. Protein phosphatases reverse the action of kinases As discussed, one way that the cell can terminate a cAMP signal is to use a phosphodiesterase to degrade cAMP. In this way, the subsequent steps along the signaling pathway can also be terminated. However, because the downstream effects of cAMP often involve phosphorylation of effector proteins at serine and threonine residues by kinases such as PKA, another powerful way to terminate the action of cAMP is to dephosphorylate these effector proteins. Such dephosphorylation events are mediated by enzymes called serine/threonine phosphoprotein phosphatases. Four groups of serine/threonine phosphoprotein phosphatases (PP) are known, 1, 2a, 2b, and 2c. These enzymes themselves are regulated by phosphorylation at their serine, threonine, and tyrosine residues. The balance between kinase and phosphatase activity plays a major role in the control of signaling events. PP1 dephosphorylates many proteins phosphorylated by PKA, including those phosphorylated in response to epinephrine (see Chapter 58). Another protein, phosphoprotein phosphatase inhibitor 1 (I-1), can bind to and inhibit PP1. Interestingly, PKA phosphorylates and thus activates I-1 (Fig. 3-7), thereby inhibiting PP1 and preserving the phosphate groups added by PKA in the first place. PP2a, which is less specific than PP1, appears to be the main phosphatase responsible for reversing the action of other protein serine/threonine kinases. The Ca2+-dependent PP2b, also known as calcineurin, is prevalent in the brain, skeletal muscle, and cardiac muscle and is also the target of the immunosuppressive reagents FK-506 and cyclosporine. The importance of PP2c is presently unclear.

cAMP

R

cGMP is another cyclic nucleotide that is involved in G protein signaling events. In the outer segments of rods and cones in the visual system, the G protein does not couple to an enzyme that generates cGMP but, as noted earlier, couples to an enzyme that breaks it down. As discussed further in Chapter 15, light activates a GPCR called rhodopsin, which activates the G protein transducin, which in turn activates the cGMP phosphodiesterase that lowers [cGMP]i. The fall in [cGMP]i closes cGMP-gated nonselective cation channels that are members of the same family of CNG ion channels that cAMP activates in olfactory signaling (see Chapter 15).

Many messengers bind to receptors that activate phosphoinositide breakdown

C R cAMP

cAMP

cGMP exerts its effect by stimulating a nonselective cation channel in the retina

G PROTEIN SECOND MESSENGERS: PRODUCTS OF PHOSPHOINOSITIDE BREAKDOWN

PKA (active)

cAMP

In addition to serine/threonine kinases such as PKA, a second group of kinases involved in regulating signaling pathways (discussed later in this chapter) are known as tyrosine kinases because they phosphorylate their substrate proteins on tyrosine residues. The enzymes that remove phosphates from these tyrosine residues are much more variable than the serine and threonine phosphatases. The first phosphotyrosine phosphatase (PTP) to be characterized was the cytosolic enzyme PTP1B from human placenta. PTP1B has a high degree of homology with CD45, a membrane protein that is both a receptor and a tyrosine phosphatase. cDNA sequence analysis has identified a large number of PTPs that can be divided into two classes: membranespanning receptor-like proteins such as CD45 and cytosolic forms such as PTP1B. A number of intracellular PTPs contain so-called Src homology 2 (SH2) domains, a peptide sequence or motif that interacts with phosphorylated tyrosine groups. Several of the PTPs are themselves regulated by phosphorylation.

C

I-1

I-1

I-1

P

P

PP1 Inactive PP1

Phosphoprotein phosphatase (active)

Figure 3-7 Activation of phosphoprotein phosphatase 1 (PP1) by PKA. I-1, inhibitor of PP1.

Although the phosphatidylinositols (PIs) are minor constituents of cell membranes, they are largely distributed in the internal leaflet of the membrane and play an important role in signal transduction. The inositol sugar moiety of PI molecules (see Fig. 2-2A) can be phosphorylated to yield the two major phosphoinositides that are involved in signal transduction: phosphatidylinositol 4,5-bisphosphate (PI4,5P2 or PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3). Certain membrane-associated receptors act though G proteins (e.g., Gq) that stimulate phospholipase C (PLC) to cleave PIP2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), as shown in Figure 3-8A. PLCs are classified into three families (β, γ, δ) that differ in their catalytic properties, cell type–specific expression, and modes of activation. PLCβ is typically activated downstream of certain G proteins (e.g., Gq), whereas PLCγ contains an SH2 domain

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PIP2

DAG 13p6

A

Binding of a hormone to a cell surface G protein–coupled receptor activates phospholipase Cβ.

O

C

PRODUCTION OF IP3 AND DAG CH2

PLC cleaves the polar head group here.

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–

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Phospholipase Cβ hydrolyzes PIP2 into IP3 and DAG.

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Plasma membrane Cytosol

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α

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Active

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C IP3 interacts with a receptor in the membrane of the ER, which allows + the release of Ca2 into the cytosol.

ER +

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BREAKDOWN OF PHOSPHATIDYLCHOLINE BY PLC AND PLD R1

R2

The SERCA Ca2+ pump transports the + Ca2 back into the SR.

2+

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CH2 C

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TIME COURSE OF IP3 AND DAG LEVELS IP3 The early DAG peak is caused by DAG released from PIP2 by PLCβ.

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CH2

P

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Choline

CH2 H3C

N

+

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CH3

Seconds

Minutes

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PLD The slow DAG wave is caused by DAG released by PLCβ and PLD from phosphatidylcholine (PC).

C

O O

Response

CH2 O

Hours

Figure 3-8 Second messengers in the DAG/IP3 pathway. ER, endoplasmic reticulum; SERCA, sarcoplasmic and endoplasmic reticulum Ca2+-ATPase.

Chapter 3 • Signal Transduction

and is activated downstream of certain tyrosine kinases. Stimulation of PLCβ results in a rapid increase in cytosolic IP3 levels as well as an early peak in DAG levels (Fig. 3-8B). Both products are second messengers. DAG remains in the plane of the membrane to activate protein kinase C, which migrates from the cytosol and binds to DAG in the membrane. The water-soluble IP3 travels through the cytosol to stimulate Ca2+ release from intracellular stores. It is within this system that Ca2+ was first identified as a messenger that mediates the stimulus-response coupling of endocrine cells. Phosphatidylcholines (PCs), which—unlike PI—are an abundant phospholipid in the cell membrane, are also a source of DAG. The cell can produce DAG from PC by either of two mechanisms (Fig. 3-8C). First, PLC can directly convert PC to phosphocholine and DAG. Second, phospholipase D (PLD), by cleaving the phosphoester bond on the other side of the phosphate, converts PC to choline and phosphatidic acid (PA; also phospho-DAG). This PA can then be converted to DAG by PA-phosphohydrolase. Production of DAG from PC, either directly (by PLC) or indirectly (by PLD), produces the slow wave of increasing cytosolic DAG shown in Figure 3-8B. Thus, in some systems, the formation of DAG is biphasic and consists of an early peak that is transient and parallels the formation of IP3, followed by a late phase that is slow in onset but sustained for several minutes. Factors such as tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), interleukin 3 (IL-3), interferon α (IFN-α), and colony-stimulating factor stimulate the production of DAG from PC. Once generated, some DAGs can be further cleaved by DAG lipase to arachidonic acid, which can have signaling activity itself or can be metabolized to other signaling molecules, the eicosanoids. We cover arachidonic acid metabolism later in this chapter. Inositol triphosphate liberates Ca2+ from intracellular stores As discussed earlier, IP3 is generated by the metabolism of membrane phospholipids and then travels through the cytosol to release Ca2+ from intracellular stores. The IP3 receptor (ITPR) is a ligand-gated Ca2+ channel located in the membrane of the endoplasmic reticulum (Fig. 3-8A). This Ca2+ channel is structurally related to the Ca2+ release channel (or ryanodine receptor), which is responsible for releasing Ca2+ from the sarcoplasmic reticulum of muscle and thereby switching on muscle contraction (see Chapter 9). The IP3 receptor is a tetramer composed of subunits of ∼260 kDa. At least three genes encode the subunits of the receptor. These genes are subject to alternative splicing, which further increases the potential for receptor diversity. The receptor is a substrate for phosphorylation by protein kinases A and C and calcium-calmodulin (Ca2+-CaM)–dependent protein kinases. Interaction of IP3 with its receptor results in passive efflux of Ca2+ from the endoplasmic reticulum and thus a rapid rise in the free cytosolic Ca2+ concentration. The IP3-induced changes in [Ca2+]i exhibit complex temporal and spatial patterns. The rise in [Ca2+]i can be brief or persistent and can oscillate repetitively, spread in spirals or waves within a cell,

or spread across groups of cells that are coupled by gap junctions. In at least some systems, the frequency of [Ca2+]i oscillations seems to be physiologically important. For example, in isolated pancreatic acinar cells, graded increases in the concentration of ACh produce graded increases in the frequency—but not the magnitude—of repetitive [Ca2+]i spikes. The mechanisms responsible for [Ca2+]i oscillations and waves are complex. It appears that both propagation and oscillation depend on positive feedback mechanisms, in which low [Ca2+]i facilitates Ca2+ release, as well as on negative feedback mechanisms, in which high [Ca2+]i inhibits further Ca2+ release. The dephosphorylation of IP3 terminates the release of Ca2+ from intracellular stores; an ATP-fueled Ca2+ pump (SERCA; see Chapter 5) then moves the Ca2+ back into the endoplasmic reticulum. Some of the IP3 is further phosphorylated to IP4, which may mediate a slower and more prolonged response of the cell or may promote the refilling of intracellular stores. In addition to IP3, cyclic ADP ribose (cADPR) can mobilize Ca2+ from intracellular stores and augment a process known as calcium-induced Ca2+ release. Although the details of these interactions have not been fully elucidated, cADPR appears to bind to the Ca2+ release channel (ryanodine receptor) in a Ca2+-CaM–dependent manner. In addition to the increase in [Ca2+]i produced by the release of Ca2+ from intracellular stores, [Ca2+]i can also rise as a result of enhanced influx of this ion through Ca2+ channels in the plasma membrane. For Ca2+ to function as a second messenger, it is critical that [Ca2+]i be normally maintained at relatively low levels (at or below ∼100 nM). Leakage of Ca2+ into the cell through Ca2+ channels is opposed by the extrusion of Ca2+ across the plasma membrane by both an ATP-dependent Ca2+ pump and the Na-Ca exchanger (see Chapter 5). As discussed later, increased [Ca2+]i exerts its effect by binding to cellular proteins and changing their activity. Some Ca2+-dependent signaling events are so sensitive to Ca2+ that a [Ca2+]i increase of as little as 100 nM can trigger a vast array of cellular responses. These responses include secretion of digestive enzymes by pancreatic acinar cells, release of insulin by β cells, contraction of vascular smooth muscle, conversion of glycogen to glucose in the liver, release of histamine by mast cells, aggregation of platelets, and DNA synthesis and cell division in fibroblasts. Calcium activates calmodulin-dependent protein kinases How does an increase in [Ca2+]i lead to downstream responses in the signal transduction cascade? The effects of changes in [Ca2+]i are mediated by Ca2+-binding proteins, the most important of which is calmodulin (CaM). CaM is a highaffinity cytoplasmic Ca2+-binding protein of 148 amino acids. Each molecule of CaM cooperatively binds four calcium ions. Ca2+ binding induces a major conformational change in CaM that allows it to bind to other proteins (Fig. 3-9). Although CaM does not have intrinsic enzymatic activity, it forms a complex with a number of enzymes and thereby confers a Ca2+ dependence on their activity. For example, binding of the Ca2+-CaM complex activates the enzyme that degrades cAMP, cAMP phosphodiesterase.

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Inactive protein

2+

Ca

Active protein

Calmodulin

Ca2+/Calmodulin

Ca2+/Calmodulin-dependent protein kinase

Many of the effects of CaM occur as the Ca2+-CaM complex binds to and activates a family of Ca2+-CaM–dependent kinases (CaM kinases). These kinases phosphorylate certain serine and threonine residues of a variety of proteins. An important CaM kinase in smooth muscle cells is myosin light chain kinase (MLCK) (see Chapter 9). Another CaM kinase is glycogen phosphorylase kinase (PK), which plays a role in glycogen degradation (see Chapter 58). MLCK, PK, and some other CaM kinases have a rather narrow substrate specificity. The ubiquitous CaM kinase II, on the other hand, has a broad substrate specificity. Especially high levels of this multifunctional enzyme are present at the synaptic terminals of neurons. One of the actions of CaM kinase II is to phosphorylate and thereby activate the rate-limiting enzyme (tyrosine hydroxylase; see Fig. 13-8C) in the synthesis of catecholamine neurotransmitters. CaM kinase can also phosphorylate itself, which allows it to remain active in the absence of Ca2+. Diacylglycerols and Ca2+ activate protein kinase C As noted earlier, hydrolysis of PIP2 by PLC yields not only the IP3 that leads to Ca2+ release from internal stores but also DAG (Fig. 3-8A). The most important function of DAG is to activate protein kinase C (PKC), a serine/threonine kinase. In mammals, the PKC family comprises at least 10 members that differ in their tissue and cellular localization. This family is further subdivided into three groups that all require membrane-associated phosphatidylserine but have different requirements for Ca2+ and DAG. The classical PKC family members PKCα, PKCβ, and PKCγ require both DAG and Ca2+ for activation, whereas the novel PKCs (such as PKCδ, PKCε, and PKCη) are independent of Ca2+, and the atypical PKCs (PKCζ and PKCλ) appear to be independent of both DAG and Ca2+. As a consequence, the signals generated by the PKC pathway depend on the isoforms of the enzyme that a cell expresses as well as on the levels of Ca2+ and DAG at specific locations at the cell membrane.

Figure 3-9 Calmodulin. After four intracellular Ca2+ ions bind to calmodulin, the Ca2+-CaM complex can bind to and activate another protein. In this example, the activated protein is a Ca2+-CaM–dependent kinase.

In its basal state, PKCα is an inactive, soluble cytosolic protein. When Ca2+ binds to cytosolic PKC, PKC can interact with DAG, which is located in the inner leaflet of the plasma membrane. This interaction with DAG activates PKCα by raising its affinity for Ca2+. This process is often referred to as translocation of PKC from the cytoplasm to the membrane. In most cells, the Ca2+ signal is transient, whereas the resulting physiological responses, such as proliferation and differentiation, often persist substantially longer. Sustained activation of PKCα may be essential for maintaining these responses. Elevated levels of active PKCα are maintained by a slow wave of elevated DAG (Fig. 3-8B), which is due to the hydrolysis of PC by PLC and PLD. Physiological stimulation of the classical and novel PKCs by DAG can be mimicked by the exogenous application of a class of tumor promoters called phorbol esters. These plant products bind to these PKCs, cause them to translocate to the plasma membrane, and thus specifically activate them even in the absence of DAG. Among the major substrates of PKC are the myristoylated, alanine-rich C kinase substrate (MARCKS) proteins. These acidic proteins contain consensus sites for PKC phosphorylation as well as CaM- and actin-binding sites. MARCKS proteins cross-link actin filaments and thus appear to play a role in translating extracellular signals into actin plasticity and changes in cell shape. Unphosphorylated MARCKS proteins are associated with the plasma membrane, and they cross-link actin. Phosphorylation of the MARCKS proteins causes them to translocate into the cytosol, where they are no longer able to cross-link actin. Thus, mitogenic growth factors that activate PKC may produce morphological changes and anchorage-independent cell proliferation, in part by modifying the activity of MARCKS proteins. PKC can also directly or indirectly modulate transcription factors and thereby enhance the transcription of specific genes (see Chapter 4). Such genomic actions of PKC explain why phorbol esters are tumor promoters.

Chapter 3 • Signal Transduction

G PROTEIN SECOND MESSENGERS: ARACHIDONIC ACID METABOLITES

Arachidonic acid COOH

As previously discussed, PLC can hydrolyze PIP2 and thereby release two important signaling molecules, IP3 and DAG. In addition, both PLC and PLD can release DAG from PC. However, other hydrolysis products of membrane phospholipids can also act as signaling molecules. The best characterized of these hydrolysis products is arachidonic acid (AA), which is attached by an ester bond to the second carbon of the glycerol backbone of membrane phospholipids (Fig. 3-10). Phospholipase A2 initiates the cellular actions of AA by releasing this fatty acid from glycerol-based phospholipids. A series of enzymes subsequently convert AA into a family of biologically active metabolites that are collectively called eicosanoids (from the Greek eikosi for 20) because, like AA, they all have 20 carbon atoms. Three major pathways can convert AA into these eicosanoids (Fig. 3-11). In the first pathway, cyclooxygenase enzymes produce thromboxanes, prostaglandins, and prostacyclins. In the second pathway, 5-lipoxygenase enzymes produce leukotrienes and some hydroxyeicosatetraenoic acid (HETE) compounds. In the third pathway, the epoxygenase enzymes, which are members of the cytochrome P-450 class, produce other HETE compounds as well as cis-epoxyeicosatrienoic acid (EET) compounds. These three enzymes catalyze the stereospecific insertion of molecular O2 into various positions in AA. The cyclooxygenases, lipoxygenases, and epoxygenases are selectively distributed in different cell types, further increasing the complexity of eicosanoid biology. Eicosanoids have powerful biological activities, including effects on allergic and inflammatory processes, platelet aggregation, vascular smooth muscle, and gastric acid secretion.

Phospholipid The arachidonic acid is always found esterified to the second carbon atom of the glycerol backbone.

Phospholipase A2 cleaves here.

O

C

C

O

O

CH2

CH2

COOH

O

–

O

H2C P

Lysophospholipid O

O

Polar head group

C

Phospholipase A2 is the primary enzyme responsible for releasing arachidonic acid The first step in the phospholipase A2 (PLA2) signal transduction cascade is binding of an extracellular agonist to a membrane receptor (Fig. 3-11). These receptors include those for serotonin (5-HT2 receptors), glutamate (mGLUR1 receptors), fibroblast growth factor-ß, IFN-α, and IFN-γ. Once the receptor is occupied by its agonist, it can activate a G protein that belongs to the Gi/Go family. The mechanism by which this activated G protein stimulates PLA2 is not well understood. It does not appear that a G protein α subunit is involved. The G protein ßγ dimer may stimulate PLA2 either directly or through mitogen-activated protein (MAP) kinase (see Chapter 4), which phosphorylates PLA2 at a serine residue. The result is rapid hydrolysis of phospholipids that contain AA. In contrast to the direct pathway just mentioned, agonists acting on other receptors may promote AA release indirectly. First, a ligand may bind to a receptor coupled to PLC, which would lead to the release of DAG (Fig. 3-11). As noted earlier, DAG lipase can cleave DAG to yield AA and a monoacylglycerol. Agonists that act through this pathway include dopamine (D2 receptors), adenosine (A1 receptors), norepinephrine (α2-adrenergic receptors), and serotonin (5-HT1 receptors). Second, any agonist that raises [Ca2+]i can promote AA formation because Ca2+ can stimulate some cytosolic forms of PLA2. Third, any signal transduction

O

O

OH

CH2

C H

H2C –

O

P

O

O

Polar head group

Figure 3-10 Release of AA from membrane phospholipids by PLA2. AA is esterified to membrane phospholipids at the second carbon of the glycerol backbone. PLA2 cleaves the phospholipid at the indicated position and releases AA as well as a lysophospholipid.

pathway that activates MAP kinase can also enhance AA release because MAP kinase phosphorylates PLA2. Cyclooxygenases, lipoxygenases, and epoxygenases mediate the formation of biologically active eicosanoids Once it is released from the membrane, AA can diffuse out of the cell, be reincorporated into membrane phospholipids, or be metabolized (Fig. 3-11).

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DIRECT PATHWAY Extracellular space Phospholipase A2

γ

α

β

Phospholipid

Lysophospholipid

MAG

Phospholipase Cβ

DAG

DAG lipase

PLA2

PLCβ

α

α

Reincorporation of AACoA

Cytosol

IP3

Ca2+

ARACHIDONIC ACID

ER

COOH

Cyclooxygenase (COX)

ASA

Epoxygenase (Cytochrome P450)

5-Lipoxygenase

Other HETEs EETs

5-HPETE COOH OOH

Peroxidase

PGG2 Dehydrase

5-HETE

COX

LTA4

PGH2

LTA4 Hydrolase

LTB4

OH O COOH

O Thromboxane synthase

Prostacyclin synthase Glutathione-S-transferase

TXA2 (unstable)

PGI2 (unstable)

PGD2

PGE2

PGF2α

LTC4

LTE4

LTD4 LEUKOTRIENES

PROSTAGLANDINS

LTE4

Prostacyclins

NH2

TxA2

6-keto-PGF1α

OH O O

γ

Receptor-G protein complex 2

Receptor-G protein complex 1

Thromboxanes

β

OH

HO

PGE2 HO

COOH

OH S COOH

COOH

HO

O

COOH

O

COOH OH

Figure 3-11 AA signaling pathways. In the direct pathway, an agonist binds to a receptor that activates PLA2, which releases AA from a membrane phospholipid (see Fig. 3-10). In one of three indirect pathways, an agonist binds to a different receptor that activates PLC and thereby leads to the formation of DAG and IP3, as in Figure 3-8; DAG lipase then releases the AA from DAG. In a second indirect pathway, the IP3 releases Ca2+ from internal stores, which leads to the activation of PLA2 (see the direct pathway). In a third indirect pathway (not shown), mitogen-activated protein kinase stimulates PLA2. Regardless of its source, the AA may follow any of three pathways to form a wide array of eicosanoids. The cyclooxygenase pathway produces thromboxanes, prostacyclins, and prostaglandins. The 5-lipoxygenase pathway produces 5-HETE and the leukotrienes. The epoxygenase pathway leads to the production of other HETEs and EETs. ASA, acetylsalicylic acid; EET, cis-epoxyeicosatrienoic acid; ER, endoplasmic reticulum; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; MAG, monoacylglycerol.

Chapter 3 • Signal Transduction

In the first pathway of AA metabolism (Fig. 3-11), cyclooxygenases catalyze the stepwise conversion of AA into the intermediates prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2). PGH2 is the precursor of the other prostaglandins, the prostacyclins and the thromboxanes. As noted in the box titled Inhibition of Cyclooxygenase Isoforms by Aspirin, cyclooxygenase exists in two isoforms, COX-1 and COX-2. In many cells, COX-1 is expressed in a constitutive fashion, whereas COX-2 levels can be induced by specific stimuli. For example, in monocytes stimulated by inflammatory agents such as IL-1β, only levels of COX-2 increase. These observations have led to the concept that expression of COX-1 is important for homeostatic prostaglandin functions such as platelet aggregation and regulation of vascular tone, whereas upregulation of COX-2 is primarily important for mediating prostaglandin-dependent inflammatory responses. However, as selective inhibitors of COX-2 have become available, it has become clear that this is an oversimplification. In the second pathway of AA metabolism, 5-lipoxygenase initiates the conversion of AA into biologically active leukotrienes. For example, in myeloid cells, 5-lipoxygenase converts AA to 5-HPETE, which is short-lived and rapidly degraded by a peroxidase to the corresponding alcohol 5HETE. Alternatively, a dehydrase can convert 5-HPETE to an unstable epoxide, LTA4, which can be either further metabolized by LTA4 hydrolase to LTB4 or coupled (“conjugated”) to the tripeptide glutathione (see Chapter 46). This conjugation—through the cysteine residue of glutathione—yields LTC4. Enzymes sequentially remove portions of the glutathione moiety to produce LTD4 and LTE4. LTC4, LTD4, and LTE4 are the “cysteinyl” leukotrienes; they participate in allergic and inflammatory responses and make up the mixture previously described as the slowreacting substance of anaphylaxis. The third pathway of AA metabolism begins with the transformation of AA by epoxygenase (a cytochrome P-450 oxidase). Molecular O2 is a substrate in this reaction. The epoxygenase pathway converts AA into two major products, HETEs and EETs. Members of both groups display a diverse array of biological activities. Moreover, the cells of different tissues (e.g., liver, kidney, eye, and pituitary) use different biosynthetic pathways to generate different epoxygenase products. Prostaglandins, prostacyclins, and thromboxanes (cyclooxygenase products) are vasoactive, regulate platelet action, and modulate ion transport The metabolism of PGH2 to generate selected prostanoid derivatives is cell specific. For example, platelets convert PGH2 to thromboxane A2 (TXA2), a short-lived compound that can aggregate platelets, bring about the platelet release reaction, and constrict small blood vessels. In contrast, endothelial cells convert PGH2 to prostacyclin I2 (also known as PGI2), which inhibits platelet aggregation and dilates blood vessels. Many cell types convert PGH2 to prostaglandins. Acting locally in a paracrine or autocrine fashion, prostaglandins are involved in such processes as platelet aggregation, airway constriction, renin release, and inflammation.

Eicosanoid Nomenclature

T

he nomenclature of the eicosanoids is not as arcane as it might first appear. The numerical subscript 2 (as in PGH2) or 4 (as in LTA4) refers to the number of double bonds in the eicosanoid backbone. For example, AA has four double bonds, as do the leukotrienes. For the cyclooxygenase metabolites, the letter (A to I) immediately preceding the 2 refers to the structure of the 5-carbon ring that is formed about halfway along the 20carbon chain of the eicosanoid. For the leukotrienes, the letters A and B that immediately precede the 4 refer to differences in the eicosanoid backbone. For the cysteinyl leukotrienes, the letter C refers to the full glutathione conjugate (see Fig. 46-8). Removal of glutamate from LTC4 yields LTD4, and removal of glycine from LTD4 yields LTE4, leaving behind only cysteine. For 5-HPETE and 5-HETE, the fifth carbon atom (counting the carboxyl group as number 1) is derivatized with a hydroperoxy- or hydroxy- group, respectively.

Inhibition of Cyclooxygenase Isoforms by Aspirin

C

yclooxygenase is a bifunctional enzyme that first oxidizes AA to PGG2 through its cyclooxygenase activity and then peroxidizes this compound to PGH2. Cyclooxygenase exists in two forms, COX-1 and COX-2. X-ray crystallographic studies of COX-1 reveal that the sites for the two enzymatic activities (i.e., cyclooxygenase and peroxidase) are adjacent but spatially distinct. The cyclooxygenase site is a long hydrophobic channel. Aspirin (acetylsalicylic acid) irreversibly inhibits COX-1 by acetylating a serine residue at the top of this channel. Several of the other nonsteroidal anti-inflammatory drugs (NSAIDs) interact, through their carboxyl groups, with other amino acids in the same region. COX-1 activation plays an important role in intravascular thrombosis as it leads to thromboxane A2 synthesis by platelets. Inhibition of this process by low-dose aspirin is a mainstay for prevention of coronary thrombosis in patients with atherosclerotic coronary artery disease. However, COX1 activation is also important for producing cytoprotective prostacyclins in the gastric mucosa. It is the loss of these compounds that can lead to the unwanted side effect of gastrointestinal bleeding after chronic aspirin ingestion. Inflammatory stimuli induce COX-2 in a number of cell types, and it is inhibition of COX-2 that provides the antiinflammatory actions of high-dose aspirin (a weak COX-2 inhibitor) and other nonselective cyclooxygenase inhibitors such as ibuprofen. Because the two enzymes are only 60% homologous, pharmaceutical companies have now generated compounds that specifically inhibit COX-2, such as rofecoxib and celecoxib. These work well as anti-inflammatory agents and have a reduced likelihood of causing gastrointestinal bleeding because they do not inhibit COX-1–dependent prostacyclin production. At least one of the selective COX-2 inhibitors has been reported to increase the risk of thrombotic cardiovascular events when it is taken for long periods.

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Prostaglandin synthesis has also been implicated in the pathophysiological mechanisms of cardiovascular disease, cancer, and inflammatory diseases. NSAIDs such as aspirin, acetaminophen, ibuprofen, indomethacin, and naproxen directly target cyclooxygenase. NSAID inhibition of cyclooxygenase is a useful tool in the treatment of inflammation and fever and, at least in the case of aspirin, in the prevention of heart disease. The diverse cellular responses to prostanoids are mediated by a family of G protein–coupled prostanoid receptors. This family currently has nine proposed members, including receptors for thromboxane/prostaglandin H2 (TP), PGI2 (IP), PGE2 (EP1-4), PGD2 (DP and CRTH2), and PGF2α (FP). These prostanoid receptors signal through Gq, Gi, or Gs, depending on cell type. These in turn regulate intracellular adenylyl cyclase and phospholipases. The leukotrienes (5-lipoxygenase products) play a major role in inflammatory responses The biological effects of many lipoxygenase metabolites of AA have led to the suggestion that they have a role in allergic and inflammatory diseases (Table 3-3). LTB4 is produced by inflammatory cells such as neutrophils and macrophages. The cysteinyl leukotrienes including LTC4 and LTE4 are synthesized by mast cells, basophils, and eosinophils, cells that are commonly associated with allergic inflammatory responses such as asthma and urticaria. The cysteinyl leukotriene receptors cysLT1 and cysLT2 are GPCRs found on airway smooth muscle cells as well as on eosinophils, mast cells, and lymphocytes. CysLT1, which couples to both pertussis toxin–sensitive and pertussis

Table 3-3

Involvement of Leukotrienes in Human Disease

Disease

Evidence

Asthma

Bronchoconstriction from inhaled LTE4; identification of LTC4, LTD4, and LTE4 in the serum or urine or both of patients with asthma

Psoriasis

LTB4 and LTE4 found in fluids from psoriatic lesions

Adult respiratory distress syndrome

Elevated levels of LTB4 detected in the plasma of patients with ARDS

Allergic rhinitis

Elevated levels of LTB4 found in nasal fluids

Gout

LTB4 detected in joint fluid

Rheumatoid arthritis

Elevated LTB4 found in joint fluids and serum

Inflammatory bowel disease (ulcerative colitis and Crohn disease)

Identification of LTB4 in gastrointestinal fluids and LTE4 in urine

toxin–insensitive G proteins, mediates phospholipase-dependent increases in [Ca2+]i. In the airways, these events produce a potent bronchoconstriction, whereas activation of the receptor in mast cells and eosinophils causes release of the proinflammatory cytokines histamine and TNF-α. In addition to their role in the inflammatory response, the lipoxygenase metabolites can also influence the activity of many ion channels, either directly or by regulating protein kinases. For example, in synaptic nerve endings, lipoxygenase metabolites decrease the excitability of cells by activating K+ channels. Lipoxygenase products may also regulate secretion. In pancreatic islet cells, free AA generated in response to glucose appears to be part of a negative feedback loop that prevents excess insulin secretion by inhibiting CaM kinase II. The HETEs and EETs (epoxygenase products) tend to enhance Ca2+ release from intracellular stores and to enhance cell proliferation The epoxygenase pathway leads to the production of HETEs other than 5-HETE as well as EETs. HETEs and EETs have

Role of Leukotrienes in Disease

S

ince the original description of the slow-reacting substance of anaphylaxis, which is generated during antigenic challenge of a sensitized lung, leukotrienes have been presumed to play a part in allergic disease of the airways (Table 3-3). The involvement of cells (mast cells, basophils, and eosinophils) that produce cysteinyl leukotrienes (LTC4 through LTF4) in these pathobiological processes supports this concept. In addition, the levels of LTC4, LTD4, and LTE4 are increased in lavage fluid from the nares of patients with allergic rhinitis after the application of specific antigens to the nasal airways. Introducing LTC4 or LTD4 into the airways as an aerosol (nebulizer concentration of only 10 μM) causes maximal expiratory airflow (a rough measure of airway resistance; see Chapter 27) to decline by ∼30%. This bronchoconstrictor effect is 1000-fold more potent than that of histamine, the “reference” agonist. Leukotrienes affect both large and small airways; histamine affects relatively smaller airways. Activation of the cysLT1 receptor in mast cells and eosinophils results in the chemotaxis of these cells to sites of inflammation. Because antagonists of the cysLT1 receptor (e.g., montelukast sodium) can partially block these bronchoconstrictive and proinflammatory effects, these agents are useful in the treatment of allergeninduced asthma and rhinitis. In addition to their involvement in allergic disease, several of the leukotrienes are associated with other inflammatory disorders. Synovial fluid from patients with rheumatoid arthritis contains 5-lipoxygenase products. Another example is the skin disease psoriasis. In patients with active psoriasis, LTB4, LTC4, and LTD4 have been recovered from skin chambers overlying abraded lesions. Leukotrienes also appear to be involved in inflammatory bowel disease. LTB4 and other leukotrienes are generated and released in vitro from intestinal mucosa obtained from patients with ulcerative colitis or Crohn disease.

Chapter 3 • Signal Transduction

Table 3-4

HETEs

Actions of Epoxygenase Products Cell/Tissue

Action

Stimulated mononuclear leukocytes

↑ Cell proliferation ↑ Ca2+ release from intracellular stores ↓ TNF production Implicated in the destruction of these cells in type 1 (juvenile-onset) diabetes mellitus ↓ Release of fibrinolytic factors ↓ Binding of antithrombin ↑ Cell proliferation ↑ Migration Formation of atherosclerotic plaque? Potent vasoconstrictors “Myogenic” vasoconstrictive response of renal and cerebral arteries

β Cells of pancreatic islets

Endothelial cells Vascular smooth muscle cells

Blood vessels EETs

Cells, general

Endocrine cells Toad bladder Blood vessels Endothelium Platelets

↑ Ca2+ release from intracellular stores ↑ Na-H exchange ↑ Cell proliferation ↓ Cyclooxygenase activity ↓ Release of somatostatin, insulin, glucagon ↓ Vasopressin-stimulated H2O permeability ↓ Renin release Vasodilation Angiogenesis ↑ Tumor cell adhesion ↓ Aggregation

been implicated in a wide variety of processes, some of which are summarized in Table 3-4. For example, in stimulated mononuclear leukocytes, HETEs enhance Ca2+ release from intracellular stores and promote cell proliferation. In smooth muscle cells, HETEs increase proliferation and migration; these AA metabolites may be one of the primary factors involved in the formation of atherosclerotic plaque. In blood vessels, HETEs can be potent vasoconstrictors. EETs enhance the release of Ca2+ from intracellular stores, increase Na-H exchange, and stimulate cell proliferation. In blood vessels, EETs primarily induce vasodilation and angiogenesis, although they have vasoconstrictive properties in the smaller pulmonary blood vessels. EETs generally tend to enhance the release of Ca2+ from intracellular stores, Na-H exchange, and cell proliferation. In blood vessels, EETs cause vasodilation and angiogenesis.

LTB4, thereby terminating its biological activity. LTC4 is metabolized through two pathways. One oxidizes the LTC4. The other pathway first removes the glutamic acid residue of the conjugated glutathione, which yields LTD4, and then removes the glycine residue, which yields LTE4, which is readily excreted into the urine. In the case of epoxygenase (cytochrome P-450) products, it has been difficult to characterize their metabolic breakdown because the reactions are so rapid and complex. Both enzymatic and nonenzymatic hydration reactions convert these molecules to the corresponding vicinyl diols. Some members of this group can form conjugates with reduced glutathione (GSH).

Degradation of the eicosanoids terminates their activity

Although it is not a member of the AA family, platelet-activating factor (PAF) is an important lipid signaling molecule. PAF is an ether lipid that the cell synthesizes either de novo or by remodeling of a membrane-bound precursor. PAF occurs in a wide variety of organisms and mediates many biological activities. In mammals, PAF is a potent inducer of platelet aggregation and stimulates the chemotaxis and degranulation of neutrophils, thereby facilitating the release of LTB4 and 5-HETE. PAF is involved in several aspects of allergic reactions; for example, it stimulates histamine release and enhances the secretion of IgE, IgA, and TNF. Endothelial cells are also an important target of PAF; PAF causes a negative shift of Vm in these cells by activating Ca2+-dependent K+ channels. PAF also enhances vascular permeability and the adhesion of neutrophils and platelets to endothelial cells.

Inactivation of the products of eicosanoids is an important mechanism for terminating their biological action. In the case of cyclooxygenase products, the enzyme 15-hydroxyprostaglandin dehydrogenase catalyzes the initial reactions that convert biologically active prostaglandins into their inactive 15-keto metabolites. This enzyme also appears to be active in the catabolism of thromboxanes. As far as the 5-lipoxygenase products are concerned, the specificity and cellular distribution of the enzymes that metabolize leukotrienes parallel the diversity of the enzymes involved in their synthesis. For example, 20-hydrolase-LTB4, a member of the P-450 family, catalyzes the ω oxidation of

Platelet-activating factor is a lipid mediator unrelated to arachidonic acid

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A

RECEPTOR GUANYLYL CYCLASES

B RECEPTOR SERINE/ THREONINE KINASES

Extracellular space

C RECEPTOR TYROSINE KINASES (RTKs)

D

TYROSINE-KINASE– ASSOCIATED RECEPTORS N

E

RECEPTOR TYROSINE PHOSPHATASES

Carbohydrate groups

N

Ligand Ligand

N

N

N

N N

N

Ligand

Serinethreonine kinase domain C C

C

C

Type I

Guanylyl cyclase domains Cytosol ANP RECEPTOR

JAK2

JAK2

C

Type II TGF-␤ RECEPTOR

C

Tyrosine kinase domains

This is the kinase that phosphorylates NGF downstream RECEPTOR effectors.

Tyrosine kinases

C

C

GROWTH HORMONE RECEPTOR

Tyrosine phosphatase domain

C

CD45

Figure 3-12 Catalytic receptors. A, Receptor guanylyl cyclases have an extracellular ligand-binding domain. B, Receptor serine/threonine kinases have two subunits. The ligand binds only to the type II subunit. C, Receptor tyrosine kinases (RTKs) similar to the NGF receptor dimerize on binding a ligand. D, Tyrosine kinase–associated receptors have no intrinsic enzyme activity but associate noncovalently with soluble, nonreceptor tyrosine kinases. E, Receptor tyrosine phosphatases have intrinsic tyrosine phosphatase activity. ANP, atrial natriuretic peptide; JAK, Janus kinase (originally “just another kinase”); NGF, nerve growth factor; TGF-β, transforming growth factor β.

PAF exerts its effects by binding to a specific receptor on the plasma membrane. A major consequence of PAF binding to its GPCR is formation of IP3 and stimulation of a group of MAP kinases. PAF acetylhydrolase terminates the action of this signaling lipid.

RECEPTORS THAT ARE CATALYTIC

Receptor tyrosine phosphatases cleave phosphate groups from tyrosine groups of cellular proteins. The receptor guanylyl cyclase transduces the activity of atrial natriuretic peptide, whereas a soluble guanylyl cyclase transduces the activity of nitric oxide Receptor (Membrane-Bound) Guanylyl Cyclase

A number of hormones and growth factors bind to cell surface proteins that have—or are associated with—enzymatic activity on the cytoplasmic side of the membrane. Here we discuss five classes of such catalytic receptors (Fig. 3-12): Receptor guanylyl cyclases catalyze the generation of cGMP from GTP. Receptor serine/threonine kinases phosphorylate serine or threonine residues on cellular proteins. Receptor tyrosine kinases (RTKs) phosphorylate tyrosine residues on themselves and other proteins. Tyrosine kinase–associated receptors interact with cytosolic (i.e., non–membrane bound) tyrosine kinases.

Some of the best characterized examples of a transmembrane protein with guanylyl cyclase activity (Fig. 3-12A) are the receptors for the natriuretic peptides. These are a family of related small proteins (∼28 amino acids) including atrial natriuretic peptide (ANP), B-type or brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). For example, in response to atrial stretch, cardiac myocytes release ANP and BNP. ANP and BNP have two major effects. First, they act on vascular smooth muscle to dilate blood vessels (see Chapter 23). Second, they enhance Na+ excretion into urine, which is termed natriuresis (see Chapter 40). Both activities contribute to lowering of blood pressure and effective circulating blood volume (see Chapter 5).

Chapter 3 • Signal Transduction

Natriuretic peptide receptors NPR-A and NPR-B are membrane proteins with a single membrane-spanning segment. The extracellular domain binds the ligand. The intracellular domain has two consensus catalytic domains for guanylyl cyclase activity. Binding of a natriuretic peptide induces a conformational change in the receptor that causes receptor dimerization and activation. Thus, binding of ANP to its receptor causes the conversion of GTP to cGMP and raises intracellular levels of cGMP. In turn, cGMP activates a cGMP-dependent kinase (PKG or cGK) that phosphorylates proteins at certain serine and threonine residues. In the renal medullary collecting duct, the cGMP generated in response to ANP may act not only through PKG but also by directly modulating ion channels (see Chapter 35). Soluble Guanylyl Cyclase In contrast to the receptor for ANP, which is an intrinsic membrane protein with guanylyl cyclase activity, the receptor for nitric oxide (NO) is a soluble (i.e., cytosolic) guanylyl cyclase. This soluble guanylyl cyclase (sGC) is totally unrelated to the receptor guanylyl cyclase and contains a heme moiety that binds NO. NO plays an important role in the control of blood flow and blood pressure. Vascular endothelial cells use the enzyme NO synthase (NOS) to cleave arginine into citrulline plus NO in response to stimuli such as ACh, bradykinin, substance P, thrombin, adenine nucleotides, and Ca2+. These agents trigger the entry of Ca2+, which binds to cytosolic CaM and then stimulates NOS. Activation of NOS also requires the cofactors tetrahydrobiopterin and NADPH. The newly synthesized NO rapidly diffuses out of the endothelial cell and crosses the membrane of a neighboring smooth muscle cell. In smooth muscle, NO stimulates its “receptor,” soluble guanylyl cyclase, which then converts GTP to cGMP. As a result, [cGMP]i may increase 50-fold and relax the smooth muscle. The importance of NO in the control of blood flow had long been exploited unwittingly to treat angina pectoris. Angina is the classic chest pain that accompanies inadequate blood flow to the heart muscle, usually as a result of coronary artery atherosclerosis. Nitroglycerin relieves this pain by spontaneously breaking down and releasing NO, which relaxes the smooth muscles of peripheral arterioles, thereby reducing the work of the heart and relieving the associated pain. In addition to its role as a chemical signal in blood vessels, NO appears to play an important role in the destruction of invading organisms by macrophages and neutrophils. NO also serves as a neurotransmitter and may play a role in learning and memory (see Chapter 13). Some of these actions may involve different forms of NOS. The importance of the NO signaling pathway was recognized by the awarding of the 1998 Nobel Prize for Physiology or Medicine to R. F. Furchgott, L. J. Ignarro, and F. Murad for their discoveries concerning NO as a signaling molecule in the cardiovascular system.

Some catalytic receptors are serine/threonine kinases Earlier in this chapter we discussed how activation of various G protein–linked receptors can initiate a cascade that even-

tually activates kinases (e.g., PKA, PKC) that phosphorylate proteins at serine and threonine residues. In addition, some receptors are themselves serine/threonine kinases—such as the one for transforming growth factor β (TGF-β)—and are thus catalytic receptors. The TGF-β superfamily includes a large group of cytokines, including five TGF-βs, antimüllerian hormone, the inhibins, the activins, bone morphogenic proteins, and other glycoproteins, all of which control cell growth and differentiation. Members of this family participate in embryogenesis, suppress epithelial cell growth, promote wound repair, and influence immune and endocrine functions. Unchecked TGF-β signaling is important in progressive fibrotic disorders (e.g., liver cirrhosis, idiopathic pulmonary fibrosis) that result in replacement of normal organ tissue by deposits of collagen and other matrix components. The receptors for TGF-β and related factors are glycoproteins with a single membrane-spanning segment and intrinsic serine/threonine kinase activity. Receptor types I and II (Fig. 3-12B) are required for ligand binding and catalytic activity. The type II receptor first binds the ligand, followed by the formation of a stable ternary complex of ligand, type II receptor, and type I receptor. Recruitment of the type I receptor into the complex results in phosphorylation of the type I receptor at serine and threonine residues, which in turn activates the kinase activity of the type I receptor and propagates the signal to downstream effectors. Receptor tyrosine kinases produce phosphotyrosine motifs recognized by SH domains of downstream effectors In addition to the class of receptors with intrinsic serine/ threonine kinase activity, other plasma membrane receptors have intrinsic tyrosine kinase activity. All receptor tyrosine kinases discovered to date phosphorylate themselves in addition to other cellular proteins. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin and insulinrelated growth factor type 1 (IGF-1), fibroblast growth factor (FGF), and nerve growth factor (NGF) can all bind to receptors that possess intrinsic tyrosine kinase activity. Creation of Phosphotyrosine (pY) Motifs

Most RTKs are single-pass transmembrane proteins that contain a single intracellular kinase domain (Fig. 3-12C). Binding of a ligand, such as NGF, induces a conformational change in the receptor that facilitates the formation of receptor dimers. Dimerization allows the two cytoplasmic catalytic domains to phosphorylate each other (“autophosphorylation”) and thereby activate the receptor complex. The activated receptors also catalyze the addition of phosphate to tyrosine (Y) residues on specific cytoplasmic proteins. The resulting phosphotyrosine motifs of the receptor and other protein substrates serve as high-affinity binding sites for a number of intracellular signaling molecules. These interactions lead to the formation of a signaling complex and the activation of downstream effectors. Activation of insulin and IGF-1 receptors occurs by a somewhat different mechanism: the complex analogous to the dimeric NGF receptor exists even before ligand binding, as we will discuss in Chapter 51.

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Table 3-5 Tyrosine Phosphopeptides of the PDGF Receptor That Are Recognized by SH2 Domains on Various Proteins Tyrosine (Y) That Is Phosphorylated in the PDGF Receptor

Phosphotyrosine (PY) Motif Recognized by the SH2-Containing Protein

SH2-Containing Protein

Y579

pYIYVD

Src family kinases

Y708

pYMDMS

p85

Y719

pYVPML

p85

Y739

pYNAPY

GTPase-activating protein

Y1021

pYIIPY

PLCγ

Recognition of pY Motifs by SH2 and SH3 Domains

The phosphotyrosine motifs created by tyrosine kinases serve as high-affinity binding sites for the recruitment of many cytoplasmic or membrane-associated proteins that contain a region such as an SH2 (Src homology 2), SH3 (Src homology 3), or PTB (phosphotyrosine-binding) domains. SH2 domains are ∼100 amino acids in length. They are composed of relatively well conserved residues that form the binding pocket for pY motifs as well as more variable residues that are implicated in binding specificity. These residues that confer binding specificity primarily recognize the three amino acids located on the C-terminal side of the phosphotyrosine. For example, the activated PDGF receptor has five such pY motifs (Table 3-5), each of which interacts with a specific SH2-containing protein. SH3 domains are ∼50 amino acids in length and bind to proline-rich regions in other proteins. Although these interactions are typically constitutive, phosphorylation at distant sites can change protein conformation and thereby regulate the interaction. Like SH2 interactions, SH3 interactions appear to be responsible for targeting of signaling molecules to specific subcellular locations. SH2- or SH3-containing proteins include growth factor receptor-bound protein 2 (GRB2), PLCγ, and the receptor-associated tyrosine kinases of the Src family.

The MAPK Pathway A common pathway by which activated RTKs transduce their signal to cytosol and even to the nucleus is a cascade of events that increase the activity of the small GTP-binding protein Ras. This Ras-dependent signaling pathway involves the following steps (Fig. 3-13):

Step 1: A ligand binds to the extracellular domain of a specific RTK, thus causing receptor dimerization. Step 2: The now-activated RTK phosphorylates itself on tyrosine residues of the cytoplasmic domain (autophosphorylation). Step 3: GRB2 (growth factor receptor-bound protein 2), an SH2-containing protein, recognizes pY residues on the activated receptor. Step 4: Binding of GRB2 recruits SOS (son of sevenless), a guanine nucleotide exchange protein.

Step 5: SOS activates Ras by causing GTP to replace GDP on Ras. Step 6: The activated GTP-Ras complex activates other proteins by physically recruiting them to the plasma membrane. In particular, the active GTP-Ras complex interacts with the N-terminal portion of the serine/threonine kinase Raf-1 (also known as MAP kinase kinase kinase), which is the first in a series of sequentially activated protein kinases that ultimately transmits the activation signal. Step 7: Raf-1 phosphorylates and activates a protein kinase called MEK (also known as MAP kinase kinase or MAPKK). MEK is a multifunctional protein kinase that phosphorylates substrates on both tyrosine and serine/ threonine residues. The JAK system (see next section) also activates MEK. Step 8: MEK phosphorylates MAP kinase (MAPK), also called extracellular signal-regulated kinase (ERK1, ERK2). Activation of MAPK requires dual phosphorylation on neighboring serine and tyrosine residues. Step 9: MAPK is an important effector molecule in Rasdependent signal transduction because it phosphorylates many cellular proteins. Step 10: Activated MAPK also translocates to the nucleus, where it phosphorylates a number of nuclear proteins that are transcription factors. Phosphorylation of a transcription factor by MAPK can enhance or inhibit binding to DNA and thereby enhance or suppress transcription. Two other signal transduction pathways (cAMP and Ca2+) can modulate the activity of some of the protein intermediates in this MAP kinase cascade, suggesting multiple points of integration for the various signaling systems. Tyrosine kinase–associated receptors activate loosely associated tyrosine kinases such as Src and JAK Some of the receptors for cytokines and growth factors that regulate cell proliferation and differentiation do not themselves have intrinsic tyrosine kinase activity but can associate with nonreceptor tyrosine kinases (Fig. 3-12D). Receptors in this class include those for several cytokines, including IL-2, IL-3, IL-4, IL-5, IL-6, leukemia inhibitory factor (LIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). The family also includes receptors for growth hormone (GH), prolactin (PRL), leptin, ciliary neurotrophin factor (CNTF), oncostatin M, and IFNα, IFN-β, and IFN-γ. The tyrosine kinase–associated receptors typically comprise multiple subunits that form homodimers (αα), heterodimers (αβ), or heterotrimers (αβγ). For example, the IL-3 and the GM-CSF receptors are heterodimers (αβ) that share common β subunits with transducing activity. However, none of the cytoplasmic portions of the receptor subunits contains kinase domains or other sequences with recognized catalytic function. Instead, tyrosine kinases of the Src family and Janus family (JAK or Janus kinases) associate noncovalently with the cytoplasmic domains of these receptors. Thus, these are receptor-associated tyrosine kinases. Ligand binding to these receptors results in receptor dimerization

Chapter 3 • Signal Transduction

6 The activated GTP-Ras recruits Raf-1 and activates it.

1 Ligand binding causes receptor dimerization. Ligand

Ligand

Extracellular space

5 SOS activates Ras by causing GTP to replace GDP on Ras.

8 MEK phosphorylates and activates MAPK. 9 MAPK works as an important effector molecule by phosphorylating many cellular proteins.

7 Raf-1 phosphorylates and activates MEK.

Receptor

Plasma membrane SH2 domain

Cytosol Tyrosine kinase domain

Ras

Ras Raf-1

P

P

MEK P

P

MAPK P

Cytosolic proteins

GRB2 SOS

2 The activated RTK phosphorylates itself.

Ra

f-1

SO

S

Nucleus

Inactive transcription factor

Modulation of transcription

MAPK P

3 GRB2, an SH2-containing protein, recognizes the phosphotyrosine residues.

4 The binding of GRB2 recruits SOS.

P

Active transcription factor

10 MAPK translocates to the nucleus where it phosphorylates a transcription factor.

Figure 3-13 Regulation of transcription by the Ras pathway. A ligand, such as a growth factor, binds to a specific RTK, leading to an increase in gene transcription in a 10-step process.

and tyrosine kinase activity. The activated kinase then phosphorylates tyrosines on both itself and the receptor. Thus, tyrosine kinase–associated receptors, together with their tyrosine kinases, function much like the RTKs discussed in the previous section. A key difference is that for the tyrosine kinase–associated receptors, the receptors and kinases are encoded by separate genes and the proteins are only loosely associated with one another. The Src family of receptor-associated tyrosine kinases includes at least nine members. Alternative initiation codons and tissue-specific splicing (see Chapter 4) result in at least 14 related gene products. The conserved regions of Src-related proteins can be divided into five domains: (1) an N-terminal myristylation site, through which the kinase is tethered to the membrane; (2) an SH3 domain, which binds to proline-rich regions of the kinase itself or to other cytosolic proteins; (3) an SH2 domain, which binds phosphorylated tyrosines; (4) the catalytic domain, which has tyrosine kinase activity; and (5) a noncatalytic C terminus. Members of this family are kept in the inactive state by tyrosine phosphorylation at a conserved residue in the C terminus, causing this pY to bind to the amino-terminal SH2 domain of the same molecule, obscuring the intervening kinase domain. Dephosphorylation of the pY residue, after the activation of such phosphatases as RPTPα or SHP-2, releases this inhibition, and the kinase domain can then phosphorylate its intracellular substrates. Many of the Src family members were first identified in transformed cells or tumors because of mutations that

caused them to be constitutively active. When these mutations result in malignant transformation of the cell, the gene in question is designated an oncogene; the normal, unaltered physiological counterpart of an oncogene is called a proto-oncogene. The Janus family of receptor-associated tyrosine kinases in mammals includes JAK1, JAK2, and Tyk2. JAK stands for “just another kinase.” Major downstream targets of the JAKs include one or more members of the STAT (signal transducers and activators of transcription) family. When phosphorylated, STATs interact with other STAT family members to form a complex that translocates to the nucleus (see Chapter 4). There, the complex facilitates the transcription of specific genes that are specialized for a rapid response, such as those that are characterized by the acute-phase response of inflammation (see Chapter 59). For example, after IL-6 binds to hepatocytes, the STAT pathway is responsible for producing acute-phase proteins. During inflammation, these acutephase proteins function to limit tissue damage by inhibiting the proteases that attack healthy cells as well as diseased ones. The pattern of STAT activation provides a mechanism for cytokine individuality. For example, EPO activates STAT5a and STAT5b as part of the early events in erythropoiesis, whereas IL-4 or IL-12 activates STAT4 and STAT6. Attenuation of the cytokine JAK-STAT signaling cascade involves the production of inhibitors that suppress tyrosine phosphorylation and activation of the STATs. For example, IL-6 and LIF both induce expression of the inhibitor SST-1, which contains an SH2 domain and

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prevents JAK2 or Tyk2 from activating STAT3 in M1 myeloid leukemia cells. Receptor tyrosine phosphatases are required for lymphocyte activation Tyrosine residues that are phosphorylated by the tyrosine kinases described in the preceding two sections are dephosphorylated by phosphotyrosine phosphatases (PTPs), which can be either cytosolic or membrane bound (i.e., the receptor tyrosine phosphatases). We discussed the cytosolic PTPs earlier. Both classes of tyrosine phosphatases have structures very different from the ones that dephosphorylate serine and threonine residues. Because the tyrosine phosphatases are highly active, pY groups tend to have brief life spans and are relatively few in number in unstimulated cells. The CD45 protein, found at the cell surface of T and B lymphocytes, is an example of a receptor tyrosine phosphatase. CD45 makes a single pass through the membrane. Its glycosylated extracellular domain functions as a receptor for antibodies, whereas its cytoplasmic domain has tyrosine phosphatase activity (Fig. 3-12E). During their maturation, lymphocytes express several variants of CD45 characterized by different patterns of alternative splicing and glycosylation. CD45 plays a critical role in signal transduction in lymphocytes. For instance, CD45 dephosphorylates and thereby activates Lck and Fyn (two receptor-associated tyrosine kinases of the Src family) and triggers the phosphorylation of other proteins downstream in the signal transduction cascade. This interaction between receptor tyrosine phosphatases and tyrosine kinase–associated receptors is another example of crosstalk between signaling pathways.

NUCLEAR RECEPTORS Steroid and thyroid hormones enter the cell and bind to members of the nuclear receptor superfamily in the cytoplasm or nucleus A number of important signaling molecules produce their effects not by binding to receptors on the cell membrane but by binding to nuclear receptors (also called intracellular receptors) that can act as transcription regulators, a concept that we will discuss in more depth in Chapter 4. This family includes receptors for steroid hormones, prostaglandins, vitamin D, thyroid hormones, and retinoic acid (Table 3-6). In addition, this family includes related receptors, known as orphan receptors, whose ligands have yet to be identified. Steroid hormones, vitamin D, and retinoic acid appear to enter the cell by diffusing through the lipid phase of the cell membrane. Thyroid hormones, which are charged amino acid derivatives, may cross the cell membrane either by diffusion or by carrier-mediated transport. Once inside the cell, these substances bind to intracellular receptors. The ligandbound receptors are activated transcription factors that regulate the expression of target genes by binding to specific DNA sequences. In addition, steroid hormones can also have nongenomic effects (see Chapter 47). The family of nuclear receptors contains at least 32 genes and has been classically divided into two subfamilies based on structural homology. One subfamily consists of receptors for steroid hormones, including the glucocorticoids and mineralocorticoids (see Chapter 50), androgens (see Chapter 50), and estrogens and progesterone (see Chapter 55). These receptors function primarily as homodimers (Table 3-2). The other group includes receptors for retinoic acid (see Chapter 4), thyroid hormone (see Chapter 49), and vitamin D (see Chapter 52). These receptors appear to act as heterodimers (Table 3-2). As we will see in Chapters 4 and 47, other nuclear

Oncogenes

T

he ability of certain viral proteins (oncogenes) to transform a cell from a normal to a malignant phenotype was initially thought to occur because these viral proteins acted as transcriptional activators or repressors. However, during the last 20 years, only a few of these viral proteins have been found to work in this manner. The majority of oncogenes harbor mutations that transform them into constitutively active forms of normal cellular signaling proteins called proto-oncogenes. Most of these aberrant proteins (i.e., the oncogenes) encode proteins important in a key signal transduction pathway. For example, expression of the viral protein v-erb B is involved in fibrosarcomas, and both v-erb A and v-erb B are associated with leukemias. v-erb B resembles a constitutively activated receptor tyrosine kinase (epidermal growth factor receptor), and the retroviral v-erb A is derived from a cellular gene encoding a thyroid hormone receptor. Other receptors and signaling molecules implicated in cell transformation include Src, Ras, and platelet-derived growth factor receptor. A mutation in protein tyrosine phosphatase 1C results in abnormal hematopoiesis and an increased incidence of lymphoreticular tumors.

Table 3-6

Nuclear Steroid and Thyroid Receptors

Receptor

Full Name

Dimeric Arrangement

GR

Glucocorticoid receptor

GR/GR

MR

Mineralocorticoid receptor

MR/MR

PR

Progesterone receptor

PR/PR

ER

Estrogen receptor

ER/ER

AR

Androgen receptor

AR/AR

VDR

Vitamin D receptor

VDR/RXR

TR

Thyroid hormone receptor

TR/RXR

RAR

Retinoic acid receptor

RAR/RXR

SXR

Steroid and xenobiotic receptor

SXR/RXR

CAR

Constitutive androstane receptor

CAR/RXR

Chapter 3 • Signal Transduction

Transactivation DNA-binding Nucleardomain 1 and dimerization localization (amino terminal) domain domain

Figure 3-14 Modular construction of intracellular (or nuclear) receptors. Members of this family exist in the cytoplasm or nucleus and include receptors for several ligands, including retinoic acid, vitamin D, thyroid hormones, and steroid hormones. These receptors have modular construction, with up to six elements. The percentages listed inside the A/B, C, and E domains refer to the degrees of amino acid identity, referenced to the glucocorticoid receptor. Thus, the DNA-binding or C domain of the retinoic acid receptor is 45% identical to the corresponding domain on the glucocorticoid receptor.

Transactivation domain 2

E 15%

Retinoic acid C receptor

42%

> Cs+ > Li+, Na+, Ca2+. Under normal physiological conditions, the permeability ratio PK/PNa is greater than 100 and Na+ can block some K+ channels. Some K+ channels can pass Na+ current in the complete absence of K+. This finding is analogous to the behavior of Ca2+ channels, which can pass Na+ and K+ currents in the absence of Ca2+.

Given such strong K+ selectivity and an equilibrium potential near −80 mV, the primary role of K+ channels in excitable cells is inhibitory. K+ channels oppose the action of excitatory Na+ and Ca2+ channels and stabilize the resting, nonexcited state. Whereas some K+ channels are major determinants of the resting potential, the voltage dependence and kinetics of other K+ channels in excitable cells have specialized functions, such as mediating the repolarization and shaping of action potentials, controlling firing frequency, and

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Section II • Physiology of Cells and Molecules

A

1,4-DIHYDROPYRIDINES

B

PHENYLALKYLAMINES

CH3O CF3 H3COOC

COOCH2CH3

H3COOC

H3C

CH3

Nitrendipine Inhibitor (antagonist)

H3C

N H

CH2CH2NCH2CH2CH2CCN Verapamil

CH3O

NO2 C

N H

CH(CH3)2

CH3

NO2

BENZOTHIAZEPINES

CH3

OCH3 OCH3

OCH3 H

Bay K8644 Activator (agonist)

S H N

OOCCH3

O CH2CH2N(CH3)2

Diltiazem

Figure 7-17 Antagonists and agonists of L-type Ca2+ channels. A, 1,4-Dihydropyridines. One, nitrendipine, is an antagonist; another, Bay K8644, is an agonist. B, Phenylalkylamines. Verapamil is an antagonist. C, Benzothiazepines. Diltiazem is an antagonist.

Ca2+ Channel and Autoimmune Genetic Defects

C

a2+ channels have been linked to a large variety of genetic diseases. In mice, an interesting mutation results in muscular dysgenesis, or failure of normal skeletal muscle to develop. These mice lack a functional Ca2+ channel α1 subunit in their skeletal muscle. They die shortly after birth, but their cultured muscle cells provide an assay system to investigate the mechanism of EC coupling. Contraction of such defective muscle cells can be rescued by expression of cloned genes for either the skeletal Cav1.1 (CACNA1S gene) or the cardiac Cav1.2 (CACNA1C gene) L-type Ca2+ channels. As discussed in Chapter 9, a physiologically distinguishing feature of EC coupling in normal skeletal versus cardiac muscle is that skeletal muscle does not require extracellular Ca2+, whereas cardiac muscle does. Indeed, when the rescue is accomplished with skeletal Cav1.1, contraction does not require extracellular Ca2+; when the rescue is accomplished with cardiac α1C, contraction does require extracellular Ca2+. Such studies have provided strong support for the concept that EC coupling in skeletal muscle takes place by direct coupling of Cav1.1 to the Ca2+ release channels of the sarcoplasmic reticulum; in cardiac muscle, EC coupling occurs as Ca2+ entering through α1Ccontaining channels induces the release of Ca2+ from internal stores. Mutagenesis experiments with chimeric α1 subunits containing artificially spliced segments of the cardiac and skeletal channel isoforms have shown that the intracellular linker region between repeats II and III is the domain of the α1 subunit that determines the skeletal versus the cardiac type of EC coupling. A human pathologic condition called Lambert-Eaton syndrome has been characterized as an impairment of

presynaptic Ca2+ channels at motor nerve terminals. LambertEaton syndrome is an autoimmune disorder that is most often seen in patients with certain types of cancer, such as small cell lung carcinoma. Patients afflicted with this condition produce antibodies against presynaptic Ca2+ channels that somehow reduce the number of such channels able to function in the depolarization-induced influx of Ca2+ for neurotransmitter release. Hypokalemic periodic paralysis (not to be confused with hyperkalemic periodic paralysis, discussed earlier in the box titled Na+ Channel Genetic Defects) is an autosomal dominant muscle disease of humans. Affected family members have a point mutation in the CACNA1S gene encoding the skeletal Cav1.1, located in transmembrane segment S4 of domain II. This finding explains the basis for a human disorder involving defective EC coupling of skeletal muscle. Certain other rare human genetic diseases result in neurologic symptoms of migraine (severe headache) and ataxia (a movement disorder). One of these diseases, familial hemiplegic migraine, is caused by point mutations at various locations in the human CACNA1A gene encoding Cav2.1. These locations include the S4 region of domain I, the P region of domain II, and the S6 helices of domains I and IV. Another such genetic disease caused by mutations in the human CACNA1A gene encoding Cav2.1 is called episodic ataxia type 2, a condition associated with the occurrence of ataxia originating from the cerebellum. Discovery of the genetic origin of such diseases has led to the realization that delicate perturbations of Ca2+ channel activity can have profound consequences on proper function of the human nervous system.

Chapter 7 • Electrical Excitability and Action Potentials

defining the bursting behavior of rhythmic firing. Such functions are broadly important in regulating the strength and frequency of all types of muscle contraction, in terminating transmitter release at nerve terminals, and in attenuating the strength of synaptic connections. Finally, in epithelia, K+ channels also function in K+ absorption and secretion. Before molecular cloning revealed the structural relationships among the various kinds of K+ channels, electrophysiologists classified K+ currents according to their functional properties and gating behavior. They grouped the macroscopic K+ currents into four major types: 1. 2. 3. 4.

delayed outward rectifiers; transient outward rectifiers (A-type currents); Ca2+-activated K+ currents; and inward rectifiers.

These four fundamental K+ currents are the macroscopic manifestation of five distinct families of genes (Table 6.2): 1. Kv channels (voltage-gated K+ channels related to the Shaker family); 2. Small conductance KCa channels (Ca2+-activated K+ channels), including, SKCa and IKCa channels; 3. Large-conductance KCa channels (Ca2+-activated K+ channels, including BKCa and Na+-activated K+ channels); 4. Kir channels (inward rectifier K+ channels); and 5. K2P channels (two-pore K+ channels). In the next three sections, we discuss the various families of K+ channels and their associated macroscopic currents. The Kv (or shaker-related) family of K+ channels mediates both the delayed outward rectifier current and the transient A-type current The K+ current in the HH voltage-clamp analysis of the squid giant axon is an example of a delayed outward rectifier. Figure 7-18A shows that this current activates with a sigmoidal lag phase (i.e., it is delayed in time, as in Fig. 7-6C). Figure 7-18B is an I-V plot of peak currents obtained in experiments such as that in Figure 7-18A; it shows that the outward current rises steeply at positive voltages (i.e., it is an outward rectifier). A second variety of K+ current that is also outwardly rectifying is the transient A-type K+ current. This current was first characterized in mollusk neurons, but similar currents are common in the vertebrate nervous system. A-type currents are activated and inactivated over a relatively rapid time scale. Because their voltage activation range is typically more negative than that of other K+ currents, they are activated in the negative Vm range that prevails during the afterhyperpolarizing phase of action potentials. In neurons that spike repetitively, this A-type current can be very important in determining the interval between successive spikes and thus the timing of repetitive action potentials. For example, if the A-type current is small, Vm rises relatively quickly toward the threshold, and consequently the interspike interval is short and the firing frequency is high (Fig. 7-18C).

However, if the A-type current is large, Vm rises slowly toward the threshold, and therefore the interspike interval is long and the firing frequency is low (Fig. 7-18D). Because the nervous system often encodes information as a frequencymodulated signal, these A-type currents play a critical role. The channels responsible for both the delayed outward rectifier and the transient A-type currents belong to the Kv channel family (where v stands for voltage-gated). The prototypic protein subunit of these channels is the Shaker channel of Drosophila. All channels belonging to this family contain the conserved S1-S6 core that is characteristic of the Shaker channel (Fig. 7-10) but may differ extensively in the length and sequence of their intracellular N-terminal and C-terminal domains. The voltage-sensing element in the S4 segment underlies activation by depolarization; the S4 segment actually moves outward across the membrane with depolarizing voltage, thus increasing the probability of the channel’s being open (see the box titled Crystal Structure of a Mammalian K+ Channel). The Kv channel family has multiple subclasses (see Table 6-2). Individual members of this Kv channel family, whether in Drosophila or humans, exhibit profound differences in gating kinetics that are analogous to delayed rectifier (slow activation) or A-type (rapid inactivation) currents. For example, Figure 7-18E shows the macroscopic currents of four subtypes of rat brain Kv1 (or Shaker) channels heterologously expressed in frog oocytes. All of these Kv1 channel subtypes (Kv1.1 to Kv1.4) exhibit sigmoidal activation kinetics when they are examined on a brief time scale—in the millisecond range (left side of Fig. 7-18E). That is, these channels display some degree of “delayed” activation. Different Kv channels exhibit different rates of activation. Thus, these currents can modulate action potential duration by either keeping it short (e.g., in nerve and skeletal muscle) when the delayed rectifier turns on quickly or keeping it long (e.g., in heart) when the delayed rectifier turns on slowly. Kv1 channels also differ markedly in their inactivation kinetics when they are observed over a long time scale—in the range of seconds (right side of Fig. 7-18E). Kv1.1 exhibits little time-dependent inactivation (i.e., the current is sustained throughout the stimulus). On the other hand, the Kv1.4 channel completely inactivates in less than 1 second. Kv1.2 and Kv1.3 show intermediate behavior. How are Kv channels inactivated? The structural basis for one particular type of K+ channel inactivation, known as Ntype inactivation, is a stretch of ∼20 amino acid residues at the N terminus of some fast-inactivating Kv channels. This domain acts like a ball to block or to plug the internal mouth of the channel after it opens, thereby resulting in inactivation (Fig. 7-18F). Thus, this process is also known as the balland-chain mechanism of K+ channel inactivation. Particular kinds of β subunits that are physically associated with some isoforms of Kv channels have structural elements that mimic this N-terminal ball domain and rapidly inactivate K+ channel α subunits that lack their own inactivation ball domain (Fig. 7-11). Various delayed rectifier K+ channels are blocked by either internal or external application of quaternary ammonium ions such as TEA. We already have described an example of how TEA can inhibit the outward rectifier K+ current (Fig. 7-5C) in pharmacological dissection of the currents underly-

201

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Section II • Physiology of Cells and Molecules

A

DELAYED ACTIVATION OF Kv CHANNELS +30 mV –60 mV The activation of the current is delayed.

1 2 Peak current

+ K current

B

3

4 5 +

6 P

N

OUTWARD RECTIFICATION OF Kv CHANNELS Peak K current (IK)

C

C A-TYPE OUTWARD RECTIFIER: SMALL CURRENT Small A-type current

Outward The current flows only in the outward direction. That is, the channel is an “outward rectifier.”

Vm

E

Long interspike interval Large A-type current

DIFFERENCES IN GATING KINETICS AMONG Kv-TYPE DELAYED OUTWARD RECTIFIERS

Kv1.1

150 pA

Kv1.1

Kv1.2

200 pA

Kv1.2

Kv1.3

6 pA

0

500 pA

300 pA

Kv1.4

F

D A-TYPE OUTWARD RECTIFIER: LARGE CURRENT

400 pA

0

25 50 Time (msec)

Kv1.3

10 pA

Kv1.4

200 pA

1

2 seconds

INACTIVATION OF Kv-TYPE CHANNELS 4 α subunits

4 α subunits

4 α subunits

β

The N-terminal domain ball moves in and blocks the channel.

4 α subunits

β

The β subunit moves in and blocks the channel.

3

Chapter 7 • Electrical Excitability and Action Potentials

ing the action potential. Many transient A-type K+ currents are inhibited by another organic cation, 4-aminopyridine. Two distinct families of peptide toxins—charybdotoxins of scorpion venom and dendrotoxins of mamba snake venom—can discriminate particular subtypes of Kv and KCa channels, depending on the particular amino acids present in the P region. Two families of KCa K+ channels mediate Ca2+-activated K+ currents Ca2+-activated K+ channels—KCa channels—appear to be present in the plasma membrane of cells in many different tissues. In patch-clamp experiments, they are easily recog-

Human Heart Defects Linked to Mutations of K+ Channels

A

congenital cardiac abnormality in some people results in lengthening of the QT interval of the electrocardiographic signal—long QT syndrome—which corresponds to a prolonged cardiac action potential. Affected children and young adults can exhibit an arrhythmic disturbance of the ventricular heartbeat that results in sudden death. As we have already seen in the box titled Na+ Channel Genetic Defects, one form of a long QT syndrome involves defects in cardiac Na+ channels. However, several forms of this syndrome are caused by mutations in cardiac K+ channel proteins. Some families have mutations in the KCNQ1 gene encoding KvLQT1, a 581-residue protein belonging to the Kv family of voltage-gated K+ channels. Another form of this disease involves mutations in the KCNH2 gene encoding HERG, which is related to the ether-a-go-go Drosophila mutant, a more distant relative of the Kv channels. Both KvLQT1 and HERG K+ channels participate in repolarization of the cardiac action potential. Such defective repolarization can lead to premature heartbeats or asynchronous ventricular contraction, with subsequent death. The KvLQT1 K+ channel also physically associates with another small membrane protein called minK. Mutations in minK also cause a form of long QT syndrome. K+ channels are also crucial for proper function of the auditory system. Thus, congenital deafness is commonly associated with mutations in some of these K+ channels.

nized because the opening probability of individual channels increases at positive values of Vm (Fig. 7-19A). Po also increases with increasing [Ca2+] on the intracellular surface of the membrane patch (Fig. 7-19B). Figure 7-19C shows how increasing [Ca2+]i causes a negative shift in the Po versus Vm plot for these channels. A particular type of KCa channel called the maxi-KCa or BK (for “big” K+) channel is noted for its large unitary conductance (∼300 pS) and distinctive gating activity. In principle, KCa channels provide a stabilizing mechanism to counteract repetitive excitation and intracellular Ca2+ loading. KCa channels mediate the afterhyperpolarizing phase of action potentials (Fig. 7-1A) in cell bodies of various neurons. They have also been implicated in terminating bursts of action potentials in bursting neuronal pacemaker cells. Thus, the gradual increase in [Ca2+]i that occurs during repetitive firing triggers the opening of KCa channels, which results in hyperpolarization and a quiescent interburst period that lasts until intracellular Ca2+ accumulation is reversed by the action of Ca2+ pumps. KCa channels are also present at high density in many types of smooth muscle cells, where they appear to contribute to the relaxation of tension by providing a hyperpolarizing counterbalance to Ca2+-dependent contraction. In a number of nonexcitable cells, KCa channels are activated during cell swelling and contribute to regulatory volume decrease (see Chapter 5). Drosophila genetics also led the way to identification of the first of several genes that encode members of the KCa channel family. Electrophysiological studies of the Slowpoke mutation in flies showed that this mutation eliminated a fast, Ca2+-activated K+ current that is present in larval muscle and neurons. Subsequent cloning and sequencing of the Slowpoke gene product revealed a channel-forming subunit that has an S1-S6 core domain similar to that of the Kv family, but it also contains a unique C-terminal domain of ∼850 residues (Fig. 7-19). Because BKCa channels—like Kv channels—have a voltage-sensing domain that is analogous to S4, they are also activated by positive voltage. Structure-function studies on this class of K+ channel indicate that the unique C-terminal domain contains the Ca2+-binding sites that function in channel activation. In addition to the BKCa family, another K+ channel gene family includes intermediate- and small-conductance Ca2+activated K+ channels, respectively termed IKCa and SKCa. Unlike BKCa channels, the closely related IKCa and SKCa chan-

Figure 7-18 Outwardly rectifying K+ channels. A, Note that in a voltage-clamp experiment, a depolarizing step in Vm activates the current, but with a delay. B, The current-voltage relationship is shown for a delayed outward rectifying K+ channel, as in A. C, This A-type K+ current is active at relatively negative values of Vm and tends to hyperpolarize the cell. In a spontaneously spiking neuron, a low level of the A-type current allows Vm to rise relatively quickly toward the threshold, which produces a relatively short interspike interval and thus a high firing rate. D, In a spontaneously spiking neuron, a high level of the A-type current causes Vm to rise relatively slowly toward the threshold, which produces a relatively long interspike interval and thus a low firing rate. E, These experiments were performed on four different types of K+ channels (Kv1.1, 1.2, 1.3, and 1.4) from mammalian brain and expressed in Xenopus oocytes. Shown are the results of voltage-clamp experiments in which Vm was stepped from −80 mV to 0 mV. The left panel, at high time resolution, shows that some of these channels activate more slowly than others. The right panel, at a longer time scale, shows that inactivation gradually speeds up from Kv1.1 to Kv1.4. F, The left panel shows N-type inactivation, so called because the N or amino terminus of the protein is essential for inactivation. Each of the four subunits is thought to have an N-terminal “ball” tethered by a “chain” that can swing into place to block the pore. The right panel shows a variant in which certain β subunits can provide the ball-and-chain for Kv channel α subunits that themselves lack this capability at their N termini. (Data from Stühmer W, Ruppersberg JP, Schroter KH, et al: Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain. EMBO J 1989; 8:3235-3244.)

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A

VOLTAGE DEPENDENCE α subunit

Two channels open

β subunit

N

One channel open

1 2 3 4 + 5

0

Closed

P

6

1 2

Vm = +80 mV 2+

Closed

Ca

C

N

C

Open Vm = –60 mV 40 pA

60 msec

B

CALCIUM DEPENDENCE

C

COMBINED EFFECTS OF CHANGING Vm AND [Ca2+]i

2+

1 μM Ca Vm = +40 mV

1.0 100 μM Ca2+ Closed Relative Po

2+

10 μM Ca Vm = +40 mV

0.5

10 μM Ca2+

Open 1 μM Ca2+ Closed 2+

100 μM Ca Vm = +40 mV

0

–60

–40 0 Membrane potential (mV)

Open Closed

20 pA

150 ms

Figure 7-19 Ca2+-activated K+ channels (KCa). A, Shown is an experiment on KCa channels that are expressed in Xenopus oocytes and studied by use of a patch pipette in an inside-out configuration. When Vm is held at −60 mV, there is very little channel activity. On the other hand, when Vm is +80 mV, both channels in the patch are open most of the time. B, The experiment is the same as in A except that Vm is always held at +40 mV and the [Ca2+] on the cytosolic side of the patch varies from 1 to 10 to 100 μM. Note that channel activity increases with increasing [Ca2+]i. C, Combined effects of changing Vm and [Ca2+]i. Shown is a plot of relative open probability (Po) of the KCa channels versus Vm at three different levels of Ca2+. The data come from experiments such as those shown in B. (Data from Butler A, Tsunoda S, McCobb DP, et al: mSlo, a complex mouse gene encoding “maxi” calcium-activated potassium channels. Science 1993; 261:221-224.)

40

Chapter 7 • Electrical Excitability and Action Potentials

nels are voltage insensitive and are activated by the Ca2+binding protein calmodulin (see Chapter 3). In some cells, IKCa and SKCa channels participate in action potential repolarization and afterhyperpolarization, thus regulating action potential firing frequency. Certain types of these channels function in the activation of lymphocytes.

The Kir K+ channels mediate inward rectifier K+ currents, and K2P channels may sense stress In contrast to delayed rectifiers and A-type currents— which are outwardly rectifying K+ currents—the inward rectifier K+ current (also known as the anomalous rectifier) actually conducts more K+ current in the inward direction than in the outward direction. Such inwardly rectifying, steady-state K+ currents have been recorded in many types of cells, including heart, skeletal muscle, and epithelia. Physiologically, these channels help clamp the resting membrane potential close to the K+ equilibrium potential and prevent excessive loss of intracellular K+ during repetitive activity and long-duration action potentials. In epithelial cells, these inwardly rectifying K+ currents are important because they stabilize Vm in the face of electrogenic ion transporters that tend to depolarize the cell (see Chapter 3). In contrast to the Kv and KCa channel families, the channelforming subunits of the inward rectifier (Kir) K+ channel family are smaller proteins (∼400 to 500 residues) that do not contain a complete S1-S6 core domain. However, they do have a conserved region that is similar to the S5-P-S6 segment of Kv channels (Fig. 7-20A; see the box titled Crystal Structure of a Mammalian K+ Channel). The conserved P region is the most basic structural element that is common to all K+ channels. The lack of an S1-S4 voltage-sensing domain in inward rectifier channels accounts for the observation that unlike Kv channels, Kir K+ channels are not steeply activated by voltage. Figure 7-20B shows a series of single-channel currents that were obtained from a Kir channel, with equal concentrations of K+ on both sides of the membrane as well as Mg2+ on the cytosolic side. Under these conditions, the channel conducts K+ current only in the inward direction. An I-V plot (Fig. 7-20C) derived from data such as these shows typical inward rectification of the unitary current. At negative values of Vm, the inward current decreases linearly as voltage becomes more positive, and no outward current is present at positive values of Vm. However, when Mg2+ is omitted from the cytosolic side of the membrane, the channel now exhibits a linear or ohmic I-V curve even over the positive range of Vm values. Thus, the inward rectification is due to intracellular block of the channel by Mg2+. Inhibition of outward K+ current in the presence of intracellular Mg2+ results from voltage-dependent binding of this divalent metal ion. Positive internal voltage favors the binding of Mg2+ to the inner mouth of this channel (Fig. 7-20D), as would be expected if the Mg2+ binding site is located within the transmembrane electrical field. Because Mg2+ is impermeant, it essentially blocks outward K+ current. However, negative values of Vm pull the Mg2+ out of the channel. Moreover, incoming K+ tends to displace any remaining Mg2+. Thus, the Kir channel

favors K+ influx over efflux. Intracellular polyamines such as spermine and spermidine—which, like Mg2+, carry a positive charge—also produce inward rectification of inward rectifier channels. These organic cations are important channelmodulating factors that also determine the current-voltage behavior of this particular class of ion channels. The Kir family of K+ channels exhibits various modes of regulation. One Kir subfamily (the G protein–activated, inwardly rectifying K+ channels or GIRKs) is regulated by the βγ subunits of heterotrimeric G proteins (see Chapter 3). For example, stimulation of the vagus nerve slows the heartbeat because the vagal neurotransmitter acetylcholine binds to postsynaptic muscarinic receptors in the heart that are coupled to G proteins. The binding of acetylcholine to its receptor causes the release of G protein βγ subunits, which diffuse to a site on neighboring GIRK channels to activate their opening. The resulting increase in outward K+ current hyperpolarizes the cardiac cell, thereby slowing the rate at which Vm approaches the threshold for firing action potentials and lowering the heart rate. GIRK channels are also activated by the membrane phospholipid PIP2. Thus, G protein–coupled receptors that activate phospholipase C lead to the release of PIP2, thereby activating GIRK channels. The members of another subfamily of Kir K+ channels, the KATP channels, are directly regulated by adenine nucleotides. KATP channels are present in the plasma membrane of many cell types, including skeletal muscle, heart, neurons, insulin-secreting β cell of the pancreas, and renal tubule. These channels are inhibited by intracellular adenosine triphosphate (ATP) and activated by adenosine diphosphate (ADP) in a complex fashion. They are believed to provide a direct link between cellular metabolism on the one hand and membrane excitability and K+ transport on the other. For example, if cellular ATP levels fall because of oxygen deprivation, such channels could theoretically open and hyperpolarize the cell to suppress firing of action potentials and further reduce energy expenditure. In the pancreatic β cell, an increase in glucose metabolism increases the ATP/ADP ratio. This increased ratio inhibits enough KATP channels to cause a small depolarization, which in turn activates voltage-gated Ca2+ channels and results in insulin secretion (see Chapter 51). KATP channels are the target of a group of synthetic drugs called sulfonylureas that include tolbutamide and glibenclamide. Sulfonylureas are used in the treatment of type 2 (or non–insulin-dependent) diabetes mellitus because they inhibit pancreatic KATP channels and stimulate insulin release. Newer and chemically diverse synthetic drugs called K+ channel openers (e.g., pinacidil and cromakalim) activate KATP channels. The therapeutic potential of K+ channel openers is being explored in light of their ability to relax various types of smooth muscle. The ability of sulfonylurea drugs to inhibit KATP channels depends on an accessory subunit called SUR (for sulfonylurea receptor). This protein is a member of the ATP-binding cassette family of proteins (see Chapter 5), which includes two nucleotide-binding domains. The newest family of K+ channels is that of the two-pore or K2P channels, which consist of a tandem repeat of the

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A

INWARDLY RECTIFYING + K CHANNEL

B

C

SINGLE-CHANNEL RECORDING OF A GIRK1 CHANNEL

DEPENDENCE OF INWARD RECTIFICATION ON INTRACELLULAR Mg2+

0 Vm=+60 mV 1

P

Mg2+ is absent.

–4 pA

2

N

0

pA

Vm=+40 mV

2

–100

+100

C

–2

Vm=+0 mV

Mg2+ is present on cytosolic side of membrane.

–4

Vm=–20 mV

mV

Vm=–40 mV D

MODEL OF Mg2+ BLOCK

K+ from outside displaces Mg2+ and enters cell.

+ K channel

Vm=–60 mV Closed Open

Vm=–80 mV 0

+

K

Extracellular space Plasma membrane Cytosol

Mg2+

–4 pA K+

Vm=–100 mV 0

50

150 250 milliseconds

350

Mg2+

K+ can't enter here.

Figure 7-20 Inwardly rectifying K+ channels. A, This family of channels has only two membrane-spanning segments that correspond to the S5-P-S6 domain of the voltage-gated K+ channels. B, The GIRK1 channels were expressed in Xenopus oocytes and studied by use of a patch pipette in the inside-out configuration. Vm was clamped to values between −100 mV and +60 mV, and [Mg2+] was 2.5 mM on the cytosolic side. Note that channel activity increases at more negative voltages but is virtually inactive at positive voltages. C, The I-V plot shows that there is inward rectification only in the presence of Mg2+ on the cytosolic side. In the absence of Mg2+, the I-V relationship is nearly linear or ohmic. D, As shown in the left panel, cytosolic Mg2+ occludes the channel pore and prevents the exit of K+. However, even in the presence of Mg2+, K+ can move into the cell by displacing the Mg2+. (Data from Kubo Y, Reuveny E, Slesinger PA, et al: Primary structure and functional expression of a rat G protein–coupled muscarinic potassium channel. Nature 1993; 364:802-806.)

basic Kir topology (see Fig. 6-21F). Because the monomeric subunit of K2P channels contains two linked S5-P-S6 pore domains of the basic Shaker Kv channel, the functional K2P channel is likely to be a dimer of the monomer subunit, which is itself a pseudodimer. K2P channels have been implicated in genesis of the resting membrane potential. K+ channels encoded by the 15 human genes for K2P channels may be activated by various chemical and physical signals including PIP2, membrane stretch, heat, intracellular pH, and general anesthetics. These channels are thought to be involved in a wide range of sensory and neuronal functions.

PROPAGATION OF ACTION POTENTIALS The propagation of electrical signals in the nervous system involves local current loops The extraordinary functional diversity of ion channel proteins provides a large array of mechanisms by which the membrane potential of a cell can be changed to evoke an electrical signal or biochemical response. However, channels alone do not control the spread of electrical current. Like electricity in a copper wire, the passive spread of current in biological tissue depends on the nature of the conducting

Chapter 7 • Electrical Excitability and Action Potentials A

UNMYELINATED AXON

+

+

+

+

–

–

+

+

+

+

+

+

+

+

+

–

–

–

–

+

+

–

–

–

–

–

–

–

–

–

Inactive B

Active

Inactive

MYELINATED AXON

Myelin sheath

A myelin sheath can have up to 300 layers of membrane. Active

Figure 7-21 Local current loops during action-potential propagation. A, In an unmyelinated axon, the ionic currents flow at one instant in time as a result of the action potential (“active” zone). In the “inactive” zones that are adjacent to the active zone, the outward currents lead to a depolarization. If the membrane is not in an absolute refractory period and if the depolarization is large enough to reach threshold, the immediately adjacent inactive zones will become active and fire their own action potential. In the more distant inactive zones, the outward current is not intense enough to cause Vm to reach threshold. Thus, the magnitudes of the outward currents decrease smoothly with increasing distance from the active zone. B, In this example, the “active” zone consists of a single node of Ranvier. In a myelinated axon, the ionic current flows only through the nodes, where there is no myelin and the density of Na+ channels is very high. Ionic current does not flow through the internodal membrane because of the high resistance of myelin. As a result, the current flowing down the axon is conserved, and the current density at the nodes is very high. This high current density results in the generation of an action potential at the node. Thus, the regenerative action potential propagates in a “saltatory” manner by jumping from node to node. Note that the action potential is actually conducted through the internodal region by capacitative current due to charge displacement across the membrane arising from the resistance-capacitance properties of the membrane (see Fig. 6-11).

and insulating medium. Important factors include geometry (i.e., cell shape and tissue anatomy), electrical resistance of the aqueous solutions and cell membrane, and membrane capacitance. Furthermore, the electrotonic spread of electrical signals is not limited to excitable cells. Efficient propagation of a change in Vm is essential for the local integration of electrical signals at the level of a single cell and for the global transmission of signals across large distances in the body. As we discussed earlier in this chapter (Fig. 7-2), action potentials propagate in a regenerative manner without loss of amplitude as long as the depolarization spreads to an adjacent region of excitable membrane and does so with sufficient strength to depolarize the membrane above its threshold. However, many types of nonregenerative, subthreshold potentials also occur and spread for short distances along cell membranes. These graded responses, which we also discussed earlier, contrast with the all-or-nothing nature of action potentials. Such nonregenerative signals include receptor potentials generated during the transduction of sensory stimuli and synaptic potentials generated by the opening of agonist-activated channels. With a graded response, the greater the stimulus, the greater the voltage response. For example, the greater the intensity of light that is shined on a mammalian photorecep-

tor cell in the retina, the greater the hyperpolarization produced by the cell. Similarly, the greater the concentration of acetylcholine that is applied at a postsynaptic neuromuscular junction, the greater the resulting depolarization (i.e., synaptic potential). Of course, if this depolarization exceeds the threshold in an excitable cell, an all-or-nothing action potential is initiated. The generation of a physiological response from a graded potential change critically depends on its electrotonic spread to other regions of the cell. Like the subthreshold voltage responses produced by injection of a current into a cell through a microelectrode, the electrotonic spread of graded responses declines with distance from the site of initiation. Graded signals dissipate over distances of a few millimeters and thus have only local effects; propagated action potentials can travel long distances through nerve axons. Electrotonic spread of voltage changes along the cell occurs by the flow of electrical current that is carried by ions in the intracellular and extracellular medium along pathways of the least electrical resistance. Both depolarizations and hyperpolarizations of a small area of membrane produce local circuit currents. Figure 7-21A illustrates how the transient voltage change that occurs during an action potential at a particular active site results in local current flow. The

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cytosol of the active region, where the membrane is depolarized, has a slight excess of positive charge compared with the adjacent inactive regions of the cytosol, which have a slight excess of negative charge. This charge imbalance within the cytosol causes currents of ions to flow from the electrically excited region to adjacent regions of the cytoplasm. Because current always flows in a complete circuit along pathways of least resistance, the current spreads longitudinally from positive to negative regions along the cytoplasm, moves outward across membrane conductance pathways (“leak channels”), and flows along the extracellular medium back to the site of origin, thereby closing the current loop. Because of this flow of current (i.e., positive charge), the region of membrane immediately adjacent to the active region becomes more depolarized, and Vm eventually reaches threshold. Thus, an action potential is generated in this adjacent region as well. Nerve and muscle fibers conduct impulses in both directions if an inactive fiber is excited at a central location, as in this example. However, if an action potential is initiated at one end of a nerve fiber, it will travel only to the opposite end and stop because the refractory period prevents backward movement of the impulse. Likewise, currents generated by subthreshold responses migrate equally in both directions. Myelin improves the efficiency with which axons conduct action potentials The flow of electrical current along a cylindrical nerve axon has often been compared with electrical flow through an undersea cable. Similar principles apply to both types of conducting fiber. An underwater cable is designed to carry an electrical current for long distances with little current loss; therefore, it is constructed of a highly conductive (low resistance) metal in its core and a thick plastic insulation wrapped around the core to prevent loss of current to the surrounding seawater. In contrast, the axoplasm of a nerve fiber has much higher resistance than a copper wire, and the nerve membrane is inherently electrically leaky because of background channel conductance. Therefore, in a biological fiber such as a nerve or muscle cell, some current is passively lost into the surrounding medium, and the amplitude of the signal rapidly dissipates over a short distance. Animal nervous systems use two basic strategies to improve the conduction properties of nerve fibers: (1) increasing the diameter of the axon, thus decreasing the internal resistance of the cable; and (2) myelination, which increases the electrical insulation around the cable. As axon diameter increases, the conduction velocity of action potentials increases because the internal resistance of the axoplasm is inversely related to the internal cross-sectional area of the axon. Unmyelinated nerve fibers of the invertebrate squid giant axon (as large as ∼1000 μm in diameter) are a good example of this type of size adaptation. These nerve axons mediate the escape response of the squid from its predators and can propagate action potentials at a velocity of ∼25 m/s. In vertebrates, myelination of smaller diameter (∼1 to 5 μm) nerve axons serves to improve the efficiency of impulse propagation, especially over the long distances that nerves traverse between the brain and the extremities. Axons are literally embedded in myelin, which consists of concentrically wound wrappings of the membranes of glial cells (see

Chapter 11). The thickness of the myelin sheath may amount to 20% to 40% of the diameter of a nerve fiber, and the sheath may consist of as many as 300 membrane layers. The glial cells that produce myelin are called Schwann cells in the periphery and oligodendrocytes in the brain. Because resistors in series add directly and capacitors in series add as the sum of the reciprocal, the insulating resistance of a myelinated fiber with 300 membrane layers is increased by a factor of 300 and the capacitance is decreased to 1/300 that of a single membrane. This large increase in membrane resistance minimizes loss of current across the leaky axonal membrane and forces the current to flow longitudinally along the inside of the fiber. In myelinated peripheral nerves, the myelin sheath is interrupted at regular intervals, forming short (∼1 μm) uncovered regions called nodes of Ranvier. The length of the myelinated axon segments between adjacent unmyelinated nodes ranges from 0.2 to 2 mm. In mammalian axons, the density of voltage-gated Na+ channels is very high in the nodal membrane. The unique anatomy of myelinated axons results in a mode of impulse propagation known as saltatory conduction. Current flow that is initiated at an excited node flows directly to adjacent nodes with little loss of transmembrane current through the internode region (Fig. 7-21B). In other words, the high membrane resistance in the internode region effectively forces the current to travel from node to node. The high efficiency of impulse conduction in such axons allows several adjacent nodes in the same fiber to fire an action potential virtually simultaneously as it is being propagated. Thus, saltatory conduction in a myelinated nerve can reach a very high velocity, up to 130 m/s. The action potential velocity in a myelinated nerve fiber can thus be severalfold greater than that in a giant unmyelinated axon, even though the axon diameter in the myelinated fiber may be more than two orders of magnitude smaller. During conduction of an action potential in a myelinated axon, the intracellular regions between nodes also depolarize. However, no transmembrane current flows in these internodal regions, and therefore no dissipation of ion gradients occurs. The nodal localization of Na+ channels conserves ionic concentration gradients that must be maintained at the expense of ATP hydrolysis by the Na-K pump. The cable properties of the membrane and cytoplasm determine the velocity of signal propagation Following the analogy of a nerve fiber as an underwater cable, cable theory allows one to model the pathways of electrical current flow along biomembranes. The approach is to use circuit diagrams that were first employed to describe the properties of electrical cables. Figure 7-22A illustrates the equivalent circuit diagram of a cylindrical electrical cable or membrane that is filled and bathed in a conductive electrolyte solution. The membrane itself is represented by discrete elements, each with a transverse membrane resistance (rm) and capacitance (cm) connected in parallel (a representation we used earlier, in Fig. 6-11A). Consecutive membrane elements are connected in series by discrete resistors, each of which represents the electrical resistance of a finite length of

Chapter 7 • Electrical Excitability and Action Potentials

A

EQUIVALENT-CIRCUIT MODEL ro ro

ro

ro

ro

ro

ri

ri

ri

ri

Membrane rm

cm ri

ri Axoplasm

Extracellular fluid

B

DISTRIBUTION OF CURRENT FLOW

C

VOLTAGE DECAY

Injection

Vo The decay is exponential. V

V = Voe

Vo = 0.37 Vo e

–x\λ

λ

0

X

Figure 7-22 Passive cable properties of an axon. A, The axon is represented as a hollow, cylindrical “cable” that is filled with an electrolyte solution. All of the electrical properties of the axon are represented by discrete elements that are expressed in terms of the length of the axon. ri is the resistance of the internal medium. Similarly, ro is the resistance of the external medium. rm and cm are the membrane resistance and capacitance per discrete element of axon length. B, When current is injected into the axon, the current flows away from the injection site in both directions. The current density smoothly decays with increasing distance from the site of injection. C, Because the current density decreases with distance from the site of current injection in B, the electrotonic potential (V) also decays exponentially with distance in both directions. Vo is the maximum change in Vm that is at the site of current injection.

the external medium (ro) or internal medium (ri). The parameters rm, cm, ro, and ri refer to a unit length of axon (Table 7-3). How do the various electrical components of the cable model influence the electrotonic spread of current along an axon? To answer this question, we inject a steady electrical current into an axon with a microelectrode to produce a constant voltage (V0) at a particular point (x = 0) along the length of the axon (Fig. 7-22B). This injection of current results in the longitudinal spread of current in both directions from point x = 0. The voltage (V) at various points along the axon decays exponentially with distance (x) from the point of current injection (Fig. 7-22C), according to the following equation:

V = V0 e − x/λ

(7-5)

The parameter λ has units of distance and is referred to as the length constant or the space constant. One length constant away from the point of current injection, V is 1/e, or ∼37% of the maximum value of V0. The decaying currents that spread away from the location of a current-passing electrode are called electrotonic currents. Similarly, the spread of subthreshold voltage changes away from a site of origin is referred to as electrotonic spread, unlike the regenerative propagation of action potentials. The length constant depends on the three resistance elements in Figure 7-22A:

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λ=

rm ro + ri

(7-6)

We can simplify this expression by noting that internal resistance is much larger than external resistance, so the contribution of ro to the denominator can be ignored. Thus, λ=

rm ri

(7-7)

The significance of the length constant is that it determines how far the electrotonic spread of a local change in membrane potential is able to influence neighboring regions of membrane. The longer the length constant, the farther down the axon a voltage change spreads. How does the diameter of an axon affect the length constant? To answer this question, we must replace rm and ri (expressed in terms of axon length) in Equation 7-7 with the specific resistances Rm and Ri (expressed in terms of the area of axon membrane or cross-sectional area of axoplasm). Making the substitutions according to the definitions in Table 7-3, we have λ=

aRm 2Ri

(7-8)

Thus, the length constant (λ) is directly proportional to the square root of the axon radius (a). Equation 7-8 confirms

TABLE 7-3

Cable Parameters

Parameter

Units

Definition or Relationship

rm

Ω × cm

Membrane resistance (per unit length of axon)

ro

Ω/cm

Extracellular resistance (per unit length of axon)

ri

Ω/cm

Intracellular resistance (per unit length of axon)

cm

μF/cm

Membrane capacitance (per unit length of axon)

Rm = rm × 2πa

Ω × cm2

Specific membrane resistance (per unit area of membrane)

Ri = ri × πa2

Ω × cm

Specific internal resistance (per unit cross-sectional area of axoplasm)

Cm = cm/(2πa)

μF/cm2

Specific membrane capacitance (per unit area of membrane)

a, radius of the axon; Ω, ohm; F, farad.

basic intuitive notions about what makes an efficiently conducting electrical cable: 1. The greater the specific membrane resistance (Rm) and cable radius, the greater the length constant and the less the loss of signal. 2. The greater the resistance of the internal conductor (Ri), the smaller the length constant and the greater the loss of signal. These relationships also confirm measurements of length constants in different biological preparations. For example, the length constant of a squid axon with a diameter of ∼1 mm is ∼13 mm, whereas that of a mammalian nerve fiber with a diameter of ∼1 μm is ∼0.2 mm. So far, we have been discussing the spatial spread of voltage changes that are stable in time. In other words, we assumed that the amount of injected current was steady. What happens if the current is not steady? For example, what happens at the beginning of a stimulus when we (or a physiological receptor) first turn the current “on”? To answer these questions, we need to know how rapidly Vm changes in time at a particular site, which is described by a second cable parameter called the membrane time constant (τm). Rather than determining the spread of voltage changes in space, as the length constant does, the time constant influences the spread of voltage changes in time and thus the velocity of signal propagation. We previously discussed the time constant with respect to the time course of the change in Vm caused by a stepwise pulse of current (see Fig. 6-12A). Because the membrane behaves like an RC circuit, the voltage response to a square current pulse across a small piece of membrane follows an exponential time course with a time constant that is equal to the product of membrane resistance and capacitance: τ m = Rm ⋅Cm

(7-9)

We introduced this expression earlier as Equation 6-17. The shorter the time constant, the more quickly a neighboring region of membrane will be brought to threshold and the sooner the region will fire an action potential. Thus, the shorter the time constant, the faster the speed of impulse propagation, and vice versa. In contrast, conduction velocity is directly proportional to the length constant. The greater the length constant, the farther a signal can spread before decaying below threshold and the greater the area of membrane that the stimulus can excite. These relationships explain why, in terms of relative conduction velocity, a highresistance, low-capacitance myelinated axon has a distinct advantage over an unmyelinated axon of the same diameter for all but the smallest axons ( > > a)

Phospholamban

Present

Absent

Absent

Present

Present

Calsequestrin

“Fast” and “cardiac”

“Fast”

“Fast”

“Cardiac”

? “Cardiac” ? “Fast”

Ca2+ release mechanisms

RYR1 (Ca2+-release channel or “ryanodine” receptor)

RYR1

RYR1

RYR2

IP3R (3 isoforms) RYR3

Ca2+ sensor

Troponin C1 (TNNC1)

Troponin C2 (TNNC2)

Troponin C2 (TNNC2)

Troponin C1 (TNNC1)

Calmodulin (multiple isoforms)

DIVERSITY AMONG MUSCLES As we have seen, each muscle type (skeletal, cardiac, and smooth) is distinguishable on the basis of its unique histology, EC coupling mechanisms, and regulation of contractile function. However, even within each of the three categories, muscle in different locations must serve markedly different purposes, with different demands for strength, speed, and fatigability. This diversity is possible because of differences in the expression of specific isoforms for various contractile and regulatory proteins (Table 9-1). Skeletal muscle is composed of slow-twitch and fast-twitch fibers Some skeletal muscles must be resistant to fatigue and be able to maintain tension for relatively long periods, although they need not contract rapidly. Examples are muscles that maintain body posture, such as the soleus muscle of the lower part of the leg. In contrast, some muscles need to contract rapidly, yet infrequently. Examples are the extraocular muscles, which must contract rapidly to redirect the eye as an object of visual interest moves about. Individual muscle fibers are classified as slow twitch (type I) or fast twitch (type II), depending on their rate of force development. These fiber types are also distinguished by their histologic appearance and their ability to resist fatigue. Slow-twitch fibers (Table 9-2) are generally thinner and have a denser capillary network surrounding them. These type I fibers also appear red because of a large amount of the oxygen-binding protein myoglobin (see Chapter 29) within the cytoplasm. This rich capillary network together with myoglobin facilitates oxygen transport to the slowtwitch fibers, which mostly rely on oxidative metabolism for

energy. The metabolic machinery of the slow-twitch fiber also favors oxidative metabolism because it has low glycogen content and glycolytic enzyme activity but a rich mitochondrial and oxidative enzyme content. Oxidative metabolism is slow but efficient, making these fibers resistant to fatigue. Fast-twitch fibers differ among themselves with respect to fatigability. Some fast-twitch fibers are fatigue resistant; they rely on oxidative metabolism (type IIa) and are quite similar to slow-twitch fibers with respect to myoglobin content (indeed, they are red) and metabolic machinery. One important difference is that fast-twitch oxidative fibers contain abundant glycogen and have a greater number of mitochondria than slow-twitch fibers do. These features ensure adequate ATP generation to compensate for the increased rate of ATP hydrolysis in fast-twitch fibers. Other fast-twitch fibers are not capable of sufficient oxidative metabolism to sustain contraction. Because these fibers must rely on the energy that is stored within glycogen (and phosphocreatine), they are more easily fatigable. Fatigable fast-twitch fibers (type IIb) have fewer mitochondria and lower concentrations of myoglobin and oxidative enzymes. Because of their low myoglobin content, type IIb muscle fibers are white. They are, however, richer in glycolytic enzyme activity than other fiber types are. In reality, slow- and fast-twitch fibers represent the extremes of a continuum of muscle fiber characteristics. Moreover, each whole muscle is composed of fibers of each twitch type, although one of the fiber types predominates in any given muscle. The differences between fiber types derive in large part from differences in isoform expression of the various contractile and regulatory proteins (Table 9-1). Differences in the rate of contraction, for example, may be directly correlated with the maximal rate of myosin ATPase activity. The human genome database lists at least 15 MHC genes, with their respective splice variants. Individual isoform expression varies among muscle types and is devel-

Chapter 9 • Cellular Physiology of Skeletal, Cardiac, and Smooth Muscle

TABLE 9-2

Properties of Fast- and Slow-Twitch Muscle Fibers Slow Twitch

Fast Twitch

Fast Twitch

Synonym

Type I

Type IIa

Type IIb

Fatigue

Resistant

Resistant

Fatigable

Color

Red (myoglobin)

Red (myoglobin)

White (low myoglobin)

Metabolism

Oxidative

Oxidative

Glycolytic

Mitochondria

High

Higher

Fewer

Glycogen

Low

Abundant

High

opmentally regulated. At least four isoforms of the MHC protein are expressed in skeletal muscle (MHC-I, MHC-IIa, MHC-IIb, MHC-IIx/d). For the most part, a muscle fiber type expresses a single MHC isoform, the ATPase activity of which appears to correspond to the rate of contraction in that fiber type. Whereas most fibers express one of these isoforms, some fibers express a combination of two different isoforms. These hybrid cells have rates of contraction that are intermediate between the two pure fiber types. Differences in the rates and strength of contraction may also result from differences in myosin light chain isoform expression or from isoform differences among other components of the EC coupling process. Three skeletal muscle isoforms have been identified. MLC-1as and MLC-1bs are expressed in slow-twitch fibers, whereas MLC-1f and MLC3f are expressed in fast-twitch fibers. Isoform differences also exist for the SR Ca2+ pump (i.e., the SERCA), calsequestrin, the Ca2+-release channel, and troponin C. Furthermore, some proteins, such as phospholamban, are expressed in one fiber type (slow twitch) and not the other. One particularly interesting feature of muscle differentiation is that fiber-type determination is not static. Through exercise training or changes in patterns of neuronal stimulation, alterations in contractile and regulatory protein isoform expression may occur. For example, it is possible for a greater proportion of fast-twitch fibers to develop in a specific muscle with repetitive training. It is even possible to induce cardiac-specific isoforms in skeletal muscle, given appropriate stimulation patterns. The properties of cardiac cells vary with location in the heart Just as skeletal muscle consists of multiple fiber types, so too does heart muscle. The electrophysiological and mechanical properties of cardiac muscle vary with their location (i.e., atria versus conducting system versus ventricle). Moreover, even among cells within one anatomical location, functional differences may exist between muscle cells near the surface of the heart (epicardial cells) and those lining the interior of the same chambers (endocardial cells). As in skeletal muscle, many of these differences reflect differences in isoform

expression of the various contractile and regulatory proteins. Although some of the protein isoforms expressed in cardiac tissue are identical to those expressed in skeletal muscle, many of the proteins have cardiac-specific isoforms (Table 9-1). The MHC in heart, for example, exists in two isoforms, α and β, which may be expressed alone or in combination. Smooth muscle cells may differ markedly among tissues and may adapt their properties with time even in a single tissue When one considers that smooth muscle has a broad range of functions, including regulating the diameter of blood vessels, propelling food through the gastrointestinal tract, regulating the diameter of airways, and delivering a newborn infant from the uterus, it is not surprising that smooth muscle is a particularly diverse type of muscle. In addition to being distinguished as unitary or multiunit muscle, smooth muscle in different organs diverges with respect to nerve and hormonal control, electrical activity, and characteristics of contraction. Even among smooth muscle cells within the same sort of tissue, important functional differences may exist. For example, vascular smooth muscle cells within the walls of two arterioles that perfuse different organs may vary in their contractile response to various stimuli. Differences may even exist between vascular smooth muscle cells at two different points along one arterial pathway. The phenotype of smooth muscle within a given organ may change with shifting demands. The uterus, for example, is composed of smooth muscle—the myometrium—that undergoes remarkable transformation during gestation as it prepares for parturition (see Chapter 56). In addition to hypertrophy, greater coupling develops between smooth muscle cells through the increased formation of gap junctions. The cells also undergo changes in their expression of contractile protein isoforms. Changes in the expression of ion channels and hormone receptors facilitate rhythmic electrical activity. This activity is coordinated across the myometrium by propagation of action potentials and increases in [Ca2+]i through the gap junctions. These rhythmic, coordinated contractions develop spontaneously, but they are strongly influenced by the hormone oxytocin, levels of which

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increase just before and during labor and just after parturition. These differences in smooth muscle function among various tissues or even over the lifetime of a single cell probably reflect differences in protein composition. Indeed, in comparison to striated muscle, smooth muscle cells express a wider variety of isoforms of contractile and regulatory proteins (Table 9-1). This variety is a result of both multiple genes and alternative splicing (see Chapter 4). This richness in diversity is likely to have important consequences for smooth muscle cell function, although the precise relationship between the structure and function of these protein isoforms is not yet clear. Smooth muscle cells express a wide variety of neurotransmitter and hormone receptors Perhaps one of the most impressive sources of diversity among smooth muscle cells relates to differences in response to neurotransmitters, environmental factors, and circulating hormones. Smooth muscle cells differ widely with respect to the types of cell surface receptors that mediate the effects of these various mediators. In general, smooth muscle cells

TABLE 9-3

each express a variety of such receptors, and receptor stimulation may lead to either contraction or relaxation. Many substances act through different receptor subtypes in different cells, and these receptor subtypes may act through different mechanisms. For example, whereas some neurotransmitter/hormone receptors may be ligand-gated ion channels, others act through heterotrimeric G proteins that either act directly on targets or act through intracellular second messengers such as cAMP, cGMP, or IP3 and DAG. The list of neurotransmitters, hormones, and environmental factors regulating the function of vascular smooth muscle cells alone is vast (see Chapter 23). A few of these vasoactive substances include epinephrine, norepinephrine, serotonin, angiotensin, vasopressin, neuropeptide Y, nitric oxide, endothelin, and oxygen. Identical stimuli, however, may result in remarkably different physiological responses by smooth muscle in different locations. For example, systemic arterial smooth muscle cells relax when the oxygen concentration around them decreases, whereas pulmonary arterial smooth muscle contracts when local oxygen decreases (see Chapter 31). A summary comparison between muscle types is presented in Table 9-3.

Summary of Comparisons Between Muscle Types Skeletal

Cardiac

Smooth

Mechanism of excitation

Neuromuscular transmission

Pacemaker potentials Electrotonic depolarization through gap junctions

Synaptic transmission Hormone-activated receptors Electrical coupling Pacemaker potentials

Electrical activity of muscle cell

Action potential spikes

Action potential plateaus

Action potential spikes, plateaus Graded membrane potential changes Slow waves

Ca2+ sensor

Troponin

Troponin

Calmodulin

Excitation-contraction coupling

L-type Ca2+ channel (DHP receptor) in T-tubule membrane coupling to Ca2+-release channel (ryanodine receptor) in SR

Ca2+ entry through L-type Ca2+ channel (DHP receptor) triggers Ca2+-induced Ca2+ release from SR

Ca2+ entry through voltage-gated Ca2+ channels Ca2+- and IP3-mediated Ca2+ release from SR Ca2+ entry through storeoperated Ca2+ channels

Terminates contraction

Breakdown of ACh by acetylcholinesterase

Action potential repolarization

Myosin light chain phosphatase

Twitch duration

20-200 ms

200-400 ms

200 ms—sustained

Regulation of force

Frequency and multifiber summation

Regulation of calcium entry

Balance between MLCK phosphorylation and dephosphorylation Latch state

Metabolism

Oxidative, glycolytic

Oxidative

Oxidative

Chapter 9 • Cellular Physiology of Skeletal, Cardiac, and Smooth Muscle

REFERENCES Books and Reviews Farah CS, Reinach FC: The troponin complex and regulation of muscle contraction. FASEB J 1995; 9:755-767. Franzini-Armstrong C, Protasi F: Ryanodine receptors of striated muscles: A complex channel capable of multiple interactions. Physiol Rev 1997; 77:699-729. Holda J, Klishin A, Sedova M, et al: Capacitative calcium entry. News Physiol Sci 1998; 13:157-163. Horowitz A, Menice CB, Laporte R, Morgan KG: Mechanisms of smooth muscle contraction. Physiol Rev 1996; 76:967-1003. Parekh AB, Penner R: Store depletion and calcium influx. Physiol Rev 1997; 77:901-930. Striggow F, Ehrlich BE: Ligand-gated calcium channels inside and out. Curr Opin Cell Biol 1996; 8:490-495.

Journal Articles Cannell MB, Cheng H, Lederer WJ: The control of calcium release in heart muscle. Science 1995; 268:1045-1049. Finer JT, Simmons RM, Spudich JA: Single myosin molecule mechanics: Piconewton forces and nanometre steps. Nature 1994; 368:113-119. Gordon AM, Huxley AF, Julian FJ: The variation in isometric tension with sarcomere length in vertebrate muscle. J Physiol 1966; 184:170-192. Mickelson JR, Louis CF: Malignant hyperthermia: Excitationcontraction coupling, Ca2+-release channel, and cell Ca2+ regulation defects. Physiol Rev 1996; 76:537-592.

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SECTION

III

T H E N E R VO U S SYST E M Chapter 10

• Organization of the Nervous System ...... 267

Chapter 11 • The Neuronal Microenvironment ...... 289 Chapter 12

• Physiology of Neurons ...... 310

Chapter 13 • Synaptic Transmission in the Nervous System ...... 323 Chapter 14 • The Autonomic Nervous System ...... 351 Chapter 15

• Sensory Transduction ...... 371

Chapter 16 • Circuits of the Central Nervous System ...... 408

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CHAPTER

10

O R G A N I Z AT I O N O F T H E N E R V O U S S Y S T E M Bruce R. Ransom

The human brain is the most complex tissue in the body. It mediates behavior ranging from simple movements and sensory perception to learning and memory. It is the organ of the mind. Many of the brain’s functions are poorly understood. In fact, the most prominent function of the human brain, its capacity to think, is hardly understood at all. Our lack of knowledge about fundamental aspects of brain function stands in marked contrast to the level of comprehension that we have about the primary functions of other organ systems, such as the heart, lungs, and kidneys. Nevertheless, tremendous strides have been made in the past few decades. While philosophers ponder the paradox of a person thinking about thinking, physiologists are trying to learn about learning. In this part of the book, we present the physiology of the nervous system in a manner that is intended to be complementary to texts on neurobiology and neuroanatomy. In this chapter, we review the basic cellular, developmental, and gross anatomy of the nervous system. In Chapter 11, we discuss the fluid environment of the neurons in the brain, how this environment interacts with the rest of the extracellular fluid of the body, and the role of glial cells. Chapters 12 and 13 focus on the broad physiological principles that underlie how the brain’s cellular elements operate. Another major goal of this section is to provide more detailed information on those parts of the nervous system that play key roles in the physiology of other systems in the body. Thus, in Chapter 14, we discuss the autonomic nervous system, which controls “viscera” such as the heart, lungs, and gastrointestinal tract. Finally, in Chapters 15 and 16, we discuss the special senses and simple neuronal circuits. The nervous system can be divided into central, peripheral, and autonomic nervous systems The manner in which the nervous system is subdivided is somewhat arbitrary. All elements of the nervous system work closely together in a way that has no clear boundaries. Nevertheless, the traditional definitions of the subdivisions provide a useful framework for talking about the brain and its connections and are important if only for that reason. The central nervous system (CNS) consists of the brain and spinal cord (Table 10-1). It is covered by three “mem-

branes”—the meninges. The outer membrane is the dura mater; the middle is the arachnoid; and the delicate inner membrane is called the pia mater. Within the CNS, some neurons that share similar functions are grouped into aggregations called nuclei. The peripheral nervous system (PNS) consists of those parts of the nervous system that lie outside the dura mater (Table 10-1). These elements include sensory receptors for various kinds of stimuli, the peripheral portions of spinal and cranial nerves, and all the peripheral portions of the autonomic nervous system (see the next paragraph). The sensory nerves that carry messages from the periphery to the CNS are termed afferent nerves (Latin, ad + ferens, or carrying toward). Conversely, the peripheral motor nerves that carry messages from the CNS to peripheral tissues are called efferent nerves (Latin, ex + ferens, or carrying away). Peripheral ganglia are groups of nerve cells concentrated into small knots or clumps that are located outside the CNS. The autonomic nervous system (ANS) is that portion of the nervous system that regulates and controls visceral functions, including heart rate, blood pressure, digestion, temperature regulation, and reproductive function. Although the ANS is a functionally distinct system, it is anatomically composed of parts of the CNS and PNS (Table 10-1). Visceral control is achieved by reflex arcs that consist of visceral afferent (i.e., sensory) neurons that send messages from the periphery to the CNS, control centers in the CNS that receive this input, and visceral motor output. Moreover, visceral afferent fibers typically travel together with visceral efferent fibers. Each area of the nervous system has unique nerve cells and a different function Nervous tissue is composed of neurons and neuroglial cells. Neurons vary greatly in their structure throughout the nervous system, but they all share certain features that tailor them for the unique purpose of electrical communication (see Chapter 12). Neuroglial cells, often simply called glia, are not primary signaling cells and have variable structures that are suited for their diverse functions (see Chapter 11). The human brain contains ∼1011 neurons and several times as many glial cells. Each of these neurons may interact

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TABLE 10-1

Subdivisions of the Nervous System

Subdivision

Components

Special Features

Central

Brain (including CN II and retina) and spinal cord

Oligodendrocytes provide myelin Axons cannot regenerate

Peripheral

Peripheral ganglia (including cell bodies); sensory receptors; peripheral portions of spinal and cranial nerves (except CN II), both afferent and efferent

Schwann cells provide myelin Axons can regenerate

Autonomic

Selected portions of the CNS and PNS

Functionally distinct system

CN, cranial nerve.

with thousands of other neurons, which helps explain the awesome complexity of the nervous system. No evidence suggests that the human brain contains receptors, ion channels, or cells that are unique to humans and not seen in other mammals. The unparalleled capabilities of the human brain are presumed to result from its unique patterns of connectivity and its large size. The brain’s diverse functions are the result of tremendous regional specialization. Different brain areas are composed of neurons that have special shapes, physiological properties, and connections. One part of the brain, therefore, cannot substitute functionally for another part that has failed. Any compensation of neural function by a patient with a brain lesion (e.g., a stroke) reflects enhancement of existing circuits or recruitment of latent circuits. A corollary is that damage to a specific part of the brain causes predictable symptoms that can enable a clinician to establish the anatomical location of the problem, a key step in diagnosis of neurological diseases.

CELLS OF THE NERVOUS SYSTEM The neuron doctrine first asserted that the nervous system is composed of many individual signaling units—the neurons In 1838, Schleiden and Schwann proposed that the nucleated cell is the fundamental unit of structure and function in both plants and animals. They reached this conclusion by microscopic observation of plant and animal tissues that had been stained to reveal their cellular composition. However, the brain proved to be more difficult to stain than other tissues, and until 1885, when Camillo Golgi introduced his silver impregnation method, “the black reaction,” there was no clear indication that the brain is composed of individual cells. The histologist Santiago Ramón y Cajal worked relent-

lessly with the silver-staining method and eventually concluded that not only is nervous tissue composed of individual cells but the anatomy of these cells also confers a functional polarization to the passage of nervous signals; the tapering branches near the cell body are the receptive end of the cell, and the long-axis cylinder conveys signals away from the cell. In the absence of any reliable physiological evidence, Cajal was nevertheless able to correctly anticipate how complex cell aggregates in the brain communicate with each other. The pathologist Heinrich von Waldeyer referred to the individual cells in the brain as neurons. He wrote a monograph in 1891 that assembled the evidence in favor of the cellular composition of nervous tissue, a theory that became known as the neuron doctrine. It is ironic that Golgi, whose staining technique made these advances possible, never accepted the neuron doctrine, and he argued vehemently against it when he received his Nobel Prize along with Cajal in 1906. The ultimate proof of the neuron doctrine was established by electron microscopic observations that definitively demonstrated that neurons are entirely separate from one another, even though their processes come into very close contact. Nerve cells have four specialized regions: cell body, dendrites, axon, and presynaptic terminals Neurons are specialized for sending and receiving signals, a purpose reflected in their unique shapes and physiological adaptations. The structure of a typical neuron can generally be divided into four distinct domains: (1) the cell body, also called the soma or perikaryon; (2) the dendrites; (3) the axon; and (4) the presynaptic terminals (Fig. 10-1). Cell Body As the name perikaryon implies, the cell body is the portion of the cell surrounding the nucleus. It contains much of the cell’s complement of endoplasmic reticular membranes as well as the Golgi complex. The cell body appears to be responsible for many of the neuronal housekeeping functions, including the synthesis and processing of proteins. Dendrites

Dendrites are tapering processes of variable complexity that arise from the cell body. The dendrites and cell body are the main areas for receiving information. Thus, their membranes are endowed with receptors that bind and respond to the neurotransmitters released by neighboring cells. The chemical message is translated by membrane receptors into an electrical or a biochemical event that influences the state of excitability of the receiving neuron. The cytoplasm of the dendrites contains dense networks of microtubules as well as extensions of the endoplasmic reticulum.

Axon

Perhaps the most remarkable feature of the neuron, the axon is a projection that arises from the cell body, like the dendrites. Its point of origin is a tapered region known as the axon hillock. Just distal to the cone-shaped hillock is an untapered, unmyelinated region known as the initial segment. This area is also called the spike initiation zone because it is where an action potential (see Chapter 7) nor-

Chapter 10 • Organization of the Nervous System

Dendrites

Presynaptic terminal

Cell soma

Synaptic cleft Dendrite

Dendrites

Axon hillock

Myelin sheath Node of Ranvier

Axon

Internode

Dendrites

Presynaptic terminal

Figure 10-1

Structure of a typical neuron.

mally arises as the result of the electrical events that have occurred in the cell body and dendrites. In contrast to the dendrites, the axon is thin, does not taper, and can extend for more than a meter. Because of its length, the typical axon contains much more cytoplasm than does the cell body, up to 1000 times as much. The neuron uses special metabolic mechanisms to sustain this unique structural component. The cytoplasm of the axon, the axoplasm, is packed with parallel arrays of microtubules and microfilaments that provide structural stability and a means to rapidly convey materials back and forth between the cell body and the axon terminus. Axons are the message-sending portion of the neuron. The axon carries the neuron’s signal, the action potential, to a specific target, such as another neuron or a muscle. Some axons have a special electrical insulation, called myelin, that consists of the coiled cell membranes of glial cells that wrap themselves around the nerve axon (see Chapter 11). If the axon is not covered with myelin, the action potential travels down the axon by continuous propagation. On the other hand, if the axon is myelinated, the action potential jumps from one node of Ranvier (the space between adjacent myelin segments) to another in a process called saltatory conduction (see Chapter 7). This adaptation greatly speeds impulse conduction. Presynaptic Terminals At its target, the axon terminates in multiple endings—the presynaptic terminals—usually designed for rapid conversion of the neuron’s electrical signal into a chemical signal. When the action potential reaches the presynaptic terminal, it causes the release of chemical signaling molecules in a complex process called synaptic transmission (see Chapters 8 and 13). The junction formed between the presynaptic terminal and its target is called a chemical synapse. Synapse is derived from the Greek for “joining together” or “junction”; this word and concept were introduced in 1897 by the neurophysiologist Charles Sherrington, whose contributions led to a share of the 1932 Nobel Prize in Medicine or Physiology. A synapse comprises the presynaptic terminal, the membrane of the target cell (postsynaptic membrane), and the space between the two (synaptic cleft). In synapses between two neurons, the presynaptic terminals primarily contact dendrites and the cell body. The area of the postsynaptic membrane is frequently amplified to increase the surface that is available for receptors. This amplification can occur either through infolding of the plasma membrane or through the formation of small projections called dendritic spines. The molecules released by the presynaptic terminals diffuse across the synaptic cleft and bind to receptors on the postsynaptic membrane. The receptors then convert the chemical signal of the transmitter molecules—either directly or indirectly—back into an electrical signal. In many ways, neurons can be thought of as highly specialized endocrine cells. They package and store hormones and hormone-like molecules, which they release rapidly into the extracellular space by exocytosis (see Chapter 2) in response to an external stimulus, in this case a nerve action potential. However, instead of entering the bloodstream to exert systemic effects, the substances secreted by neurons act

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over the very short distance of a synapse to communicate locally with a single neighboring cell (see Chapter 5). In a different sense, neurons can be thought of as polarized cells with some of the properties of epithelial cells. Like epithelial cells, neurons have different populations of membrane proteins at each of the distinct domains of the neuronal plasma membrane, an arrangement that reflects the individual physiological responsibilities of these domains. Thus, the design of the nervous system permits information transfer across synapses in a selective and coordinated way that serves the needs of the organism and summates to produce complex behavior. The cytoskeleton helps compartmentalize the neuron and also provides the tracks along which material travels between different parts of the neuron Neurons are compartmentalized in both structure and function. Dendrites are tapered, have limited length, and contain neurotransmitter receptor proteins in their membranes. Axons can be very long and have a high density of Na+ channels. Dendrites and the cell body contain mRNA, ribosomes, and a Golgi apparatus. These structures are absent in axons. How does this compartmentalization come about? The answer is not certain, but microtubule-associated proteins (MAPs) appear to play an important role. (Note that these MAPs are totally unrelated to the mitogen-activated protein [MAP] kinase introduced in Chapter 4.) Two major classes of MAPs are found in the brain: high-molecular-weight proteins such as MAP-1 and MAP-2 and lower molecular weight tau proteins. Both classes of MAPs associate with microtubules and help link them to other cell components. MAP-2 is found only in cell bodies and dendrites. Dephosphorylated tau proteins are confined entirely to axons. In cultured neurons, suppressing the expression of tau protein prevents formation of the axon without altering formation of the dendrites. Microtubules may also help create the remarkable morphological and functional divisions in neurons. In axons, microtubules assemble with their plus ends pointed away from the cell body; this orientation polarizes the flow of

TABLE 10-2

material into and out of the axon. The cytoskeletal “order” provided in part by the microtubules and the MAPs helps define what should or should not be in the axonal cytoplasm. In dendrites, the microtubules do not have a consistent orientation, which gives the dendrites a greater structural and functional similarity to the cell body. The neuron cell body is the main manufacturing site for the membrane proteins and membranous organelles that are necessary for the structural integrity and function of its processes. Axons have no protein synthetic ability, whereas dendrites have some free ribosomes and may be able to engage in limited protein production. The transport of proteins from the cell body all the way to the end of long axons is a challenging task. The neuron also has a second task: moving various material in the opposite direction, from presynaptic terminals at the end of the axon to the cell body. The neuron solves these problems by using two distinct mechanisms for moving material to the presynaptic terminals in an “anterograde” direction and a third mechanism for transport in the opposite or “retrograde” direction (Table 10-2). Fast Axoplasmic Transport

If the flow of materials from the soma to the distant axon terminus were left to the whims of simple diffusion, their delivery would be far too slow to be of practical use. It could take months for needed proteins to diffuse to the end of an axon, and the presynaptic terminals are high-volume consumers of these molecules. To overcome this difficulty, neurons exploit a rapid, pony express–style system of conveyance known as fast axoplasmic transport (Table 10-2). Membranous organelles, including vesicles and mitochondria, are the principal freight of fast axoplasmic transport. The proteins, lipids, and polysaccharides that move at fast rates in axons do so because they have caught a ride with a membranous organelle (i.e., sequestered inside the organelle, or bound to or inserted into the organellar membrane). The peptide and protein contents of dense-core secretory granules, which are found in the presynaptic axonal terminals, are synthesized as standard secretory proteins (see Chapter 2). Thus, they are cotranslationally inserted across the membranes of the rough endoplasmic reticulum and subsequently processed in the cisternae of the Golgi complex. They are shipped to the axon in the lumens of Golgi-derived carrier vesicles (Table 10-2).

Features of Axoplasmic Transport

Transport Type

Speed (mm/day)

Mechanism

Material Transported

Fast anterograde

∼400

Saltatory movement along microtubules by the motor molecule kinesin (ATP dependent)

Mitochondria Vesicles containing peptide and other neurotransmitters, some degradative enzymes

Fast retrograde

∼200–300

Saltatory movement along microtubules by the motor molecule dynein (ATP dependent)

Degraded vesicular membrane Absorbed exogenous material (toxins, viruses, growth factors)

Slow anterograde

∼0.2–8

Not clear; possibly by molecular motors

Cytoskeletal elements (e.g., neurofilament and microtubule subunits) Soluble proteins of intermediary metabolism Actin

Chapter 10 • Organization of the Nervous System

Organelles and vesicles, and their macromolecule payloads, move along microtubules with the help of a microtubule-dependent motor protein called kinesin (Fig. 10-2A). The kinesin motor is itself an ATPase that produces vectorial movement of its payload along the microtubule (see Chapter 2). This system can move vesicles down the axon at rates of up to 400 mm/day; variations in cargo speed simply reflect more frequent pauses during the journey. Kinesins always move toward the plus end of microtubules (i.e., away from the cell body), and transport function is lost if the microtubules are disrupted. The nervous system has many forms of kinesin that recognize and transport different cargo. It is not known how the motor proteins recognize and attach to their intended payloads.

these cells has a clearly defined axon that arises from the axon hillock located on the cell body or proximal dendrite and extends away from the cell body, sometimes for remarkable distances. Some neurons in the cortex, for example, project to the distal part of the spinal cord, a stretch of nearly a meter. All the other processes that a projection neuron has are dendrites. The other type of neuron that is defined in this way has all of its processes confined to one region of the brain. These neurons are called interneurons (or intrinsic neurons or Golgi type II cells). Some of these cells have very short axons, whereas others seem to lack a conventional axon altogether and may be referred to as anaxonal. The anaxonal neuron in the retina is called an amacrine (from the Greek for “no large/long fiber”) cell.

Fast Retrograde Transport

Dendritic Geometry A roughly pyramid-shaped set of dendritic branches characterizes pyramidal cells, whereas a radial pattern of dendritic branches defines stellate cells. This classification often includes mention of the presence or absence of dendritic spines, those small, protuberant projections that are sites for synaptic contact. All pyramidal cells appear to have spines, but stellate cells may have them (spiny) or not (aspiny).

Axons move material back toward the cell body with a different motor protein called dynein (Fig. 10-2B). Like kinesin, dynein (see Chapter 2) also moves along microtubule tracks and is an ATPase (Table 10-2). However, dynein moves along microtubules in the opposite direction of kinesin (Fig. 10-2C). Retrograde transport provides a mechanism for target-derived growth factors, like nerve growth factor, to reach the nucleus of a neuron where it can influence survival. How this signal is transmitted up the axon has been a persistent question. It may be endocytosed at the axon’s terminal and transported to the cell body in a “signaling endosome.” The loss of ATP production, as occurs with blockade of oxidative metabolism, causes fast axonal transport in both the anterograde and retrograde directions to fail.

Slow Axoplasmic Transport

Axons also have a need for hundreds of other proteins, including cytoskeletal proteins and soluble proteins that are used as enzymes for intermediary metabolism. These proteins are delivered by a slow anterograde axoplasmic transport mechanism that moves material at a mere 0.2 to 8 mm/day, the nervous system’s equivalent of snail mail. The slowest moving proteins are neurofilament and microtubule subunits (0.2 to 1 mm/day). The mechanism of slow axoplasmic transport is not well understood, but motor molecules appear to be involved. In fact, the difference between slow and fast axonal transport may primarily be the number of transport interruptions during the long axonal journey.

Neurons can be classified on the basis of their axonal projection, their dendritic geometry, and the number of processes emanating from the cell body The trillions of nerve cells in the CNS have great structural diversity. Typically, neurons are classified on the basis of where their axons go (i.e., where they “project”), the geometry of their dendrites, and the number of processes that emanate from the cell body (Fig. 10-3). The real significance of these schemes is that they have functional implications. Axonal Projection Neurons with long axons that connect with other parts of the nervous system are called projection neurons (or principal neurons or Golgi type I cells). Each of

Number of Processes Neurons can also be classified by the number of processes that extend from their cell bodies. The dorsal root ganglion cell is the classic unipolar neuron. The naming of the processes of primary sensory neurons, like the dorsal root ganglion cell, is often ambiguous. The process that extends into the CNS from this unipolar neuron is easily recognized as an axon because it carries information away from the cell body. On the other hand, the process that extends to sensory receptors in the skin and elsewhere is less easily defined. It is a typical axon in the sense that it can conduct an action potential, has myelin, and is characterized by an axonal cytoskeleton. However, it conveys information toward the cell body, which is usually the function of a dendrite. Bipolar neurons, such as the retinal bipolar cell, have two processes extending from opposite sides of the cell body. Most neurons in the brain are multipolar. Cells with many dendritic processes are designed to receive large numbers of synapses. Most neurons in the brain can be categorized by two or more of these schemes. For example, the large neurons in the cortical area devoted to movement (i.e., the motor cortex) are multipolar, pyramidal, projection neurons. Similarly, a retinal bipolar cell is both an interneuron and a bipolar cell.

Glial cells provide a physiological environment for neurons Glial cells are defined in part by what they lack: axons, action potentials, and synaptic potentials. They are much more numerous than neurons and are diverse in structure and function. The main types of CNS glial cells are oligodendrocytes, astrocytes, and microglial cells. In the PNS, the main types of glial cells are satellite cells in autonomic and sensory ganglia, Schwann cells, and enteric glial cells. Glial function is discussed in Chapter 11. Oligodendrocytes form the myelin sheaths of CNS axons, and Schwann cells myelinate periph-

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A

ANTEROGRADE MOVEMENT The vesicles and mitochondria are carried down the axon on microtubule “tracks” by kinesin motors that are energized by ATP.

Proteins synthesized in the “secretory pathway” are packaged by budding off in membraneenclosed vesicles from the Golgi.

Postsynaptic neuron Golgi

ER

Vesicles

Mitochondria

Axon

Myelin sheath

Nucleus Lysosomes

Soma B

Synaptic terminal

RETROGRADE MOVEMENT

Microtubules

Vesicles now move in reverse, carried by dynein motors, which also split ATP and move along microtubule “tracks.”

Dendrites

Synaptic terminal

Vesicles

Myelin sheath

Microtubule Postsynaptic neuron

C

Kinesin

MICROTUBULE Dynein

Retrograde movement

Light chains Heavy chains

–

end

Figure 10-2

Anterograde movement

+

end

Fast axoplasmic transport. ER, endoplasmic reticulum.

eral nerves. Glial cells are involved in nearly every function of the brain and are far more than simply “nerve glue,” a literal translation of the name neuroglia (from the Greek neuron, nerve, and glia, glue). In depictions of the nervous system, the presence of glial cells is sometimes minimized or neglected altogether. Glia

fills in almost all the space around neurons, with a narrow extracellular space left between neurons and glial cells that has an average width of only ∼0.02 μm. The composition of the extracellular fluid, which has a major impact on brain function, as well as the function of glial cells is taken up in detail in Chapter 11.

Chapter 10 • Organization of the Nervous System

Basis for classification

Functional implication

Example

Structure

1. Axonal projection Goes to a distant brain area

Projection neuron or Principal neuron or Golgi type I cell (cortical motor neuron)

Affects different brain areas

Dorsal root ganglion cell

Stays in a local brain area

Intrinsic neuron or Interneuron or Golgi type II cell (cortical inhibitory neuron)

Affects only nearby neurons

Retinal bipolar cell

Pyramidal cell

2. Dendritic pattern Pyramid-shaped spread of dendrites

Pyramidal cell (hippocampal pyramidal neuron)

Large area for receiving synaptic input; determines the pattern of incoming axons that can interact with the cell (i.e., pyramid-shaped)

Radial-shaped spread of dendrites

Stellate cell (cortical stellate cell)

Large area for receiving synaptic input; determines pattern of incoming axons that can interact with the cell (i.e., star-shaped)

Stellate cell

One process exits the cell body

Unipolar neuron (dorsal root ganglion cell)

Small area for receiving synaptic input: highly specialized function

Unipolar

Two processes exit the cell body

Bipolar neuron (retinal bipolar cell)

Small area for receiving synaptic input: highly specialized function

Bipolar

Many processes exit the cell body

Multipolar neuron (spinal motor neuron)

Large area for receiving synaptic input; determines the pattern of incoming axons that can interact with the cell

Multipolar

Spine

3. Number of processes

Figure 10-3

Classification of neurons based on their structure.

Soma

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Definitions of Neural Modalities

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he type of information, or neural modality, that a neuron transmits is classically categorized by three terms that refer to different attributes of the neuron.

1. The first category defines the direction of information flow. Afferent (sensory): neurons that transmit information into the CNS from sensory cells or sensory receptors outside the nervous system. Examples are the dorsal root ganglion cell and neurons in the sensory nucleus of the fifth cranial nerve. Efferent (motor): neurons that transmit information out of the CNS to muscles or secretory cells. Examples are spinal motor neurons and motor neurons in the ANS. 2. The second category defines the anatomical distribution of the information flow. Visceral: neurons that transmit information to or from internal organs or regions that arise embryologically from the branchial arch (e.g., chemoreceptors of the carotid body). Somatic: neurons that transmit information to or from all nonvisceral parts of the body, including skin and muscle. 3. The third category, which is somewhat arbitrary, defines the information flow on the basis of the embryological origin of the structure being innervated. Special: neurons that transmit information to or from a “special” subset of visceral or somatic structures. For example, in the case of special visceral neurons, information travels to or from structures derived from the branchial arch region of the embryo (e.g., pharyngeal muscles). In the case of special somatic neurons, which handle only sensory information, the neurons arise from the organs of special sense (e.g., retina, taste receptors, cochlea). General: neurons that transmit information to or from visceral or somatic structures that are not in the special group. Each axon in the body conveys information of only a single modality. In this classification scheme, a motor neuron in the spinal cord is described as a general somatic efferent neuron. A motor neuron in the brain stem that innervates branchial arch–derived chewing muscles is described as a special visceral efferent neuron. Because each of these three categories defines two options, you might expect a total of eight distinct neural modalities. In practice, however, only seven neural modalities exist. The term special somatic efferent neuron is not used.

DEVELOPMENT OF NEURONS AND GLIAL CELLS Neurons differentiate from the neuroectoderm Although the embryology of the nervous system may seem like an odd place to begin studying the physiology of the

brain, there are a number of reasons to start here. Knowledge of the embryology of the nervous system greatly facilitates comprehension of its complex organization. Events in the development of the nervous system highlight how different neuronal cell types evolve from a single type of precursor cell and how these neurons establish astonishingly specific connections. Finally, the characteristics of brain cell proliferation as well as the growth of neuronal processes during development provide insight into the consequences of brain injury. The vertebrate embryo consists of three primitive tissue layers at the stage of gastrulation: endoderm, mesoderm, and ectoderm (Fig. 10-4). The entire nervous system arises from ectoderm, which also gives rise to the skin. Underlying the ectoderm is a specialized cord of mesodermal cells called the notochord. Cells of the notochord somehow direct or “induce” the overlying ectoderm, or neuroectoderm, to form the neural tube in a complex process called neurulation. The first step in neurulation is formation of the neural plate at about the beginning of the third fetal week. Initially, the neural plate is only a single layer of neuroectoderm cells. Rapid proliferation of these cells, especially at the lateral margins, creates a neural groove bordered by neural folds. Continued cell division enlarges the neural folds, and they eventually fuse dorsally to form the neural tube. The neural tube is open at both ends, the anterior and posterior neuropores. The neural tube ultimately gives rise to the brain and spinal cord. The lumen of the neural tube, the neural canal, becomes the four ventricles of the brain and the central canal of the spinal cord. Congenital malformations of the brain commonly arise from developmental defects in the neural tube. The neural crest derives from symmetric lateral portions of the neural plate. Neural crest cells migrate to sites in the body where they form the vast majority of the PNS and most of the peripheral cells of the ANS, including the sympathetic ganglia and the chromaffin cells of the adrenal medulla. On the sensory side, these neural crest derivatives include unipolar neurons whose cell bodies are in the dorsal root ganglia, as well as the equivalent sensory cells of cranial nerves V, VII, IX, and X. Neural crest cells also give rise to several nonneuronal structures, including Schwann cells, satellite glial cells in spinal and cranial ganglia, and pigment cells of the skin. The human brain begins to exhibit some regional specialization around the fourth gestational week (Fig. 10-5A, B). By then, it is possible to discern an anterior part called the prosencephalon, a midsection called the mesencephalon, and a posterior part called the rhombencephalon. Rapid brain growth ensues, and important new regions emerge in just another week (Fig. 10-5C). Distinct regions called brain vesicles, which are destined to become separate parts of the adult brain, are set apart as swellings in the rostral-caudal plane (Fig. 10-5B, C). The prosencephalon is now divisible into the telencephalon, which will give rise to the basal ganglia and cerebral cortex, and the diencephalon, which becomes the thalamus, subthalamus, hypothalamus, and neurohypophysis (the posterior or neural portion of the pituitary). Similarly, the rhombencephalon can now be divided into the metencephalon, which will give rise to the

Chapter 10 • Organization of the Nervous System

NEURAL PLATE Ectoderm

Neural plate

Endoderm

Notochord

Neural crest

Somatic mesoderm

Cut edge of amnion Mesoderm

Splanchnic mesoderm

Brain plate NEURAL GROOVE, DAY 19

Neural plate Node of Henson Primitive streak Day 19

Paraxial mesoderm

NEURAL GROOVE, DAY 21

Cardiogenic area

Neural folds

Neural groove

Neural fold

Somatic mesoderm

Brain plate Somite I Neural groove

Splanchnic mesoderm

Node of Henson NEURAL TUBE, DAY 23

Primitive streak Day 21

Neural crest

Neural tube

Somite

Neural fold Anterior neuropore Cardiogenic area

Intermediate mesoderm

Somite I

Paraxial mesoderm

Lateral mesoderm

NEURAL TUBE, LATER Neural tube

Ependymal canal

Dorsal root ganglion

Posterior neuropore Day 23

Figure 10-4 Development of the nervous system. The left column provides a dorsal view of the developing nervous system at three different time points. The right column shows cross sections of the dorsal portion of the embryo at five different stages, three of which correspond to the dorsal views shown at the left.

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B PRIMARY VESICLES

A—28 DAYS

C SECONDARY VESICLES

28 DAYS

35 DAYS

Neural tissue

Cavities

Prosencephalon (forebrain)

Telencephalon

Cerebral hemispheres

Lateral ventricles

Thalamus Subthalamus Hypothalamus Neuropituitary

Most of third ventricle

Midbrain

Cerebral aqueduct Rostral fourth ventricle

Mesencephalon (midbrain)

Prosencephalon (forebrain)

Diencephalon Mesencephalon (midbrain)

Spinal cord

Mesencephalon

Cephalic flexure

Optic vesicle

Cervical flexure

Cranial and spinal sensory ganglia

Metencephalon

Pons Cerebellum

Myelencephalon

Medulla

Caudal fourth ventricle

Spinal cord

Central canal

Rhombencephalon (hindbrain)

Neural tissue

Rhombencephalon (hindbrain)

Cavity Spinal cord

Figure 10-5

ADULT DERIVATIVES

Spinal cord

Embryonic development of the brain.

pons and cerebellum, and the myelencephalon, which becomes the medulla. Robust development of the cerebral cortex becomes apparent in mammals, especially humans, after the seventh week. This structure gradually expands so that it enwraps the rostral structures. As the neural tube thickens with cell proliferation, a groove called the sulcus limitans forms on the inner, lateral wall of the neural tube (Fig. 10-6A). This anatomical landmark extends throughout the neural tube except in the farthest rostral area that will become the diencephalon and cortex. The sulcus limitans divides the neural tube into a ventral area called the basal plate and a dorsal area called the alar plate. Structures that derive from the basal plate mediate efferent functions, and structures that arise from the alar plate mediate afferent and associative functions. Efferent neurons are mainly motor neurons that convey information from the CNS to outside effectors (i.e., muscles or secretory cells). In a strict sense, the only true afferent neurons are those that derive from neural crest cells and that convey sensory information from various kinds of receptors to the CNS. In the CNS, these afferent neurons synapse on other neurons derived from the alar plate; these alar plate neurons may be referred to as afferent because they receive sensory information and pass it along to other parts of the CNS. However, it

is also appropriate to call these alar plate–derived neurons associative. The development of the spinal cord and medulla illustrates how this early anatomical division into alar and basal plates helps make sense of the final organization of these complex regions. Neurons of the alar and basal plates proliferate, migrate, and aggregate into discrete groups that have functional specificity. In the spinal cord (Fig. 10-6B, C), the basal plate develops into the ventral horn, which contains the cell bodies of somatic motor neurons, and the intermediolateral column, which contains the cell bodies of autonomic motor neurons. Both regions contain interneurons. The alar plate in the spinal cord develops into the dorsal horn, which contains the cell bodies onto which sensory neurons synapse. In the medulla (Fig. 10-6D, E), as well as in the rest of the brain, aggregates of neurons are called nuclei. Nuclei that develop from the alar plate are generally afferent, such as the nucleus tractus solitarii, which plays an important sensory role in the ANS. Nuclei that develop from the basal plate are generally efferent, such as the dorsal motor nucleus of the vagus nerve, which plays an important motor role in the ANS. The choroid plexus that invaginates into the lumen of the central canal is responsible for secreting cerebrospinal fluid (see Chapter 11).

Chapter 10 • Organization of the Nervous System

A NEURAL TUBE Roof plate Marginal layer

Alar plate Central canal

Sulcus limitans

Basal plate

Neuroepithelial layer

Floor plate

SPINAL CORD B

MEDULLA D ROSTRAL MEDULLA

EARLY SPINAL CORD Dorsal root ganglion

Choroid plexus

Sulcus limitans

Ventricle filled with cerebrospinal fluid (CSF)

Tela choroidea

Sulcus Special limitans somatic afferent (auditoryvestibular nerve)

Alar plate

General somatic afferent (nucleus of the spinal tract of CN V)

Central canal Neural crest

C

Basal plate

General somatic General visceral Special visceral efferents efferent (dorsal efferent (nucleus (hypoglossal nuclei) ambiguus) motor nucleus of CN X)

MATURE SPINAL CORD Dorsal root (sensory)

Special visceral afferent (nucleus solitarius) Inferior olive

E

Dorsal horn Central canal

Dorsal root ganglion

CAUDAL MEDULLA

Blood vessel

Central gray matter

Sulcus limitans

Gracile nucleus Cuneate nucleus

Alar plate Nucleus of the spinal tract of CN V Inferior olive Nucleus ambiguus

Intermediolateral column

Ventral root (motor)

Ventral horn

Sulcus limitans

Hypoglossal nucleus

Central canal Pyramids

Figure 10-6 Development of the spinal cord and medulla. A, In this cross section through the neural tube, the sulcus limitans is the landmark that separates the ventral basal plate from the dorsal alar plate. The basal plate will form efferent (or motor-type) structures, whereas the alar plate will form afferent and associative (or sensory-type) functions. B, The true afferent neurons are those in the dorsal root ganglion, which derive from neural crest cells. These afferents will contact the neurons in the alar plate, which will become associative. C, The basal plate has developed into the ventral horn and intermediolateral column (motor), whereas the alar plate has developed into the dorsal horn (associative). D, The basal plate has developed into nuclei with motor functions, whereas the alar plate has developed into nuclei with sensory functions. The roof of the rostral medulla becomes the fourth ventricle. E, This cross section shows the same gross separation between motor and associative-sensory functions as is seen with the rostral medulla and the spinal cord.

Basal plate

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Abnormalities of Neural Tube Closure

C

losure of the neural tube in humans normally occurs between 26 and 28 days of gestation. A disturbance in this process results in a midline congenital abnormality called a dysraphism (from the Greek dys, abnormal, + rhaphe–, seam or suture). The defect can be so devastating that it is incompatible with life or, alternatively, have so little consequence that it goes unnoticed throughout life. These midline embryonic abnormalities also involve the primitive mesoderm and ectoderm associated with the neural tube. Therefore, the vertebral bodies or skull (derived from mesoderm) and the overlying skin (derived from ectoderm) may be affected along with the nervous system. The most serious neural tube defect, occurring in 1 of 1000 deliveries, is anencephaly, in which the cerebral hemispheres are absent and the rest of the brain is severely malformed. Overlying malformations of the skull, brain coverings, and scalp are present (Table 10-3). Affected fetuses are often spontaneously aborted. The most common dysraphisms affect formation of the spinal vertebral bodies and are called spina bifida. The problem may be slight and cause only a minor problem in closure of the vertebral arch, called spina bifida occulta (Fig. 10-7A). This malformation affects ∼10% of the population, usually at the fifth lumbar or first sacral vertebra, and generally causes no significant sequelae. If the dura and arachnoid membranes herniate (i.e., protrude) through the vertebral defect, the malformation is called spina bifida cystica (Fig. 10-7B); if the spinal cord also herniates through the defect, it is called myelomeningocele (Fig. 10-7C). These problems are often more significant and may cause severe neurological disability. Genetic and nongenetic factors can cause dysraphism. Some severe forms of this condition appear to be inherited, although the genetic pattern suggests that multiple genes are involved. Nongenetic factors may also play a role, as in the case of folic acid deficiency. Mothers taking folic acid (see Chapter 56) before and during the periconceptional period have a decreased risk of having a fetus with a neural tube closure defect. Current medical recommendations are that women contemplating becoming pregnant receive folic acid supplementation, and it has been suggested that bread products should be enriched with folic acid to ensure that women will have the protective advantage of this vitamin if they become pregnant. Other factors that increase the risk of these defects are maternal heat exposure (e.g., from a hot tub) and certain drugs such as the anticonvulsant valproate. Neural tube disorders can be detected during pregnancy by measuring the concentration of α-fetoprotein in maternal blood or amniotic fluid. α-Fetoprotein is synthesized by the fetal liver and, for unclear reasons, increases in concentration abnormally with failure of neural tube closure.

A

SPINA BIFIDA OCCULTA Hair

Incomplete vertebral arch Dura mater

Skin Subarachnoid space Vertebra

Spinal cord

Back muscles

B SPINA BIFIDA CYSTICA OR MENINGOCELE

Subarachnoid space

Dura mater

Rudiment of vertebral arch

Vertebra

C MYELOMENINGOCELE

Membranous sac

Displaced spinal cord

Vertebra

Figure 10-7 Variations of spina bifida. A, An incomplete vertebral arch with no herniation. B, The dura and arachnoid membranes herniate through the vertebral defect. C, The spinal cord and meninges herniate through the vertebral defect.

Chapter 10 • Organization of the Nervous System

TABLE 10-3

Defects of Neural Tube Closure

Malformation

Brain Defects Anencephaly

Cephalocele Meningocele

Spina Bifida Defects Spina bifida occulta Spina bifida cystica

Myelomeningocele

Characteristics

Absence of the brain, with massive defects in the skull, meninges, and scalp Partial brain herniation through skull defect (cranium bifidum) Meningeal herniation through skull or spine defect

Vertebral arch defect only Herniation of the dura and arachnoid through a vertebral defect Herniation of the spinal cord and meninges through a vertebral defect

Neurons and glial cells originate from cells in the proliferating germinal matrix near the ventricles The trillions of neurons and glial cells that populate the brain arise from rapidly dividing stem cells called neuroepithelial cells located near the ventricles (which derive from the neural canal) of the embryonic CNS. This germinal area (Fig. 10-8A) is divided into two regions, the ventricular zone (VZ) and the subventricular zone (SVZ). Most of the neurons in the human brain are generated during the first 120 days of embryogenesis. Growth factors such as epidermal growth factor and platelet-derived growth factor and hormones such as growth hormone influence the rate of cell division of the neuroepithelial cells. The signals that direct one immature neuron to become a cortical pyramidal cell and another to become a retinal ganglion cell are not understood. Neuroepithelial cells generate different classes of neural precursor cells that develop into different mature cell types. In the developing brain, radial cells (Fig. 10-8), so called because their processes extend from the ventricular surface to the brain’s outer surface, appear very early in neurogenesis and generate most of the projection neurons in forebrain cortex. Inhibitory interneurons, in contrast, arise from neural precursor cells located in the SVZ. Neurons are probably not fully differentiated when first created and their mature characteristics may depend on their interactions with the chemical environment or other cells in a specific anatomical region of the nervous system. The VZ appears to produce separate progenitor cells that produce only neurons, oligodendrocytes, astrocytes, and ependymal cells (Fig. 10-8B). The VZ does not contribute to the population of Schwann cells, which derive from neural crest tissue, or to microglial cells, which arise from the mesodermal cells that briefly invade the brain during early postnatal development. Recent work shows that the embryonic and perinatal VZ and SVZ may give rise to the adult SVZ, which is in part responsible for limited adult neurogenesis.

Neuronal progenitor cells appear earliest and produce nearly the entire complement of adult neurons during early embryonic life. Glial cells arise later in development. Neurons are confined to specific locations of the brain, whereas glial cells are more evenly distributed. Many more neurons are created during fetal development than are present in the adult brain. Most neurons, having migrated to a final location in the brain and differentiated, are lost through a process called programmed cell death, or apoptosis (Greek for “falling off ”). Apoptosis is a unique form of cell death that requires protein synthesis and can be triggered by removal of specific trophic influences, such as the action of a growth factor. In contrast to necrotic cell death, which rapidly leads to loss of cell membrane integrity after some insult causes a toxic increase in [Ca2+]i, apoptosis evolves more slowly. For example, in the retina, ∼60% of the ganglion cells and thus ∼60% of the retinal axons are lost in the first 2 weeks of extrauterine life as a result of programmed retinal ganglion cell death. This process of sculpting the final form of a neuronal system by discarding neurons through programmed death is a common theme in developmental biology. The number of glial cells in different areas of the brain appears to be determined by signals from nearby neurons or axons. For example, in the optic nerve, the final number of glia in the nerve is closely determined by the number of axons. When programmed cell death is prevented by expression of the bcl-2 gene in transgenic animals, the number of axons in the optic nerve is dramatically increased, as well as the number of astrocytes and oligodendrocytes. Thus, glial cell–axon ratios remain relatively constant. The axon-dependent signal or signals responsible for these adjustments in glial cell number are not known, but the process appears to operate by influencing both glial cell survival and proliferation.

Neurons migrate to their correct anatomical position in the brain with the help of adhesion molecules During embryogenesis, the long processes of radial cells create an organized, cellular scaffolding on which neurons can migrate to their final position in the brain shortly after they appear. Migrating neurons contact radial cells (Fig. 10-8B) and move along their processes toward their final positions in the developing cortex. Thus, the prearranged positions of these radial processes determine the direction of neuronal migration. The importance of the radial framework for assisting neuronal migration is illustrated by the failure of neurons to populate the cortex normally when the radial processes are interrupted by hemorrhage in the fetal brain. The navigation mechanisms used by migrating cells in the nervous system and elsewhere in the body are only partially understood. Proteins that promote selective cellular aggregation are called cell-cell adhesion molecules (CAMs; see Chapter 2) and include the Ca2+-dependent cadherins and Ca2+-independent neural cell adhesion molecules (N-CAMs). These molecules are expressed by developing cells in an organized, sequential manner. Cells that express the appropriate

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A

SECTION OF DEVELOPING FOREBRAIN

B

RADIAL GLIAL CELLS

Ventricular and subventricular Developing zones cortex

To cortex

Migratory neuroblast

Oligodendrocyte Radial glial cells

Ventricle

Developing cortex

Pial surface Migrating neurons

Intermediate zone

Astrocyte

Radial cells

Subventricular zone (SVZ) Ventricular zone (VZ)

Multipotent “Transit amplifier” cells

Ventricle Radial cell

Ventricular surface

Ependymal cell with cilia

Embryonic and Perinatal VZ/SVZ

Adult SVZ

Figure 10-8 Arrangement of radial cells and migrating neurons. A, The upper portion is a coronal section of developing occipital cerebral lobe of fetal monkey brain. The lower portion is a magnified view. The ventricular zone contains the germinal cells that give rise to the neurons as well as to the cell bodies of the radial cells. These radial cells extend from the ventricular surface to the pial surface, which overlies the developing cortex. B, The more magnified view on the left shows the cell bodies of two radial cells as well as their processes that extend upward toward the cortex. Also shown are two migratory neuroblasts moving from the ventricular zone toward the cortex along the fibers of the radial cells. The black arrows indicate possible pathways of proliferation and differentiation. (Data from Rakic P: Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 1972; 145:61-84; and Tramontin AD, GarciaVerdugo JM, Lim DA, Alvarez-Buylla A: Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment. Cerebral Cortex 2003; 13:580-587.)

adhesion molecules have a strong tendency to adhere to one another. These Velcro-like molecules can assemble cells in a highly ordered fashion; experimentally, disrupted germ cells can properly reorganize themselves into a three-layered structure that replicates the normal embryonic pattern. Another mechanism that assists migrating cells is the presence of extracellular matrix molecules such as laminin and fibronectin. These glycoproteins are selectively secreted by both neurons and astrocytes and form a kind of extracellular roadway with which migrating cells can interact. Growing axons express at their surface cell matrix adhesion molecules called integrins that bind laminin and fibronectin (see Chapter 2). As a result, growing axons move together in fascicles. Perhaps the least understood mechanism related to cell migration is chemotaxis, the ability of a cell to follow a

chemical signal emitted from a target cell. The tips of developing axons, called growth cones, appear to follow such chemical cues as they grow toward their specific targets. For example, a molecule called netrin, secreted by midline cells, attracts developing axons destined to cross the midline. On the other hand, molecules like slit repel axons by interacting with specific receptors on the growth cone. Such signals steer axon growth cones, perhaps by localized changes in intracellular [Ca2+], leading to the strategic insertion of new patches of membrane on the surface of the growth cone. Neurons do not regenerate Neurons Most human neurons arise in about the first 4 months of intrauterine life. After birth, neurons do not divide, and if a neuron is lost for any reason, it is generally

Chapter 10 • Organization of the Nervous System

Axonal Degeneration and Regeneration

A

xons have their own mitochondria and produce the ATP that they need to maintain the steep ion gradients necessary for excitability and survival. In this sense, they are metabolically independent of the cell body. However, they cannot make proteins and are unable to sustain themselves if separated from the cell body (Fig. 10-2). If an axon is cut, in either the PNS or the CNS, a characteristic series of changes takes place (Fig. 10-9): Step 1: Degeneration of the synaptic terminals distal to the lesion. Synaptic transmission occurring at the axon terminal fails within hours because this complex process is dependent on material provided by axonal transport. Visible changes in the degenerating terminal are seen a few days after the lesion. The terminal retracts from the postsynaptic target. Step 2: Wallerian degeneration. The lesion divides the axon into proximal and distal segments. The distal segment degenerates slowly during a period of several weeks in a process named after its discoverer, Augustus Waller. Eventually, the entire distal segment is destroyed and removed. Step 3: Myelin degeneration. If the affected axon is myelinated, the myelin degenerates. The myelinating cell (i.e., the Schwann cell in the PNS and the oligodendrocyte in the CNS) usually survives this process. Schwann cells are immediately induced to divide, and they begin to synthesize trophic factors that may be important for regeneration.

not replaced, which is the main reason for the relatively limited recovery from serious brain and spinal cord injuries. It has been argued that this lack of regenerative ability is a design principle to ensure that learned behavior and memories are preserved in stable populations of neurons throughout life. A notable exception to this rule is olfactory bulb neurons, which are continually renewed throughout adult life by a population of stem cells or neuronal progenitor cells. As noted earlier, cells in the adult SVZ have the capacity to generate neurons and may do so to a limited extent throughout life. Learning how to induce these cells to make functional new CNS neurons after severe neural injury is the holy grail of regeneration research. Glia

Unlike neurons, glial cells can be replaced if they are lost or injured in an adult. Such repopulation depends on progenitor cells committed to the glial cell lineage. Either the progenitor cells reside in a latent state (or are slowly turning over) in adult brains or true multipotential stem cells are activated by specific conditions, such as brain injury, to produce de novo glial progenitors. The most typical reaction of mammalian brains to a wide range of injuries is the formation of an astrocytic glial scar. This scar is produced primarily by an enlargement of individual astrocytes, a process called hypertrophy, and increased expression of a particular cytoskeleton protein, glial acidic fibrillary protein. Only a small degree of astrocytic proliferation (i.e., an increase

Step 4: Scavenging of debris. Microglia in the CNS and macrophages and Schwann cells in the PNS scavenge the debris created by the breakdown of the axon and its myelin. This step is more rapid in the PNS than in the CNS. Step 5: Chromatolysis. After axonal injury, most neuron cell bodies swell and undergo a characteristic rearrangement of organelles called chromatolysis. The nucleus also swells and moves to an eccentric position. The endoplasmic reticulum, normally close to the nucleus, reassembles around the periphery of the cell body. Chromatolysis is reversible if the neuron survives and is able to re-establish its distal process and contact the appropriate target. Step 6: Retrograde transneuronal degeneration. Neurons that are synaptically connected to injured neurons may themselves be injured, a condition called transneuronal or trans-synaptic degeneration. If the neuron that synapses on the injured cell undergoes degeneration, it is called retrograde degeneration. Step 7: Anterograde transneuronal degeneration. If a neuron that received synaptic contacts from an injured cell degenerates, it is called anterograde degeneration. The magnitude of these transneuronal effects (retrograde and anterograde degeneration) is quite variable.

in cell number) accompanies this reaction. Microglial cells, which derive from cells related to the monocyte-macrophage lineage in blood and not from neuroepithelium, also react strongly to brain injury and are the main cells that proliferate at the injury site. Axons Another reason that relatively little recovery follows severe brain and spinal cord injury is that axons within the CNS do not regenerate effectively. This lack of axon regeneration in the CNS is in sharp contrast to the behavior of axons in the PNS, which can regrow and reconnect to appropriate end organs, either muscle or sensory receptors. For example, if the median nerve of the forearm is crushed by blunt trauma, the distal axon segments die off in a process called wallerian degeneration (see the box titled Axonal Degeneration and Regeneration) because the sustaining relationship with their proximal cell bodies is lost. These PNS axons can slowly regenerate and connect to muscles and sensory receptors in the hand. It is believed that the inability of CNS axons to regenerate is the fault of the local environment more than it is an intrinsic property of these axons. For example, on their surface, oligodendrocytes and myelin carry molecules, such as myelin-associated glycoprotein, that inhibit axon growth. Experiments have shown that if severed CNS axons are given the opportunity to regrow in the same environment that surrounds axons in the PNS, they are capable of regrowth and can make functional connections

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A

NORMAL NEURON

Schwann cell nucleus (PNS)

Oligodendrocyte (CNS)

Myelin Nissl substance (ER)

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DEGENERATING NEURON 7 Anterograde transneuronal degeneration: the anterograde neuron degenerates.

1 The synaptic terminals, distal to the lesion in the axon, degenerate. 2 Wallerian degeneration: Loss of axonal structure distal to lesion.

Surviving Schwann cells

3 Myelin degenerates, leaving debris behind. 4 Microglia (CNS) or macrophages (PNS) scavenge the debris of breakdown.

Lesion

5 ER degenerates (chromatolysis).

6 Retrograde transneuronal degeneration: the retrograde neuron's terminals retract and the neuron degenerates.

Figure 10-9 Nerve degeneration. A, Normal neuron. B, Degenerating neuron. ER, endoplasmic reticulum.

with CNS targets. The remarkable ability of damaged peripheral nerves to regenerate, even in mammals, has encouraged hope that CNS axons might, under the right conditions, be able to perform this same feat. It would mean that victims of spinal cord injury might walk again.

SUBDIVISIONS OF THE NERVOUS SYSTEM A rudimentary knowledge of the anatomy of the nervous system is a prerequisite to discussion of its physiology. This section provides an overview of nervous system anatomy that builds on what has already been discussed about its embryological development. We in turn consider the CNS, PNS, and ANS (Table 10-1). The directional terms used to describe brain structures can be somewhat confusing because the human nervous system, unlike that of lower vertebrates, bends during development. Thus, the dorsal surface of the cerebral cortex is also superior, whereas the dorsal surface of the spinal cord is also posterior (Fig. 10-10A).

The CNS consists of the telencephalon, cerebellum, diencephalon, midbrain, pons, medulla, and spinal cord The CNS can be conveniently divided into five major areas: (1) telencephalon, (2) cerebellum, (3) diencephalon, (4) brainstem (consisting of the midbrain, pons, and medulla), and (5) spinal cord (Fig. 10-10B). Each of these areas has symmetric right and left sides. Telencephalon

One of the crowning glories of evolution is the human cerebral cortex, the most conspicuous part of the paired cerebral hemispheres. The human cerebral cortex has a surface area of ∼2200 cm2 and is estimated to contain 1.5 to 2 × 1010 neurons. The number of synaptic contacts between these cells is ∼3 × 1014. The cortical surface area of mammals increases massively from mouse to monkey to humans in a ratio of 1 : 100 : 1000. The capacity for information processing by this neuronal machine is staggering and includes a remarkable range of functions: thinking, learning, memory, and consciousness. The cortex is topographically organized in two ways. First, certain areas of the cortex mediate specific functions. For example, the area that mediates motor control is a welldefined strip of cortex located in the frontal lobe (Fig. 10-10C). Second, within a portion of cortex that manages a specific function (e.g., motor control, somatic sensation, hearing, or vision), the parts of the body spatially map onto this cortex in an orderly way. We discuss this principle of somatotopy in Chapter 16. Another part of the telencephalon is the great mass of axons that stream into and out of the cerebral cortex and connect it with other regions. The volume of axons needed to interconnect cortical neurons increases as a power function of cortical surface area, which increases so dramatically from mice to humans. Thus, the relative volume of white matter to gray matter is 5-fold greater in humans versus mice. The final part of the telencephalon includes the basal ganglia, which comprise the striatum (caudate nucleus and putamen) and globus pallidus. These structures have indirect connections with motor portions of the cerebral cortex and are involved in motor control.

Chapter 10 • Organization of the Nervous System

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AXES OF THE CNS Dorsal (superior) Rostral (anterior)

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MAJOR COMPONENTS OF THE CNS Cerebral hemispheres Telencephalon

Cerebellum Diencephalon (thalamus, subthalamus, hypothalamus) Midbrain Pons

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Medulla Cervical Thoracic

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Lumbar Sacral

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SURFACE ANATOMY OF THE CEREBRAL CORTEX Frontal lobe

Parietal lobe

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Figure 10-10

Gross anatomy of the CNS.

Occipital lobe

Cerebellum This brain region lies immediately dorsal to the brainstem. Although the cerebellum represents only ∼10% of the CNS by volume, it contains ∼50% of all CNS neurons. The exceedingly large number of input connections to the cerebellum conveys information from nearly every type of receptor in the nervous system, including visual and auditory input. Combined, these afferent fibers outnumber the efferent projections by an estimated ratio of 40 : 1. Functionally and by virtue of its connections, the cerebellum can be divided into three parts. Phylogenetically, the vestibulocerebellum (also called the archicerebellum) is the oldest of these three parts, followed by the spinocerebellum (also called the paleocerebellum) and then by the cerebrocerebellum (also called the neocerebellum). The vestibulocerebellum is closely related to the vestibular system, whose sensors are located in the inner ear and whose way stations are located in the pons and medulla. It helps maintain the body’s balance. The spinocerebellum receives strong input from muscle stretch receptors through connections in the spinal cord and brainstem. It helps regulate muscle tone. The cerebrocerebellum, the largest part of the human cerebellum, receives a massive number of projections from sensorimotor portions of the cerebral cortex through neurons in the pons. It coordinates motor behavior. Much of the cerebellum’s output reaches the contralateral (i.e., on the opposite side of the body) motor cortex by way of the thalamus. Other efferent projections reach neurons in all three parts of the brainstem. Diencephalon This brain region consists of the thalamus, the subthalamus, and the hypothalamus, each with a very different function. The thalamus is the main integrating station for sensory information that is bound for the cerebral cortex, where it will reach the level of conscious perception. Along with the subthalamus, the thalamus also receives projections from the basal ganglia that are important for motor function. Input to the thalamus from the cerebellum (specifically, the cerebrocerebellum) is important for normal motor control. Patients with Parkinson disease, a severe movement disorder, gradually lose the ability to make voluntary movements; in some of these patients, it is possible to improve movement by stimulating certain areas of the thalamus or subthalamus. Control of arousal and certain aspects of memory function also reside in discrete areas of the thalamus. The hypothalamus is the CNS structure that most affects the ANS. It performs this function through strong, direct connections with autonomic nuclei in the brainstem and spinal cord. It also acts as part of the endocrine system in two major ways. First, specialized neurons located within specific nuclei in the hypothalamus synthesize certain hormones (e.g., arginine vasopressin and oxytocin) and transport them down their axons to the posterior pituitary gland, where the hormones are secreted into the blood. Second, other specialized neurons in other nuclei synthesize “releasing hormones” (e.g., gonadotropin-releasing hormone) and release them into a plexus of veins, called a portal system, that carries the releasing hormones to cells in the anterior pituitary. There, the releasing hormones stimulate certain cells (e.g., gonadotrophs) to secrete hormones (e.g., folliclestimulating hormone or luteinizing hormone) into the

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bloodstream. We discuss these principles in Chapter 47. The hypothalamus also has specialized centers that play important roles in controlling body temperature and hunger (see Chapters 58 and 59), thirst (see Chapter 40), and the cardiovascular system. It is the main control center of the ANS. Brainstem (Midbrain, Pons, and Medulla)

This region lies immediately above, or rostral to, the spinal cord. Like the spinal cord, the midbrain, pons, and medulla have a segmental organization, receive sensory (afferent) information, and send out motor (efferent) signals through paired nerves that are called cranial nerves. The midbrain, pons, and medulla also contain important control centers for the ANS (see Chapter 14). In addition to motor neurons, autonomic neurons, and sensory neurons present at each level, the caudal brainstem serves as a conduit for a large volume of axons traveling from higher CNS centers to the spinal cord (descending pathways) and vice versa (ascending pathways). In addition, this portion of the brainstem contains a loosely organized interconnected collection of neurons and fibers called the reticular formation. This neuronal network has diffuse connections with the cortex and other brain regions and affects the level of consciousness or arousal. The midbrain has somatic motor neurons that control eye movement. These neurons reside in the nuclei for CN III and CN IV. Other midbrain neurons are part of a system, along with the cerebellum and cortex, for motor control. The midbrain also contains groups of neurons that are involved in relaying signals related to hearing and vision. Just caudal to the midbrain is the pons, which contains the somatic motor neurons that control mastication (nucleus for CN V), eye movement (nucleus for CN VI), and facial muscles (nucleus for CN VII). The pons also receives somatic sensory information from the face, scalp, mouth, and nose (portion of the nucleus for CN V). It is also involved in processing information that is related to hearing and equilibrium (nucleus for CN VIII). Neurons in the ventral pons receive input from the cortex, and these neurons in turn form a massive direct connection with the cerebellum (see earlier) that is crucial for coordinating motor movements. The most caudal portion of the brainstem is the medulla. The organization of the medulla is most similar to that of the spinal cord. The medulla contains somatic motor neurons that innervate the muscles of the neck (nucleus of CN XI) and tongue (nucleus of CN XII). Along with the pons, the medulla is involved in controlling blood pressure, heart rate, respiration, and digestion (nuclei of CN IX and X). The medulla is the first CNS way station for information traveling from the special senses of hearing and equilibrium.

Spinal Cord

Continuous with the caudal portion of the medulla is the spinal cord. The spinal cord runs from the base of the skull to the body of the first lumbar vertebra. Thus, it does not run the full length of the vertebral column in adults. The spinal cord consists of 31 segments that each have a motor and sensory nerve root. (The sensory nerve root of the first cervical segment is very small and can be missing.) These nerve roots combine to form 31 bilaterally symmetric pairs of spinal nerves. The spinal roots, nerves, and ganglia are part of the PNS (see later).

Sensory information from the skin, muscle, and visceral organs enters the spinal cord through fascicles of axons called dorsal roots (Fig. 10-11A). The point of entry is called the dorsal root entry zone. Dorsal root axons have their cell bodies of origin in the spinal ganglia (i.e., dorsal root ganglia) associated with that spinal segment. Ventral roots contain strictly efferent fibers (Fig. 10-11B). These fibers arise from motor neurons (i.e., general somatic efferent neurons) whose cell bodies are located in the ventral (or anterior) gray horns of the spinal cord (gray because they contain mainly cell bodies without myelin) and from preganglionic autonomic neurons (i.e., general visceral efferent neurons) whose cell bodies are located in the intermediolateral gray horns (i.e., between the dorsal and ventral gray horns) of the cord. Most of the efferent fibers are somatic efferents that innervate skeletal muscle to mediate voluntary movement. The other fibers are visceral efferents that synapse with postganglionic autonomic neurons, which in turn innervate visceral smooth muscle or glandular tissue. Each segment of the spinal cord contains groups of associative neurons in its dorsal gray horns. Some but not all incoming sensory fibers synapse on these associative neurons, which in turn contribute axons to fiber paths that both mediate synaptic interactions within the spinal cord and convey information to more rostral areas of the CNS by way of several conspicuous ascending tracts of axons (Fig. 1011C). Similarly, descending tracts of axons from the cerebral cortex and brainstem control the motor neurons whose cell bodies are in the ventral horn, thus leading to coordinated voluntary or posture-stabilizing movements. The most important of these descending tracts is called the lateral corticospinal tract; ∼90% of its cell bodies of origin are in the contralateral cerebral cortex. These ascending and descending tracts are located in the white portion of the spinal cord (white because it contains mostly myelinated axons). The spatial organization of spinal cord neurons and fiber tracts is complex but orderly and varies somewhat among the 31 segments. If sensory fibers enter the spinal cord and synapse directly on motor neurons in that same segment, this connection underlies a simple segmental reflex or interaction. If the incoming fibers synapse with neurons in other spinal segments, they can participate in an intersegmental reflex or interaction. Finally, if the incoming signals travel rostrally to the brainstem before they synapse, they constitute a suprasegmental interaction. The peripheral nervous system comprises the cranial and spinal nerves, their associated sensory ganglia, and various sensory receptors The PNS serves four main purposes: (1) it transduces physical or chemical stimuli both from the external environment and from within the body into raw sensory information through receptors; (2) it conveys sensory information to the CNS along axon pathways; (3) it conveys motor signals from the CNS along axon pathways to target organs, primarily skeletal and smooth muscle; and (4) it converts the motor signals to chemical signals at synapses on target tissues in the periphery. Figure 10-11B summarizes these four functions for a simple reflex arc in which a painful stimulus to the foot

Chapter 10 • Organization of the Nervous System

A

B

SPINAL CORD AND NERVE ROOTS Posterior intermediate septum

Posterior median septum Dorsal root entry zone

Posterior gray horn Posterior funiculus

A SPINAL REFLEX ARC Interneuron Dorsal root

Dorsal horn

Primary sensory neuron Sensory axon

Lateral funiculus

Anterior gray horn Anterior funiculus Root filaments

Dorsal

Motor axon Pia mater

Mixed spinal nerve

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Spinal cord Motor neuron

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ASCENDING AND DESCENDING TRACTS Fasciculus gracilis Fasciculus cuneatus Dorsolateral tract (fasciculus) (of Lissauer)

Septomarginal fasciculus (oval bundle) Interfascicular fasciculus (comma tract)

Dorsal (posterior) spinocerebellar tract Lateral spinothalamic tract and spinoreticular tract

Lateral corticospinal (pyramidal) tract (crossed) Rubrospinal tract Lateral (medullary) reticulospinal tract

Ventral (anterior) spinocerebellar tract

Medial longitudinal (sulcomarginal) fasciculus

Spinoolivary tract

Vestibulospinal tract

Spinotectal tract

Ventral (anterior) or medial (pontine) reticulospinal tract

Ventral (anterior) spinothalamic tract Fasciculus proprius

Ascending pathways Descending pathways Short ascending and descending pathways

Tectospinal tract Ventral (anterior) corticospinal tract (direct)

Figure 10-11 Spinal cord. A, Each spinal segment has dorsal and ventral nerve roots that carry sensory and motor nerve fibers, respectively. B, The simple “flexor” reflex arc is an illustration of the four functions of the PNS: (1) a receptor transduces a painful stimulus into an action potential, (2) a primary sensory neuron conveys the information to the CNS, (3) the CNS conveys information to the target organ by a motor neuron, and (4) the electrical signals are converted to signals at the motor end plate. C, Ascending pathways, which carry information to more rostral areas of the CNS, are shown on the left. Descending pathways, which carry information in the opposite direction, are shown on the right.

results in retraction of the foot from the source of the pain. Like the CNS, the PNS can be divided into somatic and autonomic parts. The somatic division includes the sensory neurons and axons that innervate the skin, joints, and muscle as well as the motor axons that innervate skeletal muscle.

The somatic division of the PNS primarily deals with the body’s external environment, either to gather information about this environment or to interact with it through voluntary motor behavior. The ANS, discussed in the next section and in Chapter 14, is a functionally distinct part of both the CNS and PNS (Table 10-1). The autonomic portion

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Epineurium Endoneurium

Perineurium

Unmyelinated axons

Schwann cell

Blood vessels

Fascicle

Schwann cell soma Node of Ranvier Myelinated axon

Figure 10-12

Myelin sheath

Peripheral nerve.

of the PNS consists of the motor and sensory axons that innervate smooth muscle, the exocrine glands, and other viscera. This division mainly deals with the body’s internal environment. Three important aspects of the PNS are discussed in other chapters. Sensory transduction is reviewed in Chapter 15, synaptic transmission in Chapters 8 and 13, and peripheral neuronal circuits in Chapter 16. Here, we focus primarily on the system of axons that is such a prominent feature of the PNS. Axons in the PNS are organized into bundles called peripheral nerves (Fig. 10-12). These nerves contain, in a large nerve such as the sciatic nerve, tens of thousands of axons. Individual axons are surrounded by loose connective tissue called the endoneurium. Within the nerve, axons are bundled together in small groups called fascicles, each one covered by a connective tissue sheath known as the perineurium. The perineurium contributes structural stability to the nerve. Fascicles are grouped together and surrounded by a matrix of connective tissue called the epineurium. Fascicles within a nerve anastomose with neighboring fascicles. Axons shift from one fascicle to another along the length of the nerve, but they tend to remain in roughly the same general area within the nerve over long distances. The interlocking meshwork of fascicles adds further mechanical strength to the nerve. Axons range in diameter from less than 1 to 20 μm. Because axons are extremely fragile, adaptations that enhance mechanical stability are very important. The PNS is designed

to be much tougher, physically, than nervous tissue in the CNS. The PNS must be mechanically flexible, tolerant of minor physical trauma, and sustainable by a blood supply that is less dependable than the one providing for the CNS. A spinal cord transplanted to the lower part of the leg would not survive the running of a 100-meter dash. Axons in peripheral nerves are closely associated with Schwann cells. In the case of a myelinated axon, a Schwann cell forms a myelinated wrap around a single adjacent axon, a single internodal myelin segment between 250 and 1000 μm in length. Many such internodal myelin segments, and thus many Schwann cells, are necessary to myelinate the entire length of the axon. In an unmyelinated nerve, the cytoplasm of a Schwann cell envelops but does not wrap around axons. Unmyelinated axons outnumber myelinated axons by about 2 : 1 in typical human nerves. Diseases that affect the PNS can disrupt nerve function by causing either loss of myelin or axonal injury. The functional organization of a peripheral nerve is best illustrated by a typical thoracic spinal nerve and its branches. Every spinal nerve is formed by the dorsal and ventral roots joining together and emerging from the spinal cord at that segmental level (Fig. 10-11). The dorsal roots coalesce and display a spindle-shaped swelling called the spinal or dorsal root ganglion, which contains the cell bodies of the sensory axons in the dorsal roots. Individual neurons are called dorsal root ganglion cells or spinal ganglion cells and are typical unipolar neurons that give rise to a single process that

Chapter 10 • Organization of the Nervous System

C2 C4

C5

C3

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Figure 10-13 Dermatomes. A dermatome is the area of cutaneous sensory innervation that a single spinal segment provides.

bifurcates in a T-like manner into a peripheral and central branch (Fig. 10-3). The central branch carries sensory information into the CNS and the peripheral branch terminates as a sensory ending. The peripheral process, which brings information toward the cell body, meets one definition of a dendrite; however, it has all the physiological and morphological features of a peripheral axon. Spinal nerves divide into several branches that distribute motor and sensory axons to the parts of the body associated with that segment. Axons conveying autonomic motor or autonomic sensory signals also travel in these branches. These branches are said to be “mixed” because they contain both efferent and afferent axons. Further nerve division occurs as axons travel to supply their targets, such as the skin, muscle, or blood vessels. In the case of thoracic spinal nerves, the subdivision is orderly and has a similar pattern for most of the nerves. In the cervical and lumbosacral areas, however, the spinal nerves from different segments of the spinal cord intermingle to form a nerve plexus. The subsequent course of the nerves in the upper and lower extremities is complex. The pattern of cutaneous innervation of the body is shown in Figure 10-13. The area of cutaneous innervation provided by a single dorsal root and its ganglion is called a dermatome. Severing a single dorsal root does not produce anesthesia in that dermatome because of overlap between the cutaneous innervation provided by adjacent dorsal roots. The sole exception to this rule is the C2 root, sectioning of which causes a patch of analgesia on the back of the head; neither C3 nor the trigeminal nerve innervates skin in this area. Also note that no dermatomes are shown for the first cervical and the coccygeal segments because they are small

or may be missing (in the case of the first cervical segment). The autonomic nervous system innervates effectors that are not under voluntary control The nervous system regulates some physiological mechanisms in a way that is independent or autonomous of voluntary control. Control of body temperature is an example of a fundamental process that most individuals cannot consciously regulate. Other examples include blood pressure and heart rate. The absence of voluntary control means that the ANS has little cortical representation. The ANS has three divisions: sympathetic, parasympathetic, and enteric. The sympathetic and parasympathetic divisions have both CNS and PNS parts. The enteric division is entirely in the PNS. The parasympathetic and sympathetic efferent systems are composed of two neurons. The cell body of the first neuron is located in the CNS and that of the second in the PNS. The sympathetic and parasympathetic divisions innervate most visceral organs and have a yin-yang functional relationship. The enteric division regulates the rhythmic contraction of intestinal smooth muscle and also regulates the secretory functions of intestinal epithelial cells. It receives afferent input from the gut wall and is subject to modulation by the two other divisions of the ANS. All the divisions have both efferent and afferent connections, although the efferent actions of the ANS are usually emphasized. We consider the ANS in detail in Chapter 14.

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Peripheral Nerve Disease

T

he symptoms of peripheral nerve disease, or neuropathy, are numbness (i.e., a sensory deficit) and weakness (i.e., a motor deficit). Such symptoms may arise from disturbances in many parts of the nervous system. How, then, can one tell whether a problem is the result of disease in the PNS? Motor axons directly innervate and have “trophic” effects on skeletal muscle. If the axon is cut or dies, this trophic influence is lost and the muscle undergoes denervation atrophy. In addition, individual muscle fibers may twitch spontaneously (fibrillation). The cause of fibrillation is still debated, but it may be related to the observation that acetylcholine receptors spread beyond the neuromuscular junction and become “supersensitive” to their agonist. If true, these observations imply continuing exposure to acetylcholine, even if it is in smaller quantities. Schwann cells at denervated junctions may be the source of acetylcholine. When a motor axon is first damaged but has not yet lost continuity with the muscle fibers that it innervates, these muscle cells may twitch in unison. These small twitches can be seen under the skin and are called fasciculations. They are probably due to spontaneous action potentials in dying or injured motor neurons or their axons.

REFERENCES Books and Reviews Abrous DN, Koehl M, Le Moal M: Adult neurogenesis: From precursors to network and physiology. Physiol Rev 2005; 85: 523-569. Gage FH: Stem cells of the central nervous system. Curr Opin Neurobiol 1998; 8:671-676. Hirokawa N: Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 1998; 279:519-526. Kandel ER, Schwartz JH, Jessell TM: Principles of Neural Science, 4th ed. New York: McGraw-Hill, 2000.

When the PNS is affected by a diffuse or generalized disease (e.g., the result of a metabolic problem or toxin), all peripheral nerves are involved, but symptoms arise first in the longest nerves of the body (i.e., those traveling from the spinal cord to the feet). This predilection for affecting the longest nerves often causes a “stocking pattern” defect in sensation and sometimes in strength. If both the feet and hands are affected, the process is called a “stocking and glove” defect. With progression of the disease, the level of involvement moves centripetally (i.e., up the leg, toward the trunk), and the sensory or motor dysfunction comes to involve more proximal portions of the legs and arms. One of the most common causes of this diffuse pattern of PNS involvement is the sensorimotor polyneuropathy associated with diabetes. Other causes include chronic renal failure (uremia), thiamine deficiency (often seen with alcohol abuse), and heavy metal poisoning. If a patient exhibits weakness or sensory loss that is associated with muscle fibrillation and atrophy and a stocking or stocking and glove pattern of sensory disturbance, a PNS problem is likely. Patients with peripheral neuropathy may also complain of tingling sensations (paresthesias) or pain in areas of the body supplied by the diseased nerves.

Journal Articles Burne JF, Staple JK, Raff MC: Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons. J Neurosci 1996; 16:2064-2073. Chiasson BJ, Tropepe V, Morshead CM, van der Kooy D: Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics. J Neurosci 1999; 19:4462-4471. Colbert CM, Johnston D: Axonal action-potential initiation and Na+ channel densities in the soma and axon initial segment of subicular pyramidal neurons. J Neurosci 1996; 16:6676-6686.

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THE NEURONAL MICROENVIRONMENT Bruce R. Ransom

Extracellular fluid in the brain provides a highly regulated environment for central nervous system neurons Everything that surrounds individual neurons can be considered part of the neuronal microenvironment. Technically, therefore, the neuronal microenvironment includes the extracellular fluid (ECF), capillaries, glial cells, and adjacent neurons. Although the term often is restricted to just the immediate ECF, the ECF cannot be meaningfully discussed in isolation because of its extensive interaction with brain capillaries, glial cells, and cerebrospinal fluid (CSF). How the microenvironment interacts with neurons and how the brain (used here synonymously with central nervous system, or CNS) stabilizes it to provide constancy for neuronal function are the subjects of this discussion. The concentrations of solutes in brain extracellular fluid (BECF) fluctuate with neural activity, and conversely, changes in ECF composition can influence nerve cell behavior. Not surprisingly, therefore, the brain carefully controls the composition of this important compartment. It does so in three major ways. First, the brain uses the blood-brain barrier to protect the BECF from fluctuations in blood composition. Second, the CSF, which is synthesized by choroid plexus epithelial cells, strongly influences the composition of the BECF. Third, the surrounding glial cells “condition” the BECF. The brain is physically and metabolically fragile The ratio of brain weight to body weight in humans is the highest in the animal kingdom. The average adult brain weight is ∼1400 g in men and ∼1300 g in women, approximately the same weight as the liver (see Chapter 46). This large and vital structure, which has the consistency of thick pudding, is protected from mechanical injury by a surrounding layer of bone and by the CSF in which it floats. The brain is also metabolically fragile. This fragility arises from its high rate of energy consumption, absence of significant stored fuel in the form of glycogen (∼5% of the amount in the liver), and rapid development of cellular damage when ATP is depleted. However, the brain is not the greediest of

the body’s organs; both the heart and kidney cortex have higher metabolic rates. Nevertheless, although it constitutes only 2% of the body by weight, the brain receives ∼15% of resting blood flow and accounts for ∼20% and 50%, respectively, of total resting oxygen and glucose utilization. The brain’s high metabolic demands arise from the need of its neurons to maintain the steep ion gradients on which neuronal excitability depends. In addition, neurons rapidly turn over their actin cytoskeleton. Neuroglial cells, the other major cells in the brain, also maintain steep transmembrane ion gradients. More than half of the energy consumed by the brain is directed to maintain ion gradients, primarily through operation of the Na-K pump (see Chapter 5). An interruption of the continuous supply of oxygen or glucose to the brain results in rapid depletion of energy stores and disruption of ion gradients. Because of falling ATP levels in the brain, consciousness is lost within 10 seconds of a blockade in cerebral blood flow. Irreversible nerve cell injury can occur after only 5 minutes of interrupted blood flow.

CEREBROSPINAL FLUID CSF is a colorless, watery liquid. It fills the ventricles of the brain and forms a thin layer around the outside of the brain and spinal cord in the subarachnoid space. CSF is secreted within the brain by a highly vascularized epithelial structure called the choroid plexus and circulates to sites in the subarachnoid space where it enters the venous blood system. The composition of CSF is highly regulated, and because CSF is in slow diffusional equilibrium with BECF, it helps regulate the composition of BECF. The choroid plexus can be thought of as the brain’s “kidney” in that it stabilizes the composition of CSF, just as the kidney stabilizes the composition of blood plasma. CSF fills the ventricles and subarachnoid space The ventricles of the brain are four small compartments located within the brain (Fig. 11-1A). Each ventricle contains a choroid plexus and is filled with CSF. The ventricles are linked together by channels, or foramina, that allow CSF to

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VENTRICLES OF THE BRAIN Choroid plexus

Left lateral ventricle

Right lateral ventricle

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4th ventricle Foramina of Monro (right and left) 3rd ventricle

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Pia mater

Choroid plexus of 3rd ventricle Cerebral aqueduct (of Sylvius) Foramen of Luschka (right lateral aperture) Choroid plexus of 4th ventricle

Roof of 4th ventricle Foramen of Magendie (median aperture)

Chapter 11 • The Neuronal Microenvironment

move easily between them. The two lateral ventricles are the largest and are symmetrically located within the cerebral hemispheres. The choroid plexus of each lateral ventricle is located along the inner radius of this horseshoe-shaped structure (Fig. 11-1B). The two lateral ventricles each communicate with the third ventricle, which is located in the midline between the thalami, through the two interventricular foramina of Monro. The choroid plexus of the third ventricle lies along the ventricle roof. The third ventricle communicates with the fourth ventricle by the cerebral aqueduct of Sylvius. The fourth ventricle is the most caudal ventricle and is located in the brainstem. It is bounded by the cerebellum superiorly and by the pons and medulla inferiorly. The choroid plexus of the fourth ventricle lies along only a portion of this ventricle’s tent-shaped roof. The fourth ventricle is continuous with the central canal of the spinal cord. CSF escapes from the fourth ventricle and flows into the subarachnoid space through three foramina: the two laterally placed foramina of Luschka and the midline opening in the roof of the fourth ventricle, called the foramen of Magendie. We shall see later how CSF circulates throughout the subarachnoid space of the brain and spinal cord. The brain and spinal cord are covered by two membranous tissue layers called the leptomeninges, which are in turn surrounded by a third, tougher layer. The innermost of these three layers is the pia mater; the middle is the arachnoid mater (or arachnoid membrane); and the outermost layer is the dura mater (Fig. 11-2). Between the arachnoid mater and pia mater (i.e., the leptomeninges) is the subarachnoid space, which is filled with CSF that escaped from the fourth ventricle. The CSF in the subarachnoid space completely surrounds the brain and spinal cord. In adults, the subarachnoid space and the ventricles with which they are continuous contain ∼150 mL of CSF, 30 mL in the ventricles and 120 mL in the subarachnoid spaces of the brain and spinal cord. The pia mater (Latin for “tender mother”) is a thin layer of connective tissue cells that is very closely applied to the surface of the brain and covers blood vessels as they plunge through the arachnoid into the brain. A nearly complete layer of astrocytic endfeet—the glia limitans—abuts the pia from the brain side and is separated from the pia by a basement membrane. The pia adheres so tightly to the associated glia limitans in some areas that they seem to be continuous with each other; this combined structure is sometimes called the pial-glial membrane or layer. This layer does not restrict diffusion of substances between the BECF and the CSF. The arachnoid membrane (Greek for “cobweb-like”) is composed of layers of cells, resembling those that make up the pia, linked together by tight junctions. The arachnoid isolates the CSF in the subarachnoid space from blood in the overlying vessels of the dura mater. The cells that constitute the arachnoid and the pia are continuous in the trabeculae

that span the subarachnoid space. These arachnoid and pial layers are relatively avascular; thus, the leptomeningeal cells that form them probably derive nutrition from the CSF that they enclose as well as from the ECF that surrounds them. The leptomeningeal cells can phagocytose foreign material in the subarachnoid space. The dura mater is a thick, inelastic membrane that forms an outer protective envelope around the brain. The dura has two layers that split to form the intracranial venous sinuses. Blood vessels in the dura mater are outside the blood-brain barrier (see later), and substances could easily diffuse from dural capillaries into the nearby CSF if it were not for the blood-CSF barrier created by the arachnoid. The brain floats in CSF, which acts as a shock absorber An important function of CSF is to buffer the brain from mechanical injury. The CSF that surrounds the brain reduces the effective weight of the brain from ∼1400 g to less than 50 g. This buoyancy is a consequence of the difference in the specific gravities of brain tissue (1.040) and CSF (1.007). The mechanical buffering that the CSF provides greatly diminishes the risk of acceleration-deceleration injuries in the same way that wearing a bicycle helmet reduces the risk of head injury. As you strike a tree, the foam insulation of the helmet gradually compresses and reduces the velocity of your head. Thus, the deceleration of your head is not nearly as severe as the deceleration of the outer shell of your helmet. The importance of this fluid suspension system is underscored by the consequences of reduced CSF pressure, which sometimes happens transiently after the diagnostic procedure of removal of CSF from the spinal subarachnoid space (see the box titled Lumbar Puncture). Patients with reduced CSF pressure experience severe pain when they try to sit up or to stand because the brain is no longer cushioned by shock-absorbing fluid and small gravity-induced movements put strain on pain-sensitive structures. Fortunately, the CSF leak that can result from lumbar puncture is only temporary; the puncture hole easily heals itself, with prompt resolution of all symptoms. The choroid plexuses secrete CSF into the ventricles, and the arachnoid granulations absorb it Most of the CSF is produced by the choroid plexuses, which are present in four locations (Fig. 11-1): the two lateral ventricles, the third ventricle, and the fourth ventricle. The capillaries within the brain appear to form a small amount of CSF. Total CSF production is ∼500 mL/day. Therefore, the entire volume of CSF, ∼150 mL, is replaced or “turns over” about three times each day.

Figure 11-1 The brain ventricles and the cerebrospinal fluid. A, This is a transparent view, looking from the left side of the brain. The two lateral ventricles communicate with the third ventricle, which in turn communicates with the fourth ventricle. B, Each ventricle contains a choroid plexus, which secretes CSF. The CSF escapes from the fourth ventricle and into the subarachnoid space through the two lateral foramina of Luschka and the single foramen of Magendie.

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Arachnoid membrane

Dura mater

Arachnoid granulations

Superior sagittal sinus

Subarachnoid space Trabecula Leptomeninges

Arachnoid mater Pia mater Glia limitans Cerebral cortex

Dura mater

Ventricle

Ependymal cells

Figure 11-2 The meninges and ependymal cells. The figure represents a coronal section through the anterior portion of the brain. The upper inset shows the three layers of meninges: the dura mater, which here is split into two layers to accommodate the superior sagittal sinus (filled with venous blood); the arachnoid mater, which is formed by cells that are interconnected by tight junctions; and the pia mater, which closely adheres to a layer composed of astrocyte endfeet that are covered by a basement membrane (glia limitans). The lower inset shows ependymal cells lining the interior of the frontal horn of the left ventricle. Both the subarachnoid space and the cavities of the ventricles are filled with CSF.

Secretion of new CSF creates a slight pressure gradient, which drives the circulation of CSF from its ventricular sites of origin into the subarachnoid space through three openings in the fourth ventricle, as discussed earlier. CSF percolates throughout the subarachnoid space and is finally absorbed into venous blood in the superior sagittal sinus, which lies between the two cerebral hemispheres (Fig. 11-2). The sites of absorption are specialized evaginations of the arachnoid membrane into the venous sinus (Fig. 113A). These absorptive sites are called pacchionian granulations or simply arachnoid granulations when they are large (up to 1 cm in diameter) and arachnoid villi if their size is microscopic. These structures act as pressure-sensitive, one-way valves for bulk CSF clearance; CSF can cross into venous blood, but venous blood cannot enter CSF. The actual mechanism of CSF absorption may involve transcytosis (see Chapter 20), the formation of giant fluid-containing vacuoles that cross from the CSF side of the arachnoid epithelial cells to the blood side (Fig. 11-3A).

CSF may also be absorbed into spinal veins from herniations of arachnoid cells into these venous structures. Net CSF movement into venous blood is promoted by the pressure of the CSF, which is higher than that of the venous blood. When intracranial pressure (equivalent to CSF pressure) exceeds ∼70 mm H2O, absorption commences and increases with intracranial pressure (Fig. 11-3B). In contrast to CSF absorption, CSF formation is not sensitive to intracranial pressure. This arrangement helps stabilize intracranial pressure. If intracranial pressure increases, CSF absorption selectively increases as well so that absorption exceeds formation (Fig. 11-3B). This response results in a lower CSF volume and a tendency to counteract the increased intracranial pressure. However, if absorption of CSF is impaired even at an initially normal intracranial pressure, CSF volume increases and causes an increase in intracranial pressure. Such an increase in intracranial pressure can lead to a disturbance in brain function.

Chapter 11 • The Neuronal Microenvironment

Lumbar Puncture

A

MECHANISM OF CSF ABSORPTION Venous sinus

O

ne of the most important diagnostic tests in neurology is the sampling of CSF by lumbar puncture. Critical information about the composition of CSF and about intracranial pressure can be obtained from this procedure. The anatomist Vesalius noted in 1543 that the ventricles are filled with a clear fluid, but the diagnostic technique of placing a needle into the lumbar subarachnoid space to obtain CSF was not introduced until 1891 by the neurologist Heinrich Quincke. The method of lumbar puncture is dictated by spine anatomy. In adults, the spinal cord ends at the interspace between L1 and L2 (see Chapter 10). A hollow needle for sampling of CSF can be safely inserted into the subarachnoid space at the level of the L3-L4 interspace, well below the end of the spinal cord. Once the needle is in the subarachnoid space, the physician attaches it to a manometer to measure pressure. With the patient lying on the side, normal pressure varies from 100 to 180 mm H2O, or 7 to 13 mm Hg. With the subject in this position and in the absence of a block to the free circulation of CSF, lumbar CSF pressure roughly corresponds to intracranial pressure. The physician can demonstrate direct communication of the pressure in the intracranial compartment to the lumbar subarachnoid space by gently compressing the external jugular veins in the neck for 10 seconds. This maneuver, called the Queckenstedt test, rapidly increases intracranial pressure because it increases the volume of intracranial venous blood. It quickly leads to an increase in lumbar pressure, which just as rapidly dissipates when the jugular pressure is removed. CSF pressure can become elevated because of a pathological mass within the cranium, such as a tumor or collection of blood, or because the brain is swollen as a result of injury or infection (see the later box titled Cerebral Edema). If a “mass lesion” (i.e., any pathological process that occupies intracranial space) is large or critically placed, it can displace the brain and cause interference with the free circulation of CSF. For example, an expanding mass in the cerebellum can force the inferior part of the cerebellum into the foramen magnum and block flow of CSF into the spinal subarachnoid space. Under these conditions, performance of lumbar puncture can precipitate a neurological catastrophe. If a needle is placed in the lumbar subarachnoid space and fluid is removed for diagnostic examination or leaks out after the needle is removed, the ensuing decrease in pressure in the lumbar space creates a pressure gradient across the foramen magnum and potentially forces the brain down into the spinal canal. This disaster is called herniation. For this reason, a computed tomographic scan or magnetic resonance image of the head is usually obtained before a lumbar puncture is attempted; the imaging study can rule out the possibility of a large intracranial lesion that might raise intracranial pressure and increase the risk of herniation when the subarachnoid space is punctured and CSF withdrawn. The Queckenstedt test must also be avoided when an intracranial mass is suspected because it could enhance the pressure gradient and hasten herniation.

Arachnoid villus

Dura mater Subarachnoid CSF Rise in pressure

Venous sinus

CSF B

RATE OF CSF ABSORPTION 1.6 CSF volume is constant in time when absorption equals formation (steady state). 1.2 Absorption Flow (ml/min)

0.8

0.4 CSF formation

0 0

70

100 112 CSF pressure (mm H2O)

200

Figure 11-3 Absorption of CSF. A, Arachnoid villi—or the larger arachnoid granulations (not shown)—are specialized evaginations of the arachnoid membrane through the dura mater and into the lumen of the venous sinus. The absorption of CSF may involve transcytosis. Note that arachnoid villi and granulations serve as oneway valves; fluid cannot move from the vein to the subarachnoid space. B, The rate of CSF formation is virtually insensitive to changes in the pressure of the CSF. On the other hand, the absorption of CSF increases steeply at CSF pressures above ∼70 mm H2O.

The epithelial cells of the choroid plexus secrete the CSF Each of the four choroid plexuses is formed during embryological development by invagination of the tela choroidea into the ventricular cavity (Fig. 11-4). The tela choroidea consists of a layer of ependymal cells covered by the pia mater and its associated blood vessels. The choroid epithelial cells (Fig. 11-4, first inset) are specialized ependymal cells and therefore contiguous with the ependymal lining of the ventricles at the margins of the choroid plexus. Choroid

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epithelial cells are cuboidal and have an apical border with microvilli and cilia that project into the ventricle (i.e., into the CSF). The plexus receives its blood supply from the anterior and posterior choroidal arteries; blood flow to the plexuses—per unit mass of tissue—is ∼10-fold greater than the average cerebral blood flow. Sympathetic and parasympathetic nerves innervate each plexus, and sympathetic input appears to inhibit CSF formation. A high density of relatively

Normal-Pressure Hydrocephalus

I

mpaired CSF absorption is one mechanism proposed to explain a clinical form of ventricular enlargement called normal-pressure hydrocephalus. This condition is somewhat misnamed because the intracranial pressure is often intermittently elevated. Damage to the arachnoid villi can occur most commonly from infection or inflammation of the meninges or from the presence of an irritating substance, such as blood in the CSF after a subarachnoid hemorrhage. A spinal tap reveals normal pressure readings, but computed tomography or magnetic resonance imaging of the head shows enlargement of all four ventricles. Patients with normal-pressure hydrocephalus typically have progressive dementia, urinary incontinence, and gait disturbance, probably caused by stretching of axon pathways that course around the enlarged ventricles. A flexible plastic tube can be placed in one of the lateral ventricles to shunt CSF to venous blood or to the peritoneal cavity, thereby reducing CSF pressure. This procedure may reduce ventricular size and decrease neurological symptoms. The “shunting” procedure is also used for patients with obstructive hydrocephalus. In this condition, CSF outflow from the ventricles is blocked, typically at the aqueduct of Sylvius.

TABLE 11-1

leaky capillaries is present within each plexus; as discussed later, these capillaries are outside the blood-brain barrier. The choroid epithelial cells are bound to one another by tight junctions that completely encircle each cell, an arrangement that makes the epithelium an effective barrier to free diffusion. Thus, although the choroid capillaries are outside the blood-brain barrier, the choroid epithelium insulates the ECF around these capillaries (which has a composition more similar to that of arterial blood) from the CSF. Moreover, the thin neck that connects the choroid plexus to the rest of the brain isolates the ECF near the leaky choroidal capillaries from the highly protected BECF in the rest of the brain. The composition of CSF differs considerably from that of plasma; thus, CSF is not just an ultrafiltrate of plasma (Table 11-1). For example, CSF has lower concentrations of K+ and amino acids than plasma does, and it contains almost no protein. Moreover, the choroid plexuses rigidly maintain the concentration of ions in CSF in the face of large swings in ion concentration in plasma. This ion homeostasis includes K+, H+/HCO−3, Mg2+, Ca2+, and, to a lesser extent, Na+ and Cl−. All these ions can affect neural function, hence the need for tight homeostatic control. The neuronal microenvironment is so well protected from the blood by the choroid plexuses and the rest of the blood-brain barrier that essential micronutrients, such as vitamins and trace elements that are needed in very small amounts, must be selectively transported into the brain. Some of these micronutrients are transported into the brain primarily by the choroid plexus and others primarily by the endothelial cells of the blood vessels. In comparison, the brain continuously metabolizes relatively large amounts of “macronutrients,” such as glucose and some amino acids. CSF forms in two sequential stages. First, ultrafiltration of plasma occurs across the fenestrated capillary wall (see

Composition of Cerebrospinal Fluid

Solute

Plasma (mM of protein-free plasma)

CSF (mM)

CSF/Plasma Ratio

Na+

153

147

0.96

K+

4.7

2.9

0.62

Ca2+

1.3 (ionized)

1.1 (ionized)

0.85

Mg2+

0.6 (ionized)

1.1 (ionized)

1.8

Cl−

110

113

1.03

HCO3−

24

22

0.92

H2PO4− and HPO2− 4

0.75 (ionized)

0.9

1.2

pH

7.40

7.33

Amino acids

2.6

0.7

0.27

Proteins

7 g/dL

0.03 g/dL

0.004

Osmolality (mOsm)

290

290

1.00

Chapter 11 • The Neuronal Microenvironment

Epithelium

Lateral ventricle

Choroid plexus

Cilia Microvilli Choroidal epithelium Apical membrane Cerebrospinal fluid (CSF)

Third ventricle Capillaries

Basolateral membrane

Apical end

Basal end HCO3– CO2 + H+ OH– CA

K+ K+ K

+

Cl–

Na+

Na+

H2O

Cl– HCO3–

Cl–

Na+ 3 HCO–3 H2O

Cl–

Na+ HCO3– Na+ 2 HCO3–

Na+ Cl– HCO3– H2O

H2O

+

K +

K

Na+ Cl

Na+ +

K

2 Cl–

CSF

–

Transepithelial fluxes

K+ Epithelial cell

Tight junction

Extracellular space

Figure 11-4 Secretion of CSF by the choroid plexus. The top panel shows the location of the choroid plexuses in the two lateral ventricles and the third ventricle. The middle panel shows the organization of a single fold of choroidal epithelial cells, with the basolateral membranes of the epithelial cells overlying capillaries and the apical membranes facing the CSF. The bottom panel shows a single choroid epithelial cell and several of the transporters and channels that are believed to play a role in the isosmotic secretion of CSF. CA, carbonic anhydrase.

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Chapter 20) into the ECF beneath the basolateral membrane of the choroid epithelial cell. Second, choroid epithelial cells secrete fluid into the ventricle. CSF production occurs with a net transfer of NaCl and NaHCO3 that drives water movement isosmotically (Fig. 11-4, large transepithelial arrow in the right inset). The renal proximal tubule (see Chapter 35) and small intestine (see Chapter 5) also perform near-isosmotic transport, but in the direction of absorption rather than secretion. In addition, the choroid plexus conditions CSF by absorbing K+ (Fig. 11-4, small transepithelial arrow in the right inset) and certain other substances (e.g., a metabolite of serotonin, 5-hydroxyindoleacetic acid). The upper portion of the right inset of Figure 11-4 summarizes the ion transport processes that mediate CSF secretion. The net secretion of Na+ from plasma to CSF is a two-step process. The Na-K pump in the choroid plexus, unlike in other epithelia (see Chapter 5), is unusual in being located on the apical membrane, where it moves Na+ out of the cell into the CSF—the first step. This active movement of Na+ out of the cell generates an inward Na+ gradient across the basolateral membrane, energizing basolateral Na+ entry— the second step—through Na-H exchange and Na+-coupled HCO3− transport. In the case of Na-H exchange, the limiting factor is the availability of intracellular H+, which carbonic anhydrase generates, along with HCO−3 , from CO2 and H2O. Thus, blocking of the Na-K pump with ouabain halts CSF formation, whereas blocking of carbonic anhydrase with acetazolamide slows CSF formation. The net secretion of Cl−, like that of Na+, is a two-step process. The first step is the intracellular accumulation of Cl− by the basolateral Cl-HCO3 exchanger. Note that the net effect of parallel Cl-HCO3 exchange and Na-H exchange is NaCl uptake. The second step is efflux of Cl− across the apical border into the CSF through either a Cl− channel or a K/Cl cotransporter. HCO3− secretion into CSF is important for neutralizing acid produced by CNS cells. At the basolateral membrane, the epithelial cell probably takes up HCO3− directly from the plasma filtrate through electroneutral Na/HCO3 cotransporters (see Fig. 5-11F) and the Na+-driven Cl-HCO3 exchanger (see Fig. 5-13C). As noted before, HCO3− can also accumulate inside the cell after CO2 entry. The apical step, movement of intracellular HCO3− into the CSF, probably occurs by an electrogenic Na/HCO3 cotransporter (see Fig. 5-11D) and Cl− channels (which are generally permeable to HCO3−). The lower portion of the right inset of Figure 11-4 summarizes K+ absorption from the CSF. The epithelial cell takes up K+ by the Na-K pump and the Na/K/Cl cotransporter at the apical membrane (see Fig. 5-11G). Most of the K+ recycles back to the CSF, but a small amount exits across the basolateral membrane and enters the blood. The concentration of K+ in freshly secreted CSF is ∼3.3 mM. Even with very large changes in plasma [K+], the [K+] in CSF changes very little. The value of [K+] in CSF is significantly lower in the subarachnoid space than in choroid secretions, which suggests that brain capillary endothelial cells remove extracellular K+ from the brain. Water transport across the choroid epithelium is driven by a small osmotic gradient favoring CSF formation. This water movement is facilitated by expression of the water

channel aquaporin 1 on both the apical and basal membranes as in renal proximal tubule (see Chapter 35).

BRAIN EXTRACELLULAR SPACE Neurons, glia, and capillaries are packed tightly together in the CNS The average width of the space between brain cells is ∼20 nm, which is about three orders of magnitude smaller than the diameter of either a neuron or glial cell body (Fig. 11-5). However, because the surface membranes of neurons and glial cells are highly folded (i.e., have a large surface-tovolume ratio), the BECF in toto has a sizable volume fraction, ∼20%, of the total brain volume. The fraction of the brain that is occupied by BECF varies somewhat in different

At

nf

As SR

Den

Ax f

S

Den

At

Ax2 As

Ax1 Den

Ax1

S

Ax2 At S

Figure 11-5 Tight packing of neurons and astrocytes. This is an electron micrograph of a section of the spinal cord from an adult rat showing the intermingling and close apposition of neurons and glial cells, mainly astrocytes. Neurons and glial cells are separated by narrow clefts that are ∼20 nm wide and not visible at this magnification. The BECF in this space creates a tortuous path for the extracellular diffusion of solutes. Astrocyte processes are colored. As, astrocytes; At, en passant synapses; Ax, unmyelinated axons, Ax1 and Ax2, myelinated axons; Den, dendrites; f, astrocytic fibrils; nf, neurofilaments; S, synapses; SR, smooth endoplasmic reticulum. (Modified from Peters A, Palay SL, Webster H: The Fine Structure of the Nervous System. Philadelphia: WB Saunders, 1976.)

Chapter 11 • The Neuronal Microenvironment

areas of the CNS. Moreover, because brain cells can increase volume rapidly during intense neural activity, the BECF fraction can reversibly decrease within seconds from ∼20% to ∼17% of brain volume. Even though the space between brain cells is extremely small, diffusion of ions and other solutes within this thin BECF space is reasonably high. However, a particle that diffuses through the BECF from one side of a neuron to the other must take a circuitous route that is described by a parameter called tortuosity. For a normal width of the cellto-cell spacing, this tortuosity reduces the rate of diffusion by ∼60% compared with movement in free solution. Decreases in cell-to-cell spacing can further slow diffusion. For example, brain cells, especially glial cells, swell under certain pathological conditions and sometimes with intense neural activity. Cell swelling is associated with a reduction in BECF because water moves from the BECF into cells. The intense cell swelling associated with acute anoxia, for example, can reduce BECF volume from ∼20% to ∼5% of total brain volume. By definition, this reduced extracellular volume translates to reduced cell-to-cell spacing, further slowing the extracellular movement of solutes between the blood and brain cells (see the box titled Cerebral Edema). The BECF is the route by which important molecules such as oxygen, glucose, and amino acids reach brain cells and by which the products of metabolism, including CO2 and catabolized neurotransmitters, leave the brain. The BECF also permits molecules that are released by brain cells to diffuse to adjacent cells. Neurotransmitter molecules

released at synaptic sites, for example, can spill over from the synaptic cleft and contact nearby glial cells and neurons, in addition to their target postsynaptic cell. Glial cells express neurotransmitter receptors, and neurons have extrajunctional receptors; therefore, these cells are capable of receiving “messages” sent through the BECF. Numerous trophic molecules secreted by brain cells diffuse in the BECF to their targets. Intercellular communication by way of the BECF is especially well suited for the transmission of tonic signals that are ideal for longer term modulation of the behavior of aggregates of neurons and glial cells. The chronic presence of variable amounts of neurotransmitters in the BECF supports this idea. The CSF communicates freely with the BECF, thereby stabilizing the composition of the neuronal microenvironment CSF in the ventricles and the subarachnoid space can exchange freely with BECF across two borders, the pia mater and ependymal cells. The pial-glial membrane (Fig. 11-2, upper inset) has paracellular gaps (see Chapter 2) through which substances can equilibrate between the subarachnoid space and BECF. Ependymal cells (Fig. 11-2, lower inset) are special glial cells that line the walls of the ventricles and form the cellular boundary between the CSF and the BECF. These cells form gap junctions between themselves that mediate intercellular communication, but they do not create a tight epithelium (see Chapter 5). Thus, macromolecules and ions

Cerebral Edema

A

lmost any type of insult to the brain causes cell swelling. This swelling is frequently accompanied by a net accumulation of water within the brain that is referred to as cerebral edema. Cell swelling in the absence of net water accumulation in the brain does not constitute cerebral edema. For example, intense neural activity causes a rapid shift of fluid from the BECF to the intracellular space, with no net change in brain water content. In cerebral edema, the extra water comes from the blood, as shown in Figure 11-6. The mechanisms by which glial cells and neurons swell are not completely understood. Neuron cell bodies and dendrites, but not axons, swell when they are exposed to high concentrations of the neurotransmitter glutamate. This transmitter, along with others, is released to the BECF in an uncontrolled fashion with brain injury. Activation of ionotropic glutamate receptors (see Chapter 13) allows Na+ to enter neurons, and water and Cl− follow passively. Glial cells, both astrocytes and oligodendrocytes, swell vigorously under pathological conditions. One mechanism of glial swelling is an increase in [K+]o, which is a common ionic disturbance in a variety of brain pathological processes. This elevated [K+]o causes a net uptake of K+, accompanied by the passive influx of Cl− and water. Cerebral edema can be life-threatening when it is severe. The problem is a mechanical one. The skull is an inelastic container housing three relatively noncompressible substances: brain, CSF, and blood. A significant increase in the volume of CSF, blood, or brain rapidly causes increased pressure within

the skull (Fig. 11-3). If the cerebral edema is generalized, it can be tolerated until intracerebral pressure exceeds arterial blood pressure, at which point blood flow to the brain stops, with disastrous consequences. Fortunately, sensors in the medulla detect the increased intracerebral pressure and can partially compensate (Cushing reflex), to a point, by increasing arterial pressure (see Chapter 24). Focal cerebral edema (i.e., edema involving an isolated portion of the brain) causes problems by displacing nearby brain tissue. This abnormality may result in distortion of normal anatomical relationships, with selective pressure on critical structures such as the brainstem. Clinical evidence of cerebral edema results directly from the increased intracranial pressure and includes headache, vomiting, altered consciousness, and focal neurological problems such as stretching and dysfunction of the sixth cranial nerve. Hyperventilation is the most effective means of combating the acute increase in intracranial pressure associated with severe cerebral edema. Hyperventilation causes a prompt respiratory alkalosis (see Chapter 28) that is rapidly translated to an increase in the pH surrounding vascular smooth muscle, thereby triggering vasoconstriction and reduced cerebral blood flow (see Chapter 24). Thus, total intracranial blood content falls, with a rapid subsequent drop in intracranial pressure. Alternatively, the brain can be partially dehydrated by adding osmoles to the blood in the form of intravenously administered mannitol (see Chapter 5).

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A

CELLULAR UPTAKE OF WATER FROM BLOOD

B

70

Edematous neuron

ΔP2>ΔP1

60 50

Red blood cells

Inflow of fluid

RISE IN INTRACRANIAL PRESSURE WITH VOLUME

Inflow of fluid

Edematous astrocyte

Edematous endothelial cell of blood capillary

Intracranial pressure 40 (mm Hg) 30

Compliance falls as volume rises.

ΔP2 ΔV ΔP1

20

ΔV

10 0 Volume

Edematous astrocyte

Figure 11-6 Cerebral edema. A, In cerebral edema, the brain fluid that accumulates comes from the vascular compartment. Cell swelling due to the mere shift of fluid from the extracellular to the intracellular fluid is not cerebral edema. B, Although small increases in intracranial volume have little effect on pressure, additional increases in volume cause potentially life-threatening increases in pressure. Note that compliance (i.e., ΔV/ΔP) falls at increasing volumes.

can also easily pass through this cellular layer through paracellular openings (some notable exceptions to this rule are considered later) and equilibrate between the CSF in the ventricle and the BECF. Because CSF and BECF can readily exchange with one another, it is not surprising that they have a similar chemical composition. For example, [K+] is ∼3.3 mM in freshly secreted CSF and ∼3 mM in both the CSF of the subarachnoid space (Table 11-1) and BECF. The [K+] of blood is ∼4.5 mM. However, because of the extent and vast complexity of the extracellular space, changes in the composition of CSF are reflected slowly in the BECF and probably incompletely. CSF is an efficient waste management system because of its high rate of production, its circulation over the surface of the brain, and the free exchange between CSF and BECF. Products of metabolism and other substances released by cells, perhaps for signaling purposes, can diffuse into the chemically stable CSF and ultimately be removed on a continuous basis either by bulk resorption into the venous sinuses or by active transport across the choroid plexus into the blood. For example, choroid plexus actively absorbs the breakdown products of the neurotransmitters serotonin (i.e., 5-hydroxyindoleacetic acid) and dopamine (i.e., homovanillic acid). The ion fluxes that accompany neural activity cause large changes in extracellular ion concentration As discussed in Chapter 7, ionic currents through cell membranes underlie the synaptic and action potentials by which

neurons communicate. These currents lead to changes in the ion concentrations of the BECF. It is estimated that even a single action potential can transiently lower [Na+]o by ∼0.75 mM and increase [K+]o by a similar amount. Repetitive neuronal activity causes larger perturbations in these extracellular ion concentrations. Because ambient [K+]o is much lower than [Na+]o, activity-induced changes in [K+]o are proportionately larger and are of special interest because of the important effect that [K+]o has on membrane potential (Vm). For example, K+ accumulation in the vicinity of active neurons depolarizes nearby glial cells. In this way, neurons signal to glial cells the pattern and extent of their activity. Even small changes in [K+]o can alter metabolism and ionic transport in glial cells and may be used for signaling. Changes in the extracellular concentrations of certain common amino acids, such as glutamate and glycine, can also affect neuronal Vm and synaptic function by acting at specific receptor sites. If the nervous system is to function reliably, its signaling elements must have a regulated environment. Glial cells and neurons both function to prevent excessive extracellular accumulation of K+ and neurotransmitters.

THE BLOOD-BRAIN BARRIER The blood-brain barrier prevents some blood constituents from entering the brain extracellular space The unique protective mechanism called the blood-brain barrier was first demonstrated by Ehrlich in 1885. He injected aniline dyes intravenously and discovered that the soft tissues

Chapter 11 • The Neuronal Microenvironment

of the body, except for the brain, were uniformly stained. Even though aniline dyes, such as trypan blue, extensively bind to serum albumin, the dye-albumin complex passes across capillaries in most areas of the body, but not the brain. This ability to exclude certain substances from crossing CNS blood vessels into the brain tissue is due to the blood-brain barrier. We now recognize that a blood-brain barrier is present in all vertebrates and many invertebrates as well. The need for a blood-brain barrier can be understood by considering that blood is not a suitable environment for neurons. Blood is a complex medium that contains a large variety of solutes, some of which can vary greatly in concentration, depending on factors such as diet, metabolism, illness, and age. For example, the concentration of many amino acids increases significantly after a protein-rich meal. Some of these amino acids act as neurotransmitters within the brain, and if these molecules could move freely from the blood into the neuronal microenvironment, they would nonselectively activate receptors and disturb normal neurotransmission. Similarly, strenuous exercise can increase plasma concentrations of K+ and H+ substantially. If these ionic changes were communicated directly to the microenvironment of neurons, they could disrupt ongoing neural activity. Running a foot race might temporarily lower your IQ. Increases in [K+]o would depolarize neurons and thus increase their likelihood of firing and releasing transmitter. H+ can nonspecifically modulate neuronal excitability and influence the action of certain neurotransmitters. A broad range of blood constituents—including hormones, other ions, and inflammatory mediators such as cytokines—can influence the behavior of neurons or glial cells, which can express receptors for these molecules. For the brain to function efficiently, it must be spared such influences. The choroid plexus and several restricted areas of the brain lack a blood-brain barrier; that is, they are supplied by leaky capillaries. Intra-arterially injected dyes can pass into the brain extracellular space at these sites through gaps between endothelial cells. The BECF in the vicinity of these leaky capillaries is similar to blood plasma more than to normal BECF. The small brain areas that lack a blood-brain barrier are called the circumventricular organs because they surround the ventricular system; these areas include the area postrema, posterior pituitary, median eminence, organum vasculosum laminae terminalis, subfornical organ, subcommissural organ, and pineal gland (Fig. 11-7). The ependymal cells that overlie the leaky capillaries in some of these regions (e.g., the choroid plexus) are linked together by tight junctions that form a barrier between the local BECF and the CSF, which must be insulated from the variability of blood composition. Whereas dyes with molecular weights up to 5000 can normally pass from CSF across the ependymal cell layer into the BECF, they do not pass across the specialized ependymal layer at the median eminence, area postrema, and infundibular recess. At these points, the localized BECF-CSF barrier is similar to the one in the choroid plexus. These specialized ependymal cells often have long processes that extend to capillaries within the portal circulation of the pituitary. Although the function of these cells is not known, it has been suggested that they may form a special route for neurohumoral signaling; molecules secreted by hypothalamic cells into the third ventricle could be taken up by these

Subcommissural organ

Pineal gland

Subfornical organ

OVLT

Posterior pituitary

Median eminence

Area postrema

Figure 11-7 Leaky regions of the blood-brain barrier: the circumventricular organs. The capillaries of the brain are leaky in several areas: the area postrema, the posterior pituitary, the subfornical organ, the median eminence, the pineal gland, and the organum vasculosum laminae terminalis (OVLT). In these regions, the neurons are directly exposed to the solutes of the blood plasma. A midline sagittal section is shown.

cells and transmitted to the general circulation or to cells in the pituitary. Neurons within the circumventricular organs are directly exposed to blood solutes and macromolecules; this arrangement is believed to be part of a neuroendocrine control system for maintaining such parameters as osmolality (see Chapter 40) and appropriate hormone levels, among other things. Humoral signals are integrated by connections of circumventricular organ neurons to endocrine, autonomic, and behavioral centers within the CNS. In the median eminence, neurons discharge “releasing hormones,” which diffuse into leaky capillaries for carriage through the pituitary portal system to the anterior pituitary. The lack of a blood-brain barrier in the posterior pituitary is necessary to allow hormones that are released there to enter the general circulation (see Chapter 47). In the organum vasculosum laminae terminalis, leakiness is important in the action of cytokines from the periphery, which act as signals to temperature control centers that are involved in fever (see Chapter 59). Continuous tight junctions link brain capillary endothelial cells The blood-brain barrier should be thought of as a physical barrier to diffusion from blood to brain ECF and as a selective set of regulatory transport mechanisms that determine how certain organic solutes move between the blood and brain. Thus, the blood-brain barrier contributes to stabilization and protection of the neuronal microenvironment by

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facilitating the entry of needed substances, removing waste metabolites, and excluding toxic or disruptive substances. The structure of brain capillaries differs from that of capillaries in other organs. Capillaries from other organs generally have small, simple openings—or clefts—between their endothelial cells (Fig. 11-8A). In some of these other organs, windows, or fenestrae, provide a pathway that bypasses the cytoplasm of capillary endothelial cells. Thus, in most capillaries outside the CNS, solutes can easily diffuse through the clefts and fenestrae. The physical barrier to solute diffusion in brain capillaries (Fig. 11-8B) is provided by the capillary endothelial cells, which are fused to each other by continuous tight junctions (or zonula occludens; see Chapter 2). The tight junctions prevent water-soluble ions and molecules from passing from the blood into the brain through the paracellular route. Not surprisingly, the electrical resistance of the cerebral capillaries is 100 to 200 times higher than that of most other systemic capillaries. Elsewhere in the systemic circulation, molecules may traverse the endothelial cell by the process of transcytosis (see Chapter 20). In cerebral capillaries, transcytosis is uncommon, and brain endothelial cells have fewer endocytic vesicles than do systemic capillaries. However, brain endothelial cells have many more mitochondria than systemic endothelial cells do, which may reflect the high metabolic demands imposed on brain endothelial cells by active transport. Other interesting features of brain capillaries are the thick basement membrane that underlies the endothelial cells, the presence of occasional pericytes within the basement membrane sheath, and the astrocytic endfeet (or processes) that provide a nearly continuous covering of the capillaries and other blood vessels. Astrocytes may play a crucial role in forming tight junctions between endothelial cells; experiments have shown that these glial cells can induce the formation of tight junctions between endothelial cells derived from capillaries outside the CNS. The close apposition of the astrocyte endfoot to the capillary also could facilitate transport of substances between these cells and blood.

A

NONBRAIN SYSTEMIC CAPILLARY

Transcytosis

B BRAIN CAPILLARY

Astrocytic endfoot

Intercellular cleft Lipid soluble

Lipid soluble Carrier mediated

Fenestra Tight junction

C

Endothelial cells sit on a thick basal lamina basement membrane.

BRAIN-CAPILLARY ENDOTHELIAL CELL

Tight junction

+

K

Na+ O2

GLUT1 Glucose

CO2

GLUT1

Na+

Glucose

H2 O Endfoot O2

CO2

H+ Na+

K+ +

Na K+

2 Cl–

Uncharged and lipid-soluble molecules more readily pass the blood-brain barrier The capacity of the brain capillaries to exclude large molecules is strongly related to the molecular mass of the molecule and its hydrated diameter (Table 11-2). With a mass of 61 kDa, prealbumin is 14 times as concentrated in blood as in CSF (essentially equivalent to BECF for purposes of this comparison), whereas fibrinogen, which has a molecular mass of 340 kDa, is ∼5000 times more concentrated in blood than in CSF. Diffusion of a solute is also generally limited by ionization at physiological pH, by low lipid solubility, and by binding to plasma proteins. For example, gases such as CO2 and O2 and drugs such as ethanol, caffeine, nicotine, heroin, and methadone readily cross the blood-brain barrier. However, ions such as K+ or Mg2+ and protein-bound metabolites such as bilirubin have restricted access to the brain. Finally, the blood-brain barrier is permeable to water because of the presence of water channels in the endothelial cells. Thus, water moves across the blood-brain barrier in response to changes in plasma osmolarity. When dehydration raises

Brain capillary lumen

Basement membrane

BECF

Figure 11-8 The blood-brain barrier function of brain capillaries. A, Capillaries from most other organs often have interendothelial clefts or fenestrae, which makes them relatively leaky. B, Brain capillaries are not leaky and have reduced transcytosis. C, Continuous tight junctions connect the endothelial cells in the brain, making the capillaries relatively tight.

the osmolality of blood plasma (see the box titled Disorders of Extracellular Osmolality in Chapter 5), the increased osmolality of the CSF and BECF can affect the behavior of brain cells. Cerebral capillaries also express enzymes that can affect the movement of substances from blood to brain and vice versa. Peptidases, acid hydrolases, monoamine oxidase, and other enzymes are present in CNS endothelial cells and can

Chapter 11 • The Neuronal Microenvironment

TABLE 11-2

Comparison of Proteins in Blood Plasma versus Cerebrospinal Fluid

Protein

Molecular Mass (kDa)

Hydrodynamic Radius (nM)

Plasma/CSF Ratio*

Prealbumin

61

3.3

14

Albumin

69

3.6

240

Transferrin

81

3.7

140

Ceruloplasmin

152

4.7

370

IgG

150

5.3

800

IgA

150

5.7

1350

α 2-Macroglobulin

798

9.4

1100

Fibrinogen

340

11.0

4940

IgM

800

12.1

1170

2240

12.4

6210

β-Lipoprotein

*The greater the plasma/CSF ratio, the more the blood-brain barrier excludes the protein from the CSF.

degrade a range of biologically active molecules, including enkephalins, substance P, proteins, and norepinephrine. Orally administered dopamine is not an effective treatment of Parkinson disease (see Chapter 13), a condition in which CNS dopamine is depleted, because dopamine is rapidly broken down by monoamine oxidase in the capillaries. Fortunately, the dopamine precursor compound l-dopa is effective for this condition. Neutral amino acid transporters in capillary endothelial cells move l-dopa to the BECF, where presynaptic terminals take up the l-dopa and convert it to dopamine in a reaction that is catalyzed by dopa decarboxylase. Transport by capillary endothelial cells contributes to the blood-brain barrier Two classes of substances can pass readily between blood and brain. The first consists of the small, highly lipid soluble molecules discussed in the preceding section. The second group consists of water-soluble compounds—either critical nutrients entering or metabolites exiting the brain—that traverse the blood-brain barrier by specific transporters. Examples include glucose, several amino acids and neurotransmitters, nucleic acid precursors, and several organic acids. Two major transporter groups provide these functions: the SLC superfamily and ABC transporters (see Chapter 5). As is the case for other epithelial cells, capillary endothelial cells selectively express these and other membrane proteins on either the luminal or basal surface. Although the choroid plexuses secrete most of the CSF, brain endothelial cells produce some interstitial fluid with a composition similar to that of CSF. Transporters such as those shown in Figure 11-8C are responsible for this CSF-

like secretion as well as for the local control of [K+] and pH in the BECF.

GLIAL CELLS Glial cells constitute half the volume of the brain and outnumber neurons The three major types of glial cells in the CNS are astrocytes, oligodendrocytes, and microglial cells (Table 11-3). As discussed in Chapter 10, the peripheral nervous system (PNS) contains other, distinctive types of glial cells, including satellite cells, Schwann cells, and enteric glia. Glial cells represent about half the volume of the brain and are more numerous than neurons. Unlike neurons, which have little capacity to replace themselves when lost, neuroglial (or simply glial) cells can proliferate throughout life. An injury to the nervous system is the usual stimulus for proliferation. Historically, glial cells were viewed as a type of CNS connective tissue whose main function was to provide support for the true functional cells of the brain, the neurons. This firmly entrenched concept remained virtually unquestioned for the better part of a century after the early description of these cells by Virchow in 1858. Knowledge about glial cells has accumulated slowly because these cells have proved far more difficult to study than neurons. Because glial cells do not exhibit easily recorded action potentials or synaptic potentials, these cells were sometimes referred to as silent cells. However, glial cells are now recognized as intimate partners with neurons in virtually every function of the brain.

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TABLE 11-3

Glial Types

Glial Cell Type

System

Location

GFAP

Astrocytes Fibrous Protoplasmic Radial glial cells Müller cells Bergmann glia Ependymal cells

CNS CNS CNS CNS CNS CNS

White matter Gray matter Throughout brain during development Retina Cerebellum Ventricular lining

Positive Weakly positive Positive Positive Positive Positive

Oligodendrocytes

CNS

Mainly white matter

Negative

Microglial cells

CNS

Throughout the brain

Negative

Satellite cells

PNS

Sensory and autonomic ganglia

Weakly positive

Schwann cells

PNS

Peripheral axons

Negative

Enteric glial cells

ENS

Gut wall

Positive

ENS, enteric nervous system; GFAP, glial fibrillary acidic protein; PNS, peripheral nervous system.

Endfoot Brain surface

Brain surface

Pial-glial membrane

Pial-glial membrane Endfoot Capillary

Capillary Endfeet surround capillary.

Fibrous astrocyte

Protoplasmic astrocyte

Figure 11-9 Astrocytes. The endfeet of both fibrous and protoplasmic astrocytes abut the pia mater and the capillaries.

Astrocytes supply fuel to neurons in the form of lactic acid Astrocytes have great numbers of extremely elaborate processes that closely approach both blood vessels and neurons. This arrangement led to the idea that astrocytes transport substances between the blood and neurons. This notion may be true, but it has not been proved. Throughout the brain, astrocytes envelop neurons, and both cells bathe in a common BECF. Therefore, astrocytes are ideally positioned to modify and to control the immediate environment of neurons. Most astrocytes in the brain are traditionally subdivided into fibrous and protoplasmic types. Fibrous astrocytes (found mainly in white matter) have long, thin, and well-defined processes; protoplasmic astrocytes (found mainly in gray matter) have shorter, frilly processes (Fig. 11-9). Astrocytes are evenly spaced. In cortical

regions, the dense processes of an individual astrocyte define its spatial domain, into which adjacent astrocytes do not encroach. The cytoskeleton of these and other types of astrocytes contains an identifying intermediate filament (see Chapter 2) that is composed of a unique protein called glial fibrillar acidic protein (GFAP). The basic physiological properties of both types of astrocyte are similar, but specialized features, such as the expression of neurotransmitter receptors, vary among astrocytes from different brain regions. During development, another type of astrocyte called the radial glial cell (see Chapter 10) is also present. As discussed in Chapter 10, these cells create an organized “scaffolding” by spanning the developing forebrain from the ventricle to the pial surface. Astrocytes in the retina and cerebellum are similar in appearance to radial glial cells. Like astrocytes elsewhere, these cells contain the intermediate filament

Chapter 11 • The Neuronal Microenvironment

GFAP. Retinal astrocytes, called Müller cells, are oriented so that they span the entire width of the retina. Bergmann glial cells in the cerebellum have processes that run parallel to the processes of Purkinje cells. Astrocytes store virtually all the glycogen present in the adult brain. They also contain all the enzymes needed for metabolizing glycogen. The brain’s high metabolic needs are primarily met by glucose transferred from blood because the brain’s glucose supply in the form of glycogen is very limited. In the absence of glucose from blood, astrocytic glycogen could sustain the brain for only 5 to 10 minutes. As implied, astrocytes can share with neurons the energy stored in glycogen, but not by the direct release of glucose into the BECF. Instead, astrocytes break glycogen down to glucose and even further to lactate, which is transferred to nearby neurons, where it can be aerobically metabolized (Fig. 11-10). The extent to which this metabolic interaction takes place under normal conditions is not known, but it may be important during periods of intense neuronal activity, when the demand for glucose exceeds the supply from blood. Astrocytes can also provide fuel to neurons in the form of lactate derived directly from glucose, independent of glycogen. Glucose entering the brain from blood first encounters the astrocytic endfoot. Although it can diffuse past this point to neurons, glucose may be preferentially taken up by astrocytes and shuttled through astrocytic glycolysis to lactic acid, a significant portion of which is excreted into the BECF surrounding neurons. Several observations support the notion that astrocytes provide lactate to neurons. First, astrocytes have higher anaerobic metabolic rates and export much more lactate than do neurons. Second, neurons and their axons function normally when glucose is replaced by lactate, and some neurons seem to prefer lactate to glucose as fuel.

Note that when they are aerobically metabolized, the two molecules of lactate derived from the breakdown of one molecule of glucose provide nearly as much ATP as the complete oxidation of glucose itself (28 versus 30 molecules of ATP; see Table 58-4 on p. 1231). The advantage of this scheme for neuronal function is that it provides a form of substrate buffering, a second energy reservoir that is available to neurons. The availability of glucose in the neuronal microenvironment depends on moment-to-moment supply from the blood and varies as a result of changes in neural activity. The concentration of extracellular lactate, however, is buffered against such variability by the surrounding astrocytes, which continuously shuttle lactate to the BECF through the metabolism of glucose or by breaking down glycogen. Astrocytes are predominantly permeable to K+ and also help regulate [K+]o The membrane potential of glial cells is more negative than that of neurons. For example, astrocytes have a Vm of about −85 mV, whereas the resting neuronal Vm is about −65 mV. Because the equilibrium potential for K+ is about −90 mV in both neurons and glia, the more negative Vm in astrocytes indicates that glial membranes have higher K+ selectivity than neuronal membranes do (see Chapter 6). Although glial cells express a variety of K+ channels, inwardly rectifying K+ channels seem to be important in setting the resting potential. These channels are voltage gated and are open at membrane potentials that are more negative than about −80 mV, close to the observed resting potential of astrocytes. Astrocytes express many other voltage-gated ion channels that were once thought to be restricted to neurons. The significance of voltage-gated Na+ and Ca2+ channels in glial cells is

Interstitial space Transastrocyte path GLUT1

Glycogen

Direct path

Glucose

Endothelium

GLUT3

5

5

3

3 2 Pyruvate

2 Lactate

2 Lactate +

H

H

2 Pyruvate

+

25

25

CO2 + H2O MCT1 BLOOD

ASTROCYTE

MCT2

CO2 + H2O

NEURON

Figure 11-10 Role of astrocytes in providing lactate as fuel for neurons. Neurons have two fuel sources. They can obtain glucose directly from the blood plasma, or they can obtain lactate from astrocytes. In the direct path, the oxidation of one glucose molecule provides 30 ATP molecules to the neuron. In the transastrocyte path, conversion of two lactates to two pyruvates, and then the subsequent oxidation of the pyruvate, provides 28 molecules of ATP to the neuron. GLUT1 and GLUT3, glucose transporters; MCT1 and MCT3, monocarboxylate cotransporters.

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unknown. Because the ratio of Na+ to K+ channels is low in adult astrocytes, these cells are not capable of regenerative electrical responses such as the action potential. One consequence of the higher K+ selectivity of astrocytes is that the Vm of astrocytes is far more sensitive than that of neurons to changes in [K+]o. For example, when [K+]o is raised from 4 to 20 mM, astrocytes depolarize by ∼25 mV versus only ∼5 mV for neurons. This relative insensitivity of neuronal resting potential to changes in [K+]o in the “physiological” range may have emerged as an adaptive feature that stabilizes the resting potential of neurons in the face of the transient increases in [K+]o that accompany neuronal activity. In contrast, natural stimulation, such as viewing visual targets of different shapes or orientations, can cause depolarizations of up to 10 mV in astrocytes of the visual cortex. The accumulation of extracellular K+ that is secondary to neural activity may serve as a signal—to glial cells—that is

A

MECHANISMS OF K+ UPTAKE BY ASTROCYTES Neuron

Extracellular space

Cl–

proportional to the extent of the activity. For example, small increases in [K+]o cause astrocytes to increase their glucose metabolism and to provide more lactate for active neurons. In addition, the depolarization that is triggered by the increased [K+]o leads to the influx of HCO3− into astrocytes by the electrogenic Na/HCO3 cotransporter (see Chapter 5); this influx of bicarbonate in turn causes a fall in extracellular pH that may diminish neuronal excitability. Not only do astrocytes respond to changes in [K+]o, they also help regulate it (Fig. 11-11A). The need for homeostatic control of [K+]o is clear because changes in brain [K+]o can influence transmitter release, cerebral blood flow, cell volume, glucose metabolism, and neuronal activity. Active neurons lose K+ into the BECF, and the resulting increased [K+]o tends to act as a positive feedback signal that increases excitability by further depolarizing neurons. This potentially unstable situation is combated by efficient mechanisms that expedite

B

SPATIAL BUFFERING

K+ Neuron

Glucose +

K

Na+

+

2K

Na+

Astrocyte

[K+]i

[K+]o = 12 mM

3 Na+ Astrocyte

K+ Na+

K+

2 Cl–

[K+]o > 3 mM (ceiling level ~12 mM) Gap junctions

[K+]i = 108 mM

[K+]o = 4 mM

Figure 11-11

K+ handling by astrocytes. ECS, extracellular space.

Chapter 11 • The Neuronal Microenvironment

K+ removal and limit its accumulation to a maximum level of 10 to 12 mM, the so-called ceiling level. [K+]o would rise far above this ceiling with intense neural activity if K+ clearance depended solely on passive redistribution of K+ in the BECF. Neurons and blood vessels can contribute to K+ homeostasis, but glial mechanisms are probably most important. Astrocytes can take up K+ in response to elevated [K+]o by three major mechanisms: the Na-K pump, the Na/K/Cl cotransporter, and the uptake of K+ and Cl− through channels. Conversely, when neural activity decreases, K+ and Cl− leave the astrocytes through ion channels. Gap junctions couple astrocytes to one another, allowing diffusion of small solutes The anatomical substrate for cell-cell coupling among astrocytes is the gap junction, which is composed of membrane proteins called connexins that form large aqueous pores connecting the cytoplasm of two adjacent cells (see Chapter 6). Coupling between astrocytes is strong because hundreds of gap junction channels may be present between two astrocytes. Astrocytes may also be weakly coupled to oligodendrocytes. Ions and organic molecules that are up to 1 kDa in size, regardless of charge, can diffuse from one cell into another through these large channels. Thus, a broad range of biologically important molecules, including nucleotides, sugars, amino acids, small peptides, cAMP, Ca2+, and inositol 1,4,5trisphosphate (IP3), have access to this pathway. Gap junctions may coordinate the metabolic and electrical activities of cell populations, amplify the consequences of signal transduction, and control intrinsic proliferative capacity. The strong coupling among astrocytes ensures that all cells in the aggregate have similar intracellular concentrations of ions and small molecules and similar membrane potentials. Thus, the network of astrocytes functionally behaves like a syncytium, much like the myocytes in the heart (see Chapter 21). In ways that are not yet clear, gap junctional communication can be important for the control of cellular proliferation. The most common brain cell– derived tumors in the CNS arise from astrocytes. Malignant astrocyte tumors, like malignant neoplasms derived from other cells that are normally coupled (e.g., liver cells), lack gap junctions. The coupling among astrocytes may also play an important role in controlling [K+]o by a mechanism known as spatial buffering. The selective K+ permeability of glia, together with their low-resistance cell-cell connections, permits them to transport K+ from focal areas of high [K+]o, where a portion of the glial syncytium would be depolarized, to areas of normal [K+]o, where the glial syncytium would be more normally polarized (Fig. 11-11B). Redistribution of K+ proceeds by way of a current loop in which K+ enters glial cells at the point of high [K+]o and leaves them at sites of normal [K+]o, with the extracellular flow of Na+ completing this circuit. At a site of high neuronal activity, [K+]o might rise to 12 mM, which would produce a very large depolarization of an isolated, uncoupled astrocyte. However, because of the electrical coupling among astrocytes, the Vm of the affected astrocyte remains more negative than the EK predicted for a [K+]o of 12 mM. Thus, K+ would tend to passively enter coupled astrocytes through channels at sites of high

[K+]o. As discussed in the preceding section, K+ may also enter the astrocyte by transporters. Astrocytes synthesize neurotransmitters, take them up from the extracellular space, and have neurotransmitter receptors Astrocytes synthesize at least 20 neuroactive compounds, including both glutamate and γ-aminobutyric acid (GABA). Neurons can manufacture glutamate from glucose or from the immediate precursor molecule glutamine (Fig. 11-12). The glutamine pathway appears to be the primary one in the synthesis of synaptically released glutamate. Glutamine, however, is manufactured only in astrocytes by use of the astrocyte-specific enzyme glutamine synthetase to convert glutamate to glutamine. Astrocytes release this glutamine into the BECF through the SNAT3 and 5 transporters (SLC38 family; see Table 5-4) for uptake by neurons through SNAT1 and 2. Consistent with its role in the synthesis of glutamate for neurotransmission, glutamine synthetase is localized to astrocytic processes surrounding glutamatergic synapses. In the presynaptic terminals of neurons, glutaminase converts the glutamine to glutamate, for release into the synaptic cleft by the presynaptic terminal. Finally, astrocytes take up much of the synaptically released glutamate to complete this glutamate-glutamine cycle. Disruption of this metabolic interaction between astrocytes and neurons can depress glutamate-dependent synaptic transmission. Glutamine derived from astrocytes is also important for synthesis of the brain’s most prevalent inhibitory neurotransmitter, GABA. In the neuron, the enzyme glutamic acid decarboxylase converts glutamate (generated from glutamine) to GABA (see Fig. 13-8A). Because astrocytes play such an important role in the synthesis of synaptic transmitters, these glial cells are in a position to modulate synaptic efficacy. Astrocytes have high-affinity uptake systems for the excitatory transmitter glutamate and the inhibitory transmitter GABA. In the case of glutamate uptake, mediated by EAAT1 and EAAT2 (SLC1 family; see Table 5-4), astrocytes appear to play the dominant role compared with neurons or other glial cells. Glutamate moves into cells accompanied by two Na+ ions and an H+ ion, with one K+ ion moving in the opposite direction (Fig. 11-12). Because a net positive charge moves into the cell, glutamate uptake causes membrane depolarization. The presynaptic cytoplasm may contain glutamate at a concentration as high as 10 mM, and vesicles may contain as much as 100 mM glutamate. Nevertheless, the glutamate uptake systems can maintain extracellular glutamate at concentrations as low as ∼1 μM, which is crucial for normal brain function. Neurotransmitter uptake systems are important because they help terminate the action of synaptically released neurotransmitters. Astrocyte processes frequently surround synaptic junctions and are therefore ideally placed for this function. Under pathological conditions in which transmembrane ion gradients break down, high-affinity uptake systems may work in reverse and release transmitters, such as glutamate, into the BECF. Astrocytes express a wide variety of ionotropic and metabotropic neurotransmitter receptors that are similar

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Presynaptic Terminal

EAAT3

K

+

2 Na+ H+ Glutamate

Glutaminase Glutamine

Glutamate (10mM)

Postsynaptic neuron

SNAT1 and SNAT2 Na+ H+ Na+ Glutamine

[Glutamate]° ≅ 1μM

SNAT3 and SNAT5

H+ EEAT1 and EEAT2

Glutamine synthetase

2 K+ 2 Na+

Glutamate K+

3 Na+

Astrocyte

Figure 11-12 Role of astrocytes in the glutamate-glutamine cycle. Most of the glutamate of glutamatergic neurons is generated from glutamine, which the neurons themselves cannot make. However, astrocytes take up some of the glutamate that is released at synapses (or produced by metabolism) and convert it into glutamine. The glutamine then enters the neuron, where it is converted back to glutamate. This glutamate also serves as the source for γ-aminobutyric acid in inhibitory neurons.

or identical to those present on neuronal membranes. As in neurons, activation of these receptors can open ion channels or generate second messengers. In most astrocytes, glutamate produces depolarization by increasing Na+ permeability, whereas GABA hyperpolarizes cells by opening Cl− channels, similar to the situation in neurons (see Chapter 13). Transmitter substances released by neurons at synapses can diffuse in the BECF to activate nearby receptors on astrocytes, thus providing, at least theoretically, a form of neuronal-glial signaling. Astrocytes apparently can actively enhance or depress neuronal discharge and synaptic transmission by releasing neurotransmitters that they have taken up or synthesized. The release mechanisms are diverse and include stimulation by certain neurotransmitters, a fall in [Ca2+]o, or depolarization by elevated [K+]o. Applying glutamate to cultured astrocytes increases [Ca2+]i, which may oscillate. Moreover, these increases in [Ca2+]i can travel in waves from astrocyte to astrocyte through gap junctions or through a propagated front of extracellular ATP release that activates astrocytic purinergic receptors, thereby increasing [Ca2+]i and releasing more ATP. These [Ca2+]i waves—perhaps by triggering the release of a neurotransmitter from the astrocyte—can lead to changes in the activity of nearby neurons. This interaction represents another form of glial-neuronal communication. Astrocytes secrete trophic factors that promote neuronal survival and synaptogenesis Astrocytes, and other glial cell types, are a source of important trophic factors and cytokines, including brain-derived neurotrophic factor, glial-derived neurotrophic factor, basic

fibroblast growth factor, and ciliary neurotrophic factor. Moreover, both neurons and glial cells express receptors for these molecules, which are crucial for neuronal survival, function, and repair. The expression of these substances and their cognate receptors can vary during development and with injury to the nervous system. The development of fully functional excitatory synapses in the brain requires the presence of astrocytes, which act at least in part by secreting proteins called thrombospondins. Indeed, synapses in the developing CNS do not form in substantial numbers before the appearance of astrocytes. In the absence of astrocytes, only ∼20% of the normal number of synapses form. Astrocytic endfeet modulate cerebral blood flow Astrocytic endfeet surround not only capillaries but also small arteries. Neuronal activity can lead to astrocytic [Ca2+]i waves, as previously described, that spread to the astrocytic endfeet or to isolated increases in endfoot [Ca2+]i. In either case, the result is a rapid increase in blood vessel diameter and thus in local blood flow. A major mechanism of this vasodilation is the stimulation of phospholipase A2 in the astrocyte, the formation of arachidonic acid, and the liberation through cyclooxygenase 1 (see Fig. 3-11) of a potent vasodilator that acts on vascular smooth muscle. This is one mechanism of neuron-vascular coupling—a local increase in neuronal activity that leads to a local increase in blood flow. Radiologists exploit this physiological principle in a form of functional magnetic resonance imaging (fMRI) called blood oxygen level–dependent (BOLD) MRI, which uses blood flow as an index of neuronal activity.

Chapter 11 • The Neuronal Microenvironment

Excitatory Amino Acids and Neurotoxicity

T

he dicarboxylic amino acid glutamate is the most prevalent excitatory neurotransmitter in the brain (see Chapter 13). Although glutamate is present at millimolar levels inside neurons, the BECF has only micromolar levels of glutamate, except at sites of synaptic release (Fig. 11-12). Excessive accumulation of glutamate in the BECF— induced by ischemia, anoxia, hypoglycemia, or trauma—can lead to neuronal injury. Astrocytes are intimately involved in the metabolism of glutamate and its safe disposition after synaptic release. In anoxia and ischemia, the sharp drop in cellular levels of ATP inhibits the Na-K pump, thereby rapidly leading to large increases in [K+]o and [Na+]i. These changes result in membrane depolarization, with an initial burst of glutamate release from vesicles in presynaptic terminals. Vesicular release, however, requires cytoplasmic ATP and probably halts rapidly. The ability of astrocytes to remove glutamate from the BECF is impeded by the elevated [K+]o, elevated [Na+]i, and membrane depolarization. In fact, the unfavorable ion gradients can cause the transporter to run in reverse and dump glutamate into the BECF. The action of rising levels of extracellular glutamate on postsynaptic and astrocytic receptors reinforces the developing ionic derangements by opening channels permeable to Na+ and K+. This vicious cycle at the level of the astrocyte can rapidly cause extracellular glutamate to reach levels that are toxic to neurons—excitotoxicity.

Astrocytic modulation of blood flow is complex, and increases in [Ca2+]i in endfeet can sometimes lead to vasoconstriction. Oligodendrocytes and Schwann cells make and sustain myelin The primary function of oligodendrocytes as well as of their PNS equivalent, the Schwann cell, is to provide and to maintain myelin sheaths on axons of the central and peripheral nervous systems, respectively. As discussed in Chapter 7, myelin is the insulating “electrical tape” of the nervous system (see Fig. 7-21B). Oligodendrocytes are present in all areas of the CNS, although their morphological appearance is highly variable and depends on their location within the brain. In regions of the brain that are dominated by myelinated nerve tracts, called white matter, the oligodendrocytes responsible for myelination have a distinctive appearance (Fig. 11-13A). Such an oligodendrocyte has 15 to 30 processes, each of which connects a myelin sheath to the oligodendrocyte’s cell body. Each myelin sheath, which is up to 250 μm wide, wraps many times around the long axis of one axon. The small exposed area of axon between adjacent myelin sheaths is called the node of Ranvier (see Chapter 10). In gray matter, oligodendrocytes do not produce myelin and exist as perineuronal satellite cells. During the myelination process, the leading edge of one of the processes of the oligodendrocyte cytoplasm wraps around the axon many times (Fig. 11-13A, upper axon).

TABLE 11-4 Protein

Proteins in Myelin CNS (% of total myelin proteins)

PNS (% of total myelin proteins)

MBP

30

PPV

Base Lungs

0 cm H2O +3 cm H2O

–7 cm H2O

–2 cm H2O 0 cm H2O B

REGIONAL DISTRIBUTION OF PERFUSION 200

+7 cm H2O

150 · Q Unit volume 100 (arbitrary units)

+2 cm H2O at midpoint

0 Base

–1 cm H2O

Zone 3 PPA > PPV > PA 0 cm H2O

50

0

–3 cm H2O

5

10 15 Distance (cm)

0 cm H2O

+10 cm H2O

20 Apex

0 cm H2O

+5 cm H2O

C—LUNG ZONES Zone 1 conditions occur only when PA is high (e.g., positive-pressure ventilation) or when PPA is low (e.g., hemorrhage).

+20 cm H2O

Zone 2 +15 cm H2O at midpoint

Zone 3 Zone 4

PIP leads to partial collapse of extraalveolar vessels.

Lungs normally have Zones 2 through 4. Smaller regional lung volume leads to less mechanical tethering.

Figure 31-9

+10 cm H2O

Physiological nonuniformity of pulmonary perfusion.

Zone 4 PPA > PPV > PA

0 cm H2O

713

714

Section V • The Respiratory System

. Why should Q have this peculiar height dependence? The basic answers are the same as those for the similar question we raised about the regional nonuniformity of ventilation: posture and gravity. Thus, standing on your head will reverse the flow-height relationship, and we would expect height-related differences in flow to be minimal in microgravity conditions. Figure 31-9C shows how we can divide the upright lung into four zones based on the relationships among various pressures. We define the first three zones on the basis of how alveolar blood vessels are affected by the relative values of three different pressures: alveolar pressure (Pa), the pressure inside pulmonary arterioles (PPA), and the pressure inside pulmonary venules (PPV). In the fourth zone, we instead focus on how extra-alveolar vessels are affected by intrapleural pressure (PIP). Zone 1: PA > P PA > P PV These conditions prevail at the apex of the lung under certain conditions. The defining characteristic of a zone 1 alveolar vessel is that PPA and PPV are so low that they have fallen below Pa. At the level of the left atrium (the reference point for the pressure measurements), the mean PPA is ~15 mm Hg (Table 31-1), which—because mercury is 13.6-fold more dense than water—corresponds to ~20 cm H2O (Fig. 31-9C, lower panel of zone 3). Similarly, mean PPV is ~8 mm Hg, or ~10 cm H2O. As we move upward closer to the apex of an upright lung, the actual pressures in the lumens of pulmonary arterioles and venules fall by 1 cm H2O for each 1 cm of vertical ascent. In the hypothetical case in which alveoli at the lung apex are 20 cm above the level of the left atrium, the mean PPA of these alveoli would be 0 cm H2O (Fig. 31-9C, zone 1). The corresponding PPV would be about −10 cm H2O. The pressure inside the pulmonary capillary (Pc) would be intermediate, perhaps −5 cm H2O. In principle, blood would still flow through this capillary—the driving pressure would be ~10 cm H2O—were it not for the pressure inside the alveoli, which is 0 cm H2O between breaths. Therefore, because Pa is much higher than Pc, the negative PTM would crush the capillary and greatly reduce blood flow. Fortunately, zone 1 conditions do not exist for normal people at rest. However, they can arise if there is either a sufficient decrease in PPA (e.g., in hemorrhage) or a sufficient increase in Pa (e.g., in positive-pressure ventilation). Zone 2: P PA > PA > P PV

These conditions normally prevail from the apex to the midlung. The defining characteristic of zone 2 is that mean PPA and PPV are high enough so that they sandwich Pa (Fig. 31-9C, zone 2). Thus, at the arteriolar end, the positive PTM causes the alveolar vessel to dilate. Farther down the capillary, though, luminal pressure gradually falls below Pa, so that the negative PTM squeezes the vessel, raising resistance and thus reducing flow. As we move downward in zone 2, the crushing force decreases because the hydrostatic pressures in the arteriole, capillary, and venule all rise in parallel by 1 cm H2O for each 1 cm of descent (Fig. 31-9C, upper → lower panels of zone 2). Simultaneously, resistance decreases. The conversion of a closed vessel (or one that is open but not conducting) to a conducting one by increased PPA and PPV is an example of recruitment.

Zone 3: P PA > P PV > PA These conditions prevail in the middle to lower lung. The defining characteristic of zone 3 is that mean PPA and PPV are so high that they both exceed Pa (Fig. 31-9C, zone 3). Thus, PTM is positive along the entire length of the alveolar vessel, tending to dilate it. As we move downward in zone 3, the hydrostatic pressures in the arteriole, capillary, and venule all continue to rise by 1 cm H2O for each 1 cm of descent. Because Pa between breaths does not vary with height in the lung, the gradually increasing pressure of the alveolar vessel produces a greater and greater PTM, causing the vessel to dilate more and more—an example of distention (Fig. 31-9C, upper → lower panels of zone 3). This distention causes a gradual decrease in resistance of the capillaries as we move downward in zone 3. Hence, although the driving force (PPA − PPV) remains constant, perfusion increases toward the base of the lung. The arrangement in which a variable PTM controls flow is known as a Starling resistor. Keep in mind that the driving force (PPA − PPV) is constant in all of the zones. Zone 4: P PA > P PV > PA These conditions prevail at the extreme base of the lungs. In zone 4, the alveolar vessels behave as in zone 3; they dilate more as we descend toward the base of the lung. However, the extra-alveolar vessels behave differently. At the base of the lung, PIP is least negative (Fig. 31-5C). Thus, as we approach the extreme base of the lung, the distending forces acting on the extra-alveolar blood vessels fade, and the resistance of these extra-alveolar vessels increases (Fig. 31-9C, zone 4). Recall that we saw a similar effect—at the level of the whole lung (Fig. 31-7B, blue curve)—where resistance of the extra-alveolar vessels increased as lung volume fell (i.e., as PIP became less negative). Because these . extra-alveolar vessels feed or drain the alveolar vessels, Q begins to fall from its peak as we approach the extreme base of the lungs (Fig. 31-9B). These lung zones are physiological, not anatomical. The boundaries between the zones are neither fixed nor sharp. For example, the boundaries can move downward with positive-pressure ventilation (which increases Pa) and upward with exercise (which increases PPA). In our discussion of lung zones, we have tacitly assumed that Pa is always zero and that the values of PPA and PPV are stable and depend only on height in the lung. In real life, of course, things are more complicated. During the respiratory cycle, Pa becomes negative during inspiration (promoting dilation of alveolar vessels) but positive during expiration. During the cardiac cycle, the pressure inside the arterioles and pulmonary capillaries is greatest during systole (promoting dilation of the vessel) and lowest during diastole. Thus, we would expect blood flow through an alveolar vessel to be greatest when inspiration coincides with systole.

MATCHING VENTILATION AND PERFUSION The greater the ventilation-perfusion ratio, the higher the PO2 and the lower the PCO2 in the alveolar air In Figure 31-4, we saw that all other factors being equal, alveolar ventilation determines alveolar PO2 and PCO2. The

Chapter 31 • Ventilation and Perfusion of the Lungs

greater the ventilation, the more closely PaO2 and PaCO2 approach their respective values in inspired air. However, in Figure 31-4, we were really focusing on total alveolar ventilation and how this influences the average, or idealized, alveolar PO2 and PCO2. In fact, we have already learned that both ventilation and perfusion vary among alveoli. In any group of alveoli, the greater the local ventilation, the more closely the composition of local alveolar air approaches that of the inspired air. Similarly, because blood flow removes O2 from the alveolar air and adds CO2, the greater the perfusion, the more closely the composition of local alveolar air approaches that of mixed-venous . . blood. Thus, the local ventilation-perfusion ratio (VA/Q) determines the local PaO2 and PaCO2. You might view the alveoli as a sports venue where ventilation and perfusion are engaged in a continuous struggle over control of the composition of alveolar air. To the extent that ventilation gains the upper hand, PaO2 rises and PaCO2 falls. To the extent that perfusion holds sway, these parameters change in the opposite direction. As a physical analogue of this struggle over control of . alveolar PO2, consider water flowing (analogous. to VA) from a faucet into a sink (alveoli); the water exits (Q) through a drain with an adjustable opening. If the drain opening is in midposition and we begin flowing water moderately fast, then the water level (PaO2) will gradually increase . and reach a steady state. Increasing the inflow of water (VA) will cause the water level (PaO2) to rise until the product of pressure head and drain conductance is high enough to drive water down the drain as fast as the water flows in. If we increase . the drain opening and thus the outflow of water (Q), then the water level (PaO2) will fall until the decrease in the pressure head matches the increase in drain conductance, so that once again water inflow and outflow are balanced. Just as . a. high faucet/drain ratio will raise the water level, a high VA/Q ratio will increase alveolar PO2. Because of the action of gravity, the . . regional ventilation-perfusion ratio (VA/Q ) in an upright subject is greater at the apex of the lung than at the base We have already seen that when a subject is upright in a gravitational field, ventilation falls from the base to the apex

A

· · DEPENDENCE OF VA/Q ON HEIGHT IN LUNG Base Apex 4 3

· VA · 2 Q

· · VA/Q

· Q · VA

100

1 0

200

· · VA or Q Unit volume (arbitrary units)

B

of the lung (Fig. 31-5B), and perfusion also falls, but more steeply. (Fig. 31-9B). Thus, it is not surprising that the . ratio V /Q itself varies with height in .the lung (Fig. A . . . 31-10A). VA/Q is lowest near the base, where Q exceeds VA. The ratio gradually increases to 1 at about the level of the . third rib and further increases . toward the apex, where Q falls more precipitously than VA. . . Table 31-3 shows how differences in VA/Q at the apex and base of the lungs influence the regional composition of alveolar air. At the apex (the rostral 7% of lung volume in . . most this example), where VA/Q is highest, alveolar PO2 and PCO2 most closely approach their values in inspired air. Because both O2 and CO2 transport across the blood-gas barrier are perfusion limited (see Chapter 30), O2 and CO2 have completely equilibrated between the alveolar air and the blood by the end of the pulmonary capillaries. Thus, blood leaving the apex has the same high PO2 and low PCO2 as the alveolar air. Of course, the relatively low PCO2 produces a respiratory alkalosis (see Chapter 28) in the blood leaving the apex. The situation is just the opposite near the base of the lung (the most. caudal 13% of lung volume in this example). . Because VA/Q here is lowest, alveolar PO2 and PCO2 tend more . . Table 31-3 Effect of Regional Differences in VA/Q on the Composition of Alveolar Air and Pulmonary Capillary Blood . . Location Fraction of Total VA/Q Lung Volume

PO2 PCO2 pH (mm Hg) (mm Hg)

Apex

7%

3.3

132

28

7.55 0.07

Base

13%

0.6

89

42

7.38 1.3

0.84* 100

40

7.40 5.0

Overall 100%

*Because the transport of both O2 and CO2 is perfusion limited, we assume that end-capillary values of PO2 and PCO2 are the same as their respective alveolar values. If the overall alveolar ventilation for the two lungs is 4.2 L/min, if the cardiac output (i.e., perfusion) is 5 L/min, . . and then the overall VA /Q ratio for the two lungs is (4.2 L/min)/(5 L/min) = 0.84. Data from West JB: Ventilation/Blood Flow and Gas Exchange. Oxford, UK: Blackwell, 1989.

THE O2-CO2 DIAGRAM

Mixedvenous blood: · · VA/Q = 0

60 ·

40 PCO2 (mm Hg)

4 3 Rib number

2

0

0

·

Low VA/Q Arterial blood

20

5

. Q (L/min)

40

60

High · V

A /Q

100 120 PO2 (mm Hg)

80

·

Inspired air: · · VA/Q = ∞

140

160

. . Figure 31-10 Regional differences in VA/Q ratio and alveolar gas composition. (Data from West JB: Ventilation/Blood Flow and Gas Exchange. Oxford, UK, Blackwell, 1985.)

715

716

Section V • The Respiratory System

toward their values in mixed-venous blood. What impact. do. these different regions of the lung, each with its own VA/Q ratio, have on the composition of systemic arterial blood? Each region makes a contribution that is proportional to its blood flow (see the rightmost column in Table 31-3). Because the apex is poorly perfused, it makes only a small contribution to the overall composition of arterial blood. On the other hand, pulmonary tissue at the base of the lungs, which receives ~26% of total cardiac output, makes a major contribution. As a result, the average composition of blood exiting the lung more closely reflects the composition of the blood that had equilibrated with the air in the base of the lung. The O2-CO2 diagram introduced. as. Figure 29-11 is a helpful tool to depict how different VA/Q ratios throughout the lung produce different blood gas compositions. The curve in Figure 31-10B represents all possible combinations of PO2 and PCO2 in the alveolar air or end–pulmonary capillary blood. The H2O-saturated inspired air (PO2 = 149 mm Hg, PCO2 = ~0 mm Hg) represents the . .rightmost extreme of the diagram. By definition, the VA/Q ratio of inspired air is ∞ because it does not come into contact with pulmonary capillary blood. The mixed-venous blood (PO2 = 40 mm Hg, PCO2= 46. mm . Hg) represents the other extreme. By definition, the VA/Q ratio of mixed-venous blood is 0 because it has not yet come into contact with alveolar air. With the endpoints of the diagram established, we can now predict—with the help of the alveolar gas equation (Equation 31-17) and the Bohr effect and the Haldane effect (see Chapter 29)—all possible combinations of PO2 and PCO2 throughout the lung. As shown in Figure 31-10B, the base, midportion, and apex of the lungs correspond to points along the O2-CO2 diagram between mixed-venous blood at one extreme and inspired air at the other.

A

The ventilation of unperfused alveoli . . (local VA/Q = •) triggers compensatory bronchoconstriction and a fall in surfactant production The effects . gravity on ventilation and perfusion cause . of regional VA/Q to vary widely, even in idealized lungs (Fig. 31-10A). However, microscopic or local physiological and pathological variations in ventilation perfusion can . . and cause even greater mismatches of VA/Q, the extremes of which are alveolar dead-space ventilation (this section) and shunt (next section). Alveolar. Dead-Space Ventilation .

At one end of the spectrum of VA/Q mismatches is the elimination of blood flow to a group of alveoli. For example, if we ligated the pulmonary artery feeding one lung, the affected alveoli would receive no perfusion even though ventilation would initially continue normally (Fig. 31-11A). Earlier, we saw that such alveolar dead space together with the anatomical dead space constitutes the physiological dead space (Equation 31-8). The ventilation of the unperfused alveoli is called alveolar dead-space ventilation because it does not contribute to gas exchange. Thus, these alveoli behave like conducting airways. A natural cause of alveolar dead-space ventilation is a pulmonary embolism, which obstructs blood flow to a group of alveoli. Because one task of the lung is to filter small emboli from the blood (see Chapter 26), the lung must deal with small regions of alveolar dead-space ventilation on a recurring basis. At the instant the blood flow ceases, the alveoli supplied by the affected vessels contain normal alveolar air. However, each cycle of inspiration and expiration replaces some stale alveolar air with fresh, inspired air. Because no exchange of O2 and CO2 occurs between these

ALVEOLAR DEAD-SPACE VENTILATION WITHOUT COMPENSATION B

3 Perfusion of other lung increases, causing · · V/Q.

COMPENSATION: BRONCHIOLAR CONSTRICTION

1 Because perfusion to this lung stops, while ventilation continues, · · V/Q ∞. PO2 PCO2

PO2 = 149 PCO2 = 0

2 The alveolar gas assumes the composition of inspired air.

Figure 31-11

. . Extreme VA/Q mismatch and compensatory response—alveolar dead-space ventilation.

1 PO2, PCO2, and pH around smooth muscle causes bronchiolar constriction, diverting airflow to “normal” airways. 2 In response to blood flow, alveolar type II pneumocytes produce less surfactant, causing compliance and ventilation (alveoli shrink!).

Chapter 31 • Ventilation and Perfusion of the Lungs

unperfused alveoli and pulmonary capillary blood, the alveolar gas gradually achieves the composition of moist inspired air, with alveolar PO2 rising to ~149 mm Hg and PCO2 falling to ~0 mm Hg (Fig. .31-11A, step 2). By definition, alveolar . dead space has a VA/Q ratio of ∞, as described by the “inspired air” point on the x-axis of an O2-CO2 diagram (Fig. 31-10B). Redirection of Blood Flow Blocking of blood flow to one group of alveoli diverts blood to other “normal” alveoli, which then become somewhat . . hyperperfused. Thus, the blockage not only increases VA/Q .in alveoli downstream from . the blockage but also decreases VA/Q in other regions. Redirection of blood flow thus accentuates the nonuniformity of ventilation. Regulation of Local Ventilation

Because alveolar deadspace ventilation causes alveolar PCO2 to fall to ~0 mm Hg in downstream alveoli, it leads to a respiratory alkalosis (see Chapter 28) in the surrounding interstitial fluid. These local changes trigger a compensatory bronchiolar constriction in the adjacent tissues (Fig. 31-11B), so that during a period of seconds to minutes, airflow partially diverts away from the unperfused alveoli and toward normal alveoli, to which blood flow is also being diverted. This compensation . . makes teleological sense because it tends to correct the VA/Q shift in both the unperfused and normal alveoli. The precise mechanism of bronchiolar constriction is unknown, although bronchiolar smooth muscle may contract—at least in part— in response to a high extracellular pH. In addition to a local respiratory alkalosis, the elimination of perfusion has a second consequence. Downstream from the blockage, alveolar type II pneumocytes become starved for various nutrients, including the lipids they need

A

SHUNT WITHOUT COMPENSATION

to make surfactant. (These cells never become starved for O2!) As a result of the decreased blood flow, surfactant production falls during a period of hours to days. The result is a local decrease in compliance, further reducing local ventilation. These compensatory responses—bronchiolar constriction (i.e., increased resistance, a property of conducting airways) and reduced surfactant production (i.e., decreased compliance, a property of alveoli)—work well only if the alveolar dead space is relatively small, so that an ample volume of healthy tissue remains into which the airflow can be diverted. The perfusion of unventilated alveoli . . (local VA/Q = 0) triggers a compensatory hypoxic vasoconstriction Shunt Alveolar . . dead-space ventilation is at one end of the spectrum of VA/Q mismatches. At the opposite end is shunt— the flow of blood past unventilated alveoli. For example, if we ligate a mainstem bronchus, then inspired air cannot refresh alveoli distal to the obstruction (Fig. 31-12A). As a result, mixed-venous blood perfusing the unventilated alveoli “shunts” from the right side to the left side of the heart, without benefit of ventilation. When the low-O2 shunted blood mixes with high-O2 unshunted blood (which is ventilated), the result is that the mixture has a lower than normal PO2, causing hypoxia in the systemic arteries. It is possible to calculate the extent of the shunt from the degree of hypoxia. Natural causes of airway obstruction include the aspiration of a foreign body or the presence of a tumor in the lumen of a conducting airway. The collapse of alveoli (atel-

B

COMPENSATION: VASOCONSTRICTION

1 Because ventilation to this lung stops, while perfusion continues, · · V/Q 0.

3 Ventilation of other lung increases, · · causing V/Q.

PO2 PCO2

2 The alveolar gas assumes the composition of mixed-venous blood.

Figure 31-12

. . Extreme VA/Q mismatch and compensatory response—shunt.

In response to local alveolar hypoxia, the arterioles feeding the alveoli constrict: hypoxic vasoconstriction.

717

718

Section V • The Respiratory System

ectasis) also produces a right-to-left shunt, a pathological example of which is pneumothorax (see Chapter 27). Atelectasis also occurs naturally in dependent regions of the lungs, where PIP is not so negative (Fig. 31-5C) and surfactant levels gradually decline. Sighing or yawning stimulates surfactant release (see Chapter 27) and can reverse physiological atelectasis. Imagine that an infant aspirates a peanut. Initially, the air trapped distal to the obstruction has the composition of normal alveolar air. However, pulmonary capillary blood gradually extracts O2 from the trapped air and adds CO2. Eventually, the PO2 and PCO2 of the trapped air drift to their values in mixed-venous blood. If the shunt is small, so that it does not materially affect the PO2 or PCO2 of the systemic arterial blood, then the alveoli will have a PO2 of 40 mm Hg and. a .PCO2 of 46 mm Hg. By definition, shunted alveoli have a VA/Q of 0 and are represented by the “mixedvenous blood” point on an O2-CO2 diagram (Fig. 31-10B). Redirection of Airflow

Blocking of airflow to one group of alveoli simultaneously diverts air to normal parts of the lung, which then become somewhat hyperventilated. Thus, . . shunt not only decreases V /Q in unventilated alveoli but A . . also increases VA/Q in other regions. The net effect is a widen. . ing of the nonuniformity of VA/Q ratios.

Asthma

Although it is less dramatic than complete airway . . obstruction, an incomplete occlusion also decreases VA/Q. An example is asthma, in which hyperreactivity of airway smooth muscle increases local airway resistance and decreases ventilation of alveoli distal to the pathological process. Normal Anatomical Shunts

The thebesian veins drain some of the venous blood from the heart muscle, particularly the left ventricle, directly into the corresponding cardiac chamber. Thus, delivery of deoxygenated blood from thebesian veins into the left ventricle (1 kDa). Because the basement membrane contains heparan sulfate proteoglycans, it is especially restricts large, negatively charged solutes (see Fig. 34-4).

Primary processes

Foot processes

Podocyte cell body

Secondary processes

Figure 33-4 Glomerular capillaries covered by the foot processes of podocytes. This scanning electron micrograph shows a view of glomerular capillaries from the vantage point of Bowman’s space. The outer surfaces of the capillary endothelial cells are covered by a layer of interdigitating foot processes of the podocytes. The podocyte cell body links to the foot processes by leg-like connections. (Courtesy of Don W. Fawcett.)

Figure 33-5 Inner aspect of glomerular capillaries, showing fenestrations of endothelial cells (arrows). This scanning electron micrograph shows a view of the glomerular capillary wall from the vantage point of the capillary lumen. Multiple fenestrations, each ~70 nm in diameter, perforate the endothelial cells. (From Brenner BM: Brenner and Rector’s The Kidney, 7th ed, vol 1, p 10. Philadelphia: Saunders, 2004.)

753

754

Section VI • The Urinary System

Podocytes have foot interdigitating processes that cover the basement membrane (Fig. 33-4). Between the interdigitations are filtration slits (Fig. 33-3H), which are connected by a thin diaphragmatic structure—the slit diaphragm— with pores ranging in size from 4 to 14 nm. Glycoproteins with negative charges cover the podocytes, filtration slits, and slit diaphragms. These negative charges contribute to the restriction of filtration of large anions (Fig. 33-4). Nephrin, neph1, podocin, and other membranes organized on lipid rafts of podocytes form the slit diaphragm (Fig. 333I). Phosphotyrosine motifs on the intracellular domains of some of these proteins may recruit other molecules involved in signaling events that control slit permeability. The extracellular domains of nephrin, neph1, and FAT1 from adjacent podocytes may zip together to help form the filtration slit. In Finnish-type nephrosis, the genetic absence of nephrin leads to severe proteinuria. Supporting the glomerular capillary loops is a network of contractile mesangial cells, which secrete the extracellular matrix. This network is continuous with the smooth muscle cells of the afferent and efferent arterioles. The matrix extends to the extraglomerular mesangial cells (Fig. 333F). The juxtaglomerular apparatus (JGA) includes the extraglomerular mesangial cells, the macula densa, and the granular cells. The macula densa (Latin [dense spot]) is a region of specialized epithelial cells of the thick ascending limb, where it contacts its glomerulus (Fig. 333F). These cells have strikingly large nuclei and are closely packed, and thus they have a plaque-like appearance. The granular cells, also called juxtaglomerular or epithelioid cells, in the wall of afferent arterioles are specialized smooth muscle cells that produce, store, and release renin (see Chapter 40). The JGA is part of a complex feedback mechanism that regulates renal blood flow and filtration rate (see Chapter 34), and it also indirectly modulates Na+ balance (see Chapter 40) and systemic blood pressure (see Chapter 23). The tubule components of the nephron include the proximal tubule, loop of Henle, distal tubule, and collecting duct Figure 33-6 illustrates the ultrastructure of the cells of the different tubule segments. Table 33-1 lists these segments and their abbreviations. Based on its appearance at low magnification, the proximal tubule can be divided into the proximal convoluted tubule (Fig. 33-6A), and the proximal straight tubule (Fig. 33-6B). However, based on ultrastructure, the proximal tubule can alternatively be subdivided into three segments: S1, S2, and S3. The S1 segment starts at the glomerulus and includes the first portion of the proximal convoluted tubule. The S2 segment starts in the second half of the proximal convoluted tubule and continues into the first half of the proximal straight tubule. Finally, the S3 segment includes the distal half of the proximal straight tubule that extends into the medulla. Both the apical (luminal) and basolateral (peritubular) membranes of proximal tubule cells are extensively amplified (Fig. 33-6A, B). The apical membrane has infoldings in the form of a well-developed brush border. This enlargement of the apical surface area correlates with the main function

of this nephron segment, namely, to reabsorb the bulk of the filtered fluid back into the circulation. A central cilium, which may play a role in sensing fluid flow, protrudes from the apical pole of proximal tubule cells and nearly all tubule cells. The basolateral membranes of adjacent proximal tubule cells form numerous interdigitations, bringing abundant mitochondria in close contact with the plasma membrane. The interdigitations of the lateral membranes also form an extensive extracellular compartment bounded by the tight junctions at one end and by the basement membrane of the epithelium at the other end. Proximal tubule cells contain lysosomes, endocytic vacuoles, and a well-developed endoplasmic reticulum. Proximal tubule cells are also characterized by a prominent Golgi apparatus (see Chapter 2), which is important for synthesizing many membrane components, sorting them, and targeting them to specific surface sites. From the S1 to the S3 segments, cell complexity progressively declines, correlating with a gradual decrease of reabsorptive rates along the tubule. Thus, the cells exhibit a progressively less developed brush border, diminished complexity of lateral cell interdigitations, a lower basolateral cell membrane area, and a decrease in the number of mitochondria. In comparison with the S3 segment of the proximal tubule, the cells lining the descending and ascending thin limbs of the loop of Henle are far less complex (Fig. 33-6C, D), with few mitochondria and little cell membrane amplification. In superficial nephrons, the thin ascending limbs are extremely abbreviated (Fig. 33-2). However, they form a major part of the long loops of the juxtamedullary nephrons. Epithelial cells lining the thick ascending limb of the loop of Henle, which terminates at the macula densa, are characterized by tall interdigitations and numerous mitochondria within extensively invaginated basolateral membranes (Fig. 33-6E). This complex cell machinery correlates with the key role these cells play in making the medullary interstitium hyperosmotic. Until the latter part of the 20th century, morphologists defined the classic distal tubule—on the basis of light microscopic studies—as the nephron segment stretching from the macula densa to the first confluence of two nephrons in the collecting duct system. Today, we subdivide the classic distal tubule into three segments, based on ultrastructural studies: the distal convoluted tubule (starting at the macula densa), the connecting tubule, and the initial collecting tubule. What was classically termed the early distal tubule is mainly the distal convoluted tubule, whereas the classically termed late distal tubule is mainly the initial collecting tubule. The distal convoluted tubule begins at the macula densa and ends at the transition to the connecting tubule (Fig. 33-6F). The cells of the distal convoluted tubule are similar in structure to those of the thick ascending limb. However, significant cell heterogeneity characterizes the tubule segments that follow. The connecting tubule, which ends at the transition to the initial collecting tubule, consists of two cell types: connecting tubule cells and intercalated cells. Connecting tubule cells (Fig. 33-6G) are unique in that they produce and release

Chapter 33 • Organization of the Urinary System

F

DISTAL CONVOLUTED TUBULE

G CONNECTING TUBULE Connecting-tubule cell

Intercalated cell

A

H

PROXIMAL CONVOLUTED TUBULE

S1 cell

INITIAL COLLECTING TUBULE Intercalated cell

CORTEX Principal cell

B

I

PROXIMAL STRAIGHT TUBULE

CORTICAL COLLECTING TUBULE Intercalated cell

S3 cell

Principal cell C

DESCENDING THIN LIMB

OUTER MEDULLA

J

OUTER MEDULLARY COLLECTING DUCT

K

INNER MEDULLARY COLLECTING DUCT

INNER MEDULLA D

ASCENDING THIN LIMB

Early Duct of Bellini

E

THICK ASCENDING LIMB

Late (Papillary collecting duct)

Figure 33-6 Structure of tubule cells along the nephron. Because of the variability among tubule segments, the cross sections of the tubule are not to scale.

755

756

Section VI • The Urinary System

Table 33-1

Tubule Segments of the Nephron

Tubule Segment

Abbreviation

Proximal convoluted tubule

PCT

Proximal straight tubule

PST

Thin descending limb of loop of Henle

tDLH

Thin ascending limb of loop of Henle

tALH

Thick ascending limb of loop of Henle

TAL

Distal convoluted tubule

DCT

Connecting tubule

CNT

Initial collecting tubule

ICT

Cortical collecting tubule

CCT

Outer medullary collecting duct

OMCD

Inner medullary collecting duct

IMCD

renal kallikrein, a local hormone whose precise function is still uncertain. We discuss intercalated cells later. The two segments following the connecting tubule, the initial collecting tubule (up to the first confluence) and the cortical collecting tubule (after the confluence), are identical. They are composed of intercalated and principal cells, which exhibit striking morphological and functional differences. Intercalated cells, similar in structure to the intercalated cells of the connecting tubule, make up about one third of the lining of these collecting tubule segments (Fig. 33-6H, I). They are unusual among tubule cells in that they lack a central cilium. One subpopulation of these cells (A- or αintercalated cells) secretes H+ and reabsorbs K+, whereas another (B- or ß-intercalated cells) secretes HCO−3. Principal cells make up about two thirds of the cells of the initial collecting tubule and cortical collecting tubule (Fig. 33-6H, I). Compared with intercalated cells, principal cells have fewer mitochondria, only modestly developed invaginations of the basolateral membrane, and a central cilium on the apical membrane. Principal cells reabsorb Na+ and Cl− and secrete K+. The medullary collecting duct is lined mostly by one cell type that increases in cell height toward the papilla (Fig. 33-6J, K). The number of intercalated cells diminishes, beginning at the outer medullary collecting duct. Cells in this segment continue the transport of electrolytes and participate in the hormonally regulated transport of water and of urea. At the extreme end of the medullary collecting duct (i.e., the “papillary” collecting duct or duct of Bellini), the cells are extremely tall. The tightness of tubule epithelia increases from the proximal to the medullary collecting tubule Epithelia may be either “tight” or “leaky,” depending on the permeability of their tight junctions (see Chapter 5). In

general, the tightness of the tubule epithelium increases from the proximal tubule to the collecting duct. In the leaky proximal tubule, junctional complexes are shallow and, in freeze-fracture studies, show only a few strands of membrane proteins (see Chapter 2). In contrast, in the relatively tight collecting tubule, tight junctions extend deep into the intercellular space and consist of multiple strands of membrane proteins. Tubule segments with tight junctions consisting of only one strand have low electrical resistance and high solute permeability, whereas tubules with several strands tend to have high electrical resistance and low permeability. Gap junctions (see Chapter 6) provide low-resistance pathways between some, but not all, neighboring tubule cells. These gap junctions are located at various sites along the lateral cell membranes. Electrical coupling exists among proximal tubule cells, but not among heterogenous cell types, such as those found in the connecting and collecting tubules.

MAIN ELEMENTS OF RENAL FUNCTION The nephron forms an ultrafiltrate of the blood plasma and then selectively reabsorbs the tubule fluid or secretes solutes into it As they do for capillaries elsewhere in the body, Starling forces (see Chapter 20) govern the flow of fluid across the capillary walls in the glomerulus and result in net filtration. However, in the case of the glomerular capillaries, the filtrate flows not into the interstitium, but into Bowman’s space, which is contiguous with the lumen of the proximal tubule. The main function of renal tubules is to recover most of the fluid and solutes filtered at the glomerulus. If the fluid were not recovered, the kidney would excrete the volume of the entire blood plasma in less than half an hour. The retrieval of the largest fraction of glomerular filtrate occurs in the proximal tubule, which reabsorbs NaCl, NaHCO3, filtered nutrients (e.g., glucose and amino acids), divalent ions (e.g., Ca2+, HPO42−, and SO42−), and water. Finally, the proximal tubule secretes NH+4 and a variety of endogenous and exogenous solutes into the lumen. The main function of the loop of Henle (i.e., thin descending limb of loop of Henle [tDLH], thin ascending limb of loop of Henle [tALH], thick ascending limb of loop of Henle [TAL]) is to participate in forming concentrated or dilute urine. The loop does this by pumping NaCl into the interstitium of the medulla without appreciable water flow, thus making the interstitium hypertonic. Downstream, the medullary collecting duct exploits this hypertonicity by either permitting or not permitting water to flow by osmosis into the hypertonic interstitium. In humans, only ~15% of the nephrons, the juxtamedullary nephrons, have long loops that descend to the tip of the papilla. Nevertheless, this subpopulation of nephrons (Fig. 33-2) is extremely important for creating the osmotic gradients within the papilla that allow water movement out of the lumen of the entire population of medullary collecting ducts. As a result of this water movement, urine osmolality in the collecting ducts can far exceed that in the plasma.

Chapter 33 • Organization of the Urinary System

TAL cells secrete the Tamm-Horsfall glycoprotein (THP). Normal subjects excrete 30 to 50 mg/day into the urine, thus accounting—along with albumin (10 times/day) to the scrutiny of the renal tubule epithelium. If it were not for such a high turnover of the extracellular fluid, only small volumes of blood would be “cleared” per unit time (see Chapter 33) of certain solutes and water. Such a low clearance would have two harmful consequences for the renal excretion of solutes that renal tubules cannot adequately secrete. First, in the presence of a sudden increase in the plasma level of a toxic material—originating either from metabolism or from food or fluid intake—the excretion of the material would be delayed. High blood flow and a high GFR allow the kidneys to eliminate harmful materials rapidly by filtration.

A second consequence of low clearance would be that steady-state plasma levels would be very high for waste materials that depend on filtration for excretion. The following example by Robert Pitts, a major contributor to renal physiology, illustrates the importance of this concept. Consider two individuals on a diet that contains 70 g/day of protein, one person with normal renal function (e.g., GFR of 180 L/ day) and the other a renal patient with sharply reduced glomerular filtration (e.g., GFR of 18 L/day). Each individual produces 12 g/day of nitrogen in the form of urea (urea nitrogen) derived from dietary protein and must excrete this into the urine. However, these two individuals achieve urea balance at very different blood urea levels. We make the simplifying assumption that the tubules neither absorb nor secrete urea, so that only filtered urea can be excreted, and all filtered urea is excreted. The physiologically normal individual can excrete 12 g/day of urea nitrogen from 180 L of blood plasma having a blood urea nitrogen value of 12 g/180 L, or 6.7 mg/dL. In the patient whose GFR is reduced to 10% of normal, excreting 12 g/day of urea nitrogen requires that each of the 18 L of filtered blood plasma has a blood urea nitrogen that is 10 times higher, or 67 mg/ dL. Thus, excreting the same amount of urea nitrogen—to maintain a steady state—requires a much higher plasma blood urea nitrogen in the renal patient than in the normal individual. The clearance of inulin is a measure of glomerular filtration rate The ideal glomerular marker for measuring GFR would be a substance X that has the same concentration in the glomerular filtrate as in plasma and that also is not reabsorbed, secreted, synthesized, broken down, or accumulated by the tubules (Table 34-1). In Equation 33-4, we saw the following: Input into Bowman’s space

Output into urine

& PX ⋅GFR = U X ⋅ V mg mL

mL min

(34-1)

mg mL mL min

767

768

Section VI • The Urinary System

PX is the concentration of the solute in plasma, GFR is the sum of volume flow from the plasma into all Bowman’s . spaces, UX is the urine concentration of the solute, and V is the urine flow. Rearranging this equation, U X × V PX mL (mg/mL) × (mL/min) = min (mg/mL)

GFR =

(34-2)

Note that Equation 34-2 has the same form as the clearance equation (see Equation 33-3) and is identical to Equation 33-5. Thus, the plasma clearance of a glomerular marker is the GFR. Inulin is an exogenous starch-like fructose polymer that is extracted from the Jerusalem artichoke and has a molecular weight of 5000 Da. Inulin is freely filtered at the glomerulus but is neither reabsorbed nor secreted by the renal tubules (Fig. 34-1A). Inulin also fulfills the additional requirements listed in Table 34-1 for an ideal glomerular marker. Assuming that GFR does not change, three tests prove that inulin clearance is an accurate marker of GFR. First, A

HANDLING OF INULIN

Efferent arteriole

Afferent arteriole

Glomerular capillary

. as shown in Figure 34-1B, the rate of inulin excretion (UIn · V) is directly proportional to the plasma inulin concentration (PIn), as implied by Equation 34-2. The slope in Figure 34-1B is the inulin clearance. Second, inulin clearance is independent of the plasma inulin concentration (Fig. 34-1C). This conclusion was already implicit in Figure 34-1B, in which the slope (i.e., inulin clearance) does not vary with PIn. Third, inulin clearance is independent of urine flow (Fig. 34-1D). Given a particular PIn, after the renal corpuscles filter the Table 34-1 Criteria for Use of a Substance to Measure Glomerular Filtration Rate 1.

Substance must be freely filterable in the glomeruli.

2.

Substance must be neither reabsorbed nor secreted by the renal tubules.

3.

Substance must not be synthesized, broken down, or accumulated by the kidney.

4.

Substance must be physiologically inert (not toxic and without effect on renal function).

B

DEPENDENCE OF INULIN EXCRETION ON PLASMA [INULIN] 2500 The slope is the 2000 clearance of inulin. . 1500 UIn · V (mg/min) 1000 500 0

Bowman’s space

C

Amount of inulin filtered is PIn · GFR.

Peritubular capillary

0

4

8 12 PIn (mg/mL)

16

20

16

20

DEPENDENCE OF INULIN CLEARANCE ON PLASMA [INULIN] 250 200

.

UIn · V/PIn 150 (mL/min) 100

Because inulin is not reabsorbed…

50 0

…and not secreted… D …the amount excreted . in the urine (UIn · V) is the same as the amount filtered.

Renal vein

0

4

8 12 PIn (mg/mL)

DEPENDENCE OF INULIN CLEARANCE ON URINE FLOW 200

.

150

UIn · V/PIn 100 (mL/min) 50 0

Figure 34-1

Clearance of inulin.

0

1

2

3

4 5 6 . V (mL/min)

7

8

9

10

Chapter 34 • Glomerular Filtration and Renal Blood Flow

inulin, the total amount of inulin in the urine does not change. Thus, diluting this glomerular marker in a large amount of urine or concentrating it in a small volume, . does not affect the total amount of inulin excreted (UIn · V). If the urine flow is high, the urine inulin concentration . will be proportionally low, and vice versa. Because (UIn · V) is fixed, . (UIn · V)/PIn is also fixed. Two lines of evidence provide direct proof that inulin clearance represents GFR. First, by collecting filtrate from single glomeruli, Richards and coworkers showed in 1941 that the concentration of inulin in Bowman’s space of the mammalian kidney is the same as in plasma. Thus, inulin is freely filtered. Second, by perfusing single tubules with known amounts of labeled inulin, Marsh and Frasier showed that the renal tubules neither secrete nor reabsorb inulin. Although the inulin clearance is the most reliable method for measuring GFR, it is not practical for clinical use. One must administer inulin intravenously to achieve reasonably constant plasma inulin levels. Another deterrent is that the chemical analysis for determining inulin levels in plasma and urine is sufficiently demanding to render inulin unsuitable for routine use in a clinical laboratory. The normal value for GFR in a 70-kg man is ~125 mL/ min. Population studies show that GFR is proportional to body surface area. Because the surface area of an average 70-kg man is 1.73 m2, the normal GFR in men is often reported as 125 mL/min/1.73 m2 of body surface area. In women, this figure is 110 mL/min/1.73 m2. Age is a second variable. GFR is very low in the newborn, owing to incomplete development of functioning glomerular units. Beginning at ~2 years of age, GFR normalizes for body surface area and gradually falls off with age as a consequence of progressive loss of functioning nephrons. The clearance of creatinine is a useful clinical index of glomerular filtration rate Because inulin is not a convenient marker for routine clinical testing, nephrologists use other compounds that can be labeled with radioisotopes and that have clearances similar to those of inulin. The most commonly used compounds in human studies are 125I-iothalamate, radioactive vitamin B12 (57Co- or 58Co-cyanocobalamin) and 51Cr-ethylenediaminetetraacetic acid (EDTA). However, these compounds are of limited reliability in GFR measurements because of variable binding to proteins and the loss of labeled iodine from the iothalamate. The problems of intravenous infusion of a GFR marker can be completely avoided by using an endogenous substance with inulin-like properties. Creatinine is such a substance, and its clearance is a reasonable estimate of GFR in humans, but not all species. Tubules, to variable degree, secrete creatinine, which, by itself, would lead to an ~20% overestimate of GFR in humans. However, because commonly used colorimetric methods overestimate plasma creatinine concentrations, the calculated creatinine clearance turns out to be close to the inulin clearance. Thus, the effects of these two errors (i.e., tubule secretion and overestimated plasma levels) tend to cancel out each other. In clinical practice, determining the creatinine clearance is an easy and reliable means of assessing the GFR, and such determination avoids the need

to inject anything into the patient. One merely obtains samples of venous blood and urine, analyzes them for creatinine concentration, and makes a simple calculation. The source of plasma creatinine is the normal metabolism of creatine phosphate in muscle. In men, this metabolism generates creatinine at the rate of 20 to 25 mg/kg body weight/day (i.e., ~1.5 g/day in a 70-kg man). In women, the value is 15 to 20 mg/kg body weight/day (i.e., ~1.2 g/day in a 70-kg woman), owing to lower muscle mass. In the steady state, the rate of urinary creatinine excretion equals this rate of metabolic production. Therefore, to avoid errors in estimating the GFR from the creatinine clearance, one must take care to exclude non–steady-state pathologic conditions of creatinine release, such as hyperthermia or other conditions of muscle wasting or damage. Ingestion of meat, which has a high creatinine content, also produces non–steady-state conditions. To minimize the effects of such an ingestion, the patient collects urine over an entire 24-hour period, and the plasma sample is obtained by venipuncture in the morning before breakfast. Frequently, clinicians use the endogenous plasma concentration of creatinine, normally 1 mg/dL, as an instant index of GFR. This use rests on the inverse relationship between the plasma creatinine concentration (PCr) and the creatinine clearance (CCr): C Cr =

 U Cr ⋅ V PCr

(34-3)

In the steady state, when metabolic production in muscle . equals the urinary excretion rate (UCr · V) of creatinine, and both remain fairly constant, this equation predicts that a plot of PCr versus CCr (i.e., PCr versus GFR) is a rectangular hyperbola (Fig. 34-2). For example, in a healthy person whose GFR is 100 mL/min, plasma creatinine is ~1 mg/dL. The product of GFR (100 mL/min) and PCr (1 mg/dL) is thus 1 mg/min, which is the rate of both creatinine production · PCr · CCr = UCr · V = Constant 20

200

15 Plasma creatinine concentration 10 (mg/dL)

150 Blood urea 100 nitrogen (mg/dL) 50

5 0

0

0 25 50 75 100 125 GFR (mL/min)

Figure 34-2 Dependence of plasma creatinine and blood urea nitrogen on the GFR. In the steady state, the amount of creatinine . appearing in the urine per day (UCr · V ) equals the production rate. Because all fi.ltered creatinine (PCr · CCr) appears in the urine, (PCr · CCr) equals (UCr · V ), which is constant. Thus, PCr must increase as CCr (i.e., GFR) decreases, and vice versa. If we assume that the kidney handles urea in the same way that it handles inulin, then a plot of blood urea nitrogen versus GFR will have the same shape as that of creatinine concentration versus GFR.

769

770

Section VI • The Urinary System

Table 34-2

100 GFR (mL/min) 50

Substance

0

Molecular Weight (Da)

Effective Molecular Radius* (nm)

Filtrate (UFX/PX)

Na+

23

0.10

1.0

K+

39

0.14

1.0

Cl−

35

0.18

1.0

2

H2 O

18

0.15

1.0

1

Urea

60

0.16

1.0

Glucose

180

0.33

1.0

Sucrose

342

0.44

1.0

Polyethylene glycol

1,000

0.70

1.0

Inulin

5,200

1.48

0.98

Lysozyme

14,600

1.90

0.8

Myoglobin

16,900

1.88

0.75

Lactoglobulin

36,000

2.16

0.4

Egg albumin

43,500

2.80

0.22

Bence Jones protein

44,000

2.77

0.09

Hemoglobin

68,000

3.25

0.03

Serum albumin

69,000

3.55

COsm), then the difference between V and COsm is the positive CH2O. When the kidney maximally . dilutes the urine to 30 mOsm, the total urine flow (V) must be 20 L/day (see Equation 38-3): Dilute Urine

C H2O = V − C Osm Maximal dilution: CH2O = 20 L/day − 2 L/day

(38-9)

= +18 L/day Concentrated . Urine If the urine is more concentrated . than plasma (V < COsm), then the difference between V and COsm is a negative number, the negative CH2O. When the kidney maximally concentrates the urine to 1200 mOsm, the total urine flow must be 0.5 L/day (see Equation 38-4):

C H2O = V − COsm Maximal concentration: C H2O = 0.5 L/day − 2 L/day = − 1.5 L/day (38-10) Thus, the kidneys can generate CH2O of as much as +18 L/day under maximally diluting conditions, or as little as –1.5 L/ day under maximally concentrating conditions. This wide range of CH2O represents the kidneys’ attempt to stabilize the osmolality of extracellular fluid in the face of changing loads of solutes or water. From the extreme CH2O that the kidneys

Chapter 38 • Urine Concentration and Dilution

can achieve, we can conclude that the organism withstands the challenge of water load better than a water deficit.

WATER TRANSPORT BY DIFFERENT SEGMENTS OF THE NEPHRON The kidney generates concentrated urine by using osmosis to drive water from the tubule lumen, across a water-permeable epithelium, into a hypertonic interstitium The kidney generates dilute urine by pumping salts out of the lumen of tubule segments that are impermeable to water. What is left behind is tubule fluid that is hypo-osmotic (dilute) with respect to the blood. How does the kidney generate concentrated urine? One approach could be to pump water actively out of the tubule lumen. However, water pumps do not exist (see Chapter 5). Instead, the kidney uses osmosis as the driving force to concentrate the contents of the tubule lumen. The kidney generates the osmotic gradient by creating a hypertonic interstitial fluid in a confined compartment, the renal medulla. The final step for making a hyperosmotic urine is to thread a water-permeable tube—the medullary collecting duct (MCD)—through this hyperosmolar compartment. The result is that the fluid in the tubule lumen can equilibrate

with the hypertonic interstitium, thus generating concentrated urine. Although net absorption of H2O occurs all along the nephron, not all segments alter the osmolality of the tubule fluid. The proximal tubule, regardless of the final osmolality of the urine, reabsorbs two thirds of the filtered fluid isosmotically (i.e., the fluid reabsorbed has the same osmolality as plasma). The loop of Henle reabsorbs salt in excess of water, so that the fluid entering the distal convoluted tubule (DCT) is hypo-osmotic. Whether the final urine is dilute or concentrated depends on whether water reabsorption occurs in more distal segments: the initial and cortical collecting tubules (ICT and CCT) and the outer and inner MCDs (OMCD and IMCD). Arginine vasopressin (AVP)—also called antidiuretic hormone (ADH)—regulates the variable fraction of water reabsorption in these four nephron segments. Figure 13-9 shows the structure of AVP. Tubule fluid is isosmotic in the proximal tubule, becomes dilute in the loop of Henle, and then either remains dilute or becomes concentrated by the end of the collecting duct Figure 38-1 shows two examples of how tubule fluid osmolality (expressed as the ratio TFOsm/POsm) changes along the nephron. The first is a case of water restriction, in which the kidneys maximally concentrate the urine and excrete a

4

3 Water restriction (antidiuresis)

Figure 38-1 Relative osmolality of the tubule fluid along the nephron. Plotted on the y-axis is the ratio of the osmolality of the tubule fluid (TF) to the osmolality of the plasma (P); plotted on the x-axis is a representation of distance along the nephron. The red record is the profile of relative osmolality (i.e., TF/Posmolality) for water restriction, whereas the blue record is the profile for high water intake. (Data from Gottschalk CW: Physiologist 1961; 4:33-55.)

[TF/P]osmolality 2

1 High water intake (water diuresis) 0

Proximal tubule

Loop

Classic distal tubule

Cortical collecting tubule (CCT)

Urine Medullary collecting ducts (OMCD, IMCD)

837

838

Section VI • The Urinary System

minimal volume of water (antidiuresis). The second is a case of ingestion of excess water, in which the kidneys produce a large volume of dilute urine (water diuresis). In both cases, the tubule fluid does not change in osmolality along the proximal tubule, and it becomes hypotonic to plasma by the end of the thick ascending limb of the loop of Henle (TAL), also known as the diluting segment (see Chapter 35). Therefore, the fluid entering the DCT is hypo-osmotic with respect to plasma, regardless of the final urine osmolality. Under conditions of restricted water intake or hydropenia, elevated levels of AVP increase the water permeability of the nephron from the ICT to the end of the IMCD. As a result, the osmolality of the tubule fluid increases along the ICT (Fig. 38-1, red curve), achieving the osmolality of the cortical interstitium—which is the same as the osmolality of plasma (~290 mOsm)—by the end of this nephron segment. No additional increase in osmolality occurs along the CCT, because the tubule fluid is already in osmotic equilibrium with the surrounding cortical interstitium. However, in the MCDs, the luminal osmolality rises sharply as the tubule fluid equilibrates with the surrounding medullary interstitium, which becomes increasingly more hyperosmotic from the corticomedullary junction to the papillary tip. Eventually the tubule fluid reaches osmolalities that are as much as four times higher than the plasma. Thus, the MCDs are responsible for concentrating the final urine. In summary, the two key elements in producing a concentrated urine are the hyperosmotic medullary interstitium that provides the osmotic gradient and the AVP that raises the water permeability of the distal nephron. How the kidney generates this interstitial hyperosmolality is discussed in the next subchapter, and the role of AVP is discussed in the last subchapter. Under conditions of water loading, depressed AVP levels cause the water permeability of the distal nephron to remain low. However, the continued reabsorption of NaCl along the distal nephron effectively separates salt from water and leaves a relatively hypotonic fluid behind in the tubule lumen. Thus, the tubule fluid becomes increasingly hypotonic from the DCT throughout the remainder of the nephron (Fig. 38-1, blue curve).

GENERATING A HYPEROSMOTIC MEDULLA AND URINE Understanding the mechanisms involved in forming a hypertonic or hypotonic urine requires knowing (1) the solute and water permeability characteristics of each tubule segment, (2) the osmotic gradient between the tubule lumen and its surrounding interstitium, (3) the active transport mechanisms that generate the hyperosmotic medullary interstitium, and (4) the “exchange” mechanisms that sustain the hyperosmotic medullary compartment. The renal medulla is hyperosmotic to blood plasma during both antidiuresis (low urine flow) and water diuresis The loop of Henle plays a key role in both the dilution and the concentration of the urine. The main functions of the

loop are to remove NaCl—more so than water—from the lumen and to deposit this NaCl in the interstitium of the renal medulla. By separating tubule NaCl from tubule water, the loop of Henle participates directly in forming dilute urine. Conversely, because the TAL deposits this NaCl into the medullary interstitium, thus making it hyperosmotic, the loop of Henle is indirectly responsible for elaborating concentrated urine. As discussed later, urea also contributes to the hypertonicity of the medulla. Figure 38-2A shows approximate values of osmolality in the tubule fluid and interstitium during an antidiuresis produced, for example, by water restriction. Figure 38-2B, however, illustrates the comparable information during a water diuresis produced, for example, by high water intake. In both conditions, interstitial osmolality progressively rises from the cortex to the tip of the medulla (corticomedullary osmolality gradient). The difference between the two conditions is that the maximal interstitial osmolality during antidiuresis, ~1200 mOsm (Fig. 38-2A), is more than twice that achieved during water diuresis, ~500 mOsm (Fig. 38-2B). Because of the NaCl pumped out of the rather waterimpermeable TAL, the tubule fluid at the end of this segment is hypo-osmotic to the cortical interstitium during both antidiuresis and water diuresis. However, beyond the TAL, luminal osmolalities differ considerably between antidiuresis and diuresis. In antidiuresis, the fluid becomes progressively more concentrated from the ICT to the end of the nephron (Fig. 38-2A). In contrast, during water diuresis, the hypotonicity of the tubule fluid is further accentuated as the fluid passes along segments from the DCT to the end of the nephron segments that are relatively water impermeable and continue to pump NaCl out of the lumen (Fig. 38-2B). During antidiuresis, the tubule fluid in the ICT, CCT, OMCD, and IMCD more or less equilibrates with the interstitium, but it fails to do so during water diuresis. This marked difference in osmotic equilibration reflects the action of AVP, which increases water permeability in each of the previously mentioned four segments. Although NaCl transport generates a gradient of only ~200 mOsm across any portion of the ascending limb, the countercurrent system can multiply this single effect to produce a 900mOsm gradient between the cortex and the papilla Developing and maintaining the hyperosmolality of the medullary interstitium depends on the net transport of NaCl across the rather water-impermeable wall of the ascending limb of the loop of Henle, from lumen to interstitium. This salt reabsorption increases the osmolality of the interstitium and decreases the osmolality of the fluid within the lumen. The limiting NaCl concentration gradient that the tubule can develop at any point along its length is only ~200 mOsm, and this concentration alone could not explain the ability of the kidney to raise the osmolality of the papilla to 1200 mOsm. The kidney can achieve such high solute levels only because the hairpin loops of Henle create a countercurrent flow mechanism that multiplies the single transverse gradient of 200 mOsm. The result is an osmotic gradient of 900 mOsm along both the axis of the lumen of the ascending limb and

Chapter 38 • Urine Concentration and Dilution A

WATER RESTRICTION (ANTIDIURESIS)

Vasa recta

Interstitial fluid osmolality (mOsm)

Nephron 120 mOsm

AQP2,3 H2O

NKCC2 NaCl

300

CORTEX

300

300

300 AQP2,3 400

AQP1 OUTER MEDULLA

Active transport

H2O

NaCl NKCC2

H2O

600

600 AQP2–4

Passive transport

H2O

NaCl AQP1

900 Solute

Solute

H2O

AQP2–4 NaCl

INNER MEDULLA B

H2O

H2O

UT-A1, A3

H2O

1200

1200 Urea

1200 mOsm

HIGH WATER INTAKE (WATER DIURESIS)

Vasa recta

Interstitial fluid osmolality (mOsm)

Nephron 120 mOsm

300 NaCl NKCC2 NaCl

300

CORTEX

NaCl

Active transport AQP1 OUTER MEDULLA

300

100

300

NaCl NKCC2

H2O Passive transport

NaCl NaCl

400

AQP1 Solute

Solute

H2O NaCl

INNER MEDULLA

H2O

H2O

NaCl

UT-A1, A3

500 Urea

500 60 mOsm

Figure 38-2 Nephron and interstitial osmolalities. A, Water restriction (antidiuresis). B, High water intake (water diuresis). The numbers in boxes are osmolalities (mOsm) along the lumen of the nephron and along the corticomedullary axis of the interstitium. The outflow of blood from the vasa recta is greater than the inflow, a finding reflecting the uptake of water reabsorbed from the collecting ducts.

839

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Section VI • The Urinary System

the corticomedullary axis of the interstitium. In addition to the hairpin shape of the loop of Henle, osmotic multiplication also depends on a distinct pattern of salt and water permeabilities along the loop of Henle. Figure 38-3 illustrates a simplified, schematized model of a countercurrent-multiplier system. The kidney in this example establishes a longitudinal osmotic gradient of 300 mOsm from cortex (300 mOsm) to papilla (600 mOsm) by iterating (i.e., multiplying) a single effect that is capable of generating a transepithelial osmotic gradient of only 200 mOsm. Of course, if we had used more cycles, we could have generated a corticomedullary gradient that was even greater. For example, after 39 cycles in our example, the interstitial osmolality at the tip of the loop of Henle would be ~1200. Therefore, the countercurrent arrangement of the loop of Henle magnifies the osmotic work that a single ascending limb cell can perform. Among mammals, the length of the loop of Henle—compared with the thickness of the renal cortex—determines the maximal osmolality of the medulla. In the last panel of Figure 38-3, we include the collecting duct in the model to show the final event of urine concentration: allowing the fluid in the collecting duct to equilibrate osmotically with the hyperosmotic interstitium produces a concentrated urine. The single effect is the result of passive NaCl reabsorption in the thin ascending limb and active NaCl reabsorption in the thick ascending limb So far, we have treated the ascending limb as a functionally uniform epithelium that is capable of generating a 200mOsm gradient between lumen and interstitium, across a relatively water-impermeable barrier. However, the bottom of the ascending limb is “thin” (tALH), whereas the top is “thick” (TAL). Both the tALH and the TAL separate salt from water, but they transport the NaCl by very different mechanisms. The TAL moves NaCl from lumen out to interstitium using a combination of transcellular and paracellular pathways (Fig. 38-4). For the transcellular pathway, the TAL cell takes up Na+ and Cl− through an apical Na/K/Cl cotransporter and exports these ions to the blood using basolateral Na-K pumps and Cl− channels. For the paracellular pathway, the lumen-positive transepithelial voltage drives Na+ from lumen to blood through the tight junctions. Using these two pathways, the TAL can generate a single effect as large as 200 mOsm. In contrast, the movement of Na+ and Cl− from the lumen to the interstitium of the tALH appears to be an entirely passive process. During the debate on the mechanism of NaCl reabsorption in the tALH, several investigators pointed out that it was difficult to imagine how the extraordinarily thin cells of the tALH, with their paucity of mitochondria, could perform intensive active solute transport. Because the concentration of NaCl in the lumen exceeds that of the interstitium of the inner medulla, NaCl is reabsorbed passively. The key question for this model is: How did the luminal [NaCl] in the tALH become so high? The work of concentrating the NaCl in the lumen was performed earlier, when the fluid was in the thin descending

limb (tDLH) of juxtamedullary nephrons. This tDLH has three features that allow it to concentrate luminal NaCl: (1) the tDLH has a high water permeability, owing to a high expression of aquaporin 1 (AQP1); (2) the tDLH has a very low permeability to NaCl and a finite urea permeability, resulting from the presence of the UT-A2 urea transporter; and (3) the interstitium of the inner medulla has a very high [NaCl] and [urea]. The high interstitial concentrations of NaCl and urea provide the osmotic energy for passively reabsorbing water, which secondarily concentrates NaCl in the lumen of the tDLH. In the interstitium, [Na+], [Cl−], and [urea] all rise along the axis from the cortex to the papillary tip of the renal medulla (Fig. 38-4). In the outer medulla, a steep rise in interstitial [Na+] and [Cl−] occurs—owing to the pumping of NaCl out of the TAL (see Chapter 35)—that is largely responsible for producing the hypertonicity. Although urea makes only a minor contribution in the outermost portion of the outer medulla, [urea] rises steeply from the middle of the outer medulla to the papilla. At the tip of the papilla, urea and NaCl each contribute half of the interstitial osmolality. As discussed in the next section, this steep interstitial [urea] profile in the inner medulla (Fig. 38-4) is the result of the unique water and urea permeabilities of the collecting tubules and ducts (Fig. 38-2A). Knowing that NaCl and urea contribute to the high osmolality of the inner medullary interstitium, we can understand how the tDLH passively elevates [NaCl] in the lumen to levels higher than that in the interstitium. NaCl is the main solute in the lumen at the tip of the papilla but urea contributes to the luminal osmolality. As the luminal fluid turns the corner and moves up the tALH, it encounters a very different epithelium, one that is now impermeable to water but permeable to NaCl. The ClC-K1 channel is selectively localized to the tALH (overt diabetes insipidus [DI] in ClCK1 knockout mice). At the tip of the papilla interstitial [Na+] and [Cl−] are each ~300 mM (Fig. 38-4). Luminal [Na+] and [Cl−], each in excess of 300 mM, provide a substantial gradient for passive transcellular reabsorption of Na+ and Cl−. As we see in the next section, urea enters the tALH passively caused by a favorable urea gradient and by urea permeability of the tALH larger than that of the tDLH. The entry of urea opposes the osmotic work achieved by the passive reabsorption of NaCl. Even though the mechanism and magnitude of the single effect is different in the tALH and the TAL, the result is the same. At any level, osmolality in the lumen of the ascending limb is lower than it is in the interstitium. The inner medullary collecting duct reabsorbs urea and produces high levels of urea in the interstitium of the inner medulla Because urea comes from protein breakdown, urea delivery to the kidney, and therefore the contribution of urea to the medullary hyperosmolality, is larger with protein-rich diets. Indeed, investigators have long known that the higher the dietary protein content, the greater is the concentrating ability. Urea Handling The renal handling of urea is complex (see Chapter 36). The kidney filters urea in the glomerulus and

Chapter 38 • Urine Concentration and Dilution

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Figure 38-3 Stepwise generation of a high interstitial osmolality by a countercurrent multiplier. This example illustrates in a stepwise fashion how a countercurrentmultiplier system in the loop of Henle increases the osmolality of the medullary interstitium. Heavy boundaries of ascending limb and early DCT indicate that these nephron segments are rather impermeable to water, even in the presence of AVP. The numbers refer to the osmolality (mOsm) of tubule fluid and interstitium. The top panel shows the starting condition (step 0), with isosmotic fluid (~300 mOsm) throughout the ascending and descending limbs and in the interstitium. Each cycle comprises two steps. Step 1 is the “single effect”: NaCl transport from the lumen of the ascending limb to the interstitium, which instantaneously equilibrates with the lumen of the descending limb (steps 1, 3, 5, and 7). Step 2 is an “axial shift” of tubule fluid along the loop of Henle (steps 2, 4, and 6), with an instantaneous equilibration between the lumen of the descending limb and the interstitium. Beginning with the conditions in step 0, the first single effect is NaCl absorption across the rather water-impermeable ascending limb. At each level, we assume that this single effect creates a 200mOsm difference between the ascending limb (which is water impermeable) and a second compartment: the combination of the interstitium and descending limb (which is water permeable). Thus, the osmolality of the ascending limb falls to 200 mOsm, whereas the osmolality of the interstitium and descending limb rise to 400 mOsm (step 1). The shift of new isosmotic fluid (~300 mOsm) from the proximal tubule in the cortex into the descending limb pushes the column of tubule fluid along the loop of Henle, thus decreasing osmolality at the top of the descending limb and increasing osmolality at the bottom of the ascending limb. Through instantaneous equilibration, the interstitium—with an assumed negligible volume—acquires the osmolality of the descending limb, thereby diluting the top of the interstitium (step 2). A second cycle starts with net NaCl transport out of the ascending limb (step 3), again generating an osmotic gradient of 200 mOsm—at each transverse level—between the ascending limb on the one hand and the interstitium and descending limb on the other. After the axial shift of tubule fluid and instantaneous equilibration of the descending limb with the interstitium (step 4), osmolality at the bottom of the ascending limb exceeds that of the preceding cycle. With successive cycles, interstitial osmolality at tip of the loop of Henle rises progressively from 300 (step 0) to 400 (step 1) to 500 (step 3) to 550 (step 5) and then to 600 (step 7). Thus, in this example, the kidney establishes a longitudinal osmotic gradient of 300 mOsm from the cortex (300 mOsm) to the papilla (600 mOsm) by iterating (i.e., multiplying) a single effect that is capable of generating a transepithelial osmotic gradient of only 200 mOsm. Step 7A adds the collecting duct and shows the final event of urine concentration: allowing the fluid in the collecting duct to equilibrate osmotically with the hyperosmotic interstitium, producing a concentrated urine. (Based on a model by Pitts RF: Physiology of the Kidney and Body Fluids. Chicago, Year Book, 1974.)

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reabsorbs about half in the proximal tubule. In juxtamedullary nephrons, the tDLH and the tALH secrete urea into the tubule lumen. Finally, the IMCD reabsorbs urea. The net effect is that the kidney excretes less urea into the urine than it filters. Depending on urine flow (see Fig. 36-2), the fractional excretion may be as low as 15% (minimal urine flow) or as high as 60% or more (maximal urine flow). Because we are interested in understanding the role of urea in establishing a hypertonic medullary interstitium, in Figure 38-5 we consider an example in which maximal AVP produces minimal urine flow (i.e., antidiuresis), a condition already illustrated in Figure 38-2A. As the tubule fluid enters the TAL, the [urea] is severalfold higher than it is in the plasma because ~100% of the filtered load of urea remains, even though earlier nephron segments have reabsorbed water. All nephron segments from TAL to the OMCD, inclusive, have low permeabilities to urea. In the presence of AVP, however, all segments from the ICT to the end of the nephron have high water permeabilities and continuously reabsorb fluid. As a result, luminal [urea] gradually rises, beginning at the ICT and reaching a concentration as much as 8-fold to 10-fold higher than that in blood plasma by the time the tubule fluid reaches the end of the OMCD. The IMCD differs in an important way from the three upstream segments: Although AVP increases only water per-

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Figure 38-5 Urea recycling. Under conditions of water restriction (antidiuresis), the kidneys excrete ~15% of the filtered urea. The numbered yellow boxes indicate the fraction of the filtered load that various nephron segments reabsorb. The single red box indicates the fraction of the filtered load secreted by the tALH, and the single brown box indicates the fraction of the filtered load carried away by the vasa recta. The green boxes indicate the fraction of the filtered load that remains in the lumen after these segments. The values in the boxes are approximations.

Chapter 38 • Urine Concentration and Dilution

meability in the ICT, CCT, and OMCD, AVP increases water and urea permeability in the IMCD. In the IMCD, the high luminal [urea] and the high urea permeabilities of the apical membrane (through the urea transporter UT-A1; see Chapter 36) and basolateral membrane (through UT-A3) promote the outward facilitated diffusion of urea from the IMCD lumen, through the IMCD cells, and into the medullary interstitium. As a result, urea accumulates in the interstitium and contributes about half of the total osmolality in the deepest portion of the inner medulla. In addition, in the outer portion of the inner medulla, active urea reabsorption occurs through a Na-urea cotransporter in the apical membrane of the early IMCD. Because of the accumulation of urea in the inner medullary interstitium, [urea] is higher in the interstitium than it is in the lumen of the tDHL and tALH of juxtamedullary (i.e., long-loop) nephrons. This concentration gradient drives urea into the tDLH through UTA-2 and into the tALH through an as-yet unidentified transporter. The secretion of urea into the tDLH and tALH accounts for two important observations: First, more urea (i.e., a greater fraction of the filtered load) emerges from the tALH than entered the tDLH. Second, as noted earlier, [urea] in the TAL is considerably higher than that in blood plasma. Urea Recycling

The processes that we have just described— (1) absorption of urea from IMCD into the interstitium, (2) secretion of urea from interstitium into the thin limbs, and (3) delivery of urea up into the cortex and back down through nephron segments from the TAL to the IMCD—are the three elements of a loop. This urea recycling is responsible for the buildup of a high [urea] in the inner medulla. A small fraction of the urea that the IMCD deposits in the interstitium moves into the vasa recta, which removes it from the medulla and returns it either to superficial nephrons or to the general circulation. The preceding discussion focused on the situation in antidiuresis, in which AVP levels are high and the kidney concentrates urea in the inner medulla. The converse situation pertains in water diuresis, when circulating levels of AVP are low. The kidney reabsorbs less water along the ICT, CCT, OMCD, and IMCD. Furthermore, with low AVP levels, the IMCD has lower permeability to both urea and water. In addition, urea may be actively secreted by an apical Na-urea exchanger located in the apical membrane of the most distal portions of the IMCD. Therefore, during water diuresis, the interstitial [urea] is lower, and more urea appears in the urine.

The vasa recta’s countercurrent exchange mechanism and relatively low blood flow minimize the washout of the medullary hypertonicity The simplified scheme for the countercurrent multiplier presented in Figure 38-3 did not include blood vessels. If we were simply to introduce a straight, permeable blood vessel running from papilla to cortex, or vice versa, the blood flow would soon wash away the papillary hypertonicity that is critical for concentrating urine. Figure 38-6A shows a hypothetical, poorly designed kidney with only descending vasa

recta. Here, blood would flow from cortex to papilla and then exit the kidney. Because the blood vessel wall is permeable to small solutes and water, the osmolality of the blood would gradually increase from 300 to 1200 mOsm during transit from cortex to papilla, thus reflecting a loss of water or a gain of solutes. Because these movements occur at the expense of the medullary interstitium, the interstitium’s hyperosmolality would be washed out into the blood. The greater the blood flow through this straight/unlooped blood vessel, the greater the medullary washout would be. The kidney solves the medullary washout problem in two ways. First, compared with the blood flow in the renal cortex, which is one of the highest (per gram of tissue) of any tissue in the body, the blood flow through the medulla is relatively low, corresponding to no more than 5% to 10% of total renal plasma flow. This low flow represents a compromise between the need to deliver nutrients to the medulla and the need to avoid washout of medullary hypertonicity. Second, and far more significant, the kidney uses a hairpin configuration, with the descending and ascending vasa recta both entering and leaving through the same region, thus creating an efficient countercurrent exchange mechanism (Fig. 38-6B) in the blood vessels. The vasa recta have a hairpin configuration, but no capacity for active transport. We start with the osmotic stratification in the medullary interstitium that the countercurrent multiplier generated in the presence of high AVP levels. This osmotic stratification results in part from a gradient of [Na+] + [Cl−], but also from a similarly directed cortex-to-papilla gradient of [urea] (Fig. 38-4A). As isosmotic blood enters the hyperosmotic milieu of the medulla, which has high concentrations of NaCl and urea, NaCl and urea diffuse into the lumen of the descending vasa recta, whereas water moves in the opposite direction. This entry of urea into the descending vasa recta occurs through facilitated diffusion, mediated by the UT-B1 and UT-B2 urea transporter (see Chapter 36). The result is that the osmolality of the blood increases as the blood approaches the tip of the hairpin loop. As the blood rounds the curve and heads up toward the cortex inside the ascending vasa recta, that blood eventually develops a higher solute concentration than the surrounding interstitium. As a consequence, NaCl and urea now diffuse from the lumen of the vasa recta into the interstitium, whereas water moves into the ascending vasa recta. Viewed as a whole, these passive exchange processes cause the descending vasa recta to gain solute and lose water, but they cause the ascending vessels to lose solute and gain water. Thus, at any level, the descending and ascending vessels exchange solutes and water through—and at the expense of—the medullary interstitium. Solute recirculates from the ascending vessel, through the interstitium, to the descending vessel. Conversely, the countercurrent exchange mechanism also “short circuits” the water, but in the opposite direction, from the descending vessel, through the interstitium, to the ascending vessel. The net effect is that the countercurrent exchanger tends to trap solutes in and exclude water from the medulla, thereby minimizing dissipation of the corticomedullary osmolality gradient. The total mass of solute and water leaving the medulla each minute through the ascending vasa recta must exceed the total inflow of solute and water into the medulla through

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the descending vasa recta. With regard to solute balance, the renal tubules continuously deposit NaCl and urea in the medullary interstitium. Thus, in the steady state, the vasa recta must remove these solutes lest they form crystals of NaCl and urea in the medullary interstitium. Almost all the urea in the interstitium of the inner medulla comes from the IMCD, and in the steady state most of this leaves the interstitium by way of the tALH (Fig. 38-5, red box). The blood of the vasa recta carries away the balance or excess urea (Fig. 38-5, brown box). The blood also carries off the excess NaCl that enters the interstitium from the ascending limb of the loop of Henle and, to some extent, from the MCDs. With regard to water balance within the medulla, the descending limb of the loop of Henle and—in the presence of AVP—the MCD continuously gives up water to the medullary interstitium as the tubule fluid becomes more concentrated. Therefore, in the steady state, the ascending vasa recta must also remove excess water from the medulla. The net effect of managing both solute and water balance in the medulla is that the ascending vasa recta carry out more

salt and more water than the descending vasa recta carry in. Although no precise measurements have been made, it is likely that the osmolality of blood leaving the ascending vasa recta exceeds that of the blood entering the descending vessels by a fairly small amount, perhaps 10 to 30 mOsm. The medullary collecting duct produces concentrated urine by osmosis, driven by the osmotic gradient between the medullary interstitium and the lumen In contrast to the loop of Henle, which acts as a countercurrent multiplier, and the loop-shaped vasa recta, which act as a countercurrent exchanger, the MCD is an unlooped or straight-tube exchanger. The wall of the MCD has three important permeability properties: (1) in the absence of AVP, it is relatively impermeable to water, urea, and NaCl along its entire length; (2) AVP increases its water permeability along its entire length; and (3) AVP increases its urea permeability along just the terminal portion of the tube

Chapter 38 • Urine Concentration and Dilution

(IMCD). The collecting duct traverses a medullary interstitium that has a stratified, ever-increasing osmolality from the cortex to the tip of the papilla. Thus, along the entire length of the tubule, the osmotic gradient across the collecting duct epithelium favors the reabsorption of water from lumen to interstitium. A complicating factor is that two solutes—NaCl and urea—contribute to the osmotic gradient across the tubule wall. As fluid in the collecting duct lumen moves from the corticomedullary junction to the papillary tip, the [NaCl] gradient across the tubule wall always favors the osmotic reabsorption of water (Fig. 38-7). For urea, the situation is just the opposite. However, because the ICT, CCT, and OMCD are all relatively impermeable to urea, water reabsorption predominates in the presence of AVP and gradually causes luminal [urea] to increase in these segments. Because the interstitial [urea] is low in the cortex, a rising luminal [urea] in the ICT and CCT opposes water reabsorption in these segments. Even when the tubule crosses the corticomedullary junction, courses toward the papilla, and is surrounded by interstitial fluid with an ever-increasing [urea], the transepithelial urea gradient still favors water movement into the lumen. Thus, the presence of urea per se in the lumen of the collecting tubules and ducts is actually a handicap for the osmotic concentration of the urine, because the luminal urea

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tends to pull water back into the tubule lumen. Fortunately, the IMCD partially compensates for this problem by having a relatively low reflection coefficient for urea (Σurea), thus converting any transepithelial difference in urea concentration into a smaller difference in effective osmotic pressure (see Chapter 20). The Σ for urea is 0.74, whereas that for NaCl is 1.0. Thus, water reabsorption continues in the IMCD even though [urea] in tubule fluid exceeds that in the interstitium. The combination of a high interstitial [NaCl] and high ΣNaCl promotes NaCl-driven water absorption. A low Σurea minimizes urea-driven water secretion. The kidney also compensates for having a high [urea] in the lumen of the MCDs by having a high interstitial [urea], which—to some extent—osmotically balances the urea in the lumen of the papillary collecting ducts. Were it not for urea accumulation in the medullary interstitium, interstitial [NaCl] would have to be much higher, and this, in turn, would require increased NaCl transport in the TAL. If luminal urea opposes the formation of a concentrated urine, why did the mammalian kidney evolve to have high levels of urea in the lumen of the collecting tubules and ducts? At least two reasons are known. First, because urea is the body’s major excretable nitrogenous waste, the kidney’s ability to achieve high urinary [urea] reduces the necessity to excrete large volumes of water for excreting nitrogenous waste. Second, as we have already seen, the kidney actually takes advantage of urea to generate maximally concentrated urine. Thus, in the presence of AVP, the permeability of the IMCD to urea is high, so that large amounts of urea can enter the medullary interstitium. The high interstitial [urea] energizes the increase in luminal [NaCl] in the tDLH, which, in turn, fuels the single effect in the tALH, thus creating the high inner medullary [NaCl] that is directly responsible for concentrating the urine. As discussed, the composition of the inner medullary interstitium determines the composition of the final urine. However, to some extent, the composition of the final urine, as well as the rate of urine flow, also influences the composition of the interstitium. Figure 38-2 shows that the medullary interstitial osmolality is much lower, and the stratification of osmolality from cortex to papillary tip is much less, during water diuresis than during antidiuresis. Two factors contribute to the lesser degree of osmotic stratification under conditions of water diuresis, when levels of AVP are low. First, less urea moves from the IMCD lumen to the interstitium, both because of the low urea permeability of the IMCD and because of the low water permeability of the upstream segments that would otherwise concentrate urea. Second, the MCDs reabsorb some water despite the low AVP levels, and this water dilutes the medullary interstitium. The reasons for this apparent paradox are as follows: (1) even when AVP is low, the water permeability is not zero; (2) the ICT and CCT present a much larger fluid volume to the MCD, because they reabsorb less water when AVP levels are low; and (3) the tubule fluid is more hypotonic, so that a larger osmotic gradient exists for transepithelial water movement. With low AVP levels, this larger osmotic gradient overrides the effect of the lower water permeability. Table 38-2 summarizes factors that modulate urinary concentration ability.

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Table 38-2 Factors That Modulate Urinary Concentration and Dilution 1. Osmotic gradient of medullary interstitium from corticomedullary junction to papilla: a. Length of loops of Henle: Species with long loops (e.g., desert rodents) concentrate more than those with short loops (e.g., beaver). b. Rate of active NaCl reabsorption in the TAL: Increased luminal Na+ delivery to TAL (high GFR or filtration fraction, and low proximal tubule Na+ reabsorption) enhances NaCl reabsorption, whereas low Na+ delivery (low GFR, increased proximal Na+ and fluid reabsorption) reduces concentrating ability. High Na-K pump turnover enhances NaCl reabsorption, whereas inhibiting transport (e.g., loop diuretics) reduces concentrating ability. 2. Protein content of diet: High-protein diet, up to a point, promotes urea accumulation in the inner medullary interstitium and increased concentrating ability. 3. Medullary blood flow: Low blood flow promotes high interstitial osmolality. High blood flow washes out medullary solutes. 4. Osmotic permeability of the collecting tubules and ducts to water: AVP enhances water permeability and thus water reabsorption. 5. Luminal flow in the loop of Henle and the collecting duct: High flow (osmotic diuresis) diminishes the efficiency of the countercurrent multiplier and thus reduces the osmolality of the medullary interstitium. In the MCD, high flow reduces the time available for equilibration of water and urea. 6. Pathophysiology: Central DI reduces plasma AVP levels, whereas nephrogenic DI reduces renal responsiveness to AVP (see the box titled Diabetes Insipidus).

REGULATION BY ARGININE VASOPRESSIN Large-bodied neurons in the paraventricular and supraoptic nuclei of the hypothalamus synthesize AVP, a nonapeptide also known as ADH. These neurons package the AVP and transport it along their axons to the posterior pituitary, where they release AVP through a breech in the bloodbrain barrier into the systemic circulation (see p. 875). In Chapter 40, we discuss how increased plasma osmolality and decreased effective circulating volume increase AVP release. AVP has synergistic effects on two target organs. First, at rather high circulating levels, such as those seen in hypovolemic shock, AVP acts on vascular smooth muscle to cause vasoconstriction (see Chapter 23) and thus to increase blood pressure. Second, and more importantly, AVP acts on the kidney, where it is the major regulator of water excretion. AVP increases water reabsorption by enhancing the water permeabilities of the collecting tubules and ducts and also by stimulating urea transport across the cells of the IMCD.

AVP increases water permeability in all nephron segments beyond the distal convoluted tubule Of the water remaining in the DCT, the kidney reabsorbs a variable fraction in the segments from the ICT to the end of the nephron. Absorption of this final fraction of water is under the control of circulating AVP. In the kidney, AVP (1) increases water permeability in all the segments beyond the DCT, (2) increases urea permeability in the IMCD, and (3) increases active NaCl reabsorption in the TAL. Figure 38-8 summarizes the water permeability of various nephron segments. The water permeability is highest in the proximal tubule and tDHL. The high water permeability in these segments reflects the abundant presence of AQP1 water channels (see Chapter 3) in the apical and basolateral cell membranes. In marked contrast to the proximal tubule and tDLH, the following few segments—from the tALH to the connecting tubule—have very low water permeabilities. In the absence of AVP, the next tubule segments, the ICT and CCT, have rather low water permeabilities, whereas the MCDs are virtually impermeable to water. However, AVP dramatically increases the water permeabilities of the collecting tubules (ICT and CCT) and ducts (OMCD and IMCD) by causing AQP2 water channels to insert into the apical membrane (see later). A third type of water channel, AQP3, is present in the basolateral cell membranes of MCDs. Like AQP1, AQP3 is insensitive to AVP. Given the favorable osmotic gradients discussed in the preceding subchapter, high levels of AVP cause substantial water reabsorption to occur in AVP-sensitive nephron segments. In contrast, when circulating levels of AVP are low, for instance after ingestion of large amounts of water, the water permeability of these nephron segments remains low. Therefore, the fluid leaving the DCT remains hypotonic as it flows down more distal nephron segments. In fact, in the absence of AVP, continued NaCl absorption makes the tubule fluid even more hypotonic, resulting in a large volume of dilute urine (Fig. 38-1). AVP, acting through cAMP, causes vesicles containing aquaporin 2 water channels to fuse with the apical membrane of principal cells of the collecting tubules and ducts AVP binds to V2 receptors in the basolateral membrane of the principal cells from the ICT to the end of the nephron (Fig. 38-9). Receptor binding activates the Gs heterotrimeric G protein, thus stimulating adenylyl cyclase to generate cAMP (see Chapter 3). The latter activates protein kinase A, which phosphorylates unknown proteins that play a role in the trafficking of intracellular vesicles containing AQP2 and the fusion of these vesicles with the apical membrane. These water channels are AVP sensitive, not in the sense that AVP modulates their single-channel water conductance, but rather in the context of their density in the apical membrane. In conditions of low AVP, AQP2 water channels are mainly in the membrane of intracellular vesicles just beneath the apical membrane. In the membrane of these vesicles, the AQP2 water channels are present as aggregophores—aggregates of AQP2 proteins. Under the influence of AVP, the

Chapter 38 • Urine Concentration and Dilution

Proximal convoluted tubule (PCT) Proximal straight tubule (PST) Thin descending limb (tDLH) Thin ascending limb (tALH) Medullary thick ascending limb (mTAL) Nephron segments

No AVP

Cortical thick ascending limb (cTAL)

AVP Distal convoluted tubule (DCT) Connecting tubule (CNT) Cortical collecting duct (CCT) Outer medullary collecting duct (OMCD) Inner medullary collecting duct (IMCD) 10

100

10,000

1000 Osmotic water permeability (μm/sec)

Figure 38-8 Water permeability in different nephron segments. Note that the x-axis scale is logarithmic. (From Knepper MA, Rector FC: In Brenner BM [ed]: The Kidney, pp 532-570. Philadelphia: WB Saunders, 1996.)

Tubule lumen

Interstitial space

Clusters of AQP2 P

Prostaglandins Calcium Protein kinase C Other agents

Exocytosis

Vesicle

β

γ

AVP

α α

V2 receptor

AC

P

Protein phosphorylation

Other proteins

Protein kinase A

cAMP

Endocytosis Phosphodiesterase

AQP2 synthesis P

5´ AMP

DNA CRE site AP1 site

Figure 38-9

AQP3 and AQP4

CREB (CRE-binding protein)

Nucleus

Cellular mechanism of AVP action in the collecting tubules and ducts.

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Section VI • The Urinary System

vesicles containing AQP2 move to the apical membrane of principal cells of the collecting tubules and ducts. By exocytosis (see Chapter 2), these vesicles fuse with the apical membrane, thus increasing the density of AQP2. When AVP levels in the blood decline, endocytosis retrieves the water channel–containing aggregates from the apical membrane and shuttles them back to the cytoplasmic vesicle pool. The apical water permeability of principal cells depends not only on AVP levels, but also on certain other factors. For example, high [Ca2+]i and high [Li+] both inhibit adenylyl cyclase, thus decreasing [cAMP]i, reducing water permeability, and producing a diuresis. A similar inhibition of AQP2 insertion, and hence a decrease in water permeability, occurs when agents such as colchicine disrupt the integrity of the cytoskeleton. Conversely, inhibitors of phosphodiesterase (e.g., theophylline), which increase [cAMP]i, tend to increase the osmotic water permeability. AVP enhances the urinary-concentrating mechanism by stimulating the urea transporter UT-A1 in the inner medullary collecting ducts, thus increasing urea reabsorption AVP promotes water reabsorption not only by increasing the water permeability of the collecting tubules and ducts, but also by enhancing the osmotic gradients across the walls of the inner and perhaps the OMCD. In the outer medulla, AVP acts through the cAMP pathway to increase NaCl reabsorp-

tion by the TAL. AVP acts by stimulating apical Na/K/Cl cotransport and K+ recycling across the apical membrane (see Chapter 35). The net effect is to increase the osmolality of the outer medullary interstitium and thus enhance the osmotic gradient favoring water reabsorption by the OMCD. In addition, AVP stimulates the growth of TAL cells in animals that are genetically devoid of AVP. This hormone also stimulates Na+ reabsorption in the CCT, largely by activating apical Na+ channels (ENaC). These observations on the TAL and CCT were all made on rodents. In humans, these TAL and CCT mechanisms may have only minor significance. In the inner medulla, AVP enhances the urea permeability of the terminal two thirds of the IMCD. The AVP-dependent increase in [cAMP]i that triggers the apical insertion of AQP2-containing vesicles also leads to a phosphorylation of apical UT-A1 urea transporters (see Chapter 36), increasing their activity. The results are a substantial increase in urea reabsorption and thus the high interstitial [urea] that is indirectly responsible for generating the osmotic gradient that drives water reabsorption in the inner medulla. Segments of the nephron other than the IMCD have varying degrees of urea permeability. However, AVP increases urea permeability only in the apical membrane of the IMCD. In particular, AVP has no effect on other urea transporters: UT-A2 (in tDLH), UT-B1/B2 (in vasa recta), or UT-A3 (basolateral membrane of the IMCD).

Diabetes Insipidus

D

I is a fairly rare disorder that occurs in two varieties. The first, neurogenic or central DI, is caused by failure of AVP secretion. The lesion can be either at the level of the hypothalamus (where neurons synthesize AVP) or in the pituitary gland (where neurons release AVP). Central DI can be idiopathic, familial, or caused by any disorder of the hypothalamus or pituitary, such as injury, a tumor, infection, or autoimmune processes. In the second variety, nephrogenic DI, the kidneys respond inadequately to normal or even elevated levels of circulating AVP. Nephrogenic DI can also be idiopathic or familial and may be associated with electrolyte abnormalities (e.g., states of K+ depletion or high plasma [Ca2+]), the renal disease associated with sickle cell anemia, and various drugs (notably Li+ and colchicine). In both central and nephrogenic DI, patients present with polyuria and polydipsia. If allowed to progress unchecked, the disorder can result in marked hypernatremia, hypotension, and shock. Often the physician first suspects the diagnosis when the patient is deprived of access to water or other fluids. The patient may then quickly become dehydrated, and a random determination of plasma [Na+] may yield a very high value. The physician can confirm the diagnosis of DI most easily by a fluid deprivation test. The patient will continue to produce a large output of dilute urine, despite the need to conserve fluids. If the patient has central DI, administering a subcutaneous dose of AVP will rapidly increase urine osmolality by more than 50%. In patients with nephrogenic DI, conversely, the increase in urine osmolality will be less. Simultaneous measurements of plasma AVP levels may confirm the diagnosis.

The treatment for central DI is desmopressin acetate (DDAVP) (see Fig. 56-10), a synthetic AVP analogue that patients can take intranasally. Nephrogenic DI, in which the kidneys are resistant to the effects of the hormone, does not respond to DDAVP. In these patients, it is best to treat the underlying disease and also to reduce the elevated plasma [Na+] by administering a diuretic (to produce natriuresis) and by restricting dietary Na+. The high urine flow in DI is associated with low rates of solute excretion. Therefore, the physician must distinguish DI from states of polyuria accompanied by high rates of solute excretion in the urine (osmotic diuresis). The most frequent cause is chronic renal failure, when a decreasing population of nephrons is charged with excreting the daily load of solutes or other renal diseases associated with compromised proximal fluid and solute transport. Polyuria with excretion of solute-rich urine also occurs in untreated diabetes mellitus. In that case, the polyuria occurs because the high plasma [glucose] leads to the filtration of an amount of glucose that exceeds the capacity of the proximal tubule to retrieve it from the lumen (see Chapter 36). A third cause of osmotic diuresis is the administration of poorly reabsorbable solutes, such as mannitol or HCO3−. In an entirely distinct class of polyurias is primary polydipsia, a psychoneurotic disorder in which patients drink large amounts of fluid. Whereas simple water deprivation benefits a patient with primary polydipsia, it aggravates the condition of a patient with DI.

Chapter 38 • Urine Concentration and Dilution

Role of Aquaporins in Renal Water Transport

W

hereas AQP1 is the water channel responsible for a large amount of transcellular fluid movement in the proximal tubule and the tDLH, three related isoforms of the water channel protein—AQP2, AQP3, and AQP4—are present in the principal cells of the collecting ducts. These channels regulate water transport in collecting tubules and ducts. Apical AQP2 is the basis for AVP-regulated water permeability. AQP3 and AQP4 are present in the basolateral membrane of principal cells, where they provide an exit pathway for water movement into the peritubular fluid. Short-term and long-term regulation of water permeability depends on an intact AQP2 system. In short-term regulation, AVP—through cAMP—causes water channel–containing vesicles from a subapical pool to fuse with the apical membrane (Fig. 38-9). As a result, the number of channels and the water permeability sharply increase. In long-term regulation, AVP—by enhancing transcription of the AQP2 gene—increases the abundance of AQP2 protein in principal cells.

Mutations of several AQP genes lead to loss of function and marked abnormalities of water balance. Examples include sharply decreased fluid absorption along the proximal tubule in AQP1 knockout animals and nephrogenic DI (see the box on this topic) in patients with mutations of the gene for AQP2. An interesting situation may develop during the third trimester of pregnancy, when elevated plasma levels of vasopressinase— a placental aminopeptidase that degrades AVP—may lead to a clinical picture of central DI. An acquired increase of AQP2 expression often accompanies states of abnormal fluid retention, such as congestive heart failure, hepatic cirrhosis, the nephrotic syndrome, and pregnancy. Some conditions—including acute and chronic renal failure, primary polydipsia, a low-protein diet, and SIADH (see the box titled Syndrome of Inappropriate Antidiuretic Hormone Secretion)—are associated with increased AQP2 levels in the apical membrane.

Syndrome of Inappropriate Antidiuretic Hormone Secretion

T

he syndrome of inappropriate ADH secretion (SIADH) is the opposite of DI. Patients with SIADH secrete levels of ADH (i.e., AVP) or AVP-like substances that are inappropriately high, given the plasma osmolality. Thus, the urine osmolality is inappropriately high as the kidneys salvage inappropriately large volumes of water from the urine. As a result, total body water increases, the blood becomes hypo-osmolar, plasma [Na+] drops (hyponatremia), and cells swell. If plasma [Na+] falls substantially, cell swelling can cause headaches, nausea, vomiting, and behavioral changes. Eventually, stupor, coma, and seizures may ensue. Before making the diagnosis of SIADH, the physician must rule out other causes of hyponatremia in which AVP levels may be appropriate. In Chapter 40, we discuss how plasma osmolality and effective circulating volume appropriately regulate AVP secretion. SIADH has four major causes: 1. Certain malignant tumors (e.g., bronchogenic carcinoma, sarcomas, lymphomas, and leukemias) release AVP or AVPlike substances.

REFERENCES Books and Reviews Agre P, Preston GM, Smyth BL, et al: Aquaporin CHIP: The archetypal molecular water channel. Am J Physiol 1993; 265: F463-F476. Greger R: Transport mechanisms in thick ascending limb of Henle’s loop of mammalian nephron. Physiol Rev 1985; 65:760-797. Knepper MA, Saidel GM, Hascall VC, Dwyer T. Concentration of solutes in the renal inner medulla: Interstitial hyaluronan as a mechano-osmotic transducer. Am J Physiol 2003; 284: F433-F446. Sands JM: Mammalian urea transporters: Annu Rev Physiol 2003; 65:543-566. Sasaki W, Ishibashi K, Marumo F: Aquaporin-2 and -3: Representatives of two subgroups of the aquaporin family colocalized

2. Cranial disorders (e.g., head trauma, meningitis, and brain abscesses) can increase AVP release. 3. Nonmalignant pulmonary disorders (e.g., tuberculosis, pneumonia, and abscesses) and positive-pressure ventilation also can cause SIADH. 4. Several drugs can either stimulate AVP release (e.g., clofibrate, phenothiazines), increase the sensitivity of renal tubules to AVP (e.g., chlorpropamide), or both (e.g., carbamazepine). Treatment is best directed at the underlying disorder, combined, if necessary and clinically appropriate, with fluid restriction. Patients with a plasma [Na+] 200 g/day) results from either increased fluid secretion by the small or large intestine or decreased fluid reabsorption by the intestines. An important clinical example of diarrhea is cholera, especially in developing countries. Cholera can be fatal because of the water and electrolyte imbalance that it creates. Thus, the GI tract plays a crucial role in maintaining overall fluid and electrolyte balance (see Chapter 44). The GI tract also contributes to immune function. The mucosal immune system, or gut-associated lymphoid tissue (GALT), consists of both organized aggregates of lymphoid tissue (e.g., Peyer’s patches; Fig. 41-2B) and diffuse popula-

tions of immune cells. These immune cells include lymphocytes that reside between the epithelial cells lining the gut, as well as lymphocytes and mast cells in the lamina propria. GALT has two primary functions: (1) to protect against potential microbial pathogens, including bacteria, protozoans, and viruses; and (2) to permit immunologic tolerance to both the potentially immunogenic dietary substances and bacteria that normally reside primarily in the lumen of the large intestine. The mucosal immune system is important because the GI tract has the largest area of the body in potential direct contact with infectious, toxic, and immunogenic material. Approximately 80% of the immunoglobulin-producing cells are found in the small intestine. Although GALT has some interaction with the systemic immune system, GALT is operationally distinct. Finally, evidence indicates communication between the GALT and mucosal immune systems at other mucosal surfaces, such as the pulmonary epithelia. Certain nonimmunologic defense processes are also important in protecting against potential luminal pathogens and in limiting the uptake of macromolecules from the GI tract. The nonimmunologic mechanisms that are critical in maintaining the ecology of intestinal flora include gastric acid secretion, intestinal mucin, peristalsis, and the epithelial cell permeability barrier. Thus, whereas relatively low levels of aerobic bacteria are present in the lumen of the small intestine of physiologically normal subjects, individuals with impaired small intestinal peristalsis often have substantially higher levels of both aerobic and anaerobic bacteria in their small intestine. A consequence may be diarrhea or steatorrhea (i.e., increased fecal fat excretion). The clinical manifestation of impaired intestinal peristalsis is referred to as either blind loop syndrome or stagnant bowel syndrome.

REGULATION OF GASTROINTESTINAL FUNCTION The enteric nervous system is a “minibrain” with sensory neurons, interneurons, and motor neurons The ENS is the primary neural mechanism that controls GI function and, as described in Chapter 14, is one of the three divisions of the autonomic nervous system (ANS), along with the sympathetic and parasympathetic divisions. One indication of the importance of the ENS is the number of neurons consigned to it. The ENS consists of ~100 million neurons, roughly the number in the spinal cord or in the rest of the entire ANS. The ENS is located solely within GI tissue, but it can be modified by input from the brain. Neurons of the ENS are primarily, but not exclusively, clustered in one of two collections of neurons (Fig. 41-3A): the submucosal plexus and the myenteric plexus. The submucosal (or Meissner’s) plexus is found in the submucosa only in the small and large intestine. The myenteric (or Auerbach’s) plexus is located between the circular and longitudinal muscle layers throughout the GI tract from the proximal end of the esophagus to the rectum. The ENS is a complete reflex circuit and can operate totally within the GI tract, without the participation of either

Chapter 41 • Organization of the Gastrointestinal System

A

LOCATION OF THE ENS

Longitudinal muscle of muscularis externa

B Paravascular nerve

CONNECTIONS OF ENS NEURONS Longitudinal muscle

Perivascular nerve

Circular muscle

SENSORY

Myenteric (Auerbach’s) plexus

Muscularis mucosae

Blood vessels

Sensory

Tertiary plexus

Endocrine cells

PARASYMPATHETIC

Motor

Circular muscle of muscularis externa

Vagus nerve

Mechanoreceptors Motor

Deep muscular plexus

Motor

Submucosal (Meissner’s) plexus Submucosal artery

Chemoreceptors

Pelvic nerve Muscularis mucosae

Mucosal plexus

Secretory cells

SYMPATHETIC

Mucosa

Motor

Motor

Brainstem Sympathetic or spinal cord ganglia

Myenteric plexus

Submucosal plexus

Mucosa

Figure 41-3 Schematic representation of the ENS. A, The submucosal (or Meissner’s) plexus is located between the muscularis mucosae and the circular muscle of the muscularis externa. The myenteric (or Auerbach’s) plexus is located between the circular and longitudinal layers of the muscularis externa. In addition to these two plexuses that have ganglia, three others—mucosal, deep muscular, and tertiary plexus—are also present. B, The ENS consists of sensory neurons, interneurons, and motor neurons. Some sensory signals travel centrally from the ENS. Both the parasympathetic and the sympathetic divisions of the ANS modulate the ENS. This figure illustrates some of the typical circuitry of ENS neurons.

the spinal cord or the cephalic brain. As with other neurons, the activity of the ENS is the result of the generation of action potentials by single neurons and the release of chemical neurotransmitters that affect either other neurons or effector cells (i.e., epithelial or muscle cells). The ENS consists of sensory circuits, interneuronal connections, and secretomotor neurons (Fig. 41-3B). Sensory (or afferent) neurons monitor changes in luminal activity, including distention (i.e., smooth muscle tension), chemistry (e.g., pH, osmolality, specific nutrients), and mechanical stimulation. These sensory neurons, in turn, activate interneurons, which relay signals that activate efferent secretomotor neurons. These efferent secretomotor neurons stimulate or inhibit a wide range of effector cells: smooth muscle cells, epithelial cells that secrete or absorb fluid and electrolytes, submucosal blood vessels, and enteric endocrine cells. The largely independent function of the ENS has given rise to the concept of a GI “minibrain.” Because the efferent response to several different stimuli is often quite similar, a generalized concept has developed that the ENS possesses multiple preprogrammed responses. For example, both mechanical distention of the jejunum and the presence of a

bacterial enterotoxin in the jejunum can elicit identical responses: stimulation of profuse fluid and electrolyte secretion, together with propagated, propulsive, coordinated smooth muscle contractions. Such preprogrammed efferent responses are probably initiated by sensory input to the enteric interneuronal connections. However, efferent responses controlled by the ENS may also be modified by input from autonomic ganglia, which are, in turn, under the influence of the spinal cord and brain (see Chapter 14). In addition, the ENS receives input directly from the brain through parasympathetic nerves (i.e., the vagus nerve). Acetylcholine, peptides, and bioactive amines are the ENS neurotransmitters that regulate epithelial and motor function ACh is the primary preganglionic and postganglionic neurotransmitter regulating both secretory function and smooth muscle activity in the GI tract. In addition, many other neurotransmitters are present in enteric neurons. Among the peptides, vasoactive intestinal peptide (VIP) has an

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important role in both inhibition of intestinal smooth muscle and stimulation of intestinal fluid and electrolyte secretion. Although VIP was first identified in the GI tract, it is now appreciated that VIP is also an important neurotransmitter in the brain (see Table 13-1). Other substances probably also play an important role in GI regulation. These substances include the following: other peptides, such as the enkephalins, somatostatin, and substance P; amines such as serotonin (5-hydroxytryptamine [5-HT]); and nitric oxide (NO). The field of ENS neurotransmitters is rapidly evolving, and the list of agonists grows ever longer. In addition, substantial species differences exist. Frequently, chemical neurotransmitters are identified in neurons without a clear-cut demonstration of their physiological role in the regulation of organ function. More than one neurotransmitter has been identified within single neurons, a finding suggesting that regulation of some cell functions may require more than one neurotransmitter. The brain-gut axis is a bidirectional system that controls gastrointestinal function through the autonomic nervous system, gastrointestinal hormones, and the immune system Well recognized, but poorly understood, is the modification of several different aspects of GI function by the brain. In other words, neural control of the GI tract is a function not only of intrinsic nerves (i.e., the ENS), but also of nerves that are extrinsic to the GI tract. These extrinsic pathways are composed of both the parasympathetic—and, to a lesser extent, the sympathetic—nervous system and are under the control of autonomic centers in the brainstem (see Chapter 14). Parasympathetic innervation of the GI tract from the pharynx to the distal colon is through the vagus nerve; the distal third of the colon receives its parasympathetic innervation from the pelvic nerves (see Fig. 14-4). The preganglionic fibers of the parasympathetic nerves use ACh as their neurotransmitter and synapse on some neurons of the ENS (Fig. 41-3B). These ENS neurons are thus postganglionic parasympathetic fibers, and their cell bodies are, in a sense, the parasympathetic ganglion. These postganglionic parasympathetic fibers use mainly ACh as their neurotransmitter; however, as noted in the previous section, many other neurotransmitters are also present. The results of parasympathetic stimulation are—after one or more synapses in a very complex ENS network—increased secretion and motility. The parasympathetic nerves also contain afferent fibers (see Chapter 14) that carry information to autonomic centers in the medulla from chemoreceptors, osmoreceptors, and mechanical receptors in the mucosa. The loop that is initiated by these afferents, integrated by central autonomic centers, and completed by the aforementioned parasympathetic efferents is known as a vagovagal reflex. The preganglionic sympathetic fibers to the GI tract synapse on postganglionic neurons in the prevertebral ganglia (see Fig. 14-4); the neurotransmitter at this synapse is ACh (see Chapter 14). The postganglionic sympathetic fibers either synapse in the ENS or directly innervate effector cells (Fig. 41-3B).

In addition to the control that is entirely within the ENS, as well as control by autonomic centers in the medulla, the GI tract is also under the control of higher CNS centers. Examples of cerebral function that affects GI behavior include the flight-or-fight response, which reduces blood flow to the GI tract, and the sight and smell of food, which increase gastric acid secretion. Communication between the GI tract and the higher CNS centers is bidirectional. For example, cholecystokinin from the GI tract mediates, in part, the development of food satiety in the brain. In addition, gastrin-releasing peptide, a neurotransmitter made in ENS cells (see Chapter 42), inhibits gastric acid secretion when it is experimentally injected into the ventricles of the brain. Table 41-1 summarizes peptide hormones made by the GI tract, as well as their major actions. In addition to the “hard-wired” communications involved in sensory input and motor output, communication through the gut-brain axis also requires significant participation of the immune system. Neuroimmune regulation of both epithelial and motor function in the small and large intestine primarily involves mast cells in the lamina propria of the intestine. Because the mast cells are sensitive to neurotransmitters, they can process information from the brain to the ENS and can also respond to signals from interneurons of the ENS. In addition, mast cells monitor sensory input from the intestinal lumen by participating in the immune response to foreign antigens. In turn, chemical mediators released by mast cells (e.g., histamine) directly affect both intestinal smooth muscle cells and epithelial cells. Our understanding of how the immune system modulates the neural control of GI function is rapidly evolving. In conclusion, three parallel components of the gut-brain axis—the ENS, GI hormones, and the immune system— control GI function, an arrangement that provides substantial redundancy. Such redundancy permits refinement of the regulation of digestive processes and provides backup or “fail-safe” mechanisms that ensure the integrity of GI function, especially at times of impaired function (i.e., during disease).

GASTROINTESTINAL MOTILITY Tonic and rhythmic contractions of smooth muscle are responsible for churning, peristalsis, and reservoir action The motor activity of the GI tract performs three primary functions. First, it produces segmental contractions that are associated with nonpropulsive movement of the luminal contents. The result is the increased mixing (or churning) that enhances the digestion and absorption of dietary nutrients. Second, GI motor activity produces propulsion, which is a progressive wave of relaxation, followed by contraction. Peristaltic contractions cause propulsion, or the propagated movement of food and its digestive products in a caudal direction. The result is elimination of nondigested, nonabsorbed material. We discuss churning and propulsion later in this chapter. Third, motor activity allows some hollow organs—particularly the stomach and large intestine—to act

Chapter 41 • Organization of the Gastrointestinal System

Table 41-1

Gastrointestinal Peptide Hormones

Hormone

Source

Target

Action

Cholecystokinin

I cells in duodenum and jejunum and neurons in ileum and colon

Pancreas

↑ Enzyme secretion

Gallbladder

↑ Contraction

Gastric inhibitory peptide

K cells in duodenum and jejunum

Pancreas

Exocrine: ↓ fluid absorption Endocrine: ↑ insulin release

Gastrin

G cells, antrum of stomach

Parietal cells in body of stomach

↑ H+ secretion

Gastrin-releasing peptide

Vagal nerve endings

G cells in antrum of stomach

↑ Gastrin release

Guanylin

Ileum and colon

Small and large intestine

↑ Fluid absorption

Motilin

Endocrine cells in upper GI tract

Esophageal sphincter Stomach Duodenum

↑ Smooth muscle contraction

Neurotensin

Endocrine cells, widespread in GI tract

Intestinal smooth muscle

Vasoactive stimulation of histamine release

Peptide YY

Endocrine cells in ileum and colon

Stomach

↓ Vagally mediated acid secretion

Pancreas

↓ Enzyme and fluid secretion

Pancreas

↑ HCO−3 and fluid secretion by pancreatic ducts

Stomach

↓ Gastric acid secretion

Stomach

↓ Gastrin release

Intestine

↑ Fluid absorption/ ↓ secretion ↑ Smooth muscle contraction

Pancreas

↓ Endocrine/exocrine secretions

Secretin

Somatostatin

S cells in small intestine

D cells of stomach and duodenum, δ cells of pancreatic islets

Liver

↓ Bile flow

Substance P

Enteric neurons

Enteric neurons

Neurotransmitter

VIP

ENS neurons

Small intestine

↓ Smooth muscle relaxation ↑ Secretion by small intestine

Pancreas

↑ Secretion by pancreas

as reservoirs for holding the luminal content. This reservoir function is made possible by sphincters that separate the organs of the GI tract. All these functions are primarily accomplished by the coordinated activity of smooth muscle (see Chapter 9). The electrical and mechanical properties of intestinal smooth muscle needed for these functions include both tonic (i.e., sustained) contractions and rhythmic contractions (i.e., alternating contraction and relaxation) of individual muscle cells. The intrinsic rhythmic contractility is a function of the membrane voltage (Vm) of the smooth muscle cell. Vm can either oscillate in a subthreshold range at a low frequency (several per minute), referred to as slow-wave activity, or reach a threshold for initiating a true action potential (see Fig. 9-15). The integrated effect of the slow waves and action potentials determines the smooth muscle activity of the GI tract. Slow-wave activity apparently occurs as voltage-gated Ca2+ channels depolarize

the cell and increase [Ca2+]i, followed by the opening of Ca2+-activated K+ channels, which repolarize the cell (see Chapter 9). These activities are regulated, in large part, by both neural and hormonal stimuli. Modulation of intestinal smooth muscle contraction is largely a function of [Ca2+]i (see Chapter 9). Several agonists regulate [Ca2+]i by one of the following two mechanisms: (1) activation of G protein– linked receptors, resulting in the formation of inositol 1,4,5triphosphate (IP3) and the release of Ca2+ from intracellular stores; or (2) opening and closing of plasma membrane Ca2+ channels. Both excitatory and inhibitory neurotransmitters can modulate smooth muscle [Ca2+]i and thus contractility. In general, ACh is the predominant neurotransmitter of excitatory motor neurons, whereas VIP and NO are the neurotransmitters of inhibitory motor neurons. Different neural or hormonal inputs probably increase (or decrease) the frequency with which Vm exceeds threshold and produces an

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action potential and thus increases (or decreases) muscle contractility. An additional, unique factor in the aforementioned regulatory control is that luminal food and digestive products activate mucosal chemical and mechanical receptors, as discussed earlier, thus inducing hormone release or stimulating the ENS and controlling smooth muscle function. For example, gastric contents with elevated osmolality or a high lipid content entering the duodenum activate mucosal osmoreceptors and chemoreceptors that increase the release of cholecystokinin and thus delay gastric emptying (see Chapter 42). Segments of the gastrointestinal tract have both longitudinal and circular arrays of muscles and are separated by sphincters that consist of specialized circular muscles The muscle layers of the GI tract consist almost entirely of smooth muscle. Exceptions are the striated muscle of the upper esophageal sphincter ([UES] which separates the hypopharynx from the esophagus), the upper third of the esophagus, and the external anal sphincter. As shown earlier in Figure 41-2, the two smooth muscle layers are arranged as an inner circular layer and an outer longitudinal layer. The myenteric ganglia of the ENS are located between the two muscle layers. The segments of the GI tract through which food products pass are hollow, low-pressure organs that are separated by specialized circular muscles or sphincters. These sphincters function as barriers to flow by maintaining a positive resting pressure that serves to separate the two adjacent organs, in which lower pressures prevail. Sphincters thus regulate both antegrade (forward) and retrograde (reverse) movement. For example, the resting pressure of the pyloric sphincter controls, in part, the emptying of gastric contents into the duodenum. Conversely, the resting pressure of the lower esophageal sphincter (LES) serves to prevent gastric contents from refluxing back into the esophagus and causing gastroesophageal reflux disease (GERD). As a general rule, stimuli proximal to a sphincter cause sphincteric relaxation, whereas stimuli distal to a sphincter induce sphincteric contraction. Changes in sphincter pressure are coordinated with the smooth muscle contractions in the organs on either side. This coordination depends on both the intrinsic properties of sphincteric smooth muscle and neurohumoral stimuli. Sphincters effectively serve as one-way valves. Thus, the act of deglutition (or swallowing) induces relaxation of the UES, whereas the LES remains contracted. Only when the UES returns to its initial pressure does the LES begin to relax, ~3 seconds after the start of deglutition. Disturbances in sphincter activity are often associated with alterations in one or more of these regulatory processes. Location of a sphincter determines its function Six sphincters are present in the GI tract (Fig. 41-1), each with different resting pressures and different responses to various stimuli. An additional sphincter, the sphincter of Oddi, regulates movement of the contents of the common bile duct into the duodenum.

Achalasia

A

chalasia is a relatively uncommon condition associated with difficulty swallowing (dysphagia) and a dilated esophagus proximal to a narrowed, tapered area at the gastroesophageal junction. The term achalasia is derived from Greek words meaning “absence of relaxation.” The distal narrowed area of the esophagus suggests the presence of a stricture. However, it is easy to introduce an esophagoscope into the stomach through the narrowed area. Subsequent studies of esophageal motility in which investigators measured intraesophageal pressure demonstrated the presence of two defects in patients with achalasia: (1) failure of the LES to relax and (2) impaired peristalsis in the distal two thirds of the body of the esophagus (i.e., the portion that consists of smooth muscle). Peristalsis is intact in the proximal third of the esophagus, which consists of striated muscle. In essence, the smooth muscle portions of the esophagus behave as a denervated structure. The fundamental defect in achalasia is unknown but is probably related to selective loss of inhibitory neurons that regulate the LES, the neurotransmitters of which are VIP and NO. Treatment is either physical distention (or stretching) of the LES with a pneumatic bag dilator or surgical cutting of the LES (i.e., an esophageal myotomy or Heller procedure).

Upper Esophageal Sphincter

Separating the pharynx and the upper part of the esophagus is the UES, which consists of striated muscle and has the highest resting pressure of all the GI sphincters. Control of the swallowing mechanism, including the oropharynx and the UES, is largely under the control of the swallowing center in the medulla through cranial nerves V (trigeminal), IX (glossopharyngeal), X (vagus), and XII (hypoglossal). Respiration and deglutition are closely integrated (see Chapter 32). The UES is closed during inspiration, thereby diverting atmospheric air to the glottis and away from the esophagus. During swallowing, the situation reverses, with closure of the glottis and inhibition of respiration, but with relaxation of the UES (Fig. 41-4). These changes permit the entry of food contents into the esophagus and not into the airways of the respiratory tract. Lower Esophageal Sphincter

The esophagus is separated from the stomach by the LES, which is composed of specialized smooth muscle that is both anatomically and pharmacologically distinct from adjacent smooth muscle in the distal end of the esophagus and proximal portion of the stomach. The primary functions of the LES are (1) to permit coordinated movement of ingested food into the stomach from the esophagus after swallowing or deglutition and (2) to prevent reflux of gastric contents into the esophagus. Either deglutition or distention of the esophagus results in a reduction in LES pressure (Fig. 41-4) to that of intragastric pressure, thereby permitting entry of food into the stomach. Relaxation of the LES occurs after the UES has already returned to its resting pressure. The LES maintains a resting tone that is the result of both intrinsic myogenic properties of the sphincteric muscle and cholinergic regulation. Relaxation of the LES is mediated both by the vagus nerve and by

Chapter 41 • Organization of the Gastrointestinal System

Dry swallow At rest After swallowing

UES

100 mm Hg 0

1

100 mm Hg 0 2 100 mm Hg 0 3 100 mm Hg 0 4

100 mm Hg 0

Diaphragm 5 LES

6 100 mm Hg 0 0 5 sec

Figure 41-4 Esophageal pressures during swallowing. The swallowing center in the medulla that initiates deglutition includes the nucleus ambiguus (cranial nerves [CN] IX and X), the dorsal motor nucleus of the vagus (CN X), and others. Shown are recordings of intraluminal pressures at different sites along the esophagus, from the UES (record 1) to the LES (record 6). The left side of the graph shows the pressures at rest. As shown on the right side, after a dry swallow, the pressure wave of primary peristalsis moves sequentially down the esophagus. (Data from Conklin JL, Christensen J: In Johnson LR [ed]: Physiology of the Gastrointestinal Tract, 3rd ed, pp 903-928. New York: Lippincott-Raven, 1994.)

intrinsic properties of the smooth muscle, including important inhibitory effects by VIP and by NO. Abnormalities of both resting LES pressure and its relaxation in response to deglutition are often associated with significant symptoms. Thus, a reduced resting LES pressure often results in gastroesophageal reflux, which may cause esophagitis (i.e., inflammation of the esophageal mucosa). A defect in LES relaxation is a major component of a condition called achalasia (see the box titled Achalasia), which often results in dilatation of the esophagus (megaesophagus) and is associated with difficulty in swallowing (dysphagia). Swallowing and the function of the UES and the LES are closely integrated into the function of the esophagus. Under normal circumstances, esophageal muscle contractions are almost exclusively peristaltic and are initiated by swallowing. Deglutition initiates relaxation of the UES and propagated contractions, first of the UES and then of the muscles along the esophagus (Fig. 41-4). In the meantime, the LES has already relaxed. The result of the advancing peristaltic wave is the caudad propulsion of a bolus toward the stomach. Distention of the esophagus (in the absence of swallowing) also initiates propulsive esophageal contractions that are distal to the site of distention, as well as relaxation of the LES. Reflux of gastric contents into the lower part of the

esophagus also produces such a local distention, without a swallow, and elicits the same response: peristaltic contractions that clear the esophagus of refluxed gastric material. Peristalsis that is initiated by swallowing is called primary peristalsis, whereas that elicited by distention of the esophagus is referred to as secondary peristalsis. Esophageal contractions after a swallow are regulated by the medullary swallowing center, intramural esophageal plexuses, the vagus nerve, and intrinsic myogenic processes. Pyloric Sphincter The pylorus is the sphincter that separates the stomach from the duodenum. The pressure of the pyloric sphincter regulates, in part, gastric emptying and prevents duodenal-gastric reflux. However, although a specific pyloric sphincter is present, it is quite short and is a relatively poor barrier (i.e., it can resist only a small pressure gradient). The stomach, duodenum, biliary tract, and pancreas—which are closely related embryologically— function as a unit. Indeed, coordinated contraction and relaxation of the antrum, pylorus, and duodenum (which is sometimes referred to as the antroduodenal cluster unit) are probably more important than simply the pressure produced by the pyloric smooth muscle per se. Regulation of gastric emptying is discussed further in Chapter 42. Ileocecal Sphincter The valve-like structure that separates the ileum and cecum is called the ileocecal sphincter. Similar to other GI sphincters, the ileocecal sphincter maintains a positive resting pressure and is under the control of the vagus nerve, sympathetic nerves, and the ENS. Distention of the ileum results in relaxation of the sphincter, whereas distention of the proximal (ascending) colon causes contraction of the ileocecal sphincter. As a consequence, ileal flow into the colon is regulated by luminal contents and pressure, both proximal and distal to the ileocecal sphincter. Internal and External Anal Sphincters

The “anal sphincter” actually consists of both an internal and an external sphincter. The internal sphincter has both circular and longitudinal smooth muscle and is under involuntary control. The external sphincter, which encircles the rectum, contains only striated muscle but is controlled by both voluntary and involuntary mechanisms. The high resting pressure of the overall anal sphincter predominantly reflects the resting tone of the internal anal sphincter. Distention of the rectum (Fig. 41-5A), either by colonic contents (i.e., stool) or experimentally by balloon inflation, initiates the rectosphincteric reflex by relaxing the internal sphincter (Fig. 41-5B). If defecation is not desired, continence is maintained by an involuntary reflex—orchestrated by the sacral spinal cord—that contracts the external anal sphincter (Fig. 41-5C). If defecation is desired, a series of both voluntary and involuntary events occur that include relaxation of the external anal sphincter, contraction of abdominal wall muscles, and relaxation of pelvic wall muscles. Flexure of the hips and descent of the pelvic floor then facilitate defecation by minimizing the angle between the rectum and anus. In contrast, if a delay in defecation is needed or desired, voluntary contraction of the external anal sphincter is usually sufficient to override the series of reflexes initiated by rectal distention.

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A

If passive distention of the rectum is sufficiently large…

RECTUM

Rectal distention Active

40 30

Hirschsprung Disease

Passive

Change in 20 pressure (mm Hg) 10

…it triggers an active contraction of the rectal smooth muscles.

0 Time B

INTERNAL ANAL SPHINCTER Passive rectal distention also triggers relaxation of the smooth muscle of the internal anal sphincter (rectosphincteric reflex).

Change in 0 pressure (mm Hg) –10 –20

Time C

EXTERNAL ANAL SPHINCTER

30

If defecation is not desired, the skeletal muscle of the external anal sphincter contracts by an involuntary reflex.

15 Change in pressure 10 (mm Hg) 5 0 Time

Figure 41-5 A to C, Pressure changes initiated by rectal distention. (Data from Schuster MM: Johns Hopkins Med J 1965; 116:70-88.)

Motility of the small intestine achieves both churning and propulsive movement, and its temporal pattern differs in the fed and fasted states Digestion and absorption of dietary nutrients are the primary functions of the small intestine, and the motor activity of the small intestine is closely integrated with its digestive and absorptive roles. The two classes of small intestine motor activity are churning (or mixing) and propulsion of the bolus of luminal contents. Churning—which is accomplished by segmental, nonpropulsive contractions—mixes the luminal contents with pancreatic, biliary, and small intestinal secretions, thus enhancing the digestion of dietary nutrients in the lumen. These segmental contractions also decrease the unstirred water layer that is adjacent to the

T

he anal sphincter controls defecation and consists of a smooth muscle internal sphincter and a striated muscle external sphincter. Distention of the rectum by inflation of a balloon—which simulates the effect of the presence of solid feces in the rectum—results in relaxation of the internal sphincter and contraction of the external sphincter (Fig. 41-5). Voluntary control of the external sphincter regulates the timing of defecation. Hirschsprung disease is a congenital polygenic disorder. At least eight genes have been associated with Hirschsprung disease, including mutations in the endothelin-B receptor. Variable penetrance leads to variable manifestations of the disease. At the cellular level, the fundamental defect is arrest of the caudad migration of neural crest cells, which are the precursors of ganglion cells. Symptoms include constipation, megacolon, and a narrowed segment of colon in the rectum. Histologic examination of this narrowed segment reveals an absence of ganglion cells from both the submucosal (or Meissner’s) and myenteric (or Auerbach’s) plexuses (Fig. 41-3A). The patient’s constipation and resulting megacolon are secondary to failure of this “aganglionic” segment to relax in response to proximal distention. Manometric assessment of the internal and external anal sphincters reveals that the smooth muscle internal sphincter does not relax after rectal distention (Fig. 41-5), but the external anal sphincter functions normally. Treatment of this condition is usually surgical, with removal of the narrowed segment that is missing the ganglia that normally regulate relaxation of the smooth muscle of the internal anal sphincter.

apical membranes of the small intestine cells, thus promoting absorption. Churning or mixing movements occur following eating and are the result of contractions of circular muscle in segments flanked at either end by receiving segments that relax. Churning, however, does not advance the luminal contents along the small intestine. In contrast, propulsion—which is accomplished by propagated, peristaltic contractions—results in caudad movement of the intestinal luminal contents, either for absorption at more distal sites of the small or large intestine or for elimination in stool. Peristaltic propulsion occurs as a result of contraction of the circular muscle and relaxation of the longitudinal muscle in the propulsive or upstream segment, together with relaxation of the circular muscle and contraction of the longitudinal muscle in the downstream receiving segment. Thus, circular smooth muscle in the small intestine participates in both churning and propulsion. As noted earlier and in Chapter 9, the Vm changes of intestinal smooth muscle cells consist of both slow-wave activity and action potentials. The patterns of electrical and mechanical activity differ in the fasting and fed states. In the fasting state, the small intestine is relatively quiescent but exhibits synchronized, rhythmic changes in both electrical and motor activity (Fig. 41-6). The interdigestive myoelectric or migrating motor complex (MMC) is the term used to describe these rhythmic contractions of the small intestine that are observed in the fasting state. MMCs in humans

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Beginning of jejunum

Figure 41-6 Mechanical activity in the fasting and fed states. Shown are records of intraluminal pressure along the small intestine of a conscious dog. Before feeding (left side), the pattern is one of MMCs. Feeding triggers a switch to a different pattern, characterized by both segmental contractions that churn the contents and peristaltic contractions that propel the contents along the small intestine. (Data from Itoh Z, Sekiguchi T: Scand J Gastroenteral Suppl 1983; 82:121-134.)

Distance from duodenum

Duodenum

CONTRACTILE ACTIVITY Feeding Migrating motor complex

0 20 70 120 170 220 270 300

occur at intervals of 90 to 120 minutes and consist of four distinct phases: (1) a prolonged quiescent period, (2) a period of increasing action potential frequency and contractility, (3) a period of peak electrical and mechanical activity that lasts a few minutes, and (4) a period of declining activity that merges into the next quiescent period. During the interdigestive period, particles greater than 2 mm in diameter can pass from the stomach into the duodenum, thus permitting emptying of ingested material from the stomach (e.g., bones, coins) that could not be reduced in size to less than 2 mm. The slow propulsive contractions that characterize phases 2 to 4 of the MMCs clear the small intestine of its residual content, including undigested food, bacteria, desquamated cells, and intestinal and pancreatic biliary secretions. MMCs usually originate in the stomach and often travel to the distal end of the ileum, but ~25% are initiated in the duodenum and proximal part of the jejunum. Feeding terminates MMCs and initiates the appearance of the fed motor pattern (Fig. 41-6). The latter is less well characterized than MMCs but, as noted earlier, consists of both segmental contractions (churning), which enhance digestion and absorption, and peristaltic contractions (propulsion). Determination of the primary factors that regulate both MMCs and transition to the fed pattern has been hampered by both species differences and complex interactions among the multiple probable mediators. Nonetheless, clear evidence has been presented for a role of the ENS, one or more humoral factors, and extrinsic innervation. A major determinant of the MMC pattern is the hormone motilin, a 22–

Fasting 0

1

2

3 4 Time (hr)

5

6

7

8

amino acid peptide that is synthesized in the duodenal mucosa and is released just before the initiation of phase 3 of the MMC cycle. Motilin does not appear to have a role in the motor pattern that is observed in the fed state. Factors important in induction of the fed pattern include the vagus nerve (because sham feeding also both terminates MMCs and initiates a fed pattern) and the caloric content, as well as the type of food (e.g., fat more than protein) in the meal. Motility of the large intestine achieves both propulsive movement and a reservoir function The human large intestine has four primary functions. First, the colon absorbs large quantities of fluid and electrolytes and converts the liquid content of ileocecal material to solid or semisolid stool. Second, the colon avidly absorbs the short-chain fatty acids formed by the catabolism (or fermentation) of dietary carbohydrates that are not absorbed in the small intestine. The abundant colonic microflora accomplish this fermentation. Third, the storage of colonic content represents a reservoir function of the large intestine. Fourth, the colon eliminates its contents in a regulated and controlled fashion, largely under voluntary control. To accomplish these important activities, the large intestine functionally acts as two distinct organs. The proximal (or ascending and transverse) part of the colon is the site where most of the fluid and electrolyte absorption occurs and where bacterial fermentation takes place. The distal (or descending and rectosigmoid) portion of the colon provides final desiccation,

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Section VII • The Gastrointestinal System

as well as reservoir function, and serves as a storage organ for colonic material before defecation. In contrast to the motor pattern in the small intestine, no distinct fasting and fed patterns of contractions are seen in the colon. Similar to small intestinal motor activity, colonic contractions are regulated by myogenic, neurogenic, and hormonal factors. Parasympathetic control of the proximal two thirds of the colon is mediated by the vagus nerve, whereas parasympathetic control of the descending and rectosigmoid colon is mediated by pelvic nerves originating from the sacral spinal cord. The proximal colon has two types of motor activity: nonpropulsive segmentation and mass peristalsis. Nonpropulsive segmentation is generated by slow-wave activity that produces circular muscle contractions that churn the colonic contents and move them in an orad direction (i.e., toward the cecum). The segmental contractions that produce the churning give the colon its typical appearance of segments or haustra (Fig. 41-1). During this mixing phase, material is retained in the proximal portion of the large intestine for relatively long periods, and fluid and electrolyte absorption continues. One to three times a day, a so-called mass peristalsis occurs in which a portion of the colonic contents is propelled distally 20 cm or more. Such mass peristaltic contractions are the primary form of propulsive motility in the colon and may be initiated by eating. During mass peristalsis, the haustra disappear; they reappear after the completion of mass peristalsis. In the distal colon, the primary motor activity is nonpropulsive segmentation that is produced by annular or segmental contractions. It is in the distal part of the colon that the final desiccation of colonic contents occurs. It is also here that these contents are stored before an occasional mass peristalsis that propels them into the rectum. The rectum itself is kept nearly empty by nonpropulsive segmentation until it is filled by mass peristalsis of the distal end of the colon. As described in Figure 41-5, filling of the rectum trig-

gers a series of reflexes in the internal and external anal sphincters that lead to defecation. REFERENCES Books and Reviews Andrews JM, Dent J: Small intestinal motor physiology. In Feldman J, Friedman LS, Sleisenger MH (eds): Gastrointestinal and Liver Disease, vol 2, 7th ed, pp 1665-1678. Philadelphia: WB Saunders, 2002. Biancani P, Hartnett KM, Behar J: Esophageal motor function. In Yamada T (ed): Textbook of Gastroenterology, vol 1, 4th ed, pp 166-194. Philadelphia: Lippincott Williams & Wilkins, 2003. Conklin JL, Christensen J: Motor functions of the pharynx and esophagus. In Johnson LR (ed): Physiology of the Gastrointestinal Tract, 3rd ed, pp 903-928. New York: Lippincott-Raven, 1994. Cook IJ, Brookes SJ: Motility of the large intestine. In Feldman J, Friedman LS, Sleisenger MH (eds): Gastrointestinal and Liver Disease, vol 2, 7th ed, pp 1679-1691. Philadelphia: WB Saunders, 2002. Maklouf GM: Smooth muscle of the gut. In Yamada T (ed): Textbook of Gastroenterology, vol 1, 4th ed, pp 92-116. Philadelphia: Lippincott Williams & Wilkins, 2003. Rehfeld JF: The new biology of gastrointestinal hormones. Physiol Rev 1998; 78:1087-1108. Surprenant A: Control of the gastrointestinal tract by enteric neurons. Annu Rev Physiol 1994; 56:117-140. Wood JD: Enteric neuroimmunophysiology and pathophysiology. Gastroenterology 2004; 127:635-657. Wood JD: The first Nobel prize for integrated systems physiology: Ivan Petrovich Pavlov, 1904. Physiology 2004; 19:326-330. Journal Articles Itoh Z, Sekiguchi T: Interdigestive motor activity in health and disease. Scand J Gastroenterol Suppl 1983; 82:121-134. Schuster MM: Simultaneous manometric recording of internal and external anal sphincteric reflexes. Johns Hopkins Med J 1965; 116:70-88.

CHAPTER

42

GASTRIC FU NCTION Henry J. Binder

The stomach plays several important roles in human nutrition and has secretory, motor, and humoral functions. These activities are not separate and distinct, but rather represent integrated functions that are required to initiate the normal digestive process. The stomach has several specific secretory products. In addition to the stomach’s best-known product—acid, these products include pepsinogen, mucus, bicarbonate, intrinsic factor, and water. These substances continue the food digestion that was initiated by mastication and the action of salivary enzymes in the mouth. In addition, they help protect the stomach from injury. The stomach also has several important motor functions that regulate the intake of food, its mixing with gastric secretions and reduction in particle size, and the exit of partially digested material into the duodenum. Moreover, the stomach produces two important humoral agents—gastrin and somatostatin—that have both endocrine and paracrine actions. These peptides are primarily important in the regulation of gastric secretion. Although these functions are important in the maintenance of good health, the stomach is nevertheless not required for survival. Individuals who have had their entire stomach removed (i.e., total gastrectomy) for non-neoplastic reasons can maintain adequate nutrition and achieve excellent longevity.

FUNCTIONAL ANATOMY OF THE STOMACH The mucosa is composed of surface epithelial cells and glands The basic structure of the stomach wall is similar to that of other regions of the gastrointestinal (GI) tract (see Fig. 41-2); therefore, the wall of the stomach consists of both mucosal and muscle layers. The stomach can be divided, based on its gross anatomy, into three major segments (Fig. 42-1): (1) a specialized portion of the stomach called the cardia is located just distal to the gastroesophageal junction and is devoid of the acid-secreting parietal cells; (2) the body or corpus is the largest portion of the stomach; its most

proximal region is called the fundus; and (3) the distal portion of the stomach is called the antrum. The surface area of the gastric mucosa is substantially increased by the presence of gastric glands, which consist of a pit, a neck, and a base. These glands contain several cell types, including mucous, parietal, chief, and endocrine cells; endocrine cells also present in both corpus and antrum. The surface epithelial cells, which have their own distinct structure and function, secrete HCO−3 and mucus. Marked cellular heterogeneity exists not only within segments (e.g., glands versus surface epithelial cells) but also between segments of the stomach. For instance, as discussed later, the structure and function of the mucosal epithelial cells in the antrum and body are quite distinct. Similarly, although the smooth muscle in the proximal and distal portions of the stomach appear structurally similar, their functions and pharmacological properties differ substantially. With increasing rates of secretion of gastric juice, the H+ concentration rises, and the Na+ concentration falls The glands of the stomach typically secrete ~2 L/day of a fluid that is approximately isotonic with blood plasma. As a consequence of the heterogeneity of gastric mucosal function, early investigators recognized that gastric secretion consists of two distinct components: parietal cell and nonparietal cell secretion. According to this hypothesis, gastric secretion consists of (1) an Na+-rich basal secretion that originates from nonparietal cells and (2) a stimulated component that represents a pure parietal cell secretion that is rich in H+. This model helps to explain the inverse relationship between the luminal concentrations of H+ and Na+ as a function of the rate of gastric secretion (Fig. 42-2). Thus, at high rates of gastric secretion—for example, when gastrin or histamine stimulates parietal cells—intraluminal [H+] is high, whereas intraluminal [Na+] is relatively low. At low rates of secretion or in clinical situations in which maximal acid secretion is reduced (e.g., pernicious anemia; see Chapter 44 for the box on that topic), intraluminal [H+] is low but intraluminal [Na+] is high.

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Lower esophageal sphincter

Cardia

Fundus Superficial epithelial cell

Oxyntic gland mucosa Pylorus

Corpus or body

Mucous neck cell Pit

Antrum

Stem/regenerative cell

Neck Parietal (oxyntic) cell

Mucosa

Base Gland

Chief cell

Muscularis mucosae

Gastric gland in corpus

Submucosa

Endocrine cell

Figure 42-1 Anatomy of the stomach. Shown are the macroscopic divisions of the stomach, as well as two progressively magnified views of a section through the wall of the body of the stomach. 200

The proximal portion of the stomach secretes acid, pepsinogens, intrinsic factor, bicarbonate, and mucus, whereas the distal part releases gastrin and somatostatin

CI– H+

150

[Ion] 100 (mEq/L)

50 K+ 0

Na+ 0

1 2 Secretory rate (mL/min)

3

Figure 42-2 The effect of the gastric secretion rate on the composition of the gastric juice.

Corpus The primary secretory products of the proximal part of the stomach—acid (protons), pepsinogens, and intrinsic factor—are made by distinct cells from glands in the corpus of the stomach. The two primary cell types in the gastric glands of the body of the stomach are parietal cells and chief cells. Parietal cells (or oxyntic cells) secrete both acid and intrinsic factor, a glycoprotein that is required for cobalamin (vitamin B12) absorption in the ileum (see Chapter 45). The parietal cell has a very distinctive morphology (Fig. 42-1). It is a large, triangular cell with a centrally located nucleus, an abundance of mitochondria, intracellular tubulovesicular membranes, and canalicular structures. We discuss H+ secretion in the next major section and intrinsic factor in Chapter 45.

Chapter 42 • Gastric Function

Chief cells (or peptic cells) secrete pepsinogens, but not acid. These epithelial cells are substantially smaller than parietal cells. A close relationship exists among pH, pepsin secretion, and function. Pepsins are endopeptidases (i.e., they hydrolyze “interior” peptide bonds) and initiate protein digestion by hydrolyzing specific peptide linkages. The basal luminal pH of the stomach is 4 to 6; with stimulation, the pH of gastric secretions is usually reduced to less than 2. At pH values that are less than 3, pepsinogens are rapidly activated to pepsins. A low gastric pH also helps to prevent bacterial colonization of the small intestine. In addition to parietal and chief cells, glands from the corpus of the stomach also contain mucus-secreting cells, which are confined to the neck of the gland (Fig. 42-1), and five or six endocrine cells. Among these endocrine cells are enterochromaffin-like (ECL) cells, which release histamine. Antrum

The glands in the antrum of the stomach do not contain parietal cells. Therefore, the antrum does not secrete either acid or intrinsic factor. Glands in the antral mucosa contain chief cells and endocrine cells; the endocrine cells include the so-called G cells and D cells, which secrete gastrin and somatostatin, respectively (see Table 41-1). These two peptide hormones function as both endocrine and paracrine regulators of acid secretion. As discussed in more detail later, gastrin stimulates gastric acid secretion by two mechanisms and is also a major trophic or growth factor for GI epithelial cell proliferation. As discussed more fully later, somatostatin also has several important regulatory functions, but its primary role in gastric physiology is to inhibit both gastrin release and parietal cell acid secretion. In addition to the cells of the gastric glands, the stomach also contains superficial epithelial cells that cover the gastric pits, as well as the surface in between the pits. These cells secrete HCO−3.

The stomach accommodates food, mixes it with gastric secretions, grinds it, and empties the chyme into the duodenum In addition to its secretory properties, the stomach also has multiple motor functions. These functions are the result of gastric smooth muscle activity, which is integrated by both neural and hormonal signals. Gastric motor functions include both propulsive and retrograde movement of food and liquid, as well as a nonpropulsive movement that increases intragastric pressure. Similar to the heterogeneity of gastric epithelial cells, considerable diversity is seen in both the regulation and contractility of gastric smooth muscle. The stomach has at least two distinct areas of motor activity; the proximal and distal portions of the stomach behave as separate, but coordinated, entities. At least four events can be identified in the overall process of gastric filling and emptying: (1) receiving and providing temporary storage of dietary food and liquids; (2) mixing of food and water with gastric secretory products, including pepsin and acid; (3) grinding of food so that particle size is reduced to enhance digestion and to permit passage through the pylorus; and (4) regulating the exit of retained material from the stomach into the duodenum (i.e., gastric emptying of chyme) in response to various stimuli. The mechanisms by which the stomach receives and empties liquids and solids are significantly different. Emptying of liquids is primarily a function of the smooth muscle of the proximal part of the stomach, whereas emptying of solids is regulated by antral smooth muscle.

ACID SECRETION The parietal cell has a specialized tubulovesicular structure that increases apical membrane area when the cell is stimulated to secrete acid

Gastric pH and Pneumonia

M

any patients hospitalized in the intensive care unit (ICU) receive prophylactic antiulcer treatments (e.g., proton pump inhibitors, such as omeprazole) that either neutralize existing acid or block its secretion and thereby raise gastric pH. Patients in the ICU who are mechanically ventilated or who have coagulopathies are highly susceptible to hemorrhage from gastric stress ulcers, a complication that can contribute significantly to overall morbidity and mortality. These different antiulcer regimens do effectively lessen the risk of developing stress ulcers. However, by raising gastric pH, these agents also lower the barrier to gram-negative bacterial colonization of the stomach. Esophageal reflux and subsequent aspiration of these organisms are common in these very sick patients, many of whom are already immunocompromised or even mechanically compromised by the presence of a ventilator tube. If these bacteria are aspirated into the airway, pneumonia can result. The higher the gastric pH, the greater is the risk of pneumonia.

In the basal state, the rate of acid secretion is low. Tubulovesicular membranes are present in the apical portion of the resting, nonstimulated parietal cell and contain the H-K pump (or H,K-ATPase) that is responsible for acid secretion. On stimulation, cytoskeletal rearrangement causes the tubulovesicular membranes that contain the H-K pump to fuse into the canalicular membrane (Fig. 42-3). The result is a substantial increase (50- to 100-fold) in the surface area of the apical membrane of the parietal cell, as well as the appearance of microvilli. This fusion is accompanied by insertion of the H-K pumps, as well as K+ and Cl− channels, into the canalicular membrane. The large number of mitochondria in the parietal cell is consistent with the high rate of glucose oxidation and O2 consumption that is needed to support acid secretion. An H-K pump is responsible for gastric acid secretion by parietal cells The parietal cell H-K pump is a member of the gene family of P-type ATPases (see Chapter 5) that includes the

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A

RESTING

Tubulovesicles

B

ACTIVE

Lumen

Interstitial space

Canaliculus H+

Na+

H2O Cl–

3 Na+ 2 K+

HCl Parietal cell K+

Figure 42-3

Parietal cell: resting and stimulated.

+

H

K+ K

+

HCO–3

OH– CA

CO2

ubiquitous Na-K pump (Na,K-ATPase), which is present at the basolateral membrane of virtually all mammalian epithelial cells and at the plasma membrane of nonpolarized cells. Similar to other members of this ATPase family, the parietal cell H-K pump requires both an α subunit and a β subunit for full activity. The catalytic function of the H-K pump resides in the α subunit; however, the β subunit is required for targeting to the apical membrane. The two subunits form a heterodimer with close interaction at the extracellular domain. The activity of these P-type ATPases, including the gastric H-K pump, is affected by inhibitors that are clinically important in the control of gastric acid secretion. The two types of gastric H-K pump inhibitors are as follows: (1) substituted benzimidazoles (e.g., omeprazole), which act by binding covalently to cysteines on the extracytoplasmic surface; and (2) substances that act as competitive inhibitors of the K+binding site (e.g., the experimental drug Schering 28080). Omeprazole (see the box titled Gastrinoma or ZollingerEllison Syndrome) is a potent inhibitor of parietal cell H-K pump activity and is an extremely effective drug in the control of gastric acid secretion in both physiologically normal subjects and patients with hypersecretory states. In addition, H-K pump inhibitors have been useful in furthering understanding of the function of these pumps. Thus, ouabain, a potent inhibitor of the Na-K pump, does not inhibit the gastric H-K pump, whereas omeprazole does not inhibit the Na-K pump. The colonic H-K pump, whose α subunit has an amino acid sequence that is similar but not identical to that of both the Na-K pump and the parietal cell H-K pump, is partially inhibited by ouabain but not by omeprazole. According to the model presented in Figure 42-4, the key step in gastric acid secretion is extrusion of H+ into the lumen of the gastric gland in exchange for K+. The K+ taken up into the parietal cells is recycled to the lumen through K+ channels. The final component of the process is passive movement of Cl− into the gland lumen. The apical membrane H-K pump energizes the entire process, the net result of which is the active secretion of HCl. Secretion of acid across the apical membrane by the H-K pump results in a rise in parietal cell pH. The adaptive response to this rise in pH includes passive uptake of CO2 and H2O, which the

Cl– CO2 H2O

Figure 42-4 Acid secretion by parietal cells. When the parietal cell is stimulated, H-K pumps (fueled by ATP hydrolysis) extrude H+ into the lumen of the gastric gland in exchange for K+. The K+ recycles back into the lumen by K+ channels. Cl− exits through channels in the luminal membrane, thus completing the net process of HCl secretion. The H+ needed by the H-K pump is provided by the entry of CO2 and H2O, which are converted to H+ and HCO−3 by carbonic anhydrase. The HCO−3 exits across the basolateral membrane through the Cl-HCO3 exchanger.

enzyme carbonic anhydrase (see Chapter 28 for the box on this topic) converts to HCO−3 and H+. The H+ is the substrate of the H-K pump. The HCO−3 exits across the basolateral membrane through the Cl-HCO3 exchanger. This process also provides the Cl− required for net HCl movement across the apical/canalicular membrane. The basolateral Na-H exchanger may participate in intracellular pH regulation, especially in the basal state. Three secretagogues (acetylcholine, gastrin, and histamine) directly and indirectly induce acid secretion by parietal cells The action of secretagogues on gastric acid secretion occurs through at least two parallel and perhaps redundant mechanisms (Fig. 42-5). In the first, acetylcholine (ACh), gastrin, and histamine bind directly to their respective membrane receptors on the parietal cell and synergistically stimulate and potentiate acid secretion. ACh (see Fig. 14-8) is released from endings of the vagus nerve (cranial nerve X), and as we see in the next section, gastrin is released from G cells. Histamine is synthesized from histidine (see Fig. 13-8). The documented presence of ACh, gastrin, and histamine receptors, at least on the canine parietal cell, provides the primary support for this view. In the second mechanism, ACh and gastrin indirectly induce acid secretion as a result of their stimulation of histamine release from ECL cells in the lamina propria. The central role of histamine and ECL cells is consistent with the observation that histamine-2 receptor antagonists (i.e., H2 blockers), such as cimetidine and ranitidine,

Chapter 42 • Gastric Function

2 In the indirect pathway, acetylcholine and gastrin also stimulate the ECL cell, resulting in secretion of histamine. This histamine then acts on the parietal cell.

1 In the direct pathway, acetylcholine, gastrin and histamine stimulate the parietal cell, triggering the secretion of H+ into the lumen. ACh

ENS

From vagus nerve Parietal cells

ACh

M3 (ACh receptors) Histamine

+

K

+

H+

H

H2 (histamine receptor)

CCKB (gastrin receptors) Lumen of antrum

Figure 42-5

ECL cell

Gastrin Interstitial space

The direct and indirect actions of the three acid secretagogues: ACh, gastrin, and histamine.

not only block the direct action of histamine on parietal cells but also substantially inhibit the acid secretion stimulated by ACh and gastrin. The effectiveness of H2 blockers in controlling acid secretion after stimulation by most agonists is well established in studies of both humans and experimental animals. These drugs, but more importantly the proton pump inhibitors, are prescribed to treat active peptic ulcer disease. The three acid secretagogues act through either Ca2+/diacylglycerol or cAMP Stimulation of acid secretion by ACh, gastrin, and histamine, is mediated by a series of intracellular signal transduction processes similar to those responsible for the action of other agonists in other cell systems. All three secretagogues bind to specific G protein–coupled receptors on the parietal cell membrane (Fig. 42-6). ACh binds to an M3 muscarinic receptor (see Chapter 14) on the parietal cell basolateral membrane. This ACh receptor couples to a GTP-binding protein (Gaq) and activates phospholipase C (PLC), which converts phosphatidylinositol 4,5biphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG; see Chapter 3). IP3 causes internal stores to release Ca2+, which then probably acts through calmodulin-dependent protein kinase (see Chapter 3). DAG activates protein kinase C (PKC). The M3 receptor also activates a Ca2+ channel. Gastrin binds to a specific parietal cell receptor that has been identified as the gastrin-cholecystokinin B (CCKB) receptor. Two related CCK receptors have been identified: CCKA and CCKB. Their amino acid sequences are ~50% identical, and both are G protein coupled. The CCKB receptor has equal affinity for both gastrin and CCK. In contrast, the CCKA receptor’s affinity for CCK is three orders of magnitude higher than its affinity for gastrin. These observations and the availability of receptor antagonists are beginning to

CCKB Gastrin PIP2

M3 receptor

Gq

DAG

ACh

PLC

H

IP3

PKC

Lumen of antrum K+

Ca2+ +

Ca2+

ER H2

Histamine

PKA Gs

H-K pump

cAMP

Parietal cell

AC Gi

Prostaglandin receptor

Somatostatin receptor

Somatostatin

Prostaglandins

Figure 42-6 Receptors and signal transduction pathways in the parietal cell. The parietal cell has separate receptors for three acid secretagogues. ACh and gastrin each bind to specific receptors (M3 and CCKB, respectively) that are coupled to the G protein Gaq. The result is activation of PLC, which ultimately leads to the activation of PKC and the release of Ca2+. The histamine binds to an H2 receptor, coupled through Gas to adenylyl cyclase (AC). The result is production of cAMP and activation of PKA. Two inhibitors of acid secretion also act directly on the parietal cell. Somatostatin and prostaglandins bind to separate receptors that are linked to Gai. These agents thus oppose the actions of histamine. ER, endoplasmic reticulum.

clarify the parallel, but at times opposite, effects of gastrin and CCK on various aspects of GI function. The CCKB receptor couples to Gaq and activates the same PLC pathway as does ACh, and this process leads to both an increase in [Ca2+]i and activation of PKC.

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The histamine receptor on the parietal cell is an H2 receptor that is coupled to the Gas GTP-binding protein. Histamine activation of the receptor complex stimulates the enzyme adenylyl cyclase, which, in turn, generates cAMP. The resulting activation of protein kinase A leads to the phosphorylation of certain parietal cell–specific proteins, including the H-K pump. Gastrin is released by both antral and duodenal G cells, and histamine is released by enterochromaffin-like cells in the corpus The presence of a gastric hormone that stimulates acid secretion was initially proposed in 1905. Direct evidence of such a factor was obtained in 1938, and in 1964 Gregory and Tracey isolated and purified gastrin and determined its amino acid sequence. Gastrin has three major effects on GI cells: (1) stimulation of acid secretion by parietal cells (Fig. 42-5); (2) release of histamine by ECL cells; and (3) regulation of mucosal growth in the corpus of the stomach, as well as in the small and large intestine. Gastrin exists in several different forms, but the two major forms are G-17, or “little gastrin,” a 17–amino acid linear peptide (Fig. 42-7A), and G-34, or “big gastrin,” a 34–amino acid peptide (Fig. 42-7B). A single gene encodes a peptide of 101 amino acids. Several cleavage steps and C-terminal amidation (i.e., addition of a −NH2 to the C terminus) occur during gastrin’s post-translational modification, a process that occurs in the endoplasmic reticulum, trans-Golgi apparatus, and both immature and mature secretory granules. The final product of this posttranslational modification is either G-17 or G-34. The tyrosine residue may be either sulfated (so-called gastrin II) or nonsulfated (gastrin I); the two forms are equally active and are present in equal amounts. Gastrin and CCK, a related hormone, have identical C-terminal tetrapeptide sequences

A N

N

Somatostatin, released by gastric D cells, is the central mechanism of inhibition of acid secretion Gastric acid secretion is under close control of not only the stimulatory pathways discussed earlier but also the inhibitory pathways. The major inhibitory pathway involves the release of somatostatin, a polypeptide hormone made by D cells in the antrum and corpus of the stomach. Somatostatin is also made by the δ cells of the pancreatic islets (see Chapter 51) and by neurons in the hypothalamus (see Chapter 48). Somatostatin exists in two forms, SS-28 and SS-14, which have identical C termini. SS-28 is the predominant form in the GI tract.

“LITTLE GASTRIN” OR G-17 (ANTRAL AND DUODENAL) PyroGlu Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp Phe R

B

(Fig. 42-7C) that possess all the biological activities of both gastrin and CCK. Both G-17 and G-34 are present in blood plasma, and their plasma levels primarily reflect their degradation rates. Thus, although G-17 is more active than G-34, the latter is degraded at a substantially lower rate than G-17. As a consequence, the infusion of equal amounts of G-17 or G-34 produces comparable increases in gastric acid secretion. Specialized endocrine cells (G cells) in both the antrum and duodenum make each of the two gastrins. Antral G cells are the primary source of G-17, whereas duodenal G cells are the primary source of G-34. Antral G cells are unusual in that they respond to both luminal and basolateral stimuli (Fig. 42-8). Antral G cells have microvilli on their apical membrane surface and are referred to as an open-type endocrine cell. These G cells release gastrin in response to luminal peptides and amino acids, as well as in response to gastrinreleasing peptide (GRP), a 27–amino acid peptide that is released by vagal nerve endings. As discussed later, gastrin release is inhibited by somatostatin, which is released from adjacent D cells.

Amide

Minimal fragment for strong activity

PyroGlu=Pyroglutamyl Gastrin I, R=H Gastrin II, R=SO4 CCK, R=SO4

“BIG GASTRIN” OR G-34 (DUODENAL) PyroGlu Leu Gly Pro Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp Phe

Amide

R

C

CCK (DUODENAL AND JEJUNAL) Identical to gastrin

N

Lys Ala Pro Ser Gly Arg Val Ser Met Ile Lys Asn Leu Gln Lys Asn Leu Gln Ser Leu Asp Pro Ser His Arg Ile Ser Asp Arg Asp Tyr Met Gly Trp Met Asp Phe R

Minimal fragment for strong activity

Figure 42-7 Amino acid sequences of the gastrins and CCK. A, A single gene encodes a 101–amino acid peptide that is processed to both G-17 and G-34. The N-terminal glutamine is modified to create a pyroglutamyl residue. The C-terminal phenylalanine is amidated. These modifications make the hormone resistant to carboxypeptidases and aminopeptidases. B, The final 16 amino acids of G-34 are identical to the final 16 amino acids in G-17. Both G-17 and G-34 may be either not sulfated (gastrin I) or sulfated (gastrin II). C, The five final amino acids of CCK are identical to those of G-17 and G-34.

Amide

Chapter 42 • Gastric Function

Fundus Distention of stomach (from vagus nerve) Corpus or body

Corpus

Preganglionic Postganglionic

ENS

ACh M3

Histamine M3 (ACh receptor)

Parietal cells

H2 (histamine receptor Somatostatin receptor

Pa

CCKB (gastrin receptor)

e racrin

ACh

Somatostatin receptor

M3 D cell

ECL cell

Somatostatin

Endoc rine

Blood circulation

Endocrine Antrum CCKB (gastrin receptor)

Lumen of corpus

GRP

Digested protein, amino acids G cell

Somatostatin receptor

Gastrin

Somatostatin CCKB (gastrin receptor)

+

H Antrum

Luminal acid H+

D cell ACh

Figure 42-8 Regulation of gastric acid secretion. In the corpus of the stomach, the vagus nerve not only stimulates the parietal cell directly by releasing ACh but also stimulates both ECL and D cells. Vagal stimulation of the ECL cells enhances gastric acid secretion through increased histamine release. Vagal stimulation of the D cells also promotes gastric acid secretion by inhibiting the release of somatostatin, which would otherwise inhibit—by paracrine mechanisms—the release of histamine from ECL cells and the secretion of acid by parietal cells. In the antrum of the stomach, the vagus stimulates both G cells and D cells. The vagus stimulates the G cells through GRP, thus promoting gastrin release. This gastrin promotes gastric acid secretion by two endocrine mechanisms: directly through the parietal cell and indirectly through the ECL cell, which releases histamine. The vagal stimulation of D cells by ACh inhibits the release of somatostatin, which would otherwise inhibit—by paracrine mechanisms—the release of gastrin from G cells and—by an endocrine mechanism—acid secretion by parietal cells. Luminal H+ directly stimulates the D cells to release somatostatin, which inhibits gastrin release from the G cells, thereby reducing gastric acid secretion (negative feedback). In addition, products of protein digestion (i.e., peptides and amino acids) directly stimulate the G cells to release gastrin, which stimulates gastric acid secretion (positive feedback).

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Somatostatin inhibits gastric acid secretion by both direct and indirect mechanisms (Fig. 42-8). In the direct pathway, somatostatin coming from two different sources binds to a Gai-coupled receptor (SST) on the basolateral membrane of the parietal cell and inhibits adenylyl cyclase. The net effect is to antagonize the stimulatory effect of histamine and thus inhibit gastric acid secretion by parietal cells. The source of this somatostatin can either be paracrine (i.e., D cells present in the corpus of the stomach, near the parietal cells) or endocrine (i.e., D cells in the antrum). However, there is a major difference in what triggers the D cells in the corpus and antrum. Neural and hormonal mechanisms stimulate D cells in the corpus (which cannot sense intraluminal pH), whereas low intraluminal pH stimulates D cells in the antrum. Somatostatin also acts through two indirect pathways, both of which are paracrine. In the corpus of the stomach, D cells release somatostatin that inhibits the release of histamine from ECL cells (Fig. 42-8). Because histamine is an acid secretagogue, somatostatin thus reduces gastric acid secretion. In the antrum of the stomach, D cells release somatostatin, which inhibits the release of gastrin from G cells. Because gastrin is another acid secretagogue, somatostatin also reduces gastric acid secretion by this route. The gastrin released by the G cell feeds back on itself by stimulating D cells to release the inhibitory somatostatin. The presence of multiple mechanisms by which somatostatin inhibits acid secretion is another example of the redundant regulatory pathways that control acid secretion. An understanding of the regulation of somatostatin release from D cells is slowly evolving, but it appears that gastrin stimulates somatostatin release, whereas cholinergic agonists inhibit somatostatin release. Several enteric hormones (“enterogastrone”) and prostaglandins inhibit gastric acid secretion Multiple processes in the duodenum and jejunum participate in the negative feedback mechanisms that inhibit gastric acid secretion. Fat, acid, and hyperosmolar solutions in the duodenum are potent inhibitors of gastric acid secretion. Of these inhibitors, lipids are the most potent, but acid is also quite important. Several candidate hormones have been suggested as the prime mediator of this acid inhibition (Table 42-1). These include CCK, secretin, and peptide YY (see Chapter 43), as well as vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), and neurotensin. Although each inhibits acid secretion after systemic administration, none has been unequivocally established as the sole physiological “enterogastrone.” Evidence suggests that secretin, which is released by duodenal S cells, may have a prime role in inhibiting gastric acid secretion after the entry of fat and acid into the duodenum. Secretin appears to reduce acid secretion by at least three mechanisms: (1) inhibition of antral gastrin release, (2) stimulation of somatostatin release, and (3) direct downregulation of the parietal cell H+ secretory process. The presence of luminal fatty acids causes enteroendocrine cells in the duodenum and the proximal part of the small intestine to release both GIP and CCK. GIP reduces acid secretion directly by inhibiting parietal cell acid secre-

Table 42-1 Secretion

Enteric Hormones That Inhibit Gastric H+

Hormone

Source

CCK

I cells of duodenum and jejunum and neurons in ileum and colon

Secretin

S cells in small intestine

VIP

ENS neurons

GIP

K cells in duodenum and jejunum

Neurotensin

Endocrine cells in ileum

Peptide YY

Endocrine cells in ileum and colon

Somatostatin

D cells of stomach and duodenum, δ cells of pancreatic islets

tion and indirectly by inhibiting the antral release of gastrin. GIP also has the important function of stimulating insulin release from pancreatic islet cells in response to duodenal glucose and fatty acids and is therefore often referred to as glucose-dependent insulinotropic polypeptide (see Chapter 51). CCK participates in feedback inhibition of acid secretion by directly reducing parietal cell acid secretion. Finally, some evidence indicates that a neural reflex elicited in the duodenum in response to acid also inhibits gastric acid secretion. Prostaglandin E2 (PGE2) inhibits parietal cell acid secretion, probably by inhibiting histamine’s activation of parietal cell function at a site that is distal to the histamine receptor. PGE2 appears to bind to an EP3 receptor on the basolateral membrane of the parietal cell (Fig. 42-6) and stimulates Gai, which, in turn, inhibits adenylyl cyclase. In addition, prostaglandins also indirectly inhibit gastric acid secretion by reducing histamine release from ECL cells and gastrin release from antral G cells. A meal triggers three phases of acid secretion Basal State

Gastric acid secretion occurs throughout the day and night. Substantial increases in acid secretion occur after meals, whereas the rate of acid secretion between meals is low (i.e., the interdigestive phase). This interdigestive period follows a circadian rhythm; acid secretion is lowest in the morning before awakening and is highest in the evening. Acid secretion is a direct function of the number of parietal cells, which is also influenced, at least in part, by body weight. Thus, men have higher rates of basal acid secretion than do women. Considerable variability in basal acid secretion is also seen among physiologically normal individuals, and the resting intragastric pH can range from 3 to 7. In contrast to the low rate of acid secretion during the basal or interdigestive period, acid secretion is enhanced several-fold by eating (Fig. 42-9). Regulation of gastric acid

Chapter 42 • Gastric Function

Food (buffers) remaining in the stomach

As stomach empties…

Rate of + H secretion

800 40 600

30 [H+]

Rate of H+ secretion (mEq/hr) 20 + [H ] (mEq/liter)

Volume (mL) 400

10

200

Figure 42-9 Effect of eating on acid secretion. Ingesting food causes a marked fall in gastric [H+] because the food buffers the preexisting H+. However, as the food leaves the stomach and as the rate of H+ secretion increases, [H+] slowly rises to its “interdigestive” level.

0

0

1 Ingest food

secretion is most often studied in the fasting state, a state in which intragastric pH is relatively low because of the basal H+ secretory rate and the absence of food that would otherwise buffer the secreted gastric acid. Experimental administration of a secretagogue in the fasted state thus stimulates parietal cells and further lowers intragastric pH. However, the time course of intragastric pH after a meal can vary considerably despite stimulation of acid secretion. The reason is that intragastric pH depends not only on gastric acid secretion but also on the buffering power (see Chapter 28) of food and the rate of gastric emptying of both acid and partially digested material into the duodenum. Regulation of acid secretion during a meal can be best characterized by three separate, but interrelated phases: the cephalic, the gastric, and the intestinal phases. The cephalic and gastric phases are of primary importance. Regulation of acid secretion includes both the stimulatory and inhibitory mechanisms that we discussed earlier (Fig. 42-8). ACh, gastrin, and histamine all promote acid secretion, whereas somatostatin inhibits gastric acid secretion. Although dividing acid secretion during a meal into three phases has been used for decades, it is somewhat artificial because of considerable overlap in the regulation of acid secretion. For example, the vagus nerve is the central factor in the cephalic phase, but it is also important for the vagovagal reflex that is part of the gastric phase. Similarly, gastrin release is a major component of the gastric phase, but vagal stimulation during the cephalic phase also induces the release of antral gastrin. Finally, the development of a consensus model has long been hampered by considerable differences in regulation of the gastric phase of acid secretion in humans, dogs, and rodents. Cephalic Phase The smell, sight, taste, thought, and swallowing of food initiate the cephalic phase, which is primarily mediated by the vagus nerve (Fig. 42-8). Although the

2

3

4

5

Time (hr) …the rate of H secretion rises.

+

cephalic phase has long been studied in experimental animals, especially dogs by Pavlov, more recent studies of sham feeding have confirmed and extended the understanding of the mechanism of the cephalic phase of acid secretion in humans. The aforementioned sensory stimuli activate the dorsal motor nucleus of the vagus nerve in the medulla (see Chapter 14) and thus activate parasympathetic preganglionic efferent nerves. Insulin-induced hypoglycemia also stimulates the vagus nerve and in so doing promotes acid secretion. Stimulation of the vagus nerve results in four distinct physiological events (already introduced in Figure 42-8) that together result in enhanced gastric acid secretion. First, in the body of the stomach, vagal postganglionic muscarinic nerves release ACh, which stimulates parietal cell H+ secretion directly. Second, in the lamina propria of the body of the stomach, the ACh released from vagal endings triggers histamine release from ECL cells, which stimulates acid secretion. Third, in the antrum, peptidergic postganglionic parasympathetic vagal neurons, as well as other enteric nervous system (ENS) neurons, release GRP, which induces gastrin release from antral G cells. This gastrin stimulates gastric acid secretion both directly by acting on the parietal cell and indirectly by promoting histamine release from ECL cells. Fourth, in both the antrum and the corpus, the vagus nerve inhibits D cells, thereby reducing their release of somatostatin and reducing the background inhibition of gastrin release. Thus, the cephalic phase stimulates acid secretion directly and indirectly by acting on the parietal cell. The cephalic phase accounts for ~30% of total acid secretion and occurs before the entry of any food into the stomach. One of the surgical approaches for the treatment of peptic ulcer disease is cutting the vagus nerves (vagotomy) to inhibit gastric acid secretion. Rarely performed, largely because of the many effective pharmacological agents available to treat peptic ulcer disease, the technique has

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Section VII • The Gastrointestinal System

nevertheless proved effective in selected cases. Because vagus nerve stimulation affects several GI functions besides parietal cell acid secretion, the side effects of vagotomy include a delay in gastric emptying and diarrhea. To minimize these untoward events, successful attempts have been made to perform more selective vagotomies, severing only those vagal fibers leading to the parietal cell. Gastric Phase

Entry of food into the stomach initiates the two primary stimuli for the gastric phase of acid secretion (Fig. 42-10). First, the food distends the gastric mucosa, which activates a vagovagal reflex as well as local ENS reflexes. Second, partially digested proteins stimulate antral G cells. Distention of the gastric wall—both in the corpus and antrum—secondary to entry of food into the stomach elicits two distinct neurally mediated pathways. The first is activation of a vagovagal reflex (see Chapter 41), in which gastric wall distention activates a vagal afferent pathway, which, in turn, stimulates a vagal efferent response in the dorsal nucleus of the vagus nerve. Stimulation of acid secretion in response to this vagal efferent stimulus occurs through the same four parallel pathways that are operative when the vagus nerve is activated during the cephalic phase (Fig. 42-8). Second, gastric wall distention also activates a local ENS pathway that releases ACh, which, in turn, stimulates parietal cell acid secretion. The presence of partially digested proteins (peptones) or amino acids in the antrum directly stimulates G cells to release gastrin (Fig. 42-8). Intact proteins have no effect. Acid secretion and activation of pepsinogen are linked in a positive feedback relationship. As discussed later, low pH

Distention of the stomach by food

Digestion of protein

Local ENS reflexes

Vagovagal reflex

Acetylcholine

GRP

enhances the conversion of pepsinogen to pepsin. Pepsin digests proteins to peptones, which promote gastrin release. Finally, gastrin promotes acid secretion, which closes the positive feedback loop. Little evidence indicates that either carbohydrate or lipid participates in the regulation of gastric acid secretion. Components of wine, beer, and coffee stimulate acid secretion by this G cell mechanism. In addition to the two stimulatory pathways acting during the gastric phase, a third pathway inhibits gastric acid secretion by a classic negative feedback mechanism, already noted earlier in our discussion of Figure 42-8. Low intragastric pH stimulates antral D cells to release somatostatin. Because somatostatin inhibits the release of gastrin by G cells, the net effect is a reduction in gastric acid secretion. The effectiveness of low pH in inhibiting gastrin release is emphasized by the following observation: Although peptones are normally a potent stimulus for gastrin release, they fail to stimulate gastrin release either when the intraluminal pH of the antrum is maintained at 1.0 or when somatostatin is infused. The gastric phase of acid secretion, which occurs primarily as a result of gastrin release, accounts for 50% to 60% of total gastric acid secretion. Intestinal Phase The presence of amino acids and partially digested peptides in the proximal portion of the small intestine stimulates acid secretion by three mechanisms (Fig. 42-11). First, these peptones stimulate duodenal G cells to secrete gastrin, just as peptones stimulate antral G cells in the gastric phase. Second, peptones stimulate an unknown endocrine cell to release an additional humoral signal that has been referred to as entero-oxyntin. The chemical nature of this agent has not yet been identified. Third, amino acids absorbed by the proximal part of the small intestine stimulate acid secretion by mechanisms that require further definition.

Protein digestion products

Peptides and amino acids

Intestinal G cell

Antral G cell

Intestinal endocrine cell

Absorbed amino acids

Gastrin Gastrin “Entero-oxyntin”

ECL cell Parietal cell Histamine

Parietal cell H

+

H

Figure 42-10 Gastric phase of gastric acid secretion. Food in the stomach stimulates gastric acid secretion by two major mechanisms: mechanical stretch and the presence of digested protein fragments (peptones).

+

Figure 42-11 Intestinal phase of gastric acid secretion. Digested protein fragments (peptones) in the proximal small intestine stimulate gastric acid secretion by three major mechanisms.

Chapter 42 • Gastric Function

Gastrinoma or Zollinger-Ellison Syndrome

O

n rare occasion, patients with one or more ulcers have very high rates of gastric acid secretion. The increased acid secretion in these patients is most often a result of elevated levels of serum gastrin, released from a pancreatic islet cell adenoma or gastrinoma (Table 42-2). This clinical picture is also known as the ZollingerEllison syndrome. Because gastrin released from these islet cell adenomas is not under physiological control, but rather is continuously released, acid secretion is substantially increased under basal conditions. However, the intravenous administration of pentagastrin—a synthetic gastrin consisting of the last four amino acids of gastrin plus β-alanine— produces only a modest increase in gastric acid secretion. Omeprazole, a potent inhibitor of the parietal cell H-K pump, is now an effective therapeutic agent to control the marked enhancement of gastric acid secretion in patients with gastrinoma and thus helps to heal their duodenal and gastric ulcers. In contrast to patients with gastrinoma or Zollinger-Ellison syndrome, other patients with duodenal ulcer have serum gastrin levels that are near normal. Their basal gastric acid secretion rates are modestly elevated, but they increase markedly in response to pentagastrin. Patients with pernicious anemia (see Chapter 45 for the box on this topic) lack parietal cells and thus cannot secrete H+. In the absence of a low luminal pH, the antral D cell is not stimulated by acid (Fig. 42-10). Consequently, the release of somatostatin from the D cell is low, and minimal tonic inhibition of gastrin release from G cells occurs. It is not surprising, then, that these patients have very high levels of serum gastrin, but virtually no H+ secretion (Table 42-2).

Gastric acid secretion mediated by the intestinal phase is enhanced after a portacaval shunt. Such a shunt—used in the treatment of portal hypertension caused by chronic liver disease—diverts the portal blood that drains the small intestine around the liver on its return to the heart. Thus, the signal released from the small intestine during the intestinal phase is probably—in normal individuals—removed in part by the liver before reaching its target, the corpus of the stomach. Approximately 5% to 10% of total gastric acid secretion is a result of the intestinal phase.

PEPSINOGEN SECRETION Chief cells, triggered by both cAMP and Ca2+ pathways, secrete multiple pepsinogens that initiate protein digestion The chief cells in gastric glands, as well as mucous cells, secrete pepsinogens, a group of proteolytic proenyzmes (i.e., zymogens or inactive enzyme precursors) that belong to the general class of aspartic proteinases. They are activated to pepsins by cleavage of an N-terminal peptide. Pepsins are endopeptidases that initiate the hydrolysis of ingested protein in the stomach. Although eight pepsinogen isoforms were

Table 42-2 Rates

Serum Gastrin Levels and Gastric Acid Secretion

Serum Gastrin (pg/mL)

H+ SECRETION (mEq/hr) Basal

After Pentagastrin

Normal

35

0.5-2.0

20-35

Duodenal ulcer

50

1.5-7.0

25-60

Gastrinoma

500

15-25

30-75

Pernicious anemia

350

0

0

initially identified on electrophoresis, recent classifications are based on immunological identity, so pepsinogens are most often classified as group I pepsinogens, group II pepsinogens, and cathepsin E. Group I pepsinogens predominate. They are secreted from chief cells located at the base of glands in the corpus of the stomach. Group II pepsinogens are also secreted from chief cells but, in addition, are secreted from mucous neck cells in the cardiac, corpus, and antral regions. Pepsinogen secretion in the basal state is ~20% of its maximal secretion after stimulation. Although pepsinogen secretion generally parallels the secretion of acid, the ratio of maximal to basal pepsinogen secretion is considerably less than that for acid secretion. Moreover, the cellular mechanism of pepsinogen release is quite distinct from that of H+ secretion by parietal cells. Release of pepsinogen across the apical membrane is the result of a novel process called compound exocytosis, in which secretory granules fuse with both the plasma membrane and other secretory granules. This process permits rapid and sustained secretion of pepsinogen. After stimulation, the initial peak in pepsinogen secretion is followed by a persistent lower rate of secretion. This pattern of secretion has been interpreted as reflecting an initial secretion of preformed pepsinogen, followed by the secretion of newly synthesized pepsinogen. However, more recent in vitro studies suggested that a feedback mechanism may account for the subsequent reduced rate of pepsinogen secretion. Two groups of agonists stimulate chief cells to secrete pepsinogen. One group acts through adenylyl cyclase and cAMP, and the other acts through increases in [Ca2+]i. Agonists Acting Through cAMP

Chief cells have receptors for secretin/VIP, b2-adrenergic receptors, and EP2 receptors for PGE2 (see Chapter 3). All these receptors activate adenylyl cyclase. At lower concentrations than those required to stimulate pepsinogen secretion, PGE2 can also inhibit pepsinogen secretion, probably by binding to another receptor subtype. Agonists Acting Through Ca2+

Chief cells also have M3 muscarinic receptors for ACh, as well as receptors for the gastrin/CCK family of peptides. Unlike gastric acid secretion, which is stimulated by the CCKB receptor, pepsinogen secretion is stimulated by the CCKA receptor, which has a much higher affinity for CCK than for gastrin. Activation of

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both the M3 and CCKA receptors causes Ca2+ release from intracellular stores by IP3 and thereby raises [Ca2+]i. However, uncertainty exists about whether increased Ca2+ influx is also required and about whether PKC also has a role. Of the agonists just listed, the most important for pepsinogen secretion is ACh released in response to vagal stimulation. Not only does ACh stimulate chief cells to release pepsinogen, but also it stimulates parietal cells to secrete acid. This gastric acid produces additional pepsinogen secretion by two different mechanisms. First, in the stomach, a fall in pH elicits a local cholinergic reflex that results in further stimulation of chief cells to release pepsinogen. Thus, the ACh that stimulates chief cells can come both from the vagus and from the local reflex. Second, in the duodenum, acid triggers the release of secretin from S cells. By an endocrine effect, this secretin stimulates the chief cells to release more pepsinogen. The exact role or roles of histamine and gastrin in pepsinogen secretion are unclear. Low pH is required for both pepsinogen activation and pepsin activity Pepsinogen is inactive and requires activation to a protease, pepsin, to initiate protein digestion. This activation occurs by spontaneous cleavage of a small N-terminal peptide fragment (the activation peptide), but only at a pH that is less than 5.0 (Fig. 42-12). Between pH 5.0 and 3.0, spontaneous activation of pepsinogen is slow, but it is extremely rapid at a pH that is less than 3.0. In addition, pepsinogen is also autoactivated; that is, newly formed pepsin itself cleaves pepsinogen to pepsin. Once pepsin is formed, its activity is also pH dependent. It has optimal activity at a pH between 1.8 and 3.5; the precise optimal pH depends on the specific pepsin, type and concentration of substrate, and osmolality of the solution. pH values higher than 3.5 reversibly inactivate pepsin, and pH values higher than 7.2 irreversibly inactivate the enzyme. These considerations are sometimes useful for

Spontaneous breakdown pH = 3.0–5.0 Pepsinogen

Pepsin pH < 3.0 pH must be lower than 3.5 to prevent inactivation of pepsin

Figure 42-12 Activation of the pepsinogens to pepsins. At pH values from 5 to 3, pepsinogens spontaneously activate to pepsins by the removal of an N-terminal activation peptide. This spontaneous activation is even faster at pH values lower than 3. The newly formed pepsins themselves—which are active only at pH values lower than 3.5—also can catalyze the activation of pepsinogens.

establishing optimal antacid treatment regimens in peptic ulcer disease. Pepsin is an endopeptidase that initiates the process of protein digestion in the stomach. Pepsin action results in the release of small peptides and amino acids (peptones) that, as noted earlier, stimulate the release of gastrin from antral G cells; these peptones also stimulate CCK release from duodenal I cells. As previously mentioned, the peptones generated by pepsin stimulate the very acid secretion required for pepsin activation and action. Thus, the peptides that pepsin releases are important in initiating a coordinated response to a meal. However, most protein entering the duodenum remains as large peptides, and nitrogen balance is not impaired after total gastrectomy. Digestive products of both carbohydrates and lipid are also found in the stomach, although secretion of their respective digestive enzymes either does not occur or is not a major function of gastric epithelial cells. Carbohydrate digestion is initiated in the mouth by salivary amylase. However, after this enzyme is swallowed, the stomach becomes a more important site for starch hydrolysis than the mouth. No evidence indicates gastric secretion of enzymes that hydrolyze starch or other saccharides. Similarly, although lipid digestion is also initiated in the mouth by lingual lipase, significant lipid digestion occurs in the stomach as a result of both the lingual lipase that is swallowed and gastric lipase, both of which have an acid pH optimum (see Chapter 45).

PROTECTION OF THE GASTRIC SURFACE EPITHELIUM AND NEUTRALIZATION OF ACID IN THE DUODENUM At maximal rates of H+ secretion, the parietal cell can drive the intraluminal pH of the stomach to 1 or less (i.e., [H+] > 100 mM) for long periods. The gastric epithelium must maintain an H+ concentration gradient of more than a million-fold because the intracellular pH of gastric epithelial cells is ~7.2 (i.e., [H+] ≅ 60 nM) and plasma pH is ~7.4 (i.e., [H+] ≅ 40 nM). Simultaneously, a substantial plasma-to-lumen Na+ concentration gradient of ~30 is present because plasma [Na+] is 140 mM, whereas intragastric [Na+] can reach values as low as 5 mM, but only at high secretory rates (Fig. 42-2). How is the stomach able to maintain these gradients? How is it that the epithelial cells are not destroyed by this acidity? Moreover, why do pepsins in the gastric lumen not digest the epithelial cells? The answer to all three questions is the so-called gastric diffusion barrier. Although the nature of the gastric diffusion barrier had been controversial, it is now recognized that the diffusion barrier is both physiological and anatomical. Moreover, it is apparent that the diffusion barrier represents at least three components: (1) relative impermeability to acid of the apical membrane and epithelial cell tight junctions in the gastric glands, (2) a mucous gel layer varying in thickness between 50 and 200 μm overlying the surface epithelial cells, and (3) an HCO−3-containing microclimate adjacent to the

Chapter 42 • Gastric Function

surface epithelial cells that maintains a relatively high local pH. Vagal stimulation and irritation stimulate gastric mucous cells to secrete mucin, a glycoprotein that is part of the mucosal barrier The mucus layer is largely composed of mucin, phospholipids, electrolytes, and water. Mucin is the high-molecularweight glycoprotein (see Chapter 2) that contributes to the formation of a protective layer over the gastric mucosa. Gastric mucin is a tetramer consisting of four identical peptides joined by disulfide bonds. Each of the four peptide chains is linked to long polysaccharides, which are often sulfated and are thus mutually repulsive. The ensuing high carbohydrate content is responsible for the viscosity of mucus, which explains, in large part, its protective role in gastric mucosal physiology. Mucus is secreted by three different mucous cells: surface mucous cells (i.e., on the surface of the stomach), mucous neck cells (i.e., at the point where a gastric pit joins a gastric gland), and glandular mucous cells (i.e., in the gastric glands in the antrum). The type of mucus secreted by these cells differs; mucus that is synthesized and secreted in the glandular cells is a neutral glycoprotein, whereas the mucous cells on the surface and in the gastric pits secrete both neutral and acidic glycoproteins. Mucin forms a mucous gel layer in combination with phospholipids, electrolytes, and water. This mucous gel layer provides protection against injury from noxious luminal substances, including acid, pepsins, bile acids, and ethanol. Mucin also lubricates the gastric mucosa to minimize the abrasive effects of intraluminal food. The mucus barrier is not static. Abrasions can remove pieces of mucus. When mucus comes in contact with a solution with a very low pH, the mucus precipitates and sloughs off. Thus, the mucous cells must constantly secrete mucus. Regulation of mucus secretion by gastric mucosal cells is less well understood than is regulation of the secretion of acid, pepsinogens, and other substances by gastric cells. The two primary stimuli for inducing mucus secretion are vagal stimulation and physical and chemical irritation of the gastric mucosa by ingested food. The current model of mucus secretion suggests that vagal stimulation induces the release of ACh, which leads to increases in [Ca2+]i and thus stimulates mucus secretion. In contrast to acid and pepsinogen secretion, cAMP does not appear to be a second messenger for mucus secretion. Gastric surface cells secrete HCO-3, stimulated by acetylcholine, acids, and prostaglandins Surface epithelial cells both in the corpus and in the antrum of the stomach secrete HCO−3. Despite the relatively low rate of HCO−3 secretion—in comparison with acid secretion— HCO−3 is extremely important as part of the gastric mucosal protective mechanism. The mucus gel layer provides an unstirred layer under which the secreted HCO−3 remains trapped and maintains a local pH of ~of 7.0 versus an intraluminal pH in the bulk phase of 1 to 3. As illustrated in

Figure 42-13A, an electrogenic Na/HCO3 cotransporter (NBC) appears to mediate the uptake of HCO−3 across the basolateral membrane of surface epithelial cells. The mechanism of HCO−3 exit from the cell into the apical mucus layer is unknown but may be mediated by a channel. Similar to the situation for mucus secretion, relatively limited information is available about the regulation of HCO−3 secretion. The present model suggests that vagal stimulation mediated by ACh leads to an increase in [Ca2+]i, which, in turn, stimulates HCO−3 secretion. Sham feeding is a potent stimulus for HCO−3 secretion through this pathway. A second powerful stimulus of gastric HCO−3 secretion is intraluminal acid. The mechanism of stimulation by acid appears to be secondary to both activation of neural reflexes and local production of PGE2. Finally, evidence suggests that a humoral factor may also be involved in the induction of HCO−3 secretion by acid. Mucus protects the gastric surface epithelium by trapping an HCO-3-rich fluid near the apical border of these cells Mucous cells on the surface of the stomach, as well as in the gastric pits and neck portions of the gastric glands, secrete both HCO−3 and mucus. Why is this barrier so effective? First, the secreted mucus forms a mucous gel layer that is relatively impermeable to the diffusion of H+ from the gastric lumen to the surface cells. Second, beneath this layer of mucus is a microclimate that contains fluid with a high pH and high [HCO−3], the result of HCO−3 secretion by gastric surface epithelial cells (Fig. 42-13A). Thus, this HCO−3 neutralizes most acid that diffuses through the mucus layer. Mucosal integrity, including that of the mucosal diffusion barrier, is also maintained by PGE2, which—as discussed in the previous section—stimulates mucosal HCO−3 secretion. Deep inside the gastric gland, where no obvious mucus layer protects the parietal, chief, and ECL cells, the impermeability of the cells’ apical barrier appears to exclude H+ even at pH values as low as 1. The paradox of how HCl secreted by the parietal cells emerges from the gland and into the gastric lumen may be explained by a process known as viscous fingering. Because the liquid emerging from the gastric gland is both extremely acidic and presumably under pressure, it can tunnel through the mucous layer covering the opening of the gastric gland onto the surface of the stomach. However, this stream of acid apparently does not spread laterally, but rather rises to the surface as a “finger” and thus does not neutralize the HCO−3 in the microenvironment between the surface epithelial cells and the mucus. The mucous gel layer and the trapped alkaline HCO−3 solution protect the surface cells not only from H+ but also from pepsin. The mucus per se acts as a pepsin diffusion barrier. The relative alkalinity of the trapped HCO−3 inactivates any pepsin that penetrates the mucus. Recall that pepsin is reversibly inactivated at pH values higher than ~3.5 and is irreversibly inactivated by pH values higher than ~7.2. Thus, the mucus HCO−3 layer plays an important role in preventing autodigestion of the gastric mucosa.

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A

Figure 42-13 Diffusion barrier in the surface of the gastric mucosa. A, The mucus secreted by the surface cells serves two functions. First, it acts as a diffusion barrier for H+ and also pepsins. Second, the mucus layer traps a relatively alkaline solution of HCO−3. This HCO−3 titrates any H+ that diffuses into the gel layer from the stomach lumen. The alkaline layer also inactivates any pepsin that penetrates into the mucus. B, If H+ penetrates into the gastric epithelium, it damages mast cells, which release histamine and other agents, thereby setting up an inflammatory response. If the insult is mild, the ensuing increase in blood flow can promote the production of both mucus and HCO−3 by the mucus cells. If the insult is more severe, the inflammatory response leads to a decrease in blood flow and thus to cell injury.

NORMAL SURFACE EPITHELIUM

Isthmus

HCl

Neck

Breakdown of the Gastric Barrier

Base Gastric gland 1.5 Gastric lumen

[H+] = 20 mM

H+ enters

[HCO3–] = 0

HCO3– exits

Mucus gel neutralization zone Mucus gel

[H+] = 0.0001 mM

HCO3– –rich zone

H+ H2CO3

pH profile

CO2 + H2O

HCO–3 7.0

Na+

– 2 HCO3

Bloodstream

B

7.4

DAMAGED MUCOSAL BARRIER Gastric lumen HCl

Mast cell

I

ntegrity of the gastric-epithelial barrier can be conveniently judged by maintenance of a high lumen-negative transepithelial potential difference (PD) of ~−60 mV. Several agents that cause mucosal injury, including mucosal ulceration, can alter the mucosal diffusion barrier. Salicylates, bile acids, and ethanol all impair the mucosal diffusion barrier and result in H+ (acid) backdiffusion, an increase in intraluminal [Na+], a fall in PD, and mucosal damage (Fig. 42-13B). Three decades ago, Davenport proposed an attractive model to explain how H+, after having breached the mucosal diffusion barrier, produces injury to the gastric mucosa. Although several details of this original model have been modified during the ensuing years, it is still believed that entry of acid into the mucosa damages mast cells, which release histamine and other mediators of inflammation. The histamine and other agents cause local vasodilatation that increases blood flow. If the damage is not too severe, this response allows the surface cells to maintain their production of mucus and HCO−3. However, if the injury is more severe, inflammatory cells release a host of agents— including platelet-activating factor, leukotrienes, endothelins, thromboxanes, and oxidants—that reduce blood flow (ischemia) and result in tissue injury, including capillary damage. Prostaglandins play a central role in maintaining mucosal integrity. For example, prostaglandins prevent or reverse mucosal injury secondary to salicylates, bile, and ethanol. This protective effect of prostaglandins is the result of several actions, including their ability to inhibit acid secretion, stimulate both HCO−3 and mucus secretion, increase mucosal blood flow, and modify the local inflammatory response induced by acid.

Na+

K+

Fluid

Blood

Entry of acid into the duodenum induces the release of secretin from S cells, thus triggering the secretion of HCO-3 by the pancreas and duodenum, which, in turn, neutralizes gastric acid The overall process of regulating gastric acid secretion involves not only stimulation and inhibition of acid secretion (as discussed earlier), but also neutralization of the gastric acid that passes from the stomach into the duodenum. The amount of secreted gastric acid is reflected by a

HCl

Histamine

Bloodstream

Chapter 42 • Gastric Function

Helicobacter pylori

D

uring the past decade, our understanding of the etiology of duodenal and gastric ulcers has radically changed. Abundant evidence now indicates that most peptic ulcers are an infectious disease in that most (but not all) ulcers are caused by Helicobacter pylori, a gram-negative bacillus that colonizes the antral mucosa. Nonsteroidal anti-inflammatory drugs (NSAIDs) are responsible for ~20% of ulcers. Although almost all ulcers that are not associated with NSAID use are secondary to H. pylori infestation, many, if not most, individuals with evidence of H. pylori infestation do not have peptic ulcer disease. The factors responsible for H. pylori–induced inflammation or ulceration are not known. However, the increase in gastric acid secretion that is present in most patients with duodenal ulcers may occur because H. pylori–induced antral inflammation inhibits the release of somatostatin by antral D cells. Because somatostatin normally inhibits gastrin release by antral G cells, the result would be increased gastrin release and thus increased gastric acid secretion. Indeed, as noted in Table 42-2, serum gastrin levels are modestly elevated in patients with duodenal ulcers. Inhibition of acid secretion heals, but does not cure H. pylori–induced peptic ulcers. However, antibiotic therapy that eradicates H. pylori cures peptic ulcer disease.

fall in intragastric pH. We have already seen that this fall in pH serves as the signal to antral D cells to release somatostatin and thus to inhibit further acid secretion, a classic negative feedback process. Similarly, low pH in the duodenum serves as a signal for the secretion of alkali to neutralize gastric acid in the duodenum. The key factor in this neutralization process is secretin, the same secretin that inhibits gastric acid secretion and promotes pepsinogen secretion by chief cells. A low duodenal pH, with a threshold of 4.5, triggers the release of secretin from S cells in the duodenum. However, the S cells are probably not pH sensitive themselves but, instead, may respond to a signal from other cells that are pH sensitive. Secretin stimulates the secretion of fluid and HCO−3 by the pancreas, thus leading to intraduodenal neutralization of the acid load from the stomach. Maximal HCO−3 secretion is a function of the amount of acid entering the duodenum, as well as the length of duodenum exposed to acid. Thus, high rates of gastric acid secretion trigger the release of large amounts of secretin, which greatly stimulates pancreatic HCO−3 secretion; the increased HCO−3, in turn, neutralizes the increased duodenal acid load. In addition to pancreatic HCO−3 secretion, the duodenal acid load resulting from gastric acid secretion is partially neutralized by duodenal HCO−3 secretion. This duodenal HCO−3 secretion occurs in the proximal—but not the distal— part of the duodenum under the influence of prostaglandins. Attention has been focused on duodenal epithelial cells (villus or crypt cells) as the cellular source of HCO−3 secretion, but the possibility that duodenal HCO−3 originates, at least in part, from duodenal submucosal Brunner’s glands has not been excluded. The mechanism of duodenal HCO−3

secretion involves both Cl-HCO3 exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane (see Fig. 43-6). Patients with duodenal ulcer disease tend to have both increased gastric acid secretion and reduced duodenal HCO−3 secretion. Thus, the increased acid load in the duodenum is only partially neutralized, so the duodenal mucosa has increased exposure to a low-pH solution.

FILLING AND EMPTYING OF THE STOMACH Gastric motor activity plays a role in filling, churning, and emptying Gastric motor activity has three functions. First, the receipt of ingested material represents the reservoir function of the stomach and occurs as smooth muscle relaxes. This response occurs primarily in the proximal portion of the stomach. Second, ingested material is churned and is thereby altered to a form that rapidly empties from the stomach through the pylorus and facilitates normal jejunal digestion and absorption. Thus, in conjunction with gastric acid and enzymes, the motor function of the stomach helps to initiate digestion. Third, the pyloric antrum, pylorus, and proximal part of the duodenum function as a single unit for emptying into the duodenum the modified gastric contents (chyme), consisting of both partially digested food material and gastric secretions. Gastric filling and emptying are accomplished by the coordinated activity of smooth muscle in the esophagus, lower esophageal sphincter, and proximal and distal portions of the stomach, as well as the pylorus and duodenum. The pattern of gastric smooth muscle activity is distinct during fasting and after eating. The pattern during fasting is referred to as the migrating myoelectric (or motor) complex (MMC), as discussed in Chapter 41 in connection with the small intestine. This pattern is terminated by eating, at which point it is replaced by the so-called fed pattern. Just as the proximal and distal regions of the stomach differ in secretory function, they also differ in the motor function responsible for storing, processing, and emptying liquids and solids. The proximal part of the stomach is the primary location for storage of both liquids and solids. The distal portion of the stomach is primarily responsible for churning the solids and generating smaller liquid-like material, which then exits the stomach in a manner similar to that of ingested liquids. Thus, the gastric emptying of liquids and of solids is closely integrated. Filling of the stomach is facilitated by both receptive relaxation and gastric accommodation Even a dry swallow relaxes both the lower esophageal sphincter and the proximal part of the stomach. Of course, the same happens when we swallow food. These relaxations facilitate the entry of food into the stomach. Relaxation in the fundus is primarily regulated by a vagovagal reflex and has been called receptive relaxation. In a vagovagal reflex,

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afferent fibers running with the vagus nerve carry information to the central nervous system (CNS), and efferent vagal fibers carry the signal from the CNS to the stomach and cause relaxation by a mechanism that is neither cholinergic nor adrenergic. The result is that intragastric volume increases without an increase in intragastric pressure. If vagal innervation to the stomach is interrupted, gastric pressure rises much more rapidly. Quite apart from the receptive relaxation of the stomach that anticipates the arrival of food after swallowing and esophageal distention, the stomach can also relax in response to gastric filling per se. Thus, increasing intragastric volume, as a result of either entry of food into the stomach or gastric secretion, does not produce a proportionate increase in intragastric pressure. Instead, small increases in volume do not cause increases in intragastric pressure until a threshold is reached, after which intragastric pressure rises steeply (Fig. 42-14A). This phenomenon is the result of active dilatation of the fundus and has been called gastric accommodation. Vagotomy abolishes a major portion of gastric accommodation, so increases in intragastric volume produce greater increases in intragastric pressure. However, the role of the

A

GASTRIC ACCOMMODATION Stomach is empty.

Food is in stomach.

Food is in stomach after vagotomy.

80 Vagotomy

60 Intraluminal pressure 40 (cm H2O) 20 0

B

Normal 0

200

400 600 Volume (mL)

800

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GASTRIC EMPTYING 100 Oleate meal Acid meal Saline meal

75 Percent of test meal remaining 50 in stomach 25

0 0

10

20 Time (min)

30

40

Figure 42-14 Gastric filling and emptying. (B, Data from Dooley CP, Reznick JB, Valenzuela JE: Variations in gastric and duodenal motility during gastric emptying of liquid meals in humans. Gastroenterology 1984; 87:1114-1119.)

vagus nerve in gastric accommodation is one of modulation. It is generally believed that the ENS (see Chapter 41) is the primary regulator permitting the storage of substantial amounts of solids and liquids in the proximal part of the stomach without major increases in intragastric pressure. The stomach churns its contents until the particles are small enough to be gradually emptied into the duodenum The substance most rapidly emptied by the stomach is isotonic saline or water. Emptying of these liquids occurs without delay and is faster the greater the volume of fluid. Acidic and caloric fluids leave the stomach more slowly, whereas fatty materials exit even more slowly (Fig. 42-14B).

Vomiting

V

omiting, a frequent sign and symptom in clinical medicine, represents a complex series of multiple afferent stimuli coordinated by one or more brain centers, leading to a coordinated neuromuscular response. Nausea is the sensation that vomiting may occur. The act of emesis involves several preprogrammed coordinated smooth and striated muscle responses. The initial event is the abolition of intestinal slow-wave activity that is linked to propulsive peristaltic contractions. As the normal peristaltic contractions of the stomach and small intestine wane, they are replaced by retrograde contractions, beginning in the ileum and progressing to the stomach. These retrograde contractions are accompanied by contraction of abdominal and inspiratory muscles (external intercostal muscles and diaphragm) against a closed glottis, thus resulting in an increase in intra-abdominal pressure. Relaxation of the diaphragmatic crural muscle and lower esophageal sphincter permits transmission of this increase in intra-abdominal pressure into the thorax, with expulsion of the gastric contents into the esophagus. Movement of the larynx upward and forward and relaxation of the upper esophageal sphincter are required for oral propulsion, whereas closure of the glottis prevents aspiration. Three major categories of stimuli can potentially induce the foregoing series of events that lead to vomiting. First, gastric irritants and peritonitis, for example, probably act by vagal afferent pathways, presumably to rid the body of the irritant. Second, inner ear dysfunction or motion sickness acts through the vestibular nerve and vestibular nuclei. Third, drugs such as digitalis and certain cancer chemotherapeutic agents activate the area postrema in the brain (see Chapter 11). Pregnancy can also cause nausea and vomiting, by an unknown mechanism. Although several central loci receive these emetic stimuli, the primary locus is the area postrema, also called the chemoreceptor trigger zone. Although no single brainstem site coordinates vomiting, the nucleus tractus solitarii plays an important role in the initiation of emesis. Neurotransmitter receptors that are important in various causes of vomiting include neurokinin NK1 and substance P receptors in the nucleus tractus solitarii, 5-HT3 receptors in vagal afferents, and dopamine D2 receptors in the vestibular nucleus.

Chapter 42 • Gastric Function

A

PROPULSION

Peristaltic wave Bolus is pushed toward the closed pylorus.

Pylorus P P B

GRINDING The antrum churns the trapped material.

P P C

RETROPULSION

Bolus is pushed back into the proximal stomach. P

particles, a process known as retropulsion (Fig. 42-15C). These processes of propulsion, grinding, and retropulsion repeat multiple times until the gastric contents are emptied. Particles larger than 2 mm are initially retained in the stomach but are eventually emptied into the duodenum by MMCs during the interdigestive period that begins ~2 hours or more after eating. Modification of gastric contents is associated with the activation of multiple feedback mechanisms. This feedback usually arises from the duodenum (and beyond) and almost always results in a delay in gastric emptying. Thus, as small squirts of gastric fluid leave the stomach, chemoreceptors and mechanoreceptors—primarily in the proximal but also in the distal portion of the small intestine—sense low pH, a high content of calories, lipid, or some amino acids (i.e., tryptophan), or changes in osmolarity. These signals all decrease the rate of gastric emptying by a combination of neural and hormonal signals, including the vagus nerve, secretin, CCK, and GIP released from duodenal mucosa. Delayed gastric emptying represents the following: the coordinated function of fundic relaxation; inhibition of antral motor activity; stimulation of isolated, phasic contractions of the pyloric sphincter; and altered intestinal motor activity.

P

Figure 42-15 Mechanical actions of the stomach on its contents.

Solids do not leave the stomach as such, but must first be reduced in size (i.e., trituration). Particles larger than 2 mm do not leave the stomach during the immediate postprandial digestive period. The delay in gastric emptying of solids occurs because solids must be reduced to less than 2 mm; at that point, they are emptied by mechanisms similar to those of liquids. Movement of solid particles toward the antrum is accomplished by the interaction of propulsive gastric contractions and occlusion of the pylorus, a process termed propulsion (Fig. 42-15A). Gastric contractions are initiated by the gastric pacemaker, which is located on the greater curvature, approximately at the junction of the proximal and middle portions of the stomach. These contractions propel the luminal contents toward the pylorus, which is partially closed by contraction of the pyloric musculature before delivery of the bolus. This increase in pyloric resistance represents the coordinated response of antral, pyloric, and duodenal motor activity. Once a bolus of material is trapped near the antrum, it is churned to help reduce the size of the particles, a process termed grinding (Fig. 42-15B). Only a small portion of gastric material—that containing particles smaller than 2 mm—is propelled through the pylorus to the duodenum. Thus, most gastric contents are returned to the body of the stomach for pulverization and shearing of solid

REFERENCES Books and Reviews DelValle J, Todisco A: Gastric secretion. In Yamada T (ed): Textbook of Gastroenterology, vol 1, 4th ed, pp 266-307. Philadelphia: Lippincott Williams & Wilkins, 2003. Dockray G, Dimaline, R, Varro A: Gastrin: Old hormone, new functions. Pflugers Arch 2005; 449:344-355. Dockray G, Varro A, Dimaline R: Gastric endocrine cells: Gene expression, processing and targeting of active products. Physiol Rev 1996; 76:767-798. Hasler WL: The physiology of gastric motility and gastric emptying. In Yamada T (ed): Textbook of Gastroenterology, vol 1, 4th ed, pp 266-307. Philadelphia: Lippincott Williams & Wilkins, 2003. Hersey SJ, Sachs G: Gastric acid secretion. Physiol Rev 1995; 75:155-189. Lichtenberger LM: The hydrophobic barrier properties of gastrointestinal mucus. Annu Rev Physiol 1995; 57:565-583. Sachs G, Prinz C: Gastric enterochromaffin-like cells and the regulation of acid secretion. News Physiol Sci 1996; 11:57-62. Journal Articles Dooley CP, Reznick JB, Valenzuela JE: Variations in gastric and duodenal motility during gastric emptying of liquid meals in humans. Gastroenterology 1984; 87:1114-1119. Lambrecht NW, Yakubov I, Scott D, Sachs G: Identification of the efflux channel coupled to the gastric H,K-ATPase during acid secretion. Physiol Genomics 2005; 21:81-91. Waisbren SJ, Geibel JP, Modlin IM, Boron WF: Unusual permeability properties of gastric gland cells. Nature 1994; 368: 332-335.

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P A N C R E AT I C A N D S A L I V A R Y G L A N D S Christopher R. Marino and Fred S. Gorelick

OVERVIEW OF EXOCRINE GLAND PHYSIOLOGY The pancreas and major salivary glands are compound exocrine glands The exocrine pancreas and major salivary glands are compound exocrine glands—specialized secretory organs that contain a branching ductular system through which they release their secretory products. The principal function of these exocrine glands is to aid in the digestion of food. The saliva produced by the salivary glands lubricates ingested food and initiates the digestion of starch. Pancreatic juice, rich in HCO−3 and digestive enzymes, neutralizes the acidic gastric contents that enter the small intestine and also completes the intraluminal digestion of ingested carbohydrate, protein, and fat. Each of these exocrine glands is under the control of neural and humoral signals that generate a sequential and coordinated secretory response to an ingested meal. We discuss the endocrine pancreas in Chapter 51. Morphologically, the pancreas and salivary glands are divided into small but visible lobules, each of which represents a subdivision of the parenchyma and is drained by a single intralobular duct (Fig. 43-1A). Groups of lobules separated by connective tissue septa are drained by larger interlobular ducts. These interlobular ducts empty into a main duct that connects the entire gland to the lumen of the gastrointestinal tract. Within the lobules reside the microscopic structural and functional secretory units of the gland. Each secretory unit is composed of an acinus and a small intercalated duct. The acinus represents a cluster of 15 to 100 acinar cells that synthesize and secrete proteins into the lumen of the epithelial structure. In the pancreas, acinar cells secrete ~20 different digestive zymogens (inactive enzyme precursors) and enzymes. In the salivary glands, the principal acinar cell protein products are α-amylase, mucins, and proline-rich proteins. Acinar cells from both the pancreas and salivary glands also secrete an isotonic, plasma-like fluid that accompanies the secretory proteins. In all, the final acinar secretion is a protein-rich product known as the primary secretion.

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Each acinar lumen is connected to the proximal end of an intercalated duct. Distally, the intercalated ducts fuse with other small ducts to form progressively larger ducts that ultimately coalesce to form the intralobular duct that drains the lobule. Although the ducts provide a conduit for the transport of secretory proteins, the epithelial cells lining the ducts also play an important role in modifying the fluid and electrolyte composition of the primary secretion. Thus, the final exocrine gland secretion represents the combined product of two distinct epithelial cell populations, the acinar cell and the duct cell. In addition to acini and ducts, exocrine glands contain a rich supply of nerves and blood vessels. Postganglionic parasympathetic and sympathetic fibers contribute to the autonomic regulation of secretion through the release of cholinergic, adrenergic, and peptide neurotransmitters that bind to receptors on the acinar and duct cells. Both central and reflex pathways contribute to the neural regulation of exocrine secretion. The autonomic nerves also carry afferent pain fibers that are activated by glandular inflammation and trauma. The vasculature not only provides oxygen and nutrients for the gland but also carries the hormones that help to regulate secretion. Acinar cells are specialized protein-synthesizing cells Acinar cells—such as those in the pancreas (Fig. 43-1B) and salivary glands—are polarized epithelial cells that are specialized for the production and export of large quantities of protein. Thus, the acinar cell is equipped with extensive rough endoplasmic reticulum (ER). However, the most characteristic feature of the acinar cell is the abundance of electron-dense secretory granules at the apical pole of the cell. These granules are storage pools of secretory proteins, and they are poised for releasing their contents after stimulation of the cell by neurohumoral agents. The secretory granules of pancreatic acinar cells contain the mixture of zymogens and enzymes required for digestion. The secretory granules of salivary acinar cells contain either α-amylase (in the parotid gland) or mucins (in the sublingual glands). Secretory granules in the pancreas appear uniform, whereas

Chapter 43 • Pancreatic and Salivary Glands

A

ORGANIZATION OF THE PANCREAS

Common bile duct

Pancreas

Intralobular ducts

Main pancreatic duct

Intercalated duct Interlobular duct Main pancreatic duct

Lobule (secretory unit)

Acinus

B PANCREATIC ACINAR CELL Zymogen Golgi granules

Intralobular duct

Pancreatic duct epithelial cell Centroacinar cell Pancreatic acinar cell

Intercalated duct

Mitochondrion C

Rough ER

PANCREATIC DUCT EPITHELIAL CELL Mitochondria

Acinus

Lobule

Figure 43-1 Acinus duct morphology. A, The fundamental secretory unit is composed of an acinus and an intercalated duct. Intercalated ducts merge to form intralobular ducts, which, in turn, merge to form interlobular ducts, and then the main pancreatic duct. B, The acinar cell is specialized for protein secretion. Large condensing vacuoles are gradually reduced in size and form mature zymogen granules that store digestive enzymes in the apical region of the acinar cell. C, The duct cell is a cuboidal cell with abundant mitochondria. Small microvilli project from its apical membrane.

those in the salivary glands often exhibit focal nodules of condensation within the granules known as spherules. The pancreatic acinar cell has served as an important model for elucidating protein synthesis and export through the secretory pathway. Synthesis of secretory proteins (see Chapter 2) begins with the cellular uptake of amino acids and their incorporation into nascent proteins in the rough ER (Fig. 43-2). Vesicular transport mechanisms then shuttle the newly synthesized proteins to the Golgi complex. Within the Golgi complex, secretory proteins are segregated away from lysosomal enzymes. Most lysosomal enzymes require the mannose 6-phosphate receptor for sorting to the lysosome (see Chapter 2). However, the signals

required to direct digestive enzymes into the secretory pathway remain unclear. Secretory proteins exit the Golgi complex in condensing vacuoles. These large membrane-bound structures are acidic and maintain the lowest pH within the secretory pathway. Maturation of the condensing vacuole to a secretory or zymogen granule is marked by condensation of the proteins within the vacuole and pinching off of membrane vesicles. The diameter of a zymogen granule is about two thirds that of a condensing vacuole, and its content is more electron dense. Secretory proteins are stored in zymogen granules that are located in the apical region of the acinar cell. The bottom portion of Figure 43-2 shows the results of a pulsechase experiment that follows the cellular itinerary of radio-

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Rough ER

Golgi

Condensing vacuoles

Zymogen granules

100 Rough ER

network appears to be required for delivery of the secretory granules to the apical region of the cell. A second actin network, located immediately below the apical membrane, acts as a barrier that blocks fusion of the granules with the apical plasma membrane. On stimulation, this second network reorganizes and then releases the blockade to permit the secretory granules to approach the apical plasma membrane. Fusion of the granules with the plasma membrane probably requires the interaction of proteins on both the secretory granules and the apical plasma membrane, as well as various cytosolic factors (see Chapter 2). After fusion, the granule contents are released into the acinar lumen and are carried down the ducts into the gastrointestinal tract.

Golgi vesicles

75

Condensing vacuoles % Radiolabeled grains

Duct cells are epithelial cells specialized for fluid and electrolyte transport

Zymogen granules 50

25

0

0

30

60 90 Minutes after pulse

120

Radioactive pulse

Figure 43-2 Movement of newly synthesized proteins through the secretory pathway. The cell model at the top illustrates the vectoral movement of nascent proteins through the compartments of the secretory pathway. The four records in the graph show the time course of secretory proteins moving through these compartments. To label newly synthesized proteins radioactively, the investigators briefly pulsed the pancreatic acinar cells with 3H-labeled amino acids. At specific times after the pulse, tissues were fixed, and the distribution of the radioactive amino acid was determined using autoradiography. Each of the four records shows the number of radiographic grains—as a fraction of all of the grains—found in each compartment at various times after the pulse. (Data from Jamieson J, Palade G: J Cell Biol 1967; 34:597-615; and Jamieson J, Palade G: J Cell Biol 1971; 50:135-158.)

labeled amino acids as they move sequentially through the four major compartments of the secretory pathway. Exocytosis, the process by which secretory granules release their contents, is a complex series of events that involves the movement of the granules to the apical membrane, fusion of these granules with the membrane, and release of their contents into the acinar lumen. Secretion is triggered by either hormones or neural activity. At the onset of secretion, the area of the apical plasma membrane increases as much as 30-fold. Thereafter, activation of an apical endocytic pathway leads to retrieval of the secretory granule membrane for recycling and a decrease in the area of the apical plasma membrane back to its resting value. Thus, during the steady state of secretion, the secretory granule membrane is simultaneously delivered to and retrieved from the apical membrane. The cytoskeleton of the acinar cell plays an important role in the regulation of exocytosis. A component of the actin

Pancreatic and salivary duct cells are polarized epithelial cells specialized for the transport of electrolytes across distinct apical and basolateral membrane domains. Duct epithelial cells contain specific membrane transporters and an abundance of mitochondria to provide energy for active transport, and they exhibit varying degrees of basolateral membrane infolding that increases membrane surface areas of pancreatic duct cells (Fig. 43-1C) and salivary duct cells. Although some duct cells contain prominent cytoplasmic vesicles, or storage granules—an indication of an additional protein secretory function, the synthetic machinery (i.e., ER and Golgi complex) of the duct cell is, in general, much less developed than that of the acinar cell. Duct cells exhibit a considerable degree of morphologic heterogeneity along the length of the ductal tree. At the junction between acinar and duct cells in the pancreas are small cuboidal epithelial cells known as centroacinar cells. These cells express very high levels of carbonic anhydrase and presumably play a role in HCO−3 secretion. The epithelial cells of the most proximal (intercalated) duct are squamous or low cuboidal, have an abundance of mitochondria, and tend to lack cytoplasmic vesicles. These features suggest that the primary function of these cells is fluid and electrolyte transport. Progressing distally, the cells become more cuboidal columnar and contain more cytoplasmic vesicles and granules. These features suggest that these cells are capable of both transport of fluid and electrolytes and secretion of proteins. Functional studies indicate that the types of solute transport proteins within duct cells differ depending on the cell’s location in the ductal tree. Ion transport in duct cells is regulated by neurohumoral stimuli that act through specific receptors located on the basolateral membrane. As is the case for cells elsewhere in the body, duct cells can increase transcellular electrolyte movement either by activating individual transport proteins or by increasing the number of transport proteins in the plasma membrane. Goblet cells contribute to mucin production in exocrine glands In addition to acinar and duct cells, exocrine glands contain varying numbers of goblet cells. These cells secrete high-

Chapter 43 • Pancreatic and Salivary Glands

molecular-weight glycoproteins known as mucins. When hydrated, mucins form mucus (see Chapter 2). Mucus has several important functions, including lubrication, hydration, and mechanical protection of surface epithelial cells. Mucins also play an important immunologic role by binding to pathogens and interacting with immune-competent cells. These properties may help to prevent infections. In the pancreas, mucin-secreting goblet cells are primarily found among the epithelial cells that line the large, distal ducts. They can account for as many as 25% of the epithelial cells in the distal main pancreatic duct of some species. In the salivary gland, goblet cells are also seen in the large distal ducts, although in less abundance than in the pancreas. However, in many salivary glands, mucin is also secreted by acinar cells.

PANCREATIC ACINAR CELL The acinar cell secretes digestive proteins in response to stimulation To study secretion at the cellular level, investigators use enzymes to digest pancreatic connective tissue and obtain single acini (small groups of 15 to 100 acinar cells), or they mechanically dissect single lobules (groups of 250 to 1000 cells). The measure of secretion is the release of digestive proteins into the incubation medium. The amount released over a fixed time interval is expressed as a percentage of the total content at the outset of the experiment. Because amylase is released in a fully active form, it is common to use the appearance of amylase activity as a marker for secretion by acinar cells. When the acinar cells are in an unstimulated state, they secrete low levels of digestive proteins through a constituA

PANCREATIC SECRETAGOGUES

tive secretory pathway. Acinar cells stimulated by neurohumoral agents secrete proteins through a regulated pathway. Regulated secretion by acini and lobules in vitro is detected within 5 minutes of stimulation and is energy dependent. During a 30- to 60-minute stimulation period, acinar cells typically secrete 5 to 10 times more amylase than with constitutive release. However, during this period of regulated secretion, the cells typically secrete only 10% to 20% of the digestive proteins stored in their granules. Moreover, acinar cells are able to increase their rate of protein synthesis to replenish their stores. The acinar cell may exhibit two distinct patterns of regulated secretion: monophasic and biphasic (Fig. 43-3A). An agonist that generates a monophasic dose-response relationship (e.g., gastrin-releasing peptide [GRP]) causes secretion to reach a maximal level that does not fall with higher concentrations of the agent. In contrast, a secretagogue that elicits a biphasic dose-response relationship (e.g., cholecystokinin [CCK] and carbachol) causes secretion to reach a maximal level that subsequently diminishes with higher concentrations of the agent. As discussed later, this biphasic response may reflect the presence of functionally separate high-affinity and low-affinity receptors and is related to the pathogenesis of acute pancreatitis (see the box entitled Acute Pancreatitis). Regulated secretion of proteins by pancreatic acinar cells is mediated through cholecystokinin and muscarinic receptors Although at least a dozen different receptors have been found on the plasma membrane of the pancreatic acinar cell, the most important in regulating protein secretion are the CCK receptors and the muscarinic acetylcholine (ACh) receptors. These receptors have many similarities: both are B

POTENTIATED SECRETAGOGUE RESPONSE

35 30

50 CCK CCK + VIP

40

GRP 25 Amylase 20 release (%) 15

VIP Amylase secretion (%) Carbachol

30

20

10

CCK alone 10

CGRP 5 0

0 –10 –9 –8 –7 –6 –5 –4 Concentration of secretagogue (log M)

–3

0

3

10 30 100 Concentration of CCK (pM)

Figure 43-3 Neurohumoral agents elicit different secretory responses from the pancreatic acinar cell. (A, Data from Jensen RT: In Johnson LR [ed]: Physiology of the Gastrointestinal Tract, pp 1377-1446. New York: Raven Press, 1994; B, Data from Burnham DB, McChesney DJ, Thurston KC, Williams JA: J Physiol 1984; 349:475-482.)

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linked to the Gαq heterotrimeric G protein, both use the phospholipase C (PLC)/Ca2+ signal transduction pathway (see Chapter 3), and both lead to increased enzyme secretion from the acinar cell. Two closely related CCK receptors are distinguished by their structure, affinity for ligands, and tissue distribution (see Chapter 42). Although both CCK receptors may be activated by CCK or gastrin, the CCKA receptor has a much higher affinity for CCK than for gastrin, whereas the CCKB receptor has approximately equal affinities for CCK and gastrin. In some species, both forms of the CCK receptor are present on the acinar cell. An important feature of both CCK receptors is their ability to exist in both a high-affinity and a low-affinity state. Low (picomolar) concentrations of CCK activate the high-affinity forms of the CCK receptors and stimulate secretion. Conversely, supraphysiological (10- to 100-fold higher) concentrations of CCK activate the low-affinity forms of the receptors and inhibit secretion. As we explain in the next section, these two affinity states (i.e., activated by different concentrations of CCK) of each of the two CCK receptors generate distinct second-messenger signaling patterns. It is likely that, under physiological conditions, only the high-affinity states of the CCK or muscarinic receptor are activated. Stimulation of the lower-affinity states by supraphysiological concentrations of either CCK or ACh not only inhibits enzyme secretion but also may injure the acinar cell (see the box titled Acute Pancreatitis).

Ca2+ is the major second messenger for the secretion of proteins by pancreatic acinar cells Ca2+

Much of the pioneering work on the role of intracellular Ca2+ in cell signaling has been performed in the pancreatic acinar cell (Fig. 43-4A). Generation of a cytosolic Ca2+

SIGNAL-TRANSDUCTION PATHWAYS VIP receptor

Acinar cell Lumen of the acinus

VIP

Gs

AC cAMP

Secretin

PKA PK

CaM

Ca2+

ER

PP

B CHANGES IN INTRACELLULAR [Ca2+] 1250 1000 [Ca2+], nM

A

The muscarinic receptor on the acinar cell is probably of the M3 subtype (see Chapter 14) found in many glandular tissues. Like the CCK receptor, the M3 receptor is localized to the basolateral membrane of the cell. Numerous other receptors, including those for GRP, somatostatin, and vasoactive intestinal polypeptide (VIP; see Chapter 42); calcitonin gene–related peptide (CGRP; see Chapter 52), insulin (see Chapter 51), and secretin are also found on the pancreatic acinar cell. Although these other receptors may also play a role in regulating secretion, protein synthesis, growth, and transformation, their precise physiological functions remain to be clearly defined. Activation of receptors that stimulate different signal transduction pathways may lead to an enhanced secretory response. For example, as shown in Figure 43-3B, simultaneous stimulation of the high-affinity CCK receptor (which acts through [Ca2+]i) and the VIP receptor (which acts through cAMP) generates an additive effect on secretion. Alternatively, acinar cells that have previously been stimulated may become temporarily refractory to subsequent stimulation. This phenomenon is known as desensitization.

750 500

CCK 250 ACh

IP3

10 pM CCK

DAG PKC

PIP2 PLC

Altered phosphorylation of structural and regulatory proteins leads to vesicle insertion and protein secretion.

0

Gq

Muscarinic receptor

0

5

10 15 Time (min)

Interstitial space

Figure 43-4 Stimulation of protein secretion from the pancreatic acinar cell. A, The pancreatic acinar cell has at least two pathways for stimulating the insertion of zymogen granules and thus releasing digestive enzymes. ACh and CCK both activate Gαq, which stimulates PLC, which ultimately leads to the activation of PKC and the release of Ca2+. Elevated [Ca2+]i also activates calmodulin (CaM), which can activate protein kinases (PK) and phosphatases (PP). Finally, VIP and secretin both activate Gαs, which stimulates adenylyl cyclase (AC), leading to the production of cAMP and the activation of PKA. B, Applying a physiological dose of CCK (i.e., 10 pM) triggers a series of [Ca2+]i oscillations, as measured by a fluorescent dye. However, applying a supraphysiological concentration of CCK (1 nM) elicits a single large [Ca2+]i spike and halts the oscillations. Recall that high levels of CCK also are less effective in causing amylase secretion. (B, Data from Tsunoda Y, Stuenkel EL, Williams JA: Am J Physiol 1990; 259:G792-G801.)

1nM CCK 20

25

Chapter 43 • Pancreatic and Salivary Glands

signal is a complex summation of cellular events (see Chapter 3) that regulates cytosolic free Ca2+ levels ([Ca2+]i). Even when the acinar cell is in the resting state, [Ca2+]i oscillates slowly. In the presence of maximal stimulatory (i.e., physiological) concentrations of CCK or ACh, the frequency of the oscillations increases (Fig. 43-4B), but little change in their amplitude is noted. This increase in the frequency of [Ca2+]i oscillations is required for protein secretion by acinar cells. In contrast, supramaximal (i.e., hyperstimulatory) concentrations of CCK or ACh generate a sudden, large spike in [Ca2+]i and eliminate additional [Ca2+]i oscillations. This [Ca2+]i spike and the subsequent absence of oscillations are associated with an inhibition of secretion that appears to be mediated by disruption of the cytoskeletal components that are required for secretion. cGMP

Physiological stimulation of the acinar cell by either CCK or ACh also generates a rapid and prominent increase in [cGMP]i levels. The increase in [cGMP]i has been linked to nitric oxide metabolism; inhibition of nitric oxide synthase (see Chapter 3) blocks the increase in [cGMP]i after secretagogue stimulation. Some evidence suggests that cGMP may be involved in regulating Ca2+ entry and storage in the acinar cell. cAMP

Secretin, VIP, and CCK increase cAMP production and thus activate protein kinase A (PKA) activity in pancreatic acinar cells. Low concentrations of CCK cause transient stimulation of PKA, whereas supraphysiological concentrations of CCK cause a much more prominent and prolonged

increase in [cAMP]i and PKA activity. ACh, however, has little, if any, effect on the cAMP signaling pathway. Effectors As summarized in Figure 43-4A, the most important effectors of intracellular second messengers are the protein kinases. Stimulation of CCK and muscarinic receptors on the acinar cell leads to the generation of similar Ca2+ signals and activation of calmodulin-dependent protein kinases and members of the protein kinase C (PKC) family (see Chapter 3). Activation of secretin or VIP receptors increases [cAMP]i and thus activates PKA. These second messengers probably also activate protein phosphatases, as well as other protein kinases not depicted in Figure 43-4A. The protein targets of activated kinases and phosphatases in the pancreatic acinar cell are largely unknown. However, some are involved in regulating secretion, whereas others mediate protein synthesis, growth, transformation, and cell death.

In addition to proteins, the pancreatic acinar cell also secretes a plasma-like fluid In addition to protein, acinar cells in the pancreas secrete an isotonic, plasma-like fluid (Fig. 43-5). This NaCl-rich fluid hydrates the dense, protein-rich material that the acinar cells secrete. The fundamental transport event is the secretion of Cl− across the apical membrane. For transcellular (plasma to lumen) movement of Cl− to occur, Cl− must move into the cell across the basolateral membrane. As in many other Cl−secreting epithelial cells (see Chapter 5), basolateral Cl−

5 The movement of Cl– into the lumen makes the transepithelial voltage more lumen-negative, driving Na+ into the lumen via the tight junctions. Interstitial space

H2O H2O 3 Na+

Pancreatic acinar cell Lumen of the acinus 6 Cl–

+ 6 Na+ 2 K+

3 Na+ 6 Cl– 3 K+

Stimulates protein kinases

5 K+ [Ca2+]i

M3 receptor ACh

4 The intracellular accumulation of Cl– establishes the electrochemical gradient that drives Cl– secretion into the acinar lumen through apical-membrane Cl– channels.

CCK

1 The Na–K pump creates the inwardly directed Na+ gradient across the basolateral membrane. 2 The Na/K/Cl cotransporter produces the net Cl– uptake, driven by the Na+ gradient, which is generated by the Na–K pump. 3 The rise in intracellular [K+] that results from the activity of the pump and cotransporter is shunted by basolateral K+ channels that provide an exit pathway for K+.

The hormone CCK and the cholinergic neurotransmitter acetylcholine are potent stimulators of Cl– secretion.

Figure 43-5 Stimulation of isotonic NaCl secretion by the pancreatic acinar cell. Both ACh and CCK stimulate NaCl secretion, probably through phosphorylation of basolateral and apical ion channels.

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uptake by the acinar cell occurs through an Na/K/Cl cotransporter. The Na-K pump generates the Na+ gradient that energizes the Na/K/Cl cotransporter. The K+ entering through the Na-K pump and through the Na/K/Cl cotransporter exits through K+ channels that are also located on the basolateral membrane. Thus, a pump, a cotransporter, and a channel are necessary to sustain the basolateral uptake of Cl− into the acinar cell. The rise in [Cl−]i produced by basolateral Cl− uptake drives the secretion of Cl− down its electrochemical gradient through channels in the apical membrane. As the transepithelial voltage becomes more lumen negative, Na+ moves through the cation-selective paracellular pathway (i.e., tight junctions) to join the Cl− secreted into the lumen. Water also moves through this paracellular pathway, as well as through aquaporin water channels on the apical and basolateral membranes. Therefore, the net effect of these acinar cell transport processes is the production of an isotonic, NaCl-rich fluid that accounts for ~25% of total pancreatic fluid secretion. Like the secretion of protein by acinar cells, secretion of fluid and electrolytes is stimulated by secretagogues that raise [Ca2+]i. In the pancreas, activation of muscarinic receptors by cholinergic neural pathways and activation of CCK receptors by humoral pathways increase the membrane conductance of the acinar cell. A similar effect is seen with GRP. Apical membrane Cl− channels and basolateral membrane K+ channels appear to be the effector targets of the activated Ca2+ signaling pathway. Phosphorylation of these channels by Ca2+-dependent kinases is one likely mechanism that underlies the increase in open-channel probability that accompanies stimulation.

PANCREATIC DUCT CELL The pancreatic duct cell secretes isotonic NaHCO3 The principal physiological function of the pancreatic duct cell is to secrete an HCO−3 -rich fluid that alkalinizes and hydrates the protein-rich primary secretions of the acinar cell. The apical step of transepithelial HCO−3 secretion (Fig. 43-6) is mediated in part by a Cl-HCO3 exchanger, a member of the SLC26 family (see Chapter 5) that secretes intracellular HCO−3 into the duct lumen. Luminal Cl− must be available for this exchange process to occur. Although some luminal Cl− is present in the primary secretions of the acinar cell, anion channels on the apical membrane of the duct cell provide additional Cl− to the lumen in a process called Cl− recycling. The most important of these anion channels is the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl− channel that is present on the apical membrane of pancreatic duct cells (see Chapter 5). Cl− recycling is facilitated by the co-activation of CFTR and SLC26 exchangers through direct protein-protein interactions. In some species, such as the rat and mouse, pancreatic duct cells also contain a Ca2+-activated Cl− channel on the apical membrane; this channel also provides Cl− to the lumen for recycling. Apical Cl− channels may also directly serve as conduits for HCO−3 movement from the duct cell to the lumen.

The intracellular HCO−3 that exits the duct cell across the apical membrane arises from two pathways. The first is direct uptake of HCO−3 through an electrogenic Na/HCO3 cotransporter (NBCe1), which presumably operates with an Na+/HCO−3 stoichiometry of 1 : 2. The second mechanism is the generation of intracellular HCO−3 from CO2 and OH−, catalyzed by carbonic anhydrase (see Chapter 28 for the box on this topic). The OH− in this reaction is derived, along with H+, from H2O. Thus, the H+ that accumulates in the cell must be extruded across the basolateral membrane. One mechanism of H+ extrusion is Na-H exchange. The second mechanism for H+ extrusion across the basolateral membrane, at least in some species, is an ATP-dependent H+ pump. Pancreatic duct cells contain acidic intracellular vesicles (presumably containing vacuolar-type H+ pumps) that are mobilized to the basolateral membrane of the cell after stimulation by secretin, a powerful secretagogue (see later). Indeed, H+ pumps are most active under conditions of neurohumoral stimulation. Thus, three basolateral transporters directly or indirectly provide the intracellular HCO−3 that pancreatic duct cells need for secretion: (1) the electrogenic Na/HCO3 cotransporter, (2) the Na-H exchanger, and (3) the H+ pump. The physiological importance of these three acid-base transporters in humans has yet to be fully established. The pancreatic duct cell accounts for ~75% of total pancreatic fluid secretion. Secretin (through cAMP) and acetylcholine (through Ca2+) both stimulate HCO3- secretion by the pancreatic duct When stimulated, the epithelial cells of the pancreatic duct secrete an isotonic NaHCO3 solution. The duct cells have receptors for secretin, ACh, GRP (all of which stimulate HCO−3 secretion) and substance P (which inhibits it). Although some evidence indicates that CCK modulates ductular secretory processes, CCK receptors have not been identified on these cells. Secretin is the most important humoral regulator of ductal HCO−3 secretion. Activation of the secretin receptor on the duct cell stimulates adenylyl cyclase, which raises [cAMP]i. Because forskolin and cAMP analogues stimulate ductal HCO−3 secretion, the secretin response has been attributed to its effect on [cAMP]i and activation of PKA. However, even low concentrations of secretin that do not measurably increase [cAMP]i can stimulate HCO−3 secretion. This observation suggests that the secretin response may be mediated by (1) unmeasurably small increases in total cellular cAMP, (2) cAMP increases that are localized to small intracellular compartments, or (3) activation of alternative secondmessenger pathways. Secretin acts by stimulating the apical CFTR Cl− channel and the basolateral Na/HCO3 cotransporter, without affecting the Na-H exchanger. HCO−3 secretion is also regulated by the parasympathetic division of the autonomic nervous system (see Chapter 14). The postganglionic parasympathetic neurotransmitter ACh in-creases [Ca2+]i and activates Ca2+-dependent protein kinases (PKC and the calmodulin-dependent protein kinases) in pancreatic duct cells. The ACh effect is inhibited by atropine, a finding indicating that this neurotransmitter is acting through muscarinic receptors on the duct cell. Although

Chapter 43 • Pancreatic and Salivary Glands

2 Some of the HCO3– that enters the lumen directly enters the cell across the basolateral membrane via an Na/HCO3 cotransporter.

Lumen of the duct Na+ 1 Bicarbonate secretion into the lumen occurs via a Cl-HCO3 exchange mechanism.

H2O Na+

Pancreatic duct cell

Cl–

Cystic fibrosis transmembrane regulator (CFTR)

+

– 2 HCO3

HCO3–

CO2

CA

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CO2

Interstitial space 3 Additional intracellular HCO3– is generated by the activity of cytoplasmic carbonic anhydrase (CA), which catalyzes the formation of HCO3– from CO2 and OH–.

H2O Na+ H

ATP receptor

4 The OH– needed by the CA arises from the splitting of H2O. This reaction is driven by the extrusion of H+ by both an Na–H exchanger and an H+ pump.

+

H+ Cl– Na+

Outward rectifying Cl– channel (ORCC)

K+ K+

The most powerful stimulus for HCO3– secretion is secretin, which activates adenylyl cyclase, raises [cAMP]i, stimulates protein kinase A, and phosphorylates CFTR.

[cAMP]

Secretin

CaM kinase PKC

ACh also stimulates HCO3– secretion. ACh activates Gq, which in turn stimulates PLC to release DAG (which stimulates PKC) and IP3 (which releases Ca2+ from internal stores).

[Ca2+]

M3 receptor ACh

Na+ +

H2O

5 The lumen-negative voltage pulls Na+ into the lumen, via the tight junctions.

Figure 43-6 HCO3− secretion by the cells of the pancreatic duct. Secretin activates the cAMP signaling pathway and opens the CFTR Cl− channels through phosphorylation. Cl− movement out of the cell leads to basolateral membrane depolarization, thus generating the electrical gradient that favors NaHCO3 cotransport.

ductular secretion in the rat is also stimulated by GRP, the second messenger mediating this effect is not known. Unlike the effect on the acinar cell, GRP does not increase [Ca2+]i in the duct cell. GRP also does not raise [cAMP]i. In the rat, both basal and stimulated ductular HCO−3 secretion is inhibited by substance P. The second messenger mediating this effect is also unknown. Because substance P inhibits HCO−3 secretion regardless of whether the secretagogue is secretin, ACh, or GRP—which apparently act through three different signal transduction mechanisms— substance P probably acts at a site that is distal to the generation of second messengers, such as by inhibiting the Cl-HCO3 exchanger. Apical membrane chloride channels are important sites of neurohumoral regulation In the regulation of pancreatic duct cells by the neurohumoral mechanisms just discussed, the only effector proteins

that have been identified as targets of the protein kinases and phosphatases are the apical Cl− channels, basolateral K+ channels, and the Na/HCO3 cotransporter. CFTR functions as a low-conductance, apical Cl− channel (see Chapter 5). CFTR has nucleotide-binding domains that control channel opening and closing as well as a regulatory domain with multiple potential PKA and PKC phosphorylation sites. Neurohumoral agents that control fluid and electrolyte secretion by the pancreatic duct cells act at this site. Agents that activate PKA are the most important regulators of CFTR function. PKC activation enhances the stimulatory effect of PKA on CFTR Cl− transport, but alone it appears to have little direct effect on CFTR function. Thus, the CFTR Cl− channel is regulated by ATP through two types of mechanisms: interaction with the nucleotide-binding domains and protein phosphorylation (see Fig. 5-10B). In addition to CFTR, pancreatic duct cells in some species have an outwardly rectifying Cl− channel (ORCC) on the apical membrane. This channel, which has been identified

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in a variety of epithelial cells, can be activated by increases in [cAMP]i or [Ca2+]i. Studies suggest that part of the effect of cAMP on ORCCs may be indirect and may occur through CFTR. The working hypothesis is that stimulation of CFTR somehow promotes ATP efflux from the cell to the lumen and that the ATP binds to an apical purinergic receptor to activate ORCCs in an autocrine/paracrine fashion (Fig. 43-6). In rat pancreatic duct cells, Ca2+-sensitive basolateral K+ channels seem to be targets of neurohumoral stimulation. Activators of the cAMP pathway stimulate phosphorylation by PKA, thus enhancing the responsiveness of these channels to [Ca2+]i and increasing their probability of being open. Pancreatic duct cells may also secrete glycoproteins Although the primary function of the pancreatic duct cells is to secrete HCO−3 and water, these cells may also synthesize and secrete various high-molecular-weight proteoglycans. Some of these proteins are structurally distinct from the mucin that is produced by the specialized goblet cells in the duct. Unlike the proteins that are secreted by acinar cells, the glycoproteins synthesized in duct cells are not accumulated in large secretion granules. Rather, they appear to be continuously synthesized and secreted from small cytoplasmic vesicles. Secretin increases the secretion of glycoproteins

from these cells, but this action appears to result from stimulation of glycoprotein synthesis, rather than from stimulation of vesicular transport or exocytosis itself. The role of these proteins may be to protect against protease-mediated injury to mucosal cells.

COMPOSITION, FUNCTION, AND CONTROL OF PANCREATIC SECRETION Pancreatic juice is a protein-rich, alkaline secretion Humans produce ~1.5 L of pancreatic fluid each day. The pancreas has the highest rates of protein synthesis and secretion of any organ in the body. Each day, the pancreas delivers between 15 and 100 g of protein into the small intestine. The level of pancreatic secretion is determined by a balance between factors that stimulate secretion and those that inhibit it. The human pancreas secretes more than 20 proteins, some of which are listed in Table 43-1. Most of these proteins are either inactive digestive enzyme precursors—zymogens—or active digestive enzymes. The secretory proteins responsible for digestion can be classified according to their substrates: proteases hydrolyze proteins, amylases digest carbohydrates, lipases and phospholipases break down

Cystic Fibrosis

C

F is the most common lethal genetic disease in whites, in whom it affects ~1 in 2000. Approximately 1 in 20 whites carry the autosomal recessive genetic defect. Clinically, CF is characterized by progressive pancreatic and pulmonary insufficiency resulting from the complications of organ obstruction by thickened secretions. The disease results from mutations in the CF gene (located on chromosome 7) that alter the function of its product, CFTR (see Fig. 5-10). CFTR is a cAMP-activated Cl− channel that is present on the apical plasma membrane of many epithelial cells. In the pancreas, CFTR has been localized to the apical membrane of duct cells, where it functions to provide the luminal Cl− for Cl-HCO3 exchange (Fig. 43-6). Most CF gene mutations result in the production of a CFTR molecule that is abnormally folded after its synthesis in the ER. The ER quality control system recognizes these molecules as defective, and most mutant CFTR molecules are prematurely degraded before they reach the plasma membrane. Subsequent loss of CFTR expression at the plasma membrane disrupts the apical transport processes of the duct cell and results in decreased secretion of HCO3− and water by the ducts. As a result, protein-rich primary (acinar) secretions thicken within the duct lumen and lead to ductal obstruction and eventual tissue destruction. Pathologically, the ducts appear dilated and obstructed, and fibrotic tissue and fat gradually replace the pancreatic parenchyma—hence the original cystic fibrosis designation. The subsequent deficiency of pancreatic enzymes that occurs leads to the maldigestion of nutrients and thus the excretion of fat in the stool (steatorrhea) by patients with CF. Before the development of oral enzyme replacement therapy, many patients with CF died of complications of malnutrition.

Now, the major cause of morbidity and mortality in CF is progressive pulmonary disease. The pathophysiology of lung disease in CF is more complex than that of pancreatic disease. A major finding is that the airway mucus is thick and viscous as a result of insufficient fluid secretion into the airway lumen. The pulmonary epithelium probably both secretes fluid (in a mechanism that requires CFTR) and absorbs fluid (in a mechanism that requires apical ENaC Na+ channels). In CF, the reduced activity of CFTR shifts the balance more toward absorption, and a thick mucous layer is generated that inhibits the ciliary clearance of foreign bodies (see Chapter 26). The results are increased rate and severity of infections and thus inflammatory processes that contribute to the destructive process in the lung. The pulmonary symptoms most commonly bring the patient to the physician’s attention in early childhood. Cough and recurrent respiratory infections that are difficult to eradicate are usually the first indications of the illness. The child’s sputum is particularly thick and viscous. Pulmonary function progressively declines over the ensuing years, and patients may also experience frequent and severe infections, atelectasis (collapse of lung parenchyma), bronchiectasis (chronic dilatation of the bronchi), and recurrent pneumothoraces (air in the intrapleural space). In addition to the pancreatic and pulmonary manifestations, CF also causes a characteristic increase in the [NaCl] of sweat, which is intermediate in heterozygotes. Pharmacological approaches that bypass the Cl− transport defect in a lung with CF are currently being evaluated, and considerable effort is being directed toward the development of in vivo gene transfer techniques to correct the underlying genetic defect.

Chapter 43 • Pancreatic and Salivary Glands

Table 43-1

Pancreatic Acinar Cell Secretory Products 160 DIGESTIVE PROTEINS

+

Zymogens

Function

140

Trypsinogens

Digestion

120

Chymotrypsinogen

Digestion

Proelastase

Digestion

Proprotease E

Digestion

Procarboxypeptidase A

Digestion

[Ion], (mEq/ liter)

Na HCO3–

100 80 60 Cl–

40

Procarboxypeptidase B

Digestion 20

K+

ACTIVE ENZYMES

α-Amylase

Digestion

Carboxyl ester lipase

Digestion

Lipase

Digestion

RNAase

Digestion

DNAase

Digestion

Colipase

Digestion

0.2

0.4

0.6

0.8 1.0 1.2 Secretory rate (mL/min)

1.4

1.6

Figure 43-7 Flow dependence of the electrolyte composition of pancreatic fluid. In this experiment on a cat, increasing the level of secretin not only increases the rate at which fluids flow out of the pancreas but also changes the composition of the fluid. (Data from Case RM, Harper AA, Scratcherd T: J Physiol 1969; 201:563-596.)

OTHERS

Trypsin inhibitor

Blockade of trypsin activity

Lithostathine

Possible prevention of stone formation; constituent of protein plugs

GP2

Endocytosis?; formation of protein plugs

Pancreatitis-associated protein

Bacteriostasis?

Na+, Cl−, H2O

Hydration of secretions

Ca2+

?

lipids, and nucleases digest nucleic acids. The functions of other secretory proteins—such as glycoprotein II (GP2), lithostathine, and pancreatitis-associated protein—are less well defined. GP2 is an unusual protein with an N-terminal glycosyl phosphatidylinositol moiety that links it to the inner leaflet of the zymogen granule membrane. GP2 has been implicated in the regulation of endocytosis. After exocytosis, luminal cleavage of the GP2 linkage to the zymogen granule membrane seems to be necessary for proper trafficking of the zymogen granule membrane back into the cell from the plasma membrane. Under certain circumstances, the released GP2—and also lithostathine—may form protein aggregates in the pancreatic juice. This finding is not surprising inas-

much as GP2 is structurally related to the Tamm-Horsfall protein, which is secreted by the renal thick ascending limb (see Chapter 33). The tendency of GP2 and lithostathine to form aggregates may have detrimental clinical consequences in that both proteins have been implicated in the pathologic formation of protein plugs that can obstruct the lumen of acini in patients with cystic fibrosis and chronic pancreatitis. Pancreatitis-associated protein is a secretory protein that is present in low concentrations in the normal state. However, levels of this protein may increase up to several hundred-fold during the early phases of pancreatic injury. Pancreatitis-associated protein is a bacteriostatic agent that may help to prevent pancreatic infection during bouts of pancreatitis. Pancreatic juice is also rich in Ca2+ and HCO−3 . Calcium concentrations are in the millimolar range inside the organelles of the secretory pathway of the acinar cells. These high levels of Ca2+ may be required to induce the aggregation of secretory proteins and to direct them into the secretory pathway. Bicarbonate secreted by duct cells neutralizes the acidic gastric secretions that enter the duodenum and allows digestive enzymes to function properly; HCO−3 also facilitates the micellar solubilization of lipids and mucosal cell function. The [HCO−3] in pancreatic juice increases with increases in the secretory flow rate (Fig. 43-7). In the unstimulated state, the flow is low, and the electrolyte composition of pancreatic juice closely resembles that of blood plasma. As the gland is stimulated and flow increases, exchange of Cl− in the pancreatic juice for HCO−3 across the apical membrane of the duct cells produces a secretory product that is more

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alkaline (pH of ~8.1) and has a lower [Cl−]. Concentrations of Na+ or K+, however, are not significantly altered by changes in flow. In the fasting state, levels of secreted pancreatic enzymes oscillate at low levels Pancreatic secretion is regulated in both the fasted and fed states. Under basal conditions, the pancreas releases low levels of pancreatic enzymes (Fig. 43-8). However, during the digestive period (eating a meal), pancreatic secretion increases in sequential phases to levels that are 5- to 20-fold higher than basal levels. The systems that regulate secretion appear to be redundant; if one system fails, a second takes its place. These mechanisms ensure that the release of pancreatic enzymes corresponds to the amount of food in the small intestine. Like other organs in the upper gastrointestinal tract, the pancreas has a basal rate of secretion even when food is not being eaten or digested. During this interdigestive (fasting) period, pancreatic secretions vary cyclically and correspond to sequential changes in the motility of the small intestine (see Chapter 41). Pancreatic secretion is minimal when intestinal motility is in its quiescent phase (phase I); biliary and gastric secretions are also minimal at this time. As duodenal motility increases (phase II), so does pancreatic secretion. During the interdigestive period, enzyme secretion is maximal when intestinal motility—the migrating motor complexes (MMCs; see Fig. 41-6)—is also maximal (phase III). However, even this maximal interdigestive secretory rate is only 10% to 20% of that stimulated by a meal. The peak phases of interdigestive intestinal motor activity and pancreatic secretory activity are followed by a declining period (phase IV). Fluid and electrolyte secretion rates during

Migrating motor complexes 15,000

the interdigestive phase are usually less than 5% of maximum levels. The cyclic pattern of interdigestive pancreatic secretion is mediated by intrinsic and extrinsic mechanisms. The predominant mechanism of pancreatic regulation is through parasympathetic pathways. Telenzepine, an antagonist of the M1 muscarinic ACh receptor, reduces interdigestive enzyme secretion by more than 85% during phases II and III. Although cholinergic pathways are the major regulators of interdigestive pancreatic secretion, CCK and adrenergic pathways also play a role. CCK appears to stimulate pancreatic enzyme secretion during phases I and II. In contrast, basal α-adrenergic tone appears to suppress interdigestive pancreatic secretion. Although human and canine pancreas denervated during transplantation exhibits cyclic secretion, this secretion is no longer synchronous with duodenal motor activity. These observations support a role for the autonomic nervous system in regulating basal (resting) pancreatic secretion. Cholecystokinin from duodenal I cells stimulates enzyme secretion by the acini, and secretin from S cells stimulates HCO3- and fluid secretion by the ducts CCK plays a central role in regulating pancreatic secretion. CCK is released from neuroendocrine cells (I cells; see Table 41-1) present in the duodenal mucosa and acts on pancreatic acinar cells to increase protein secretion (Fig. 43-4). In response to a meal, plasma CCK levels increase 5- to 10-fold within 10 to 30 minutes. Three lines of evidence show that CCK is a physiological mediator of pancreatic protein secretion: (1) CCK levels increase in the serum in response to a meal, (2) administration of exogenous CCK at the same

Phase I Phase II Phase III Phase IV

10,000 Trypsin output (units/10 min) 5,000

0 Midnight

6 AM Interdigestive period

Noon Fed state

Figure 43-8 Time course of pancreatic secretion during fasting and feeding. The interdigestive output of secretory products (e.g., trypsin) by the pancreas varies cyclically and in rough synchrony with the four phases of motor activity (MMCs) of the small intestine, shown by colored vertical bands. During the fed state, one notes a massive and sustained increase in trypsin release by the pancreas, as well as a switch of small intestine motility to the fed state. (Data from DiMagno EP, Layer P: In Go VLW, DiMagno EP, Gardner JD, et al [eds]: The Pancreas: Biology, Pathobiology and Disease, 2nd ed, pp 275-300. New York: Raven Press, 1993.)

Chapter 43 • Pancreatic and Salivary Glands

levels produced by a meal stimulates pancreatic protein secretion to higher levels than those generated by a meal (the meal may also stimulate the release of inhibitory factors in addition to CCK), and (3) a specific CCK inhibitor reduces pancreatic protein secretion by more than 50%. The most potent stimulator of CCK release from I cells is lipid. Protein digestive products (i.e., peptones, amino acids) also increase CCK release, but carbohydrate and acid have little effect. CCK secretion may also be stimulated by CCKreleasing factors, which are peptides released by mucosal cells of the duodenum or secreted by the pancreas. The level of these releasing factors may reflect a balance between the relative amounts of nutrients and digestive enzymes that are present in the gut lumen at any one time, so the level of the factors reflects the digestive milieu of the duodenum. In the fasting state, luminal CCK releasing factors are degraded by digestive enzymes that accompany basal pancreatic secretion, so little releasing factor remains to stimulate the I cells. However, during a meal, the digestive enzymes are diverted to the digestion of ingested nutrients entering the gut lumen, and the CCK-releasing factors are spared degradation. Hence, the relative level of proteins to proteases in the small intestine determines the amount of CCK-releasing factor available to drive CCK release and thus pancreatic secretion. CCK acts on the acinar cell through both direct and indirect pathways: it directly stimulates enzyme secretion through a CCKA receptor on the acinar cell (Fig. 43-4), and it may indirectly stimulate enzyme secretion by activating the parasympathetic (cholinergic) nervous system. As we see later, the parasympathetic pathway plays a major role in mediating the intestinal phase of pancreatic secretion. Vagal stimulation can drive pancreatic secretion to nearly maximum levels. Atropine, an antagonist of muscarinic ACh receptors (see Chapter 14), reduces the secretion of enzymes and HCO−3 during the intestinal phase of a meal. Atropine also inhibits secretion in response to stimulation by physiological levels of exogenous CCK. Together, these findings suggest that CCK somehow stimulates the parasympathetic pathway, which, in turn, stimulates muscarinic receptors on the acinar cell. Like CCK, GRP—which is structurally related to bombesin—may also be a physiological regulator of pancreatic enzyme secretion. Stimulation of acinar cells with GRP leads to enzyme secretion. In contrast to the hormone CCK, the major source of GRP appears to be the vagal nerve terminals. Secretin is the most potent humoral stimulator of fluid and HCO−3 secretion by the pancreas (Fig. 43-6). Secretin is released from neuroendocrine cells (S cells) in the mucosa of the small intestine in response to duodenal acidification and, to a lesser extent, bile acids and lipids. To stimulate secretin secretion, duodenal pH must fall to less than 4.5. Like CCK, secretin levels increase after the ingestion of a meal. However, when these levels are reached experimentally by administration of exogenous secretin, pancreatic HCO−3 secretion is less than that generated by a meal. These findings suggest that secretin is acting in concert with CCK, ACh, and other agents to stimulate HCO−3 secretion. In addition to hormones of intestinal origin, insulin and other hormones secreted by the islets of Langerhans within

the pancreas (see Chapter 51) may also influence pancreatic exocrine secretion. Blood flow from the pancreatic islets moves to the exocrine pancreas through a portal system. This organization allows high concentrations of islet hormones to interact with pancreatic acinar cells. One result of this arrangement may be that insulin modifies the composition of digestive enzymes within the acinar cell and increases the relative levels of amylase. Regulation of exocrine pancreatic secretion is complex, and understanding this process has been made difficult by the following: (1) tissue levels of an exogenously infused hormone may not match those generated physiologically; (2) because several neurohumoral factors are released in response to a meal, the infusion of a single agent may not accurately reflect its physiological role; (3) specific neurohumoral inhibitors are often unavailable; and (4) pancreatic responses may differ depending on the species. A meal triggers cephalic, gastric, and intestinal phases of pancreatic secretion that are mediated by a complex network of neurohumoral interactions The digestive period has been divided into three phases (Table 43-2) based on the site at which food acts to stimulate pancreatic secretion, just as for gastric secretion (see Chapter 42). These three phases (cephalic, gastric, and intestinal) are sequential and follow the progression of a meal from its initial smell and taste to its movement through the gastrointestinal tract (Fig. 43-9). These phases act in a coordinated fashion to maximize efficiency of the digestive process. For example, stimulation of secretion before the entry of food into the small intestine during the cephalic and gastric phases ensures that active enzymes are present when food arrives. Conversely, suppression of secretion during the late digestive phase suppresses the release of pancreatic enzymes when nutrients are no longer present in the proximal end of the small intestine. The Cephalic Phase During the cephalic phase, the sight, taste, and smell of food usually generate only a modest

Table 43-2

The Three Phases of Pancreatic Secretion

Phase

Stimulant

Regulatory Pathway

Percentage of Maximum Enzyme Secretion

Cephalic

Sight Smell Taste Mastication

Vagal pathways

25%

Gastric

Distention Gastrin?

Vagal-cholinergic

10%-20%

Intestinal

Amino acids Fatty acids H+

Cholecystokinin Secretin Enteropancreatic reflexes

50%-80%

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A

Brain

Sight, taste, smell of food (cephalic phase)

CEPHALIC AND GASTRIC PHASES

Dorsal motor nucleus of vagus

Stomach Food in stomach (gastric phase)

Food

GRP Peptides and amino acids

Gastrin G cell

Antrum

Antrum of stomach (gastric phase only)

Small intestine

H2 O

+

ACh M3 receptor

Basal lamina

Pancreas CCKA

HCO3– ACh

M3 receptor Acinus H2O

Pancreatic duct cells

Enzymes

B

HCO3–

Acinar cells

Enzymes INTESTINAL PHASE

Stomach

Duodenum

Pancreas Brain

Vagus nerve

Protein and lipid breakdown products stimulate a vagovagal reflex that stimulates primarily the acinar cells.

ACh M3 Receptor

Fat Protein ACh M3 receptor

Pancreas

Pancreatic duct cells

Acinus +

H+ stimulates S cells in the duodenum to secrete secretin, which acts on receptors on duct cells, stimulating HCO3– secretion.

H

Secretin receptor CCK receptor

S cell I cell

Secretin

CCK

Protein and lipid breakdown products stimulate I cells in duodenum to secrete CCK, which acts on receptors on acinar cells, stimulating enzyme secretion.

H2O Enzymes HCO3– Protein Fat

Chapter 43 • Pancreatic and Salivary Glands

Figure 43-9 Three phases of pancreatic secretion. A, During the cephalic phase, the sight, taste, or smell of food stimulates pancreatic acinar cells, through the vagus nerve and muscarinic cholinergic receptors, to release digestive enzymes and, to a lesser extent, stimulates duct cells to secrete HCO3− and fluid. The release of gastrin from G cells is not important during this phase. During the gastric phase, the presence of food in the stomach stimulates pancreatic secretions—primarily from the acinar cells—through two routes. First, distention of the stomach activates a vagovagal reflex. Second, protein digestion products (peptones) stimulate G cells in the antrum of the stomach to release gastrin, which is a poor agonist of the CCKA receptors on acinar cells. B, The arrival of gastric acid in the duodenum stimulates S cells to release secretin, which stimulates duct cells to secrete HCO3− and fluid. Protein and lipid breakdown products have two effects. First, they stimulate I cells to release CCK, which causes acinar cells to release digestive enzymes. Second, they stimulate afferent pathways that initiate a vagovagal reflex that primarily stimulates the acinar cells through M3 cholinergic receptors.

increase in fluid and electrolyte secretion (Fig. 43-9A). However, these factors have prominent effects on enzyme secretion. In most animal species, enzyme secretion increases to 25% to 50% of the maximum rate evoked by exogenous CCK. In humans, the cephalic phase is short-lived and dissipates rapidly when food is removed. The cephalic phase is mediated by neural pathways. In the dog, stimulation of several regions of the hypothalamus (dorsomedial and ventromedial nuclei and the mammillary body) enhances pancreatic secretion. The efferent signal travels along vagal pathways to stimulate pancreatic secretion through ACh, an effect blocked by atropine. The cephalic phase does not depend on gastrin or CCK release, but it is probably mediated by the stimulation of muscarinic receptors on the acinar cell. The Gastric Phase During the gastric phase (Fig. 43-9A), the presence of food in the stomach modulates pancreatic secretion by (1) affecting the release of hormones, (2) stimulating neural pathways, and (3) modifying the pH and availability of nutrients in the proximal part of the small intestine. The presence of specific peptides or amino acids (peptones) stimulates gastrin release from G cells in the antrum of the stomach and, to a much lesser extent, G cells in the proximal part of the duodenum. The gastrin/CCKB receptor and the CCKA receptor are closely related (see Chapter 42). Although in some species the gastrin/CCKB receptor is not present on the pancreatic acinar cell, gastrin can still act—albeit not as well—through the CCKA receptor. Although physiological concentrations of gastrin can stimulate pancreatic secretion in some species, the importance of gastrin in regulating secretion in the human pancreas remains unclear. As far as local neural pathways are concerned, gastric distention stimulates low levels of pancreatic secretion, probably through a vagovagal gastropancreatic reflex. Although the presence of food in the stomach affects pancreatic secretion, the most important role for chyme in controlling pancreatic secretion occurs after the gastric contents enter the small intestine. The Intestinal Phase During the intestinal phase, chyme entering the proximal region of the small intestine stimulates a major pancreatic secretory response by three major mechanisms (Fig. 43-9B). First, gastric acid entering the duodenum and, to a lesser extent, bile acids and lipids stimulate duodenal S cells to release secretin, which stimulates duct cells to secrete HCO−3 and fluid. The acid stimulates fluid and electrolyte secretion to a greater extent than it stimulates protein secretion. Second, lipids and, to a lesser degree, peptones stimulate duodenal I cells to release CCK, which stim-

ulates acinar cells to release digestive enzymes. Finally, the same stimuli that trigger I cells also activate a vagovagal enteropancreatic reflex that predominantly stimulates acinar cells. The pattern of enzyme secretion—mediated by the CCK and vagovagal pathways—depends on the contents of the meal. For example, a liquid meal elicits a response that is only ~60% of maximal. In contrast, a solid meal, which contains larger particles and is slowly released from the stomach, elicits a prolonged response. Meals rich in calories cause the greatest response. The chemistry of the ingested nutrients also affects pancreatic secretion through the CCK and vagovagal pathways. For example, perfusion of the duodenum with carbohydrates has little effect on secretion, whereas lipids are potent stimulators of pancreatic enzyme secretion. As far as lipids are concerned, triglycerides do not stimulate pancreatic secretion, but their hydrolytic products—monoglycerides and free fatty acids—do. The longer the chain length of the fatty acid, the greater is the secretory response; C-18 fatty acids generate protein secretion that is near the maximum produced by exogenous CCK. Some fatty acids also stimulate pancreatic HCO−3 secretion. Because fatty acids also reduce gastric acid secretion and delay gastric emptying, they may play an important role in modulating pH conditions in the proximal part of the small intestine. Protein breakdown products are intermediate in their stimulatory effect. Nonessential amino acids have little effect on protein secretion, whereas some essential amino acids (see Chapter 58) stimulate secretion. The most potent amino acid stimulators are phenylalanine, valine, and methionine. Short peptides containing phenylalanine stimulate secretion to the same extent as the amino acid itself. Because gastric digestion generates more peptides than amino acids, it is likely that peptides provide the initial pancreatic stimulation during the intestinal phase. The relative potency of the different nutrients in stimulating secretion is inversely related to the pancreatic reserves of digestive enzymes. Thus, the pancreas needs to release only a small portion of its amylase to digest the carbohydrate in a meal and to release only slightly greater portions of proteolytic enzymes to digest the proteins. However, a greater fraction of pancreatic lipase has to be released to efficiently digest the fat in most meals. The exocrine pancreas has the ability to respond to long-term changes in dietary composition by modulating the reserves of pancreatic enzymes. For example, a diet that is relatively high in carbohydrates may lead to a relative increase in pancreatic amylase content.

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The pancreas has large reserves of digestive enzymes for carbohydrates and proteins, but not for lipids The exocrine pancreas stores more enzymes than are required for digesting a meal. The greatest pancreatic reserves are those required for carbohydrate and protein digestion. The reserves of enzymes required for lipid digestion— particularly for triglyceride hydrolysis—are more limited. Even so, nutrient absorption studies after partial pancreatic resection show that maldigestion of dietary fat does not occur until 80% to 90% of the pancreas has been removed. Similar reserves exist for pancreatic endocrine function. These observations have important clinical implications because they indicate that individuals can tolerate large pancreatic resections for tumors without fear of developing maldigestion or diabetes postoperatively. When fat maldigestion or diabetes does develop because of pancreatic disease, the gland must have undergone extensive destruction. Fat in the distal part of the small intestine inhibits pancreatic secretion Once maximally stimulated, pancreatic secretion begins to decrease after several hours. Nevertheless, the levels of secretion remain adequate for digestion. Regulatory systems only gradually return secretion to its basal (interdigestive) state. The regulatory mechanisms responsible for this feedback inhibition are less well characterized than those responsible for stimulating pancreatic secretion. The presence of fat in the distal end of the small intestine reduces pancreatic secretion in most animals, including humans. This inhibition may be mediated by peptide YY (PYY), which is present in neuroendocrine cells in the ileum and colon. PYY may suppress pancreatic secretion by acting on inhibitory neural pathways, as well as by decreasing pancreatic blood flow. Somatostatin (particularly SS-28; see Chapter 48), released from intestinal D cells, and glucagon, released from pancreatic islet α cells, may also be factors in returning pancreatic secretion to the interdigestive state after a meal. Several mechanisms protect the pancreas from autodigestion Premature activation of pancreatic enzymes within acinar cells may lead to autodigestion and could play a role in initiating pancreatitis. To prevent such injury, the acinar cell has certain mechanisms for preventing enzymatic activity (Table 43-3). First, many digestive proteins are stored in secretory granules as inactive precursors or zymogens. Under normal conditions, zymogens become activated only after entering the small intestine. There, the intestinal enzyme enterokinase converts trypsinogen to trypsin, which initiates the conversion of all other zymogens to their active forms (see Chapter 45). Second, the secretory granule membrane is impermeable to proteins. Thus, the zymogens and active digestive enzymes are sequestered from proteins in the cytoplasm and other intracellular compartments. Third, enzyme inhibitors such as pancreatic trypsin inhibitor are co-packaged in the secretory granule. Sufficient pancreatic

Acute Pancreatitis

A

cute pancreatitis is an inflammatory condition that may cause extensive local damage to the pancreas, as well as compromise the function of other organs such as the lungs. The most common factors that initiate human acute pancreatitis are alcohol ingestion and gallstones. However, other insults may also precipitate acute pancreatitis. Hypertriglyceridemia, an inherited disorder of lipid metabolism, is one such culprit. Less commonly, toxins that increase ACh levels, such as cholinesterase inhibitors (some insecticides) or the sting of scorpions found in the Caribbean and South and Central America, may lead to pancreatitis. Supraphysiological levels of ACh probably cause pancreatitis by overstimulating the pancreatic acinar cell. Experimental models of pancreatitis suggest a primary defect in protein processing and acinar cell secretory function. More than 100 years ago, it was found that treating animals with doses of ACh 10 to 100 times greater than those that elicited maximal enzyme secretion caused “hyperstimulation” pancreatitis. The same type of injury can be generated by CCK. The injury in this model appears to be linked to two events within the acinar cell: (1) zymogens, in particular proteases, are pathologically processed within the acinar cell into active forms; in this model, the protective mechanisms outlined in Table 43-3 are overwhelmed, and active enzymes are generated within the acinar cell; and (2) acinar cell secretion is inhibited, and the active enzymes are retained within the cell. Although premature activation of zymogens is probably an important step in initiating pancreatitis, other events are important for perpetuating injury, including inflammation, induction of apoptosis, vascular injury, and occlusion that results in decreased blood flow and reduced tissue oxygenation (ischemia). Knowledge of the mechanisms of acute pancreatitis may lead to effective therapies. In experimental models, serine protease inhibitors that block the activation of pancreatic zymogens improve the course of the acute pancreatitis. In some clinical forms of pancreatitis, prophylactic administration of the protease inhibitor gabexate appears to reduce the severity of the disease.

Table 43-3 Mechanisms That Protect the Acinar Cell from Autodigestion Protective Factor

Mechanism

Packaging of many digestive proteins as zymogens

Precursor proteins lack enzymatic activity

Selective sorting of secretory proteins and storage in zymogen granules

Restricts the interaction of secretory proteins with other cellular compartments

Protease inhibitors in the zymogen granule

Block the action of prematurely activated enzymes

Condensation of secretory proteins at low pH

Limits the activity of active enzymes

Nondigestive proteases

Degrade active enzymes

Chapter 43 • Pancreatic and Salivary Glands

Table 43-4

Autonomic Control of Salivary Secretion

Autonomic Pathway

Neurotransmitter

Receptor

Signaling Pathway

Cellular Response

Parasympathetic

Acetylcholine Substance P

Muscarinic (M3) Tachykinin NK-1

Ca2+ Ca2+

Fluid > protein secretion Fluid > protein secretion

Sympathetic

Norepinephrine Norepinephrine

α-Adrenergic β-Adrenergic

Ca2+ cAMP

Fluid > protein secretion Protein > fluid secretion

trypsin inhibitor is present in the secretory granules to block 10% to 20% of the potential trypsin activity. Fourth, the condensation of zymogens, the low pH, and the ionic conditions within the secretory pathway may further limit enzyme activity. Fifth, enzymes that become prematurely active within the acinar cell may themselves be degraded by other enzymes or be secreted before they can cause injury. Degradation of prematurely active enzymes may be mediated by other enzymes that are present within the secretory granule or by mixing secretory granule contents with lysosomal enzymes that can degrade active enzymes. Three mechanisms lead to the combination of digestive proteases and lysosomal enzymes: (1) lysosomal enzymes may be copackaged in the secretory granule; (2) secretory granules may selectively fuse with lysosomes (a process called crinophagy); and (3) secretory granules, as well as other organelles, may be engulfed by lysosomes (a process called autophagy). Failure of these protective mechanisms may result in the premature activation of digestive enzymes within the pancreatic acinar cell and may initiate pancreatitis.

SALIVARY ACINAR CELL Different salivary acinar cells secrete different proteins The organizational structure of the salivary glands is similar to that of the pancreas (Fig. 43-1A); secretory acinar units drain into progressively larger ducts. Unlike the pancreas, the salivary glands are more heterogeneous in distribution and contain two distinct acinar cell populations that synthesize and secrete different protein products. The acinar cells of the parotid glands in most species secrete a serous (i.e., watery) product that contains an abundance of α-amylase. Many acinar cells of the sublingual glands secrete a mucinous product that is composed primarily of mucin glycoproteins. The morphologic appearance of these two acinar cell populations differs as well. The submandibular gland of many species contains both mucus-type and serous-type acinar cells. In some species, these two distinct cell types are dispersed throughout the submandibular gland, whereas in other species such as humans, distinct mucus and serous acinar units are the rule. In addition to α-amylase and mucin glycoproteins, salivary acinar cells also secrete many prolinerich proteins. Like mucin proteins, proline-rich proteins are highly glycosylated, and like other secreted salivary proteins,

they are present in the acinar secretory granules and are released by exocytosis. Cholinergic and adrenergic neural pathways are the most important physiological activators of regulated secretion by salivary acinar cells Unlike the pancreas, in which humoral stimulation plays an important role in stimulating secretion, the salivary glands are mostly controlled by the autonomic nervous system (see Chapter 14). The major agonists of salivary acinar secretion are ACh and norepinephrine, which are released from postganglionic parasympathetic and sympathetic nerve terminals, respectively (see Fig. 14-8; Table 43-4). The cholinergic receptor on the salivary acinar cell is the muscarinic M3 glandular subtype. The adrenergic receptors identified on these cells include both the α and β subtypes. Other receptors identified in salivary tissue include those for substance P (NK1 receptors), VIP, purinergic agonists (P2z receptors), neurotensin, prostaglandin, and epidermal growth factor (EGF). However, some of these other receptors are found only on specific salivary glands and may be present on duct cells rather than acinar cells. Significant species variation with regard to surface receptor expression is also seen. Thus, for the salivary glands, it is difficult to discuss the regulation of acinar cell secretion in general terms. It is fair to say, however, that both cholinergic and adrenergic neurotransmitters can stimulate exocytosis by salivary acinar cells. Both cAMP and Ca2+ mediate salivary acinar secretion Protein secretion by the salivary acinar cell, as in the pancreatic acinar cell, is associated with increases in both [cAMP]i and [Ca2+]i. Activation of cAMP through the β-adrenergic receptor is the most potent stimulator of amylase secretion in the rat parotid gland. Activation of Ca2+ signaling pathways through the α-adrenergic, muscarinic, and substance P receptors also stimulates amylase secretion by the parotid gland. Increases in [Ca2+]i cause G protein–dependent activation of PLC and thus lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca2+ from intracellular stores and stimulates Ca2+dependent protein kinases such as PKC and calmodulin kinase, whereas DAG directly activates PKC (see Chapter 3). The repetitive spikes in [Ca2+]i in salivary acinar cells, as in pancreatic acinar cells, depend on Ca2+-induced Ca2+ release from intracellular stores (see Chapter 9) and on the

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influx of extracellular Ca2+. ATP co-released with norepinephrine (see Chapter 14) activates a P2z receptor, which is a receptor-gated cation-selective channel that allows Ca2+ to enter across the plasma membrane and thus increase [Ca2+]i. Fluid and electrolyte secretion is the second major function of salivary acinar cells, accounting for ~90% of total salivary volume output under stimulatory conditions. The mechanisms in salivary acinar cells are similar to those in pancreatic acinar cells (Fig. 43-5). The primary secretion of the salivary acinar cell is isotonic and results largely from the basolateral uptake of Cl− through Na/K/Cl cotransporters, working in conjunction with Na-K pumps and basolateral K+ channels. Secretion of Cl− and water into the lumen is mediated by apical Cl− and aquaporin water channels. Na+ and some water reach the lumen through paracellular routes. The salivary acinar cells in some species express carbonic anhydrase as well as parallel basolateral Cl-HCO3 and Na-H exchangers, a finding suggesting that other pathways may also contribute to the primary secretion. Stimulation of fluid and electrolyte secretion by salivary acinar cells is largely mediated by cholinergic and αadrenergic stimulation. Substance P, acting through its own receptor, also initiates conductance changes in the salivary acinar cell. All these effects seem to be mediated by rises in [Ca2+]i. Apical Cl− channels and basolateral K+ channels appear to be the effector targets of the activated Ca2+ signaling pathway. Phosphorylation of these channels by Ca2+-dependent kinases may affect the probability that these channels will be open and may thus increase conductance.

SALIVARY DUCT CELL Salivary duct cells produce a hypotonic fluid that is poor in NaCl and rich in KHCO3 In the salivary glands, as in the pancreas, the duct modifies the composition of the isotonic, plasma-like primary secretion of the acinar cells (Fig. 43-10). The active transport activity of these cells is reflected by numerous basolateral membrane infoldings and abundant mitochondria, which give the basal portion of the cells a characteristic striated appearance—hence the term striated duct epithelial cell (Fig. 43-10C). In general, salivary duct cells absorb Na+ and Cl− and, to a lesser extent, secrete K+ and HCO−3 . Because the epithelium is not very water permeable, the lumen thus becomes hypotonic. However, significant differences are seen in the various types of salivary glands. Reabsorption of Na+ by salivary duct cells is a two-step transcellular process. First, Na+ enters the cell from the lumen through apical epithelial Na+ channels (ENaCs; see Chapter 5). Second, the basolateral Na-K pump extrudes this Na+. Elevated [Na+]i provides feedback inhibition by downregulating ENaC activity, presumably through the ubiquitin-protein ligase Nedd4 (see Chapter 2). Reabsorption of Cl − is also a two-step transcellular process. Entry of Cl− across the apical membrane occurs through a

Lumen of duct

Interstitial space

K+ H

Na

+

3 Na+

+

2 K+

ATP

Na+ H+

ENaC HCO3–

Cl

Na+

–

Na Cl– Cl–

Cl–

CFTR channel

K+ H+

[Ca++]i

+

– 2 HCO3

M3 receptor ACh

H2O

Figure 43-10

Salivary duct transporters.

Cl-HCO3 exchanger. To a certain extent, apical Cl− channels, including CFTR, recycle the Cl− that is absorbed by the ClHCO3 exchanger. Duct cells also have basolateral Cl− channels that provide an exit pathway for Cl−. Secretion of HCO3− occurs through the apical Cl-HCO3 exchanger mentioned earlier. This process depends on functional CFTR, thereby confirming the coupling of CFTR to the Cl-HCO3 exchanger in salivary duct cells. HCO−3 accumulation inside the salivary duct cell may follow the same routes as in the pancreatic duct cell (Fig. 43-6). Indeed, Na/ HCO3 cotransporters in the identification of rat and human salivary duct epithelial cells support this possibility. Secretion of K+ occurs through the basolateral uptake of K+ through the Na-K pump. The mechanism of the apical K+ exit step is not well established, but it may involve K-H exchange or other pathways.

Parasympathetic stimulation decreases Na+ absorption, whereas aldosterone increases Na+ absorption by duct cells Regulation of duct cell transport processes is less well understood in the salivary glands than in the pancreas. In the intact salivary gland (i.e., acini and ducts), secretion is stimulated primarily by parasympathetic input through ACh. In the duct cell, cholinergic agonists, acting through muscarinic receptors, increase [Ca2+]i and presumably activate Ca2+-

Chapter 43 • Pancreatic and Salivary Glands

dependent regulatory pathways. The effector targets of this Ca2+ signaling pathway are not known. The role played by duct cells in the increased saliva production that occurs in response to cholinergic stimulation is limited and may reflect decreased Na-Cl absorption more than increased K-HCO3 secretion. The specific effects of adrenergic stimulation on duct cell transport activity are unclear. Nevertheless, activation of the β-adrenergic receptor increases [cAMP]i and activates the CFTR Cl− channel. Salivary duct cell function is also regulated by circulating hormones. The mineralocorticoid hormone aldosterone stimulates the absorption of Na-Cl and secretion of K+ by salivary duct cells in several species. Although its role has not been well examined in salivary duct cells, aldosterone in other Na+-absorbing epithelia (e.g., kidney and colon) stimulates Na+ transport by increasing both ENaC and Na-K pump activity (see Chapter 35). Salivary duct cells may also have receptors for certain neuropeptides such as VIP, although their physiological significance remains unknown.

Salivary duct cells also secrete and take up proteins Duct cells handle proteins in three ways. Some proteins that are synthesized by duct cells are secreted into the lumen, others are secreted into blood, and still others are reabsorbed from the lumen to the cell. Intralobular duct epithelial cells in rodent submandibular glands synthesize various proteins that are stored in intracellular granules and are secreted in response to neurohumoral stimuli. EGF, nerve growth factor, and kallikrein are among the most abundant proteins that are packaged for secretion by these cells. Salivary duct cells may also synthesize, store, and secrete some digestive enzymes (α-amylase and ribonucleases). Degranulation of intralobular duct cells occurs primarily in response to α-adrenergic stimulation, a finding suggesting that protein secretion by duct cells is regulated primarily by the sympathetic division. Although regulatory peptides (i.e., glucagon and somatostatin) have also been detected in salivary duct cells, no evidence indicates that they are stored in granules or are secreted into the lumen (i.e., they may be basolaterally secreted as peptide hormones). In addition, duct cells synthesize polymeric IgA receptors that are responsible for the basolateral endocytosis of IgA, and they also synthesize a secretory component that facilitates the apical release of IgA. Salivary duct cells can also remove organic substances from the duct lumen. Endocytosis of acinar proteins and other materials (e.g., ferritin) at the apical pole of the duct cell has been demonstrated immunocytochemically. In addition, salivary duct cells express the transferrin receptor (see Chapter 2) on the apical membrane, a finding indicating that some regulated endocytosis also occurs in these cells. The latter process may function to take up specific luminal substances or to traffic ion transporters to and from the apical plasma membrane.

COMPOSITION, FUNCTION, AND CONTROL OF SALIVARY SECRETION Depending on protein composition, salivary secretions can be serous, seromucous, or mucous Most saliva (~90%) is produced by the major salivary glands: the parotid, the sublingual, and the submandibular glands. The remaining 10% of saliva comes from numerous minor salivary glands that are scattered throughout the submucosa of the oral cavity. Each salivary gland produces either a serous, a seromucous, or a mucous secretion; the definition of these three types of saliva is based on the glycoprotein content of the gland’s final secretory product. In humans and most other mammals, the parotids produce a serous (i.e., low glycoprotein content) secretion, the sublingual and submandibular glands produce a seromucous secretion, and the minor salivary glands produce a mucous secretion. Serous secretions are enriched in α-amylase, and mucous secretions are enriched in mucin. However, the most abundant proteins in parotid and submandibular saliva are members of the group of proline-rich proteins, in which one third of all amino acids are proline. These proteins exist in acidic, basic, and glycosylated forms. They have antimicrobial properties and may play an important role in neutralizing dietary tannins, which can damage epithelial cells. In addition to these protective functions, proline-rich salivary proteins contribute to the lubrication of ingested foods and may enhance tooth integrity through their interactions with Ca2+ and hydroxyapatite. Saliva also contains smaller amounts of lipase, nucleases, lysozyme, peroxidases, lactoferrin, secretory IgA, growth factors, regulatory peptides, and vasoactive proteases such as kallikrein and renin (Table 43-5). Saliva functions primarily to prevent dehydration of the oral mucosa and to provide lubrication for the mastication and swallowing of ingested food. The sense of taste and, to a lesser extent, smell depend on an adequate supply of saliva. Saliva plays a very important role in maintaining proper oral hygiene. It accomplishes this task by washing away food particles, killing bacteria (lysozyme and IgA activity), and contributing to overall dental integrity. Although α-amylase is a major constituent of saliva and digests a significant amount of the ingested starch, salivary amylase does not appear to be essential for effective carbohydrate digestion in the presence of a normally functioning pancreas. The same can be said for lingual lipase. However, in cases of pancreatic insufficiency, these salivary enzymes can partially compensate for the maldigestion that results from pancreatic dysfunction. At low flow rates, the saliva is hypotonic and rich in K+, whereas at higher flow rates, its composition approaches that of plasma The composition of saliva varies from gland to gland and from species to species. The primary secretion of the salivary acinar cell at rest is plasma-like in composition. Its osmolal-

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Table 43-5 Saliva

Major Organic Components of Mammalian

Components

Cell Type

Glands

Possible Function

Proline-rich proteins

Acinar

P, SM

Enamel formation Ca2+ binding Antimicrobial Lubrication

Mucin glycoproteins

Acinar

SL, SM

Electrolyte Components of Human Parotid Saliva

Component

Unstimulated or Basal State (mM)

Stimulated (Cholinergic Agonists) (mM)

Na+

15

90

K+

30

15

Cl−

15

50

Total CO2

15

60

Lubrication

Enzymes

α-Amylase Lipase Ribonuclease Kallikrein

Table 43-6

Data from Thaysen JH, Thorn NA, Schwartz IL: Am J Physiol 1954; 178:155-159.

Acinar Acinar Duct Duct

P, SM SL SM P, SM, SL

Starch digestion Fat digestion RNA digestion Unknown

Lactoperoxidase Lactoferrin Lysozyme IgA receptor IgA secretory piece

Acinar Acinar Duct Duct Duct

SM Unknown SM Unknown Unknown

Antimicrobial Antimicrobial Antimicrobial Antimicrobial Antimicrobial

than plasma [K+]. In humans, increased salivary flow alkalinizes the saliva and increases its [HCO−3]. This salivary alkalinization and net HCO−3 secretion in humans neutralize the gastric acid that normally refluxes into the esophagus.

Growth factors

Duct

SM

Unknown

Parasympathetic stimulation increases salivary secretion

Miscellaneous

P, parotid; SL, sublingual; SM, submandibular.

ity, which is mostly the result of Na+ and Cl−, is ~300 mosmol/ kg. The only significant difference from plasma is that the [K+] of the salivary primary secretion is always slightly higher than that of plasma. In some species, acinar cells may help to generate a Cl−-poor, HCO−3-rich primary secretion after salivary gland stimulation. In most species, however, salivary gland stimulation does not significantly alter acinar cell transport function or the composition of the primary secretion. The leakiness of the tight junctions between acinar cells contributes to the formation of a plasma-like primary secretory product (see Chapter 5). The composition of the primary salivary secretion is subsequently modified by the transport processes of the duct cell (Fig. 43-10). At low (basal) flow rates, Na+ and Cl− are absorbed and K+ is secreted by the duct cells of most salivary glands (Table 43-6). These transport processes generate a K+-rich, hypotonic salivary secretion at rest. The tightness of the ductal epithelium inhibits paracellular water movement and therefore contributes to the formation of a hypotonic secretory product. At higher flow rates, the composition of the final secretory product begins to approach that of the plasma-like primary secretion (Table 43-6). This observation suggests that, as in the case of the renal tubules, the ductular transport processes have limited capacity to handle the increased load that is presented to them as the flow rate accelerates. However, the extent to which the transporters are flow dependent varies from gland to gland and from species to species. Human saliva is always hypotonic, and salivary [K+] is always greater

Humans produce ~1.5 L of saliva each day. Under basal conditions, the salivary glands produce saliva at a rate of ~0.5 mL/min, with a much slower flow rate during sleep. After stimulation, flow increases 10-fold over the basal rate. Although the salivary glands respond to both cholinergic and adrenergic agonists in vitro, the parasympathetic nervous system is the most important physiological regulator of salivary secretion in vivo. Parasympathetic Control Parasympathetic innervation to the salivary glands originates in the salivatory nuclei of the brainstem (see Fig. 14-5). Both local input and central input to the salivatory nuclei can regulate the parasympathetic signals transmitted to the glands. Taste and tactile stimuli from the tongue are transmitted to the brainstem, where their signals can excite the salivatory nuclei and stimulate salivary gland secretion. Central impulses triggered by the sight and smell of food also excite the salivatory nuclei and can induce salivation before food is ingested. These central effects were best illustrated by the classic experiments of Pavlov, who conditioned dogs to salivate at the sound of a bell. For his work on the physiology of digestion, Ivan Pavlov received the 1904 Nobel Prize in Physiology or Medicine. Preganglionic parasympathetic fibers travel in cranial nerve (CN) VII to the submandibular ganglia, from which postganglionic fibers reach the sublingual and submandibular glands (see Fig. 14-4). Preganglionic parasympathetic fibers also travel in CN IX to the otic ganglia, from which postganglionic fibers reach the parotid glands. In addition, some parasympathetic fibers reach their final destination through the buccal branch of CN V to the parotid glands or through the lingual branches of CN V to the sublingual and submandibular glands. Postganglionic parasympathetic

Chapter 43 • Pancreatic and Salivary Glands

Sjögren Syndrome

S

jögren syndrome is a chronic and progressive autoimmune disease that affects salivary secretion. Patients with Sjögren syndrome generate antibodies that react primarily with the salivary and lacrimal glands. Lymphocytes infiltrate the glands, and subsequent immunologic injury to the acini leads to a decrease in net secretory function. Expression of the Cl-HCO3 exchanger is lost in the striated duct cells of the salivary gland. Sjögren syndrome can occur as a primary disease (salivary and lacrimal gland dysfunction only) or as a secondary manifestation of a systemic autoimmune disease, such as rheumatoid arthritis. The disease primarily affects women; systemic disease usually does not develop. Individuals with Sjögren syndrome have xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Loss of salivary function causes these patients to have difficulty tasting, as well as chewing and swallowing dry food. They also have difficulty with continuous speech and complain of a chronic burning sensation in the mouth. On physical examination, patients with Sjögren syndrome have dry, erythematous oral mucosa with superficial ulceration and poor dentition (dental caries, dental fractures, and loss of dentition). Parotid gland enlargement is commonly present. The proteins that are the targets of the immunologic attack in Sjögren syndrome are not known. Therefore, no specific therapy for the disorder is available. Until the underlying cause of Sjögren syndrome is discovered, patients will have to rely on eyedrops and frequent oral fluid ingestion to compensate for their deficiencies in lacrimal and salivary secretion. Various stimulants of salivary secretion (sialogogues), such as methylcellulose and sour candy, can also be helpful. Patients with severe involvement and functional disability are sometimes treated with corticosteroids and immunosuppressants.

cervical ganglia that travel along blood vessels to the salivary glands (see Fig. 14-4). Although sympathetic (adrenergic) stimulation increases saliva flow, interruption of sympathetic nerves to the salivary glands has no major effect on salivary gland function in vivo. However, the sympathetic nervous system is the primary stimulator of the myoepithelial cells that are closely associated with cells of the acini and proximal (intercalated) ducts. These stellate cells have structural features of both epithelial and smooth muscle cells. They support the acinar structures and decrease the flow resistance of the intercalated ducts during stimulated secretion. Thus, the net effect of myoepithelial cell activation is to facilitate secretory flow in the proximal regions of the gland, thus minimizing the extravasation of secretory proteins that could otherwise occur during an acute increase in secretory flow. The sympathetic division can also indirectly affect salivary gland function by modulating blood flow to the gland. However, the relative contribution of this vascular effect to the overall secretory function of the salivary glands is difficult to determine. In addition to cholinergic and adrenergic regulation of salivary secretion, some autonomic fibers that innervate the salivary glands contain VIP and substance P. Although acinar cells in vitro respond to stimulation by substance P, the physiological significance of these neurotransmitters in vivo has not been established. Salivary secretion is also regulated, in part, by mineralocorticoids. The adrenal hormone aldosterone produces saliva that contains relatively less Na+ and more K+. The opposite effect on saliva is seen in patients with adrenal insufficiency caused by Addison disease. The mineralocorticoid effect represents the only well-established example of a humoral (i.e., non-neural) agent regulating salivary secretion.

REFERENCES

fibers from these ganglia directly stimulate the salivary glands through their release of ACh. The prominent role of the parasympathetic nervous system in salivary function can be readily appreciated by examining the consequences of cholinergic blockage. Disruption of the parasympathetic fibers to the salivary glands can lead to glandular atrophy. This observation suggests that parasympathetic innervation is necessary for maintaining the normal mass of salivary glands. Clinically, some medications (particularly psychiatric drugs) have “anticholinergic” properties that are most commonly manifested as “dry mouth.” For some medications, this effect is so uncomfortable for the patient that use of the medication must be discontinued. Conversely, excessive salivation is induced by some anticholinesterase agents that can be found in certain insecticides and “nerve gases.” Sympathetic Control

The salivary glands are also innervated by postganglionic sympathetic fibers from the superior

Books and Reviews Beger HG, Warshaw AL, Büchler M, et al. (eds): The Pancreas, 2nd ed. Cambridge, MA: Blackwell Publishing, 2008. Dobrosielski-Vergona K (ed): Biology of the Salvary Glands. Boca Raton, FL: CRC Press, 1993. Go VLW, DiMagno EP, Gardner JD, et al: The Pancreas: Biology, Pathobiology, and Disease, 2nd ed. New York: Raven Press, 1993. Johnson LR, et al. (eds): Physiology of the Gastrointestinal Tract, 4th ed. New York: Raven Press, 2006. Turner RJ, Sugiya H: Understanding salivary fluid and protein secretion. Oral Diseases 2002; 8:3-11. Williams JA: Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 2001; 63:77-97. Journal Articles Ishiguro H, Naruse S, Steward MC, et al: Fluid secretion in interlobular ducts isolated from guinea pig pancreas. J Physiol 1998; 511:407-422. Jamieson J, Palade G: Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells. J Cell Biol 1971; 50:135-158.

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Petersen OH: Stimulus-secretion coupling: Cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. J Physiol 1992; 448:1-51. Sohma Y, Gray MA, Imai Y, Argent BE: HCO−3 transport in a mathematical model of the pancreatic ductal epithelium. J Membr Biol 2000; 176:77-100.

Thaysen JH, Thorn NA, Schwartz IL: Excretion of sodium, potassium, chloride, and carbon dioxide in human parotid saliva. Am J Physiol 1954; 178:155-159. Zhao H, Xu X, Diaz J, Muallem S: Na+, K+, and H+/HCO−3 transport in submandibular salivary ducts. J Biol Chem 1995; 270: 19599-19605.

CHAPTER

44

I NTESTI NAL FLU I D AN D E L E C T R O LY T E M O V E M E N T Henry J. Binder

FUNCTIONAL ANATOMY The small intestine and large intestine have many similarities in structure and function. In some cases, different regions of the intestinal tract carry out certain functions in much the same manner. In other cases, however, substantial heterogeneity exists between different intestinal segments (e.g., ileum versus jejunum) or between different mucosal areas (i.e., villus versus crypt) in one intestinal segment. As discussed in Chapter 41, the basic structure of the intestine is a hollow cylinder with columnar epithelial cells lining the lumen, with circular and longitudinal layers of smooth muscle in the wall, and with endocrine and neural elements (see Fig. 41-2). Enteric neurons, as well as endocrine and paracrine agonists, regulate both epithelial transport and motor activity during both the interdigestive and the postprandial periods. As a result, the intestines propagate their contents in a caudad direction while either removing fluid and electrolytes from the intestinal lumen (i.e., absorption) or adding these substances to the lumen (i.e., secretion). Both the small intestine and large intestine absorb and secrete fluid and electrolytes, whereas only the small intestine absorbs nutrients Among mammals, absorption of dietary nutrients is an exclusive function of the small intestine. Only during the neonatal period does significant nutrient absorption take place in the large intestine. The small intestine absorbs nonelectrolytes after extensive digestion of dietary nutrients by both luminal and brush border enzymes, as discussed in Chapter 45. In contrast, both the small intestine and the large intestine absorb fluid and electrolytes by several different cellular transport processes, which may differ between the small intestine and the large intestine and are the subject of this chapter. Another vitally important function of the intestinal epithelium is the secretion of intestinal fluid and electrolytes. Teleologically, fluid secretion may be considered an adaptive mechanism used by the intestinal tract to protect itself from

noxious agents, such as bacteria and bacterial toxins. In general, the cellular mechanisms of intestinal electrolyte secretion in the small intestine and colon are similar, if not identical. Frequently, the adaptive signal that induces the secretory response also stimulates a simultaneous motor response from the intestinal muscle; together, these factors result in a propagated propulsive response in an attempt to dilute and eliminate the offending toxin. The small intestine has a villus crypt organization, whereas the colon has surface epithelial cells with interspersed crypts Both the small intestine and the large intestine have a specialized epithelial structure that correlates well with epithelial transport function. The small intestine (Fig. 441A) consists of finger-like projections—villi—surrounded by the openings of glandular structures called crypts of Lieberkühn, or simply crypts. Both villi and crypts are covered by columnar epithelial cells. The cells lining the villi are considered to be the primary cells responsible for both nutrient and electrolyte absorption, whereas the crypt cells primarily participate in secretion. The colon (Fig. 44-1B) does not have villi. Instead, the cells lining the large intestine are surface epithelial cells, and interspersed over the colonic surface are numerous apertures of colonic crypts (or glands) that are similar in function and structure to the small intestinal crypts. Not surprisingly, the surface epithelial cells of the colon are the primary cells responsible for colonic electrolyte absorption, whereas colonic gland cells are generally believed to mediate ion secretion. The intestinal mucosa is a dynamic organ with continuous cell proliferation and migration. The zone of cell proliferation is at the base of the crypt in both the small and large intestine, and the program of events is similar in both organs. The progenitor cell is a stem cell that differentiates into several specialized cells (e.g., vacuolated, goblet, and Paneth cells) that line the villi and crypts in the small intestine and the surface and glands in the colon. The vacuolated cell migrates along the crypt-villus axis and becomes a villous absorptive cell after undergoing substantial changes in its

933

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Section VII • The Gastrointestinal System

A

SMALL INTESTINE

B LARGE INTESTINE Haustra

Circular folds (of Kerckring)

Semilunar folds Surface epithelium

Villi Lamina propria

Crypt of ¨ Lieberkuhn Lamina propria

Crypt of ¨ Lieberkuhn

Muscularis mucosae

Muscularis mucosae

Submucosa

Submucosa Circular muscle of muscularis externa

Lacteal

Circular muscle of muscularis externa

Lymphoid Longitudinal nodule muscle of muscularis externa

Longitudinal muscle of muscularis externa

Villous absorptive cell Surface absorptive cell Goblet cell

Villus

Goblet cell Enteric endocrine cell

Enteric endocrine cell Stem/progenitor cell Crypt Stem/progenitor cell Crypt Paneth cell

Undifferentiated crypt cell

Undifferentiated crypt cell

Figure 44-1 Microscopic view of the anatomy of small and large intestine. A, The surface area of the small intestine is amplified at three levels: (1) macroscopic folds of Kerckring, (2) microscopic villi and crypts of Lieberkühn, and (3) submicroscopic microvilli. B, The surface area of the colon is amplified at the same three levels as the small intestine: (1) macroscopic semilunar folds, (2) crypts (but not villi), and (3) microvilli.

Chapter 44 • Intestinal Fluid and Electrolyte Movement

morphologic and functional characteristics. In the small intestine, these villous cells migrate until they reach the tips of the villi and then slough into the lumen of the intestine. The overall period from the initiation of cell proliferation to sloughing is ~48 to 96 hours. The overall rate of cell migration may increase or decrease: decreased cell turnover occurs during starvation, whereas increased cell turnover occurs during feeding and lactation, as well as after intestinal resection. The compensatory response that follows intestinal resection involves both luminal and hormonal factors. The surface area of the small intestine is amplified by folds, villi, and microvilli; amplification is less marked in the colon An additional hallmark of both the small and large intestine is the presence of structures that amplify function by increasing the luminal surface area. These structures exist at three levels. In the small intestine, the first level consists of the macroscopic folds of Kerckring. The second level consists of the microscopic villi and crypts that we have already discussed. The third level is the submicroscopic microvilli on the apical surfaces of the epithelial cells. Thus, if the small intestine is thought of as a hollow cylinder, the net increase in total surface area of the small intestine (versus that of a smooth cylinder) is 600-fold. The total surface area of the human small intestine is ~200 m2, or the surface area of a doubles tennis court (Table 44-1). The colonic surface area is also amplified, but to a more limited extent. Because the colon lacks villi, amplification is a result of only the presence of colonic folds, crypts, and microvilli. Amplification is an effective means of increasing the surface area that is available for intestinal absorption, the primary function of the small and large intestine.

Table 44-1 Structural and Functional Differences Between the Small and the Large Intestine Small Intestine

Large Intestine

Length (m)

6

2.4

Area of apical plasma membrane (m2)

~200

~25

Folds

Yes

Yes

Villi

Yes

No

Crypts or glands

Yes

Yes

Microvilli

Yes

Yes

Nutrient absorption

Yes

No

Active Na+ absorption

Yes

Yes

Active K+ secretion

No

Yes

OVERVIEW OF FLUID AND ELECTROLYTE MOVEMENT IN THE INTESTINES The small intestine absorbs ~6.5 L/day of an ~8.5-L fluid load that is presented to it, and the colon absorbs ~1.9 L/day The fluid content of the average diet is typically 1.5 to 2.5 L/ day. However, the fluid load to the small intestine is considerably greater—8 to 9 L/day. The difference between these two sets of figures is accounted for by salivary, gastric, pancreatic, and biliary secretions, as well as the secretions of the small intestine itself (Fig. 44-2). Similarly, the total quantity of electrolytes (Na+, K+, Cl−, and HCO−3) that enter the lumen of the small intestine also consists of dietary sources in addition to endogenous secretions from the salivary glands, stomach, pancreas, liver, and small intestine. We can calculate the absorption of water and electrolytes from the small intestine by comparing the total load that is presented to the lumen of the small intestine (i.e., 7.5 L/day entering from other organs +1.0 L/day secreted by the small intestine = 8.5 L/day) with that leaving the small intestine (i.e., ileocecal flow). The latter is ~2.0 L/day in normal subjects. Thus, overall small intestinal water absorption is 8.5 to 2.0, or ~6.5 L/day. Na+ absorption is ~600 mEq/day. Maximal small intestinal fluid absorption has not been directly determined but has been estimated to be as great as 15 to 20 L/day. Colonic fluid absorption is the difference between ileocecal flow (~2.0 L/day) and stool water, which is usually less than 0.2 L/day (~0.1 L/day). Thus, colonic water absorption is ~2.0 to 0.1, or 1.9 L/day. In contrast, the maximal colonic water absorptive capacity is between 4 and 5 L/day. As a result, a significant increase in ileocecal flow (e.g., up to perhaps 5 L/day, as occurs with a decrease in small intestinal fluid absorption) will not exceed the absorptive capacity of the large intestine. Thus, a compensatory increase in colonic fluid absorption can prevent an increase in stool water (i.e., diarrhea) despite substantial decreases in fluid absorption by the small intestine. The small intestine absorbs net amounts of water, Na+, Cl-, and K+ and secretes HCO3-, whereas the colon absorbs net amounts of water, Na+, and Cl- and secretes both K+ and HCO3Net ion movement represents the summation of several events. At the level of the entire small or large intestine, substantial movement of ions occurs from the intestinal lumen into the blood and from the blood into the lumen. The net ion movement across the entire epithelium is the difference between these two unidirectional fluxes. Fluid and electrolyte transport in the intestine varies considerably in two different axes, both along the length of the intestines (segmental heterogeneity) and from the bottom of a crypt to the top of a villus or to the surface cells (cryptvillus/surface heterogeneity). A comparison of two different segments of intestine (e.g., duodenum versus ileum) shows that they differ substantially in function. These differences

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Inflow

Contents of GI tract

Outflow

Mouth, esophagus Food 2.0 L /day Saliva 1.5 L /day

Stomach Gastric secretion 2.0 L /day Bile Pancreatic secretion 1.5 L /day

Duodenum

Bile secretion 0.5 L /day

Pancreas

Jejunum Secreted by small intestine 1 L /day HCO3–

H2O

Na+ K+

Cl–

Ileum

Presented to lumen of small intestine: 8.5 L/day.

Proximal colon

Presented to colon: 2.0 L/day.

Secreted by colon K+

Reabsorbed by small intestine 6.5 L /day

Reabsorbed by colon 1.9 L /day

Distal colon

Na+

HCO3–

Cl–

H2O

Anus

Excreted in feces ~0.1 L /day

Figure 44-2 Fluid balance in the gastrointestinal (GI) tract. For each segment of the GI tract, the figure shows substances flowing into the lumen on the left and substances flowing out of the lumen on the right. Of the ~8.5 L/day presented to the small intestine, the small intestine removes ~6.5 L/day, delivering ~2 L/day to the colon. The large intestine removes ~1.9 L/day, leaving ~0.1 L/day in the feces.

in function reflect segmental heterogeneity of ion transport processes along the longitudinal axis of the intestine in different macroscopic regions of both the small and the large intestine; these differences are both qualitative and quantitative. For example, HCO−3 stimulation of Na+ absorption occurs only in the proximal part of the small intestine. In contrast, the so-called electrogenic Na+ absorption (i.e., absorption associated with the development of a transepithelial potential difference) is restricted to the rectosigmoid segment of the colon. Within an intestinal segment (e.g., a piece of ileum), crypt-villus/surface heterogeneity leads to differences in transport function along the radial axis of the intestine wall. For example, it is generally believed that absorptive function is located in villous cells in the small intestine (and surface epithelial cells in the large intestine), whereas secretory processes reside in the crypt cells. Finally, at a certain level within a single villus or crypt—or within a very small area of the colonic surface epithelium—individual cells may demonstrate further heterogeneity (cellular heterogeneity), with specific transport mechanisms restricted to different cells. Overall ion movement in any segment of the intestine represents the summation of these various absorptive and secretory events. These events may be paracellular or transcellular, may occur in the villus or crypt, and may be mediated by a goblet cell or an absorptive cell. Despite the segmental heterogeneity of small intestinal electrolyte transport, overall water and ion movement in the proximal and distal portions of the small intestine is similar: in health, the small intestine is a net absorber of water, Na+, Cl−, and K+, but it is a net secretor of HCO−3 (Fig. 44-2). Fluid absorption is isosmotic in the small intestine, similar to that observed in the renal proximal tubule (see Chapter 35). In general, absorptive processes in the small intestine are enhanced in the postprandial state. The human colon carries out net absorption of water, Na+, and Cl− with few exceptions, but it carries out net secretion of K+ and HCO−3 . The intestines absorb and secrete solutes by both active and passive mechanisms As discussed in Chapter 5, intestinal epithelial cells are polar; that is, they have two very different membranes—an apical membrane and a basolateral membrane—separated from one another by tight junctions. The transport processes present in the small and large intestine are quite similar to those present in other epithelia, such as the renal tubules, with only some organ-specific specialization to distinguish them. The transepithelial movement of a solute across the entire epithelium can be either absorptive or secretory. In each case, the movement can be either transcellular or paracellular. In transcellular movement, the solute must cross the two cell membranes in series. In general, movement of the solute across at least one of these membranes must be active (i.e., against an electrochemical gradient). In paracellular movement, the solute moves passively between adjacent epithelial cells through the tight junctions. All transcellular Na+ absorption is mediated by the Na-K pump (i.e., Na,K-ATPase) located at the basolateral membrane. This enzyme is responsible for Na+ extrusion across

Chapter 44 • Intestinal Fluid and Electrolyte Movement

the basolateral membrane and results in a relatively low [Na+]i (~15 mM) and an intracellular-negative membrane potential. This Na+ gradient serves as the driving force, in large part, for Na+ entry into the epithelial cell across the luminal (apical) membrane, a process mediated either by Na+ channels or by Na+-coupled transporters (e.g., Na/ glucose cotransport, Na-H exchange). The epithelial cell may also use this Na+ gradient to energize other transport processes at the apical or basolateral membrane. Intestinal fluid movement is always coupled to solute movement, whereas solute movement may be coupled to fluid movement by “solvent drag” Fluid movement is always coupled to active solute movement. The model of the osmotic coupling of fluid movement to solute movement in the intestine is similar to that in all or most epithelial cells (see Chapter 5). It is likely that the water movement occurs predominantly by a paracellular route rather than by a transcellular route. Solute movement is the driving force for fluid movement. However, the converse may also be true: solute movement may be coupled to fluid movement by solvent drag, a phenomenon in which the dissolved solute is swept along by bulk movement of the solvent (i.e., water). Solvent drag accounts for a significant fraction of the Na+ and urea absorbed in the human jejunum (but not in the more distal segments of the small intestine or the large intestine). For all intents and purposes, solvent drag occurs through the paracellular route, and it depends on the permeability properties of the tight junctions (reflection coefficient; see Chapter 20) and the magnitude of the convective water flow. Thus, solvent drag contributes primarily to the absorption of relatively small, water-soluble molecules, such as urea and Na+, and it does so mainly in epithelia with relatively high permeability. The transepithelial permeability of the jejunum is considerably greater than that of the ileum or colon, as evidenced by its lower spontaneous transepithelial voltage difference (VTE), higher passive movement of NaCl, and larger apparent pore size. The resistance of the tight junctions primarily determines the transepithelial resistance of intestinal epithelia Epithelial permeability is an inverse function of transepithelial resistance. In epithelial structures such as the small and large intestine, transepithelial resistance is determined by cellular resistance and paracellular resistance, which are arranged in parallel (see Chapter 5). Paracellular resistance is considerably lower than transcellular resistance; therefore, overall mucosal resistance depends mainly on paracellular resistance, which, in turn, depends primarily on the properties of the tight junctions. Therefore, intestinal permeability is essentially a function of tight junction structure. Just as transport function varies greatly throughout the intestine, major differences in transepithelial permeability and the properties of tight junctions are also present throughout the intestinal tract. In general, resistance increases in the aboral direction (i.e., moving away from the mouth). Thus, the

resistance of the jejunum is considerably lower than that of the distal end of the colon. Evidence also indicates that the permeability of the tight junctions in the crypt is greater than that in the villus.

CELLULAR MECHANISMS OF NA+ ABSORPTION Both the small intestine and the large intestine absorb large amounts of Na+ and Cl− daily, but different mechanisms are responsible for this extremely important physiological process in different segments of the intestine. The villous epithelial cells in the small intestine and the surface epithelial cells in the colon are responsible for absorbing most of the Na+. Absorption of Na+ is the result of a complex interplay of both apical and basolateral membrane transport processes. Figure 44-3 summarizes the four fundamental mechanisms by which Na+ may enter the cell across the apical membrane. In each case, the Na-K pump is responsible, at least in part, for the movement of Na+ from cell to blood. Also in each case, the driving force for apical Na+ entry is provided by the large, inwardly directed electrochemical gradient for Na+, which, in turn, is provided by the Na-K pump. The following four sections describe these four apical membrane transport processes. Na/glucose and Na/amino acid cotransport in the small intestine is a major mechanism for postprandial Na+ absorption Nutrient-coupled Na+ absorption (Fig. 44-3A) occurs throughout the small intestine. Although glucose- and amino acid–coupled Na+ absorption also takes place in the colon of the newborn, it disappears during the neonatal period. Glucose- and amino acid–coupled Na+ absorption occurs only in villous epithelial cells and not in crypt epithelial cells (Fig. 44-3A). This process is the primary mechanism for Na+ absorption after a meal, but it makes little contribution during the interdigestive period, when only limited amounts of glucose and amino acids are present in the intestinal lumen. Glucose- and amino acid–coupled Na+ absorption is mediated by specific apical membrane transport proteins. The Na/glucose cotransporter SGLT1 (see Chapter 5) is responsible for glucose uptake across the apical membrane, as discussed in Chapter 45. Several distinct Na/amino acid cotransporters, each specific for a different class of amino acids (see Table 36-1), are responsible for the Na+-coupled uptake of amino acids across the apical membrane. Because these transporters couple the energetically downhill movement of Na+ to the uphill movement of glucose or an amino acid, the transporter processes are examples of secondary active transport (see Chapter 5). The glucoseand amino acid–coupled uptake of Na+ entry across the apical membrane increases [Na+]i, which, in turn, increases Na+ extrusion across the basolateral membrane through the Na-K pump. Because the apical Na/glucose and Na/amino acid cotransporters are electrogenic, as is the Na-K pump, the overall transport of Na+ carries net charge and makes VTE more lumen negative. Thus, glucose- and

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Section VII • The Gastrointestinal System

A

Na/GLUCOSE OR Na/AMINO ACID COTRANSPORTERS

SGLT1 Glucose

GLUT2 Stomach Jejunum Duodenum

3 Na+

2 Na+

Ileum 2K

Jejunum

+

Amino acid Ileum B

Na–H EXCHANGER

NHE3 Duodenum

3 Na+

Na+

2 K+

+

H

Proximal colon

Jejunum

H+

Distal colon Na+

Amiloride (mM)

NHE1

High absorption Moderate absorption

C

PARALLEL Na–H AND Cl–HCO3 EXCHANGERS

Low absorption Very low absorption

NHE3 Na+

+

3 Na +

H Cl

–

2 K+ H 2O – OH CO 2

HCO3–

CA

CO2

Cl–

CIC-2 (?)

Ileum Proximal colon

D EPITHELIAL Na+ CHANNEL

Na+

3 Na+

2 K+ Amiloride (µM)

Distal colon

Figure 44-3 Modes of active Na+ absorption by the intestine. A, Nutrient-coupled Na+ absorption occurs in the villous cells of the jejunum and ileum and is the primary mechanism for postprandial Na+ absorption. The thickness of the arrows in the inset indicates the relative magnitude of the Na+ absorptive flux through this pathway. B, Electroneutral Na-H exchange at the apical membrane, in the absence of Cl-HCO3 exchange, is stimulated by the high pH of the HCO3−-rich luminal contents. C, Na-H and Cl-HCO3 exchange is coupled by a change in intracellular pH that results in electroneutral NaCl absorption, which is the primary mechanism for interdigestive Na+ absorption. D, In electrogenic Na+ absorption, the apical step of Na+ movement occurs through the ENaC. CA, carbonic anhydrase.

Chapter 44 • Intestinal Fluid and Electrolyte Movement

amino acid–stimulated Na+ absorption is an electrogenic process. As discussed later, the increase in the lumen-negative VTE provides the driving force for the parallel absorption of Cl−. Nutrient-coupled Na+ transporters, unlike other small intestinal Na+ absorptive mechanisms, are not inhibited by either cAMP or [Ca2+]i. Thus, agonists that increase [cAMP]i (i.e., Escherichia coli or cholera enterotoxin) or [Ca2+]i (i.e., serotonin) do not inhibit glucose- or amino acid–stimulated Na+ absorption. Electroneutral Na-H exchange in the duodenum and jejunum is responsible for Na+ absorption that is stimulated by luminal alkalinity Luminal HCO−3—the result of pancreatic, biliary, and duodenal secretion—increases Na+ absorption in the proximal portion of the small intestine by stimulating apical membrane Na-H exchange (Fig. 44-3B). The Na-H exchanger couples Na+ uptake across the apical membrane to proton extrusion into the intestinal lumen, a process that is enhanced by both decreases in intracellular pH (pHi) and increases in luminal pH. The energy for Na-H exchange comes from the Na+ gradient, a consequence of the ability of the Na-K pump to extrude Na+, thereby lowering [Na+]i. This process is characteristically inhibited by millimolar concentrations of the diuretic amiloride. Several isoforms of the Na-H exchanger exist (see Chapter 5), and different isoforms are present on the apical and basolateral membranes. Intestinal epithelial cells also have Na-H exchangers on their basolateral membranes. However, this NHE1 isoform, like its counterpart in nonepithelial cells, regulates pHi (a “housekeeping” function) and does not contribute to the transepithelial movement of Na+. In contrast, both the NHE2 and NHE3 exchanger isoforms present on the apical membrane are responsible for both transepithelial Na+ movement and pHi regulation. Although Na-H exchangers are present on the apical membrane of villous epithelial cells throughout the entire intestine, only in the duodenum and jejunum (i.e., the proximal part of the small intestine) is Na-H exchange present without the parallel presence of Cl-HCO3 exchangers (see next section). Thus, in the proximal portion of the small intestine, the Na-H exchanger solely mediates the Na+ absorption that is stimulated by the alkalinity of the HCO−3 -rich intraluminal contents. Parallel Na-H and Cl-HCO3 exchange in the ileum and proximal part of the colon is the primary mechanism of Na+ absorption during the interdigestive period Electroneutral NaCl absorption occurs in portions of both the small and large intestine (Fig. 44-3C). Electroneutral NaCl absorption is not the result of an Na/Cl cotransporter, but rather of parallel apical membrane Na-H and Cl-HCO3 exchangers that are closely linked by small changes in pHi. In the human colon, DRA (downregulated-in-adenoma; SLC26A3; see Chapter 5) mediates this Cl-HCO3 exchange. This mechanism of NaCl absorption is the primary method of Na+ absorption between meals (i.e., the interdigestive

Oral Rehydration Solution

T

he therapeutic use of oral rehydration solution (ORS) provides an excellent demonstration of applied physiology. Many diarrheal illnesses (see the box titled Secretory Diarrhea) are caused by bacterial exotoxins that induce fluid and electrolyte secretion by the intestine. Hence such a toxin is referred to as an enterotoxin. Despite the massive toxin-induced fluid secretion, both intestinal morphology and nutrient-coupled Na+ absorption are normal. Because nutrient-coupled (e.g., glucose or amino acid) fluid absorption is intact, therapeutically increasing the concentration of glucose or amino acids in the intestinal lumen can enhance absorption. ORS contains varying concentrations of glucose, Na+, Cl−, and HCO3− and is extremely effective in enhancing fluid and electrolyte absorption in secretory diarrhea when the intestine secretes massive amounts of fluid. Administration of ORS can reverse the dehydration and metabolic acidosis that may occur in severe diarrhea and that are often the primary cause of morbidity and mortality, especially in children younger than 5 years. ORS is the major advance of the past half century in the treatment of diarrheal disease, especially in developing countries. The development of ORS was a direct consequence of research on the physiology of glucose- and amino acid–stimulated Na+ absorption.

period), but it does not contribute greatly to postprandial Na+ absorption, which is mediated primarily by the nutrient-coupled transporters described previously. Electroneutral NaCl absorption occurs in the ileum and throughout the large intestine, with the exception of the most distal segment. It is not affected by either luminal glucose or luminal HCO−3 . However, aldosterone inhibits electroneutral NaCl absorption. The overall electroneutral NaCl absorptive process is regulated by both cAMP and cGMP, as well as by intracellular Ca2+. Increases in each of these three intracellular messengers reduce NaCl absorption. Conversely, decreases in [Ca2+]i increase NaCl absorption. Decreased NaCl absorption is important in the pathogenesis of most diarrheal disorders. For example, one of the common causes of traveler’s diarrhea (see the box titled Secretory Diarrhea) is the heat-labile enterotoxin produced by the bacterium E. coli. This toxin activates adenylyl cyclase and increases [cAMP]i, which, in turn, decreases NaCl absorption and stimulates active Cl− secretion, as discussed later. This toxin does not affect glucose-stimulated Na+ absorption. Epithelial Na+ channels are the primary mechanism of electrogenic Na+ absorption in the distal part of the colon In electrogenic Na+ absorption (Fig. 44-3D), Na+ entry across the apical membrane occurs through epithelial Na+ channels (ENaCs) that are highly specific for Na+ (see Chapter 5). Like the Na-H exchanger, these ENaCs are blocked by the diuretic amiloride, but at micromolar rather than millimolar concen-

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trations. Na+ absorption in the distal part of the colon is highly efficient. Because this segment of the colon is capable of absorbing Na+ against large concentration gradients, it plays an important role in Na+ conservation. Na+ movement through electrogenic Na+ absorption is not affected by luminal glucose or by HCO−3 , nor is it regulated by cyclic nucleotides. However, it is markedly enhanced by mineralocorticoids (e.g., aldosterone). Mineralocorticoids increase Na+ absorption in the colon—as in other aldosterone-responsive epithelia, notably the renal collecting duct (see Chapter 35)—through multiple mechanisms. Aldosterone increases electrogenic Na+ absorption by increasing Na+ entry through the apical Na+ channel and by stimulating activity of the Na-K pump. The increase in apical Na+ uptake can occur (1) rapidly (i.e., within seconds) as a consequence of an increase in the opening of apical Na+ channels, (2) more gradually (within minutes) because of the insertion of preformed Na+ channels from subapical epithelial vesicle pools into the apical membrane, or (3) very slowly (within hours) as a result of an increase in the synthesis of both new apical Na+ channels and Na-K pumps.

CELLULAR MECHANISMS OF CLABSORPTION AND SECRETION Cl− absorption occurs throughout the small and large intestine and is often closely linked to Na+ absorption. Cl− and Na+ absorption may be coupled through either an electrical potential difference or by pHi. However, sometimes no coupling takes place, and the route of Cl− movement may be either paracellular or transcellular. Voltage-dependent Cl- absorption represents coupling of Cl- absorption to electrogenic Na+ absorption in both the small intestine and the large intestine Cl− absorption can be a purely passive process (Fig. 44-4A), driven by the electrochemical gradient for Cl− either across the tight junctions (paracellular route) or across the individual membranes of the epithelial cell (transcellular route). In either case, the driving force for Cl− absorption derives from either of the two electrogenic mechanisms of Na+ absorption described previously (namely, nutrientcoupled transport in the small intestine and the ENaCs in the distal end of the colon), which, in turn, are energized by the Na-K pump. This process is referred to as voltagedependent Cl − absorption; it is not an active transport process. Within the small intestine, induction of a lumen-negative potential difference by glucose- and amino acid–induced Na+ absorption (Fig. 44-3A) provides the driving force for Cl− absorption that occurs following a meal. As noted earlier, nutrient-coupled Na+ absorption primarily represents a villous cell process that occurs in the postprandial period and is insensitive to cyclic nucleotides and changes in [Ca2+]i. Voltage-dependent Cl− absorption shares these properties. It is most likely that the route of voltage-dependent Cl− absorption is paracellular.

PASSIVE Cl– ABSORPTION

A

– 3 Na+ Jejunum 2K

–

+

Cl

Cl–

B

Ileum

Distal colon Cl–HCO3 EXCHANGER

3 Na+

CO2

H 2O

CA

Cl–

2 K+

–

OH

Cl

HCO3–

–

H

+

DRA

Na+

Ileum

Proximal colon Distal CIC-2 (?) colon

PARALLEL Na–H AND Cl–HCO3 EXCHANGERS

C

Na+

3 Na+ +

H Cl

–

NHE3 –

HCO3

DRA

2K H2O – OH CO 2

CA

+

CO2

Cl–

CIC-2 (?)

Ileum Proximal colon

High absorption Moderate absorption Low absorption Very low absorption

Figure 44-4 Modes of Cl− absorption by the intestine. A, In voltage-dependent Cl− absorption, Cl− may passively diffuse from lumen to blood across the tight junctions, driven by the lumennegative transepithelial voltage (paracellular route). Alternatively, Cl− may diffuse through apical and basolateral Cl− channels. The thickness of the arrows in the inset indicates the relative magnitude of the Cl− absorptive flux through this pathway. B, In the absence of a parallel Na-H exchanger, electroneutral Cl-HCO3 exchange at the apical membrane results in Cl− absorption and HCO3− secretion. C, Electroneutral NaCl absorption (see Fig. 44-3C) can mediate Cl− absorption in the interdigestive period. pHi couples the two exchangers. CA, carbonic anhydrase.

In the large intestine, especially in the distal segment, electrogenic Na+ absorption through the ENaC (Fig. 44-3D) also induces a lumen-negative potential difference that provides the driving force for colonic voltage-dependent Cl− absorption. Factors that increase or decrease the voltage difference similarly affect Cl− absorption.

Chapter 44 • Intestinal Fluid and Electrolyte Movement

Congenital Chloridorrhea Duodenum

T

he congenital absence of an apical Cl-HCO3 exchanger (which mediates the Cl-HCO3 involved in electroneutral NaCl absorption) is an autosomal recessive disorder known as congenital chloridorrhea or congenital Cl- diarrhea (CLD). Affected children have diarrhea with an extremely high stool [Cl−], a direct consequence of absence of the apical membrane Cl-HCO3 exchanger. In addition, because HCO3− secretion is reduced, patients are alkalotic (i.e., have an increased plasma [HCO3−]). The gene for congenital chloridorrhea is located on chromosome 7q31. The gene product is the same as that of the DRA gene. DRA (SLC26A3; see Chapter 5) •• and mediates ClHCO3 exchange. In addition, DRA transports sulfate and other anions. However, DRA is distinct from the AE (anion exchanger) gene family that encodes the Cl-HCO3 exchangers in erythrocytes and several other tissues. Indeed, ClHCO3 exchange in the renal tubule, erythrocytes, and other cells is unaffected in individuals with CLD, as are other intestinal transport processes.

Electroneutral Cl-HCO3 exchange results in Clabsorption and HCO3- secretion in the ileum and colon Electroneutral Cl-HCO3 exchange, in the absence of parallel Na-H exchange, occurs in villous cells in the ileum and in surface epithelial cells in the large intestine (Fig. 44-4B). It is not known whether this process occurs in the cells lining the crypts. A Cl-HCO3 exchanger in the apical membrane is responsible for the 1 : 1 exchange of apical Cl− for intracellular HCO−3 . In humans, this Cl-HCO3 exchanger is DRA (see Chapter 5). The details of Cl− movement across the basolateral membrane are not well understood, but the process may involve a ClC-2 Cl− channel (see Chapter 6). Parallel Na-H and Cl-HCO3 exchange in the ileum and the proximal part of the colon mediates Cl- absorption during the interdigestive period Electroneutral NaCl absorption, discussed in connection with Na+ absorption (Fig. 44-3C), also mediates Cl− absorption in the ileum and proximal part of the colon (Fig. 44-4C). The apical step of Cl− absorption by this mechanism is mediated by parallel Na-H exchange (NHE3 or SLC9A3) and Cl-HCO3 exchange (DRA or SLC26A3), which are coupled through pHi. Electrogenic Cl- secretion occurs in crypts of both the small intestine and the large intestine In the previous three sections, we saw that intestinal Cl− absorption occurs through three mechanisms. The small intestine and the large intestine are also capable of active Cl− secretion, although Cl− secretion is believed to occur mainly in the crypts rather than in either the villi or surface cells.

IK1 and BK 3 Na

+ +

2K

Subapical vesicles

CFTR

+

K

cAMP

Na

Cl– NKCC1

–

Ileum Proximal colon Distal colon

2+

Ca

Jejunum

+

2 Cl– +

Na

High secretion Moderate secretion Low secretion Very low secretion

Figure 44-5 Cellular mechanism of electrogenic Cl− secretion by crypt cells. The basolateral Na/K/Cl cotransporter brings Cl− into the crypt cell; the Cl− exits across the apical Cl− channel. Secretagogues may open preexisting Cl− channels or may cause subapical vesicles to fuse with the apical membrane, thus delivering new Cl− channels. The paracellular pathway allows Na+ movement from blood to lumen, driven by the lumen-negative transepithelial voltage. The thickness of the arrows in the inset indicates that the magnitude of the Cl− secretory flux through this pathway is the same throughout the intestine.

A small amount of Cl− secretion probably occurs in the “basal” state but is masked by the higher rate of the three Cl− absorptive processes that are discussed earlier in this subchapter. However, Cl− secretion is markedly stimulated by secretagogues such as acetylcholine and other neurotransmitters. Moreover, Cl− secretion is the major component of the ion transport events that occur during most clinical and experimental diarrheal disorders. The cellular model of active Cl− secretion is outlined in Figure 44-5 and includes three transport pathways on the basolateral membrane: (1) an Na-K pump, (2) an Na/K/Cl cotransporter (NKCC1 or SLC12A2), and (3) two types of K+ channels (IK1 and BK). In addition, a Cl− channel (cystic fibrosis transmembrane regulator [CFTR]) is present on the apical membrane. This complex Cl− secretory system is energized by the Na-K pump, which generates a low [Na+]i and provides the driving force for Cl− entry across the basolateral membrane through Na/K/Cl cotransport. As a result, [Cl−]i is raised sufficiently that the Cl− electrochemical gradient favors the passive efflux of Cl− across the apical membrane. One consequence of these many transport processes is that the transepithelial voltage becomes more lumen negative, thereby promoting voltage-dependent Na+ secretion. This Na+ secretion that accompanies active Cl− secretion presumably occurs through the tight junctions (paracellular pathway). Thus, the net result is stimulation of NaCl and fluid secretion. Normally (i.e., in the unstimulated state), the crypts secrete little Cl− because the apical membrane Cl− channels are either closed or not present. Cl− secretion requires activa-

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tion by cyclic nucleotides or [Ca2+], which are increased by any of several secretagogues, including (1) bacterial exotoxins (i.e., enterotoxins), (2) hormones and neurotransmitters, (3) products of cells of the immune system (e.g., histamine), and (4) laxatives (Table 44-2). Some secretagogues initially bind to membrane receptors and stimulate the activation of adenylyl cyclase (vasoactive intestinal peptide [VIP]), guanylyl cyclase (the heat-stable toxin of E. coli), or phospholipase C (acetylcholine). Others increase [Ca2+]i by opening Ca2+ channels at the basolateral membrane. The resulting activation of one or more protein kinases—by any of the aforementioned pathways—increases the Cl− conductance of the apical membrane either by activating preexisting Cl− channels or by inserting into the apical membrane Cl− channels that—in the unstimulated state— are stored in subapical membrane vesicles. In either case, Cl− is now able to exit the cell through apical Cl− channels. The resulting decrease in [Cl−]i leads to increased uptake of Na+, Cl−, and K+ across the basolateral membrane through the Na/K/Cl cotransporter (NKCC1). The Na+ is recycled out of the cell through the Na-K pump. The K+ is recycled through basolateral K+ channels that are opened by the same protein kinases that increase Cl− conductance. The net result of all these changes is the initiation of active Cl− secretion across the epithelial cell. The induction of apical membrane Cl− channels is extremely important in the pathophysiology of many diarrheal disorders. The box titled Secretory Diarrhea discusses the changes in ion transport that occur in secretory diarrheas such as cholera. A central role in cystic fibrosis has been posited for the CFTR Cl− channel in the apical membrane (see Chapter 43). However, more than one (and possibly several) Cl− channels are present in the intestine, and CFTR may not be the only Cl− channel associated with active Cl− secretion.

Table 44-2

Mode of Action of Secretagogues

Category

Secretagogue

Second Messenger

Bacterial enterotoxins

Cholera toxin Escherichia coli toxins: heat labile E. coli toxins: heat stable Yersinia toxin Clostridium difficile toxin

cAMP cAMP

Hormones and neurotransmitters

VIP Guanylin Acetylcholine Bradykinin Serotonin (5-HT)

cAMP cGMP Ca2+ Ca2+ Ca2+

Immune cell products

Histamine Prostaglandins

cAMP cAMP

Laxatives

Bile acids Ricinoleic acid

Ca2+ ?

CELLULAR MECHANISMS OF K+ ABSORPTION AND SECRETION Overall net transepithelial K+ movement is absorptive in the small intestine and is secretory in the colon The gastrointestinal tract participates in overall K+ balance, although when compared with the role of the kidneys, the small intestine and large intestine play relatively modest roles, especially in healthy individuals. The pattern of intestinal K+ movement parallels that of the kidney: (1) the intestines have the capacity for both K+ absorption and secretion, and (2) the intestines absorb K+ in the proximal segments but secrete it in the distal segments. Dietary K+ furnishes 80 to 120 mmol/day, whereas stool K+ output is only ~10 mmol/day. The kidney is responsible for disposal of the remainder of the daily K+ intake (see Chapter 37). Substantial quantities of K+ are secreted in gastric, pancreatic, and biliary fluid. Therefore, the total K+ load presented to the small intestine is considerably greater than that represented by the diet. The concentration of K+ in stool is frequently more than 100 mM. This high stool [K+] is the result of several factors, including both colonic K+ secretion and water absorption, especially in the distal part of the colon. K+ absorption in the small intestine probably occurs through solvent drag Studies in which a plasma-like solution is perfused through segments of the intestine established that K+ is absorbed in the jejunum and ileum of the small intestine and is secreted in the large intestine. Although the small intestine absorbs substantial amounts of K+, no evidence has been presented to suggest that K+ absorption in the jejunum and ileum is an active transport process or even carrier mediated. Thus, K+ absorption in the small intestine is probably passive, most likely a result of solvent drag (i.e., pulled along by bulk water movement), as illustrated in Figure 44-6A. Although changes in dietary Na+ and K+ and alterations in hydration influence K+ movement in the colon, similar physiological events do not appear to affect K+ absorption in the small intestine.

cGMP cGMP Ca2+

Passive K+ secretion is the primary mechanism for net colonic secretion In contrast to the small intestine, the human colon is a net secretor of K+. This secretion occurs by two mechanisms: a passive transport process that is discussed in this section and an active process that is discussed in the next. Together, these two K+ secretory pathways are greater than a modest component of active K+ absorption in the distal part of the colon and thus account for the overall secretion of K+ by the colon. Passive K+ secretion, which is the pathway that is primarily responsible for overall net colonic K+ secretion, is driven by the lumen-negative VTE of 15 to 25 mV. The route of passive K+ secretion is predominantly paracellular, not transcellular (Fig. 44-6B). Because VTE is the primary determinant

Chapter 44 • Intestinal Fluid and Electrolyte Movement

PASSIVE K+ ABSORPTION

A

3 Na+ Jejunum

2 K+

H2O

Ileum

K+

Figure 44-6 Cellular mechanisms of K+ secretion and absorption. A, This mechanism pertains only to the small intestine, which is a net absorber of K+ through solvent drag across tight junctions. The thickness of the arrows in the inset indicates the relative magnitude of the K+ flux through this pathway. B, The colon is a net secretor of K+. The primary mechanism is passive K+ secretion through tight junctions, which occurs throughout the colon. The driving force is a lumen-negative transepithelial voltage. C, Another mechanism of K+ secretion throughout the colon is a transcellular process that involves the basolateral uptake of K+ through the Na-K pump and the Na/K/Cl cotransporter, followed by the efflux of K+ through apical K+ channels. D, Confined to the distal colon is a transcellular mechanism of K+ absorption that is mediated by an apical H-K pump.

PASSIVE K+ SECRETION

B

3 Na+ 2 K+ +

–

Proximal colon

K

The lumen potential is –25 mV.

C

Distal colon

ACTIVE K+ SECRETION

K

+

3 Na

Active K+ secretion is also present throughout the large intestine and is induced both by aldosterone and by cAMP

+ +

2K

BK

Proximal colon Na

+

K

+

Distal colon

2 Cl– NKCC1

D

+ ACTIVE K ABSORPTION

3 Na+ H+ K+

of passive K+ secretion, it is not surprising that passive K+ secretion is greatest in the distal end of the colon, where VTE difference is most negative. Similarly, increases in the lumennegative VTE that occur as an adaptive response to dehydration—secondary to an elevation in aldosterone secretion (see the next section)—result in an enhanced rate of passive K+ secretion. Information is not available regarding the distribution of passive K+ secretion between surface epithelial and crypt cells.

2 K+ ? Distal colon

High transport Moderate transport Low transport Very low transport

In addition to passive K+ secretion, active K+ transport processes—both secretory and absorptive—are also present in the colon. However, active transport of K+ is subject to considerable segmental variation in the colon. Whereas active K+ secretion occurs throughout the colon, active K+ absorption is present only in the distal segments of the large intestine. Thus, in the rectosigmoid colon, active K+ absorption and active K+ secretion are both operative and appear to contribute to total body homeostasis. The model of active K+ secretion in the colon is quite similar to that of active Cl− secretion (Fig. 44-5) and is also parallel to that of active K+ secretion in the renal distal nephron (see Chapter 37). The general paradigm of active K+ transport in the colon is a pump-leak model (Fig. 44-6C). Uptake of K+ across the basolateral membrane is a result of both the Na-K pump and the Na/K/Cl cotransporter (NKCC1), which is energized by the low [Na+]i that is created by the Na-K pump. Once K+ enters the cell across the basolateral membrane, it may exit either across the apical membrane (K+ secretion) or across the basolateral membrane (K+ recycling). The cell controls the extent to which secretion occurs, in part by K+ channels present in both the apical and the basolateral membranes. When apical K+ channel activity is less than basolateral channel activity, K+ recycling dominates. Indeed, in the basal state, the rate of active K+ secretion is low because the apical K+ channel activity is minimal in comparison with the K+ channel activity in the basolateral membrane. It is likely that aldosterone stimulates active K+ secretion in surface epithelial cells of the large intestine, whereas cAMP enhances active K+ secretion in crypt cells. In both cases, the rate-limiting step is the apical BK K+ channel, and both secretagogues act by increasing K+ channel activity.

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Aldosterone This mineralocorticoid enhances overall net K+ secretion by two mechanisms. First, it increases passive K+ secretion by increasing Na-K pump activity and thus increasing electrogenic Na+ absorption (Fig. 44-3D). The net effects are to increase the lumen-negative VTE and to enhance passive K+ secretion (Fig. 44-6B). Second, aldosterone stimulates active K+ secretion by increasing the activity of both apical K+ channels and basolateral Na-K pumps (Fig. 44-6C). cAMP and Ca2+

VIP and cholera enterotoxin both increase [cAMP]i and thus stimulate K+ secretion. Increases in [Ca2+]i—induced, for example, by serotonin (or 5-hydroxytryptamine [5-HT])—also stimulate active K+ secretion. In contrast to aldosterone, neither of these second messengers has an effect on the Na-K pump; rather, they increase the activity of both the apical and the basolateral K+ channels. Because the stimulation of K+ channels is greater at the apical than at the basolateral membrane, the result is an increase in K+ exit from the epithelial cell across the apical membrane (i.e., secretion). Stimulation of K+ secretion by cAMP and Ca2+, both of which also induce active Cl− secretion (Fig. 44-5), contributes to the significant fecal K+ losses that occur in many diarrheal diseases. Active K+ absorption is located only in the distal portion of the colon and is energized by an apical H-K pump As noted earlier, not only does the distal end of the colon actively secrete K+, but also it actively absorbs K+. The balance between the two processes plays a role in overall K+ homeostasis. Increases in dietary K+ enhance both passive and active K+ secretion (Fig. 44-6B, C). However, dietary K+ depletion enhances active K+ absorption (Fig. 44-6D). The mechanism of active K+ absorption appears to be an exchange of luminal K+ for intracellular H+ across the apical membrane, mediated by an H-K pump (see Chapter 5). The colonic H-K pump is ~60% identical at the amino acid level to both the Na-K pump and the gastric parietal cell H-K pump. Thus, colonic K+ movement through the active K+ absorption process occurs through a transcellular route, in contrast to the paracellular route that characterizes K+ absorption in the small intestine (Fig. 44-6A). The mechanism of K+ exit across the basolateral membrane may involve K/Cl cotransport. Not known is whether active K+ secretion (Fig. 44-6C) and active K+ absorption (Fig. 44-6D) occur in the same cell or in different cells.

REGULATION OF INTESTINAL ION TRANSPORT Chemical mediators from the enteric nervous system, endocrine cells, and immune cells in the lamina propria may be either secretagogues or absorptagogues Numerous chemical mediators from several different sources regulate intestinal electrolyte transport. Some of these agonists are important both in health and in diarrheal disorders,

and at times only quantitative differences separate normal regulatory control from the pathophysiology of diarrhea. These mediators may function in one or more modes: neural, endocrine, paracrine, and perhaps autocrine (see Chapter 3). Most of these agonists (i.e., secretagogues) promote secretion, whereas some others (i.e., absorptagogues) enhance absorption. The enteric nervous system (ENS), discussed in Chapters 14 and 41, is important in the normal regulation of intestinal epithelial electrolyte transport. Activation of enteric secretomotor neurons results in the release of acetylcholine from mucosal neurons and in the induction of active Cl− secretion (Fig. 44-5). Additional neurotransmitters, including VIP, 5HT, and histamine, mediate ENS regulation of epithelial ion transport. An example of regulation mediated by the endocrine system is the release of aldosterone from the adrenal cortex and the subsequent formation of angiotensin II; both dehydration and volume contraction stimulate this reninangiotensin-aldosterone axis (see Chapter 40). Both angiotensin and aldosterone regulate total body Na+ homeostasis by stimulating Na+ absorption, angiotensin in the small intestine and aldosterone in the colon. Their effects on cellular Na+ absorption differ. In the small intestine, angiotensin enhances electroneutral NaCl absorption (Fig. 44-3C), probably by upregulating apical membrane Na-H exchange. In the colon, aldosterone stimulates electrogenic Na+ absorption (Fig. 44-3D). The response of the intestine to angiotensin and aldosterone represents a classic endocrine feedback loop: dehydration results in increased levels of angiotensin and aldosterone, the primary effects of which are to stimulate fluid and Na+ absorption by both the renal tubules (see Chapter 35) and the intestines, thus restoring total body fluid and Na+ content. Regulation of intestinal transport also occurs by paracrine effects. Endocrine cells constitute a small fraction of the total population of mucosal cells in the intestines. These endocrine cells contain several peptides and bioactive amines that are released in response to various stimuli. Relatively little is known about the biology of these cells, but gut distention can induce the release of one or more of these agonists (e.g., 5-HT). The effect of these agonists on adjacent surface epithelial cells represents a paracrine action. Another example of paracrine regulation of intestinal fluid and electrolyte transport is the influence of immune cells in the lamina propria (Fig. 44-1). Table 44-3 presents these immune cells and a partial list of the agonists that they release. The same agonist may be released from more than one cell, and individual cells produce multiple agonists. These agonists may activate epithelial cells directly or may activate other immune cells or enteric neurons. For example, reactive oxygen radicals released by mast cells affect epithelial cell function by acting on enteric neurons and fibroblasts, and they also have direct action on surface and crypt epithelial cells. A single agonist usually has multiple sites of action. For example, the histamine released from mast cells can induce fluid secretion as a result of its interaction with receptors on surface epithelial cells (Fig. 44-7). However, histamine can also activate ENS motor neurons, which can, in turn, alter

Chapter 44 • Intestinal Fluid and Electrolyte Movement

epithelial cell ion transport, as well as intestinal smooth muscle tone and blood flow. As a consequence, the effects of histamine on intestinal ion transport are multiple and amplified. Secretagogues can be classified by their type and by the intracellular second-messenger system they stimulate Several agonists induce the accumulation of fluid and electrolytes in the intestinal lumen (i.e., net secretion). These secretagogues are a diverse, heterogeneous group of compounds, but they can be effectively classified in two different ways: by the type of secretagogue and by the intracellular second messenger that these agonists activate. Grouped according to type, the secretagogues fall into four categories: (1) bacterial exotoxins (i.e., enterotoxins), (2) hormones and neurotransmitters, (3) products of cells of the immune system, and (4) laxatives. Table 44-2 provides

Table 44-3 Products of Lamina Propria Cells That Affect Intestinal Ion Transport Cell

Product

Macrophages

Prostaglandins O2 radicals

Mast cells

Histamine

Neutrophils

Eicosanoids Platelet-activating factor

Fibroblasts

Eicosanoids Bradykinin

Exterior milieu

a partial list of these secretagogues. A bacterial exotoxin is a peptide that is produced and excreted by bacteria that can produce effects independently of the bacteria. An enterotoxin is an exotoxin that induces changes in intestinal fluid and electrolyte movement. For example, E. coli produces two distinct enterotoxins (the so-called heat-labile and heatstable toxins) that induce fluid and electrolyte secretion through two distinct receptors and second-messenger systems. We can also classify secretagogues according to the signal transduction system that they activate after binding to a specific membrane receptor. As summarized in Table 44-2, the second messengers of these signal transduction systems include cAMP, cGMP, and Ca2+. For example, the heatlabile toxin of E. coli binds to apical membrane receptors, becomes internalized, and then activates basolateral adenylyl cyclase. The resulting increase in [cAMP]i activates protein kinase A. VIP also acts by this route (Fig. 44-8). The heatstable toxin of E. coli binds to and activates an apical receptor guanylyl cyclase, similar to the atrial natriuretic peptide (ANP) receptor (see Chapter 3). The newly produced cGMP activates protein kinase G and may also activate protein kinase A. The natural agonist for this pathway is guanylin, a 15–amino acid peptide secreted by mucosal cells of the small and large intestine. Still other secretory agonists (e.g., 5-HT) produce their effects by increasing [Ca2+]i and thus activating protein kinase C or Ca2+-calmodulin–dependent protein kinases. One way that secretagogues can increase [Ca2+]i is by stimulating phospholipase C, which leads to the production of inositol 1,4,5-triphosphate (IP3) and the release of Ca2+ from intracellular stores (see Chapter 3). Secretagogues can also increase [Ca2+]i by activating protein kinases, which may stimulate basolateral Ca2+ channels. Although the secretagogues listed in Table 44-2 stimulate fluid and electrolyte secretion through one of three distinct second messengers (i.e., cAMP, cGMP, and Ca2+), the end

Interstitial space Antigen

Epithelial cell

Antibody Receptor

Cl–

PGE2 cAMP

Myofibroblast IL-1

Histamine

EP receptor ACh Cl–

Enteric neuron

Ca2+

M3 receptor

Intestinal smooth muscle

Mast cell in lamina propria

Figure 44-7 Mast cell activation. Activation of mast cells in the lamina propria triggers the release of histamine, which directly affects epithelial cells, or which stimulates an enteric neuron and thus has an indirect effect. The neuron modulates the epithelium (secretion), intestinal smooth muscle (motility), or vascular smooth muscle (blood flow). ACh, acetylcholine.

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Section VII • The Gastrointestinal System

PKA catalytic subunits phosphorylate apical membrane proteins.

Epithelial cell

External milieu

Interstitial space PKA regulatory subunit P

Active PKG phosphorylates apical membrane proteins. Guanylyl cyclase receptor

cAMP

cAMP

cAMP

cAMP

PKA (active)

Secretagogue (e.g., VIP) Gs cAMP

PKA catalytic subunit

PKA

AC

P Active PKG type II

PKG II cGMP

cGMP

Heat-stable toxin (STa)

PKG II

CaM kinase P

Secretagogue (e.g., serotonin)

Calmodulin

Calcium calmodulin

Gq

PLC

Active CaM kinase phosphorylates apical membrane proteins.

Active CaM kinase

Ca2+

PIP2

Ca2+

ER IP3 DAG PKC

PKC

Active PKC

Figure 44-8 Action of secretagogues. Secretagogues (agents that stimulate the net secretion of fluid and electrolytes into the intestinal lumen) act by any of three mechanisms. Some (e.g., VIP, heat-labile toxin) activate adenylyl cyclase, which, in turn, generates cAMP and thus stimulates protein kinase A (PKA). Others (e.g., a heat-stable toxin, also known as STa) bind to the guanylin receptor, which is a receptor guanylyl cyclase that generates cGMP and results in the stimulation of protein kinase G (PKG). Others (e.g., serotonin) stimulate the phospholipase C (PLC) pathway, which leads to the generation of IP3 and diacylglycerol (DAG). The DAG activates protein kinase C (PKC). The increased [Ca2+]i stimulates PKC and Ca2+-calmodulin–dependent protein kinase (CaM kinase). These activated kinases stimulate net secretion by phosphorylating apical membrane transporters or other proteins. AC, adenylyl cyclase; Gq and Gs, a-subunit types of G proteins; PIP2, phosphatidylinositol 4,5-biphosphate.

effects are quite similar. As summarized in Table 44-4, all three second-messenger systems stimulate active Cl− secretion (Fig. 44-5) and inhibit electroneutral NaCl absorption (Fig. 44-3C). The abilities of cAMP and Ca2+ to stimulate Cl− secretion and to inhibit electroneutral NaCl absorption are almost identical. In contrast, cGMP’s ability to stimulate Cl− secretion is somewhat less, although its effects on electroneutral NaCl absorption are quantitatively similar to those of cAMP and Ca2+. Both stimulation of Cl− secretion and inhibition of electroneutral NaCl absorption have the

Table 44-4 Transport

End Effects of Second Messengers on Intestinal

Second Messenger

Increased Anion Secretion

Inhibited NaCl Absorption

cAMP

+++

+++

cGMP

+

+++

Ca2+

+++

+++

Chapter 44 • Intestinal Fluid and Electrolyte Movement

same overall effect: net secretion of fluid and electrolytes. It is uncertain whether the observed decrease in electroneutral NaCl absorption is the result of inhibiting Na-H exchange, Cl-HCO3 exchange, or both, inasmuch as electroneutral NaCl absorption represents the coupling of separate Na-H and Cl-HCO3 exchange processes through pHi (Fig. 44-3C). Mineralocorticoids, glucocorticoids, and somatostatin are absorptagogues Although multiple secretagogues exist, relatively few agonists can be found that enhance fluid and electrolyte absorption. The cellular effects of these absorptagogues are less well understood than those of the secretagogues. Those few absorptagogues that have been identified increase intestinal fluid and electrolyte absorption by either a paracrine or an endocrine mechanism.

Secretory Diarrhea

D

iarrhea is a common medical problem and can be defined as a symptom (i.e., an increase in the number of bowel movements or a decrease in stool consistency) or as a sign (i.e., an increase in stool volume of more than 0.2 L/24 hours). Diarrhea has many causes and can be classified in various ways. One classification divides diarrheas by the causative factor. The causative factor can be a dietary nutrient that is not absorbed, in which case the result is osmotic diarrhea. An example of osmotic diarrhea is primary lactase deficiency. Alternatively, the causative factor may not be a dietary nutrient, but rather endogenous secretions of fluid and electrolytes from the intestine, in which case the result is secretory diarrhea. The leading causes of secretory diarrhea include infections with E. coli (the major cause of traveler’s diarrhea) and cholera (a substantial cause of morbidity and mortality in developing countries). In these infectious diarrheas, an enterotoxin produced by one of many bacterial organisms raises [cAMP]i, [cGMP]i, or [Ca2+]i (see Table 44-2). A second group of secretory diarrheas includes those produced by different, although relatively uncommon, hormone-producing tumors. Examples include tumors that produce VIP (the Verner-Morrison syndrome), glucagon (glucagonomas), and serotonin (the carcinoid syndrome). These secretagogues act by raising either [cAMP]i or [Ca2+]i (Table 44-2). When a tumor produces these secretagogues in abundance, the resulting diarrhea can be copious and explosive. As we have seen, the secretory diarrheas have in common their ability to increase [cAMP]i, [cGMP]i, or [Ca2+]i. Table 44-4 summarizes the mechanisms by which these second messengers produce the secretory diarrhea. Because the second messengers do not alter the function of nutrient-coupled Na+ absorption, administration of an ORS containing glucose and Na+ is effective in the treatment of enterotoxin-mediated diarrhea (see the earlier box titled Oral Rehydration Solution).

Corticosteroids are the primary hormones that enhance intestinal fluid and electrolyte absorption. Mineralocorticoids (e.g., aldosterone) stimulate Na+ absorption and K+ secretion in the distal end of the colon; they do not affect ion transport in the small intestine. Their cellular actions are outlined in Chapter 50. Aldosterone induces both apical membrane Na+ channels (a process that is inhibited by the diuretic amiloride) and basolateral Na-K pumps; this action results in substantial enhancement of colonic electrogenic Na+ absorption. Although the effects of glucocorticoids on ion transport have most often been considered a result of crossover binding to the mineralocorticoid receptor (see Chapter 35), it is now evident that glucocorticoids also have potent actions on ion transport through their own receptor and that these changes in ion transport are distinct from those of the mineralocorticoids. Glucocorticoids stimulate electroneutral NaCl absorption (Fig. 44-3C) throughout the large and small intestine without any effect on either K+ secretion or electrogenic Na+ absorption. Both corticosteroids act, at least in part, by genomic mechanisms (see Chapter 4). Other agonists appear to stimulate fluid and electrolyte absorption by stimulating electroneutral NaCl absorption and inhibiting electrogenic HCO−3 secretion; both these changes enhance fluid absorption. Among these absorptagogues are somatostatin, which is released from endocrine cells in the intestinal mucosa (see Chapter 42), and the enkephalins and norepinephrine, which are neurotransmitters of enteric neurons. The limited information available suggests that these agonists affect ion transport by decreasing [Ca2+]i, probably by blocking Ca2+ channels. Thus, it appears that fluctuations in [Ca2+]i regulate Na+ and Cl− transport in both the absorptive (low [Ca2+]i) and secretory (high [Ca2+]i) directions. Therefore, Ca2+ is clearly a critical modulator of intestinal ion transport. REFERENCES Books and Reviews Binder HJ, Sandle GI: Electrolyte transport in the mammalian colon. In Johnson LR (ed): Physiology of the Gastrointestinal Tract, 3rd ed, pp 2133-2172. New York: Raven Press, 1994. Greger R, Bleich M, Leipziger J, et al: Regulation of ion transport in colonic crypts. News Physiol Sci 1997; 12:62-66. Montrose MH, Keely SJ, Barrett KE: Electrolyte secretion and absorption: Small intestine and colon. In Yamada T (ed): Textbook of Gastroenterology, vol 1, 4th ed, pp 308-340. Philadelphia: Lippincott Williams & Wilkins, 2003. Palacin M, Estevez R, Bertran J, Zorzano A: Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 1998; 78:969-1054. Rao MC: Oral rehydration therapy: New explanations for an old remedy. Annu Rev Physiol 2004; 66: 385-417. Zachos NC, Tse M, Donowitz M: Molecular physiology of intestinal Na/H exchange. Annu Rev Physiol 2005; 67: 411-443. Journal Articles Canessa CM, Horisberger J-D, Rossier BC: Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 1993; 361:467-470. Knickelbein RG, Aronson PS, Schron CM, et al: Sodium and chloride transport across rabbit ileal brush border. II. Evidence for

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Cl-HCO3 exchange and mechanism of coupling. Am J Physiol 1985; 249:G236-G245. Moseley RH, Hoglund P, Wu GD, et al: Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea. Am J Physiol 1999; 276:G185-G192.

Schulz S, Green CK, Yuen PST, Garbers DL: Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell 1990; 63:941-948. Singh SK, Binder HJ, Boron WF, Geibel JP: Fluid absorption in isolated perfused colonic crypts. J Clin Invest 1995; 96: 2373-2379.

CHAPTER

45

N UTRI ENT DIGESTION AN D ABSORPTION Henry J. Binder and Adrian Reuben

In general, the digestive-absorptive processes for most of the constituents of our diet are highly efficient. For example, normal adult intestine absorbs ~95% of dietary lipid. However, we ingest most of the constituents of dietary food in a form that the intestine cannot readily absorb. Multiple digestive processes convert dietary food to a form that can be absorbed, primarily in the small intestine, but also, to a much smaller extent, in the colon. The digestive process—the enzymatic conversion of complex dietary substances to a form that can be absorbed— is initiated by the sight, smell, and taste of food. Although some digestion (that of carbohydrates) begins in the mouth and additional digestion may occur within the lumen of the stomach, most digestive processes occur in the small intestine. Digestion within the small intestine occurs either in the lumen, mediated by pancreatic enzymes, or at the small intestine brush border membrane (membrane digestion), mediated by brush border enzymes. Several different patterns of luminal, brush border, and cytosolic digestion exist (Fig. 45-1). Some of the dietary carbohydrate and protein that escape digestion and absorption in the small intestine are altered in the large intestine by bacterial enzymes to short-chain fatty acids that are absorbed by the colon. The digestive processes for carbohydrates, proteins, and lipids result in the conversion of dietary nutrients to a chemical form for which intestinal absorptive processes exist. As a consequence, the digestive-absorptive processes for the several dietary constituents are closely integrated and regulated biological events that ensure survival. Multiple diseases can alter these digestive-absorptive processes and can thereby impair nutrient assimilation (i.e., the overall process of digestion and absorption). Because of the substantial segmental distribution of nutrient absorption along the gastrointestinal tract (Fig. 45-2), the clinical manifestations of disease (Table 45-1) often reflect these segmental differences.

CARBOHYDRATE DIGESTION Carbohydrates, which provide ~45% of the total energy needs of Western diets, require hydrolysis to monosaccharides before absorption We classify dietary carbohydrates into two major groups: (1) the monosaccharides (monomers) and (2) the oligosac-

charides (short polymers) and polysaccharides (long polymers). The small intestine can directly absorb the monomers but not the polymers. Some polymers are digestible, that is, the body can digest them to form the monomers that the small intestine can absorb. Other polymers are nondigestible, or “fiber.” The composition of dietary carbohydrate is quite varied and is a function of culture. The diet of so-called developed countries contains considerable amounts of “refined” sugar and, compared with most developing countries, less fiber. Such differences in the fiber content of the Western diet may account for several diseases that are more prevalent in these societies (e.g., colon carcinoma and atherosclerosis). As a consequence, the consumption of fiber by the health-conscious public in the United States has increased during the past 2 decades. In general, increased amounts of fiber in the diet are associated with increased stool weight and frequency. Approximately 45% to 60% of dietary carbohydrate is in the form of starch, which is a polysaccharide. Starch is a storage form for carbohydrates that is primarily found in plants, and it consists of both amylose and amylopectin. In contrast, the storage form of carbohydrates in animal tissues is glycogen, which is consumed in much smaller amounts. Amylose is a straight-chain glucose polymer that typically contains multiple glucose residues, connected by α-1,4 linkages. In contrast, amylopectin is a massive branched glucose polymer that may contain 1 million glucose residues. In addition to the α-1,4 linkages, amylopectin has frequent α1,6 linkages at the branch points. Amylopectins are usually present in much greater quantities (perhaps 4-fold) than amylose. Glycogen—the “animal starch”—also has α-1,4 and α-1,6 linkages like amylopectin. However, glycogen is more highly branched (i.e., α-1,6 linkages). Most dietary oligosaccharides are the disaccharides sucrose and lactose, which represent 30% to 40% of dietary carbohydrates. Sucrose is table sugar, derived from sugar cane and sugar beets, whereas lactose is the sugar found in milk. The remaining carbohydrates are the monosaccharides fructose and glucose, which make up 5% to 10% of total carbohydrate intake. There is no evidence of any intestinal absorption of either starches or disaccharides. Because the small intestine can absorb only monosaccharides, all dietary carbohydrate must be digested to monosaccharides before absorption. The colon cannot absorb monosaccharides. Dietary fiber consists of both soluble and insoluble forms and includes lignins, pectins, and cellulose. These fibers are primarily present in fruits, vegetables, and cereals. Cellulose

949

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Section VII • The Gastrointestinal System

Lumen DIGESTION

Epithelium

Interstitial space

EXAMPLE

None Glucose Luminal hydrolysis of polymer to monomers

Protein

Glucose

Amino acids (AA)

AA

Glucose

Glucose

Fructose

Fructose

Peptide

AA

Sucrose Brushborder hydrolysis of oligomer to monomer

Intracellular hydrolysis

Glycerol

Luminal hydrolysis followed by intracellular resynthesis

Triacylglycerol

Triacylglycerol Fatty acids

Figure 45-1 General mechanisms of digestion and absorption. Digestion-absorption can follow any of five patterns. First, the substance (e.g., glucose) may not require digestion; the intestinal cells may absorb the nutrient as ingested. Second, a polymer (e.g., protein) may be digested in the lumen to its constituent monomers (e.g., amino acids) by pancreatic enzymes before absorption. Third, an oligomer (e.g., sucrose) is digested into its constituent monomers (e.g., monosaccharides) by brush border enzymes before absorption. Fourth, an oligomer (e.g., oligopeptide) may be directly absorbed by the cell and then broken down into monomers (e.g., amino acids) inside the cell. Finally, a substance (e.g., TAG) may be broken down into its constituent components before absorption; the cell may then resynthesize the original molecule.

is a glucose polymer connected by β-1,4 linkages, which cannot be digested by mammalian enzymes. However, enzymes from colonic bacteria may degrade fiber. This process is carried out with varying efficiency; pectins, gum, and mucilages are metabolized to a much greater degree than either cellulose or hemicellulose. In contrast, lignins, which are aromatic polymers and not carbohydrates, are not altered by microbial enzymes in the colonic lumen and are excreted unaltered in stool. As we discuss later, the digestive process for dietary carbohydrates has two steps: (1) intraluminal hydrolysis of starch to oligosaccharides by salivary and pancreatic amylases (Fig. 45-3) and (2) so-called membrane digestion of oligosaccharides to monosaccharides by brush border disaccharidases. The resulting carbohydrates are absorbed by transport processes that are specific for certain monosaccharides. These transport pathways are located in the apical membrane of the small intestine villous epithelial cells.

Luminal digestion begins with the action of salivary amylase and finishes with pancreatic amylase Both salivary and pancreatic acinar cells (see Chapter 43) synthesize and secrete α-amylases. Salivary and pancreatic amylases, unlike most of the pancreatic proteases that we discuss later, are secreted not in an inactive proenzyme form, but rather in an active form. Salivary and pancreatic α-amylases have similar enzymatic function, and their amino acid sequences are 94% identical. Salivary amylase in the mouth initiates starch digestion; in healthy adults, this step is of relatively limited importance. Salivary amylase is inactivated by gastric acid, but it can be partially protected by complexing with oligosaccharides. Pancreatic α-amylase completes starch digestion in the lumen of the small intestine. Although amylase binds to the apical membrane of enterocytes, this localization does not provide any kinetic advantage for starch hydrolysis. Chole-

Chapter 45 • Nutrient Digestion and Absorption

A

CARBOHYDRATES, PROTEINS AND LIPIDS

B

CALCIUM, IRON AND FOLATE Calcium Iron

Carbohydrates, proteins, lipids

Folate

Duodenum Jejunum Ileum

Duodenum Calcium Calcium

High absorption Moderate absorption C

Low absorption

D COBALAMIN

BILE ACIDS

Very low absorption

Duodenum Jejunum

Bile acids

Ileum

Cobalamin

Ileum

Figure 45-2 Sites of nutrient absorption. A, The entire small intestine absorbs carbohydrates, proteins, and lipids. However, the absorption is greatest in the duodenum, somewhat less in the jejunum, and much less in the ileum. The thickness of the arrows in the inset indicates the relative magnitude of total absorption at the indicated site in vivo. The maximal absorptive capacity of a specific segment under optimized experimental conditions (e.g., substrate concentrations) may be greater. B, Some substances are actively absorbed only in the duodenum. C, Bile acids are absorbed along the entire small intestine, but active absorption occurs only in the ileum. D, The vitamin cobalamin is absorbed only in the ileum.

Table 45-1 Major Gastrointestinal Diseases and Nutritional Deficiencies Disease

Organ Site of Predominant Disease

Defects in Nutrient Digestion/Absorption

Celiac sprue

Duodenum and jejunum

Fat absorption, lactose hydrolysis

Chronic pancreatitis

Exocrine pancreas

Fat digestion

Surgical resection of ileum; Crohn disease of ileum

Ileum

Cobalamin and bile acid absorption

Primary lactase deficiency

Small intestine

Lactose hydrolysis

cystokinin (CCK) stimulates the secretion of pancreatic αamylase by pancreatic acinar cells (see Chapter 43). α-Amylase is an endoenzyme that hydrolyzes internal α-1,4 linkages (Fig. 45-3A). α-Amylase does not cleave terminal α-1,4 linkages, α-1,6 linkages (i.e., branch points), or

α-1,4 linkages that are immediately adjacent to α-1,6 linkages. As a result, starch hydrolysis products are maltose, maltotriose, and α-limit dextrins. Because α-amylase has no activity against terminal α-1,4 linkages, glucose is not a product of starch digestion. The intestine cannot absorb these products of amylase digestion of starch, and thus further digestion is required to produce substrates (i.e., monosaccharides) that the small intestine can absorb by specific transport mechanisms. Membrane digestion involves hydrolysis of oligosaccharides to monosaccharides by brush border disaccharidases The human small intestine has three brush border oligosaccharidases: lactase, glucoamylase (most often called maltase), and sucrase-isomaltase. These enzymes are all integral membrane proteins whose catalytic domains face the intestinal lumen (Fig. 45-3B). Sucrase-isomaltase is actually two enzymes—sucrase and isomaltase (also known as α-dextrinase or debranching enzyme)—bound together. Thus, four oligosaccharidases are present at the brush border. Lactase has only one substrate; it breaks lactose into glucose and galactose. The other three enzymes have more complicated

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Lumen

A

Epithelium

DIGESTION OF STARCH IN LUMEN

Interstitial space

B DIGESTION OF OLIGOSACCHARIDES AT BRUSH BORDER Lumen

α-Amylase

Lactase Amylose

Amylopectin

Cytoplasm Lactase splits lactose. Both monomers are transported via SGLT1.

Lactose Terminal α-1,4 link Cannot be cut by amylase

Adjacent α-1,6 linkage Adjacent Terminal α-1,4 link (branching) α-1,4 link α-1,4 link Cannot be cut by amylase

SGLT1

+ Glucoamylase (also known as maltase) removes glucose monomers for transport.

2 Na+ +

Glucoamylase

Maltotriose +

Maltotriose or maltose

α-Limit dextrins

Maltose

C

SGLT1

+

Sucrase-isomaltase is actually two enzymes. The sucrase moiety splits sucrose, as well as maltose and maltotriose.

+

2 Na

GLUT5

ABSORPTION OF MONOSACCHARIDES Lumen

Epithelium

SGLT1 Galactose Glucose +

2 Na

Interstitial space

Glucose 3 Na+

+

Sucrase-isomaltase Sucrose

Sucrase Isomaltase

Maltose

Sucrase Isomaltase

GLUT2 Maltotriose +

2K

Fructose GLUT5

Fructose

GLUT2

SGLT1 α-limit dextrins Maltose Maltotriose

+ 2 Na+

The isomaltase moiety splits α-limit dextrins, as well as maltose and maltotriose.

Figure 45-3 Digestion of carbohydrates to monosaccharides. A, Salivary and pancreatic α-amylase are endoenzymes. They can digest the linear internal α-1,4 linkages between glucose residues, but they cannot break terminal α-1,4 linkages (i.e., between the last two sugars in the chain). They also cannot split the α-1,6 linkages at the branch points of amylopectin or the adjacent α-1,4 linkages. As a result, the products of αamylase action are linear glucose oligomers, maltotriose (a linear glucose trimer), maltose (a linear glucose dimer), and α-limit dextrins (which contain an α-1,6 branching linkage). B, The brush border oligosaccharidases are intrinsic membrane proteins with their catalytic domains facing the lumen. The sucrase-isomaltase is actually two enzymes, and, therefore, four oligosaccharidases split the oligosaccharides produced by αamylase into monosaccharides. C, SGLT1 is the Na+-coupled transporter that mediates the uptake of glucose or galactose from the lumen of the small intestine into the enterocyte. GLUT5 mediates the facilitated diffusion of fructose into the enterocyte. Once the monosaccharides are inside the enterocyte, GLUT2 mediates their efflux across the basolateral membrane into the interstitial space.

Chapter 45 • Nutrient Digestion and Absorption

substrate spectra. All cleave the terminal α-1,4 linkages of maltose, maltotriose, and α-limit dextrins. In addition, each of these three enzymes has at least one other activity. Maltase can also degrade the α-1,4 linkages in straight-chain oligosaccharides up to nine monomers in length. However, maltase cannot split either sucrose or lactose. The sucrase moiety of sucrase-isomaltase is required to split sucrose into glucose and fructose. The isomaltase moiety of sucraseisomaltase is critical; it is the only enzyme that can split the branching α-1,6 linkages of α-limit dextrins. The action of the four oligosaccharidases generates several monosaccharides. Maltose is hydrolyzed to two glucose residues, whereas the hydrolysis products of sucrose are glucose and fructose. The hydrolysis of lactose by lactase yields glucose and galactose. The activities of the hydrolysis reactions of sucrase-isomaltase and maltase are considerably greater than the rates at which the various transporters can absorb the resulting monosaccharides. Thus, uptake, not hydrolysis, is the rate-limiting step. In contrast, lactase activity is considerably less than that of the other oligosaccharidases and is rate limiting for overall lactose digestion-absorption. The oligosaccharidases have a varying spatial distribution throughout the small intestine. In general, peak oligosac-

charidase distribution and activity occur in the proximal jejunum (i.e., at the ligament of Treitz). Considerably less activity is noted in the duodenum and distal ileum, and none is reported in the large intestine. The distribution of oligosaccharidase activity parallels that of active glucose transport. These oligosaccharidases are affected by developmental and dietary factors in different ways. In many nonwhite ethnic groups, as well as in almost all other mammals, lactase activity markedly decreases after weaning in the postnatal period. The regulation of this decreased lactase activity is genetically determined. The other oligosaccharidases do not decrease in the postnatal period. In addition, long-term feeding of sucrose upregulates sucrase activity. In contrast, sucrase activity is greatly reduced much more by fasting than is lactase activity. In general, lactase activity is both more susceptible to enterocyte injury (e.g., following viral enteritis) and is slower to recover from damage than is other oligosaccharidase activity. Thus, reduced lactase activity (as a consequence of both genetic regulation and environmental effects) has substantial clinical significance in that lactose ingestion may result in a range of symptoms in affected individuals (Fig. 45-4).

A PRESENCE OF LACTASE ACTIVITY Plasma glucose rises after glucose or lactose ingestion…

140

Plasma glucose

…and subsequent H2 excreted by lungs is low.

120

Breath H2

Glucose ingested 100

Lactose ingested

Lactose

80 0

Glucose 0

1

2

3

0

1

Hours

2

3

Hours

B LACTASE DEFICIENCY …and colonic bacteria metabolize the lactose that enters the colon, resulting in higher H2 excretion.

Lactase-deficient individuals hydrolyze less lactose to glucose… 140

Plasma glucose

Glucose ingested

120

Lactose Breath H2

100 Glucose Lactose ingested

80 0 0

1

2 Hours

3

0

1

2 Hours

Figure 45-4 Effects of lactase deficiency on levels of glucose in the plasma and H2 in the breath. A, In an individual with normal lactase activity, blood glucose levels rise after the ingestion of either glucose or lactose. Thus, the small intestine can split the lactose into glucose and galactose and can absorb the two monosaccharides. At the same time, H2 in the breath is low. B, In an adult with low lactase activity, the rise in blood levels is less pronounced after ingesting lactose. Because the rise is normal after ingesting glucose, we can conclude that the difference is the result of lactase activity. Conversely, the individual with lactase deficiency excretes large amounts of H2 into the breath. This H2 is the product of lactose catabolism by colonic bacteria.

3

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Section VII • The Gastrointestinal System

Lactase Deficiency

A

STRUCTURE OF SGLT1

Extracellular space

P

rimary lactase deficiency is extremely common in nonwhites, and it also occurs in some whites. Lactase activity decreases after weaning; the time course of its reduction is determined by hereditary factors. Ingestion of lactose in the form of milk and milk products by individuals with decreased amounts of small intestinal lactase activity may be associated with a range of gastrointestinal symptoms, including diarrhea, cramps, and flatus, or with no discernible symptoms. Several factors determine whether individuals with lactase deficiency experience symptoms after ingestion of lactose, including rate of gastric emptying, transit time through the small intestine, and, most importantly, the ability of colonic bacteria to metabolize lactose to short-chain fatty acids, CO2, and H2. Figure 45-4A shows the rise of plasma [glucose] following the ingestion of either lactose or glucose in adults with normal lactase levels. This figure also shows that the [H2] in the breath rises only slightly following the ingestion of either lactose or glucose in these individuals with normal lactase levels. Figure 45-4B shows that in individuals with primary lactase deficiency, the ingestion of lactose leads to a much smaller rise in plasma [glucose], although the ingestion of glucose itself leads to a normal rise in plasma [glucose]. Thus, no defect in glucose absorption per se is present, but simply a markedly reduced capacity to hydrolyze lactose to glucose and galactose. In lactase-deficient individuals, breath H2 is increased after lactose ingestion, because nonabsorbed lactose is metabolized by colonic bacteria to H2, which is absorbed into the blood and is subsequently excreted by the lungs. In contrast, the rise in breath H2 is normal after the ingestion of glucose in these individuals. Treatment for symptomatic individuals with primary lactase deficiency is reduction or elimination of milk and milk products or the use of milk products treated with a commercial lactase preparation. No other defects in intestinal function or structure are associated with primary lactase deficiency.

Oligosaccharide side chain

N

C

1

2

3

4

5

6

7

8

9

10 11 12 13 14

Cytosol B

STRUCTURAL REQUIREMENTS OF SUGAR 6

H

5 4

HO

CH2OH

OH 3

H

O H 2

H 1

OH

OH

Pyranose ring in configuration.

D

Figure 45-5 SGLT1. A, The SGLT family of proteins is believed to have 12 membrane-spanning segments. The deduced amino acid sequence has an open reading frame of 662 amino acids, predicting a molecular mass of 73 kDa. SGLT1 has a Na+-sugar stoichiometry of 2 : 1. B, SGLT1 transports only hexoses in a d-configuration and with a pyranose ring. This figure shows d-glucose; d-galactose is identical, except the H and OH on C-4 are inverted.

CARBOHYDRATE ABSORPTION The three monosaccharide products of carbohydrate digestion—glucose, galactose, and fructose—are absorbed by the small intestine in a two-step process involving their uptake across the apical membrane into the epithelial cell and their coordinated exit across the basolateral membrane (Fig. 45-3C). The Na/glucose transporter 1 (SGLT1) is the membrane protein responsible for glucose and galactose uptake at the apical membrane. The exit of all three monosaccharides across the basolateral membrane uses a facilitated sugar transporter (GLUT2). Because SGLT1 cannot carry fructose, the apical step of fructose absorption occurs by the facilitated diffusion of fructose through GLUT5. Thus, although two different apical membrane transport mechanisms exist for glucose and fructose uptake, a single transporter (GLUT2) is responsible for the movement of both monosaccharides across the basolateral membrane.

SGLT1 is responsible for the Na+-coupled uptake of glucose and galactose across the apical membrane The uptake of glucose across the apical membrane through SGLT1 (Fig. 45-5A) represents active transport, because the glucose influx occurs against the glucose concentration gradient (see Chapter 5). Glucose uptake across the apical membrane is energized by the electrochemical Na+ gradient, which, in turn, is maintained by the extrusion of Na+ across the basolateral membrane by the Na-K pump. This type of Na+-driven glucose transport is an example of secondary active transport (see Chapter 5). Inhibition of the Na-K pump reduces active glucose absorption by decreasing the apical membrane Na+ gradient and thus decreasing the driving force for glucose entry. The affinity of SGLT1 for glucose is markedly reduced in the absence of Na+. The varied affinity of SGLT1 for

Chapter 45 • Nutrient Digestion and Absorption

Glucose-Galactose Malabsorption

PROTEIN DIGESTION

M

Proteins require hydrolysis to oligopeptides or amino acids before absorption in the small intestine

olecular studies have been performed with jejunal mucosa from patients with so-called glucosegalactose malabsorption (or monosaccharide malabsorption). These individuals have diarrhea when they ingest dietary sugars that are normally absorbed by SGLT1. This diarrhea results from both reduced small intestine Na+ and fluid absorption (as a consequence of the defect in Na+-coupled monosaccharide absorption) and fluid secretion secondary to the osmotic effects of nonabsorbed monosaccharide. Eliminating the monosaccharides glucose and galactose, as well as the disaccharide lactose (i.e., glucose + galactose), from the diet eliminates the diarrhea. The monosaccharide fructose, which crosses the apical membrane through GLUT5, does not induce diarrhea. Early studies identified the abnormality in this hereditary disorder as a defect at the apical membrane that is presumably related to defective or absent SGLT1. Molecular studies of SGLT1 have revealed multiple mutations that result in single amino acid substitutions in SGLT1, each of which prevents the transport of glucose by SGLT1 in affected individuals. Patients with glucose-galactose malabsorption do not have glycosuria (i.e., glucose in the urine), because glucose reabsorption by the proximal tubule normally occurs through both SGLT1 and SGLT2 (see Chapter 36).

different monosaccharides reflects its preference for specific molecular configurations. SGLT1 has two structural requirements for monosaccharides: (1) a hexose in a d-configuration and (2) a hexose that can form a six-membered pyranose ring (Fig. 45-5B). SGLT1 does not absorb l-glucose, which has the wrong stereochemistry, and it does not absorb d-fructose, which forms a fivemembered ring.

With the exception of antigenic amounts of dietary protein that are absorbed intact, proteins must first be digested into their constituent oligopeptides and amino acids before being taken up by the enterocytes. Digestion-absorption occurs through four major pathways. First, several luminal enzymes (i.e., proteases) from the stomach and pancreas may hydrolyze proteins to peptides and then to amino acids, which are then absorbed (Fig. 45-6). Second, luminal enzymes may digest proteins to peptides, but enzymes present at the brush border digest the peptides to amino acids, which are then absorbed. Third, luminal enzymes may digest proteins to peptides, which are themselves taken up as oligopeptides by Epithelium Lumen

Interstitial space One of many brush-border peptidases (AA)4

Gastric and pancreatic peptidases

Tripeptidase

(AA)3

Oligopeptides (AA)n

AA

+

H

AA PepT1

+ Amino acids Proteins

AA

(AA)2

(AA)2

+ AA

The GLUT transporters mediate the facilitated diffusion of fructose at the apical membrane and of all three monosaccharides at the basolateral membrane Early work showed that fructose absorption is independent of Na+ but has characteristics of both a carrier-mediated and a passive process. These observations show that the small intestine has separate transport systems for glucose and fructose. Subsequent studies established that facilitated diffusion is responsible for fructose absorption. Fructose uptake across the apical membrane is mediated by GLUT5 (see Chapter 5), a member of the GLUT family of transport proteins. GLUT5 is present mainly in the jejunum. The efflux of glucose, fructose, and galactose across the basolateral membrane also occurs by facilitated diffusion. The characteristics of the basolateral sugar transporter, identified as GLUT2, are similar to those of other sugar transport systems in erythrocytes, fibroblasts, and adipocytes. GLUT2 has no homology to SGLT1 but is 41% identical to GLUT5, which is responsible for the uptake of fructose from the lumen.

AA

(AA)3

H+

AA

+ AA

One of many AA transporters

Dipeptidase

AA Na+

Figure 45-6 Action of luminal, brush border, and cytosolic peptidases. Pepsin from the stomach and the five pancreatic proteases hydrolyze proteins—both dietary and endogenous—to single amino acids, AA, or to oligopeptides, (AA)n. These reactions occur in the lumen of the stomach or small intestine. Various peptidases at the brush borders of enterocytes then progressively hydrolyze oligopeptides to amino acids. The amino acids are directly taken up by any of several transporters. The enterocyte directly absorbs some of the small oligopeptides through the action of the H+/oligopeptide cotransporter (PepT1). These small peptides are digested to amino acids by peptidases in the cytoplasm of the enterocyte. Several Na+-independent amino acid transporters move amino acids out of the cell across the basolateral membrane.

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Section VII • The Gastrointestinal System

the enterocytes. Further digestion of the oligopeptides by cytosolic enzymes yields intracellular amino acids, which are moved by transporters across the basolateral membrane into the blood. Fourth, luminal enzymes digest dietary proteins to oligopeptides, which are taken up by enterocytes and moved directly into the blood. Overall protein digestionabsorption is very efficient; less than 4% of ingested nitrogen is excreted in the stool. The protein that is digested and absorbed in the small intestine comes from both dietary and endogenous sources. Dietary protein in developed countries amounts to 70 to 100 g/day. This amount is far in excess of minimum daily requirements and represents 10% to 15% of energy intake. In contrast, dietary protein content in developing countries in Africa is often 50 g/day. Deficiency states are rare unless intake is markedly reduced. Proteins are encoded by mRNA and consist of 20 amino acids. Nine of these amino acids are essential (see Chapter 58); that is, they are not synthesized in adequate amounts by the body and thus must be derived from either animal or plant protein sources. In addition, cells synthesize additional amino acids by post-translational modifications: γcarboxyglutamic acid, hydroxylysine, 4-hydroxyproline, and 3-hydroxyproline. Protein digestion is influenced by the amino acid composition of the protein, by the source of protein, and by food processing. Thus, proteins rich in proline and hydroxyproline are digested relatively less completely. Cooking, storage, and dehydration also reduce the completeness of digestion. In general, protein derived from animal sources is digested more completely than plant protein. In addition to dietary sources of protein, significant amounts of endogenous protein are secreted into the gastrointestinal tract, then conserved by protein digestion and absorption. Such endogenous sources represent ~50% of the total protein entering the small intestine and include enzymes, hormones, and immunoglobulins present in salivary, gastric, pancreatic, biliary, and jejunal secretions. A second large source of endogenous protein is desquamated intestinal epithelial cells as well as plasma proteins that the small intestine secretes. Neonates can absorb substantial amounts of intact protein from colostrum (see Chapter 57) through the process of endocytosis. This mechanism is developmentally regulated and in humans remains active only until ~6 months of age.

Table 45-2

In adults, proteins are almost exclusively digested to their constituent amino acids and dipeptides and tripeptides or tetrapeptides before absorption. However, even adults absorb small amounts of intact proteins. These absorbed proteins can be important in inducing immune responses to dietary proteins. Luminal digestion of protein involves both gastric and pancreatic proteases, thus yielding amino acids and oligopeptides Both gastric and pancreatic proteases, unlike the digestive enzymes for carbohydrates and lipids, are secreted as proenzymes that require conversion to their active form for protein hydrolysis to occur. The gastric chief cells secrete pepsinogen. We discuss the pH-dependent activation of pepsinogen in Chapter 42. The hydrolytic activity of pepsin is maximal at a pH of 1.8 to 3.5, and pepsin is irreversibly inactivated at a pH of less than 7. Pepsin is an endopeptidase with primary specificity for peptide linkages of aromatic and larger neutral amino acids. Although pepsin in the stomach partially digests 10% to 15% of dietary protein, pepsin hydrolysis is not absolutely necessary; patients with either total gastrectomies or pernicious anemia (who do not secrete acid and thus whose intragastric pH is always >7) do not have increased fecal nitrogen excretion. Five pancreatic enzymes (Table 45-2) participate in protein digestion and are secreted as inactive proenzymes. Trypsinogen is initially activated by a jejunal brush border enzyme, enterokinase (enteropeptidase), by the cleavage of a hexapeptide, thereby yielding trypsin. Trypsinogen is also autoactivated by trypsin. Trypsin also activates the other pancreatic proteolytic proenzymes. The secretion of proteolytic enzymes as proenzymes, with subsequent luminal activation, prevents pancreatic autodigestion before enzyme secretion into the intestine. Pancreatic proteolytic enzymes are either exopeptidases or endopeptidases and function in an integrated manner. Trypsin, chymotrypsin, and elastase are endopeptidases with affinity for peptide bonds adjacent to specific amino acids, thus resulting in the production of oligopeptides with two to six amino acids. In contrast, the exopeptidases—carboxypeptidase A and carboxypeptidase B—hydrolyze peptide bonds adjacent to the carboxy terminus, thereby resulting in the release of individual amino acids. The coordinated action

Pancreatic Peptidases

Proenzyme

Activating Agent

Active Enzyme

Action

Products

Trypsinogen

Enteropeptidase (i.e., enterokinase from jejunum) and trypsin

Trypsin

Endopeptidase

Oligopeptides (2-6 amino acids)

Chymotrypsinogen

Trypsin

Chymotrypsin

Endopeptidase

Oligopeptides (2-6 amino acids)

Proelastase

Trypsin

Elastase

Endopeptidase

Oligopeptides (2-6 amino acids)

Procarboxypeptidase A

Trypsin

Carboxypeptidase A

Exopeptidase

Single amino acids

Procarboxypeptidase B

Trypsin

Carboxypeptidase B

Exopeptidase

Single amino acids

Chapter 45 • Nutrient Digestion and Absorption

of these pancreatic proteases converts ~70% of luminal amino nitrogen to oligopeptides and ~30% to free amino acids. Brush border peptidases fully digest some oligopeptides to amino acids, whereas cytosolic peptidases digest oligopeptides that directly enter the enterocyte Small peptides present in the small intestinal lumen after digestion by gastric and pancreatic proteases undergo further hydrolysis by peptidases at the brush border (Fig. 45-6). Multiple peptidases are present on both the brush border and in the cytoplasm of villous epithelial cells. This distribution of cell-associated peptidases stands in contrast to that of the oligosaccharidases, which are found only at the brush border. Because each peptidase recognizes only a limited repertoire of peptide bonds, and because the oligopeptides to be digested contain 24 different amino acids, large numbers of peptidases are required to ensure the hydrolysis of peptides. As we discuss later, a transporter on the apical membrane of enterocytes can take up small oligopeptides, primarily dipeptides and tripeptides. Once inside the cell, these oligopeptides may be further digested by cytoplasmic peptidases. The brush border and cytoplasmic peptidases have substantially different characteristics. For example, the brush border peptidases have affinity for relatively larger oligopeptides (three to eight amino acids), whereas the cytoplasmic peptidases primarily hydrolyze dipeptides and tripeptides. Because the brush border and cytoplasmic enzymes often have different biochemical properties (e.g., heat lability and electrophoretic mobility), it is evident that the peptidases in the brush border and cytoplasm are distinct, independently regulated molecules. Like the pancreatic proteases, each of the several brush border peptidases is an endopeptidase, an exopeptidase, or a dipeptidase and has affinity for specific peptide bonds. The exopeptidases are either carboxypeptidases, which release carboxy-terminal amino acids, or aminopeptidases, which hydrolyze the amino acids at the aminoterminal end. Cytoplasmic peptidases are relatively less numerous.

PROTEIN, PEPTIDE, AND AMINO ACID ABSORPTION Absorption of whole protein by apical pinocytosis occurs primarily during the neonatal period During the postnatal period, intestinal epithelial cells absorb protein by endocytosis, a process that provides a mechanism for transfer of passive immunity from mother to child. The uptake of intact protein by the epithelial cell ceases by the sixth month; the cessation of this protein uptake, called closure, is hormonally mediated. For example, administration of corticosteroids during the postnatal period induces closure and reduces the time that the intestine can absorb significant amounts of whole protein.

The adult intestine can absorb finite amounts of intact protein and polypeptides. Uncertainty exists regarding the cellular route by which these substances are absorbed, as well as the relationship of the mechanism of protein uptake in adults to that in neonates. Enterocytes can take up by endocytosis a small amount of intact protein, most of which is degraded in lysosomes (Fig. 45-7). A small amount of intact protein appears in the interstitial space. The uptake of intact proteins also occurs through a second, more specialized route. In the small intestine, immediately overlying Peyer’s patches (follicles of lymphoid tissue in the lamina propria), M cells replace the usual enterocytes on the surface of the gut. M cells have few microvilli and are specialized for protein uptake. They have limited ability for lysosomal protein degradation; rather, they package ingested proteins (i.e., antigens) in clathrin-coated vesicles, which they secrete at their basolateral membranes into the lamina propria. There, immunocompetent cells process the target antigens and transfer them to lymphocytes to initiate an immune response. Although protein uptake in adults may not have nutritional value, such uptake is clearly important in mucosal immunity and probably is involved in one or more disease processes. The apical absorption of dipeptides, tripeptides, and tetrapeptides occurs through an H+-driven cotransporter Virtually all absorbed protein products exit the villous epithelial cell and enter the blood as individual amino acids.

Lumen

Phagocytosis of proteins 3200 ng

Interstitial space

10% Direct pathway Enterocyte 90% Degradative pathway

Phagocytosis of proteins 400 ng

Intact protein 200 ng/(h·cm2)

Direct pathway Degradative pathway M cell

Processed protein 2 3000 ng/(h·cm )

Intact protein 2 200 ng/(h·cm ) Processed protein 2 200 ng/(h·cm ) Underlying lymphocytes

Figure 45-7 Absorption of whole proteins. Both enterocytes and specialized M cells can take up intact proteins. The more abundant enterocytes can endocytose far more total protein than can the M cells. However, the lysosomal proteases in the enterocytes degrade ~90% of this endocytosed protein. The less abundant M cells take up relatively little intact protein, but approximately half of this emerges intact at the basolateral membrane. There, immunocompetent cells process the target antigens and then transfer them to lymphocytes, thus initiating an immune response.

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Section VII • The Gastrointestinal System

Substantial portions of these amino acids are released in the lumen of the small intestine by luminal proteases and brush border peptidases and, as we discuss later, move across the apical membranes of enterocytes through several amino acid transport systems (Fig. 45-6). However, substantial amounts of protein are absorbed from the intestinal lumen as dipeptides, tripeptides, or tetrapeptides and are then hydrolyzed to amino acids by intracellular peptidases. The transporter responsible for the uptake of luminal oligopeptides (Fig. 45-8A) is distinct from the various amino acid transporters. Furthermore, administering an amino acid as a peptide (e.g., the dipeptide glycylglycine) results in a higher blood level of the amino acid than administering an equivalent amount of the same amino acid as a monomer (e.g., glycine; Fig. 45-8B). One possible explanation for this effect is that the oligopeptide cotransporter, which carries multiple amino acids rather than a single amino acid into the cell, may simply be more effective than amino acid transporters in transferring amino acid monomers into the cell. This accelerated peptide absorption has been referred to as a kinetic advantage and raises the question of the usefulness of the enteral administration of crystalline amino acids to patients with impaired intestinal function or catabolic deficiencies. The evidence for a specific transport process for dipeptides, tripeptides, and tetrapeptides comes from direct measurements of oligopeptide transport, molecular identification of the transporter, and studies of the hereditary disorders of amino acid transport, cystinuria, and Hartnup disease. Oligopeptide uptake is an active process driven not by a Na+ gradient, but by a proton gradient. Oligopeptide uptake occurs through an H+/oligopeptide cotransporter known as PepT1 (SLC15A1; see Chapter 5), which is also present in

the renal proximal tubule. PepT1 also appears to be responsible for the intestinal uptake of certain dipeptide-like antibiotics (e.g., oral amino-substituted cephalosporins). As noted earlier, after their uptake, dipeptides, tripeptides, and tetrapeptides are usually hydrolyzed by cytoplasmic peptidases to their constituent amino acids, the forms in which they are transported out of the cell across the basolateral membrane. Because peptides are almost completely hydrolyzed to amino acids intracellularly, few peptides appear in the portal vein. Proline-containing dipeptides, which are relatively resistant to hydrolysis, are the primary peptides present in the circulation. Amino acids enter enterocytes through one or more group-specific apical membrane transporters Multiple amino acid transport systems have been identified and characterized in various nonepithelial cells. The absorption of amino acids across the small intestine requires sequential movement across both the apical and basolateral membranes of the villous epithelial cell. Although the amino acid transport systems have overlapping affinities for various amino acids, the general consensus is that at least seven distinct transport systems are present at the apical membrane (see Table 36-1); we discuss the basolateral amino acid transporters in the next section. Whereas many apical amino acid transporters are probably unique to epithelial cells, some of those at the basolateral membrane are probably the same as in nonepithelial cells. The predominant apical amino acid transport system is system B0 (SLC6A19; see Table 36-1), and it results in Na+dependent uptake of neutral amino acids. As is the case for

B OLIGOPEPTIDE ABSORPTION Epithelium Lumen

“KINETIC ADVANTAGE” OF PEPTIDE ABSORPTION

Interstitial space PepT1 3 Na+

Peptide

+

Peptidases

yc

ine

H+

lgl

Na+

2K

+

Gl

yc y

H

Glycine appearance in blood

yc ine

A

Gl

958

Figure 45-8 Absorption of oligopeptides. A, The H+/oligopeptide cotransporter PepT1 moves dipeptides, tripeptides, and tetrapeptides into the enterocyte, across the apical membrane. Peptidases in the cytoplasm hydrolyze the oligopeptides into their constituent amino acids, which then exit across the basolateral membrane through one of three Na+-independent amino acid transporters. B, If glycine is present in the lumen only as a free amino acid, then the enterocyte absorbs it only through apical amino acid transporters. However, if the same amount of glycine is present in the lumen in the form of the dipeptide glycylglycine, the rate of appearance of glycine in the blood is about twice as high. Thus, PepT1, which moves several amino acid monomers for each turnover of the transporter, is an effective mechanism for absorbing “amino acids.”

Chapter 45 • Nutrient Digestion and Absorption

glucose uptake, uphill movement of neutral amino acids is driven by an inwardly directed Na+ gradient that is maintained by the basolateral Na-K pump. The uptake of amino acids by system B0 is an electrogenic process and represents another example of secondary active transport. It transports amino acids with an l-stereo configuration and an amino group in the α position. System B0+ (SLC6A14) is similar to system B0 but has broader substrate specificity. System b0+ (SLC7A9/SLC3A1 dimer) differs from B0+ mainly in being independent of Na+. Other carrier-mediated transport mechanisms exist for anionic (i.e., acidic), cationic (i.e., basic), β amino acids, and imino acids (see Table 36-1). Because the apical amino acid transporters have overlapping affinities for amino acids, and because of species differences as well as segmental and developmental differences among the transporters, it has been difficult to establish a comprehensive model of apical membrane amino acid transport in the mammalian small intestine. At the basolateral membrane, amino acids exit enterocytes through Na+-independent transporters and enter through Na+-dependent transporters Amino acids appear in the cytosol of intestinal villous cells as the result either of their uptake across the apical membrane or of the hydrolysis of oligopeptides that had entered the apical membrane (Fig. 45-6). The enterocyte subsequently uses ~10% of the absorbed amino acids for intracellular protein synthesis. Movement of amino acids across the basolateral membrane is bidirectional; the movement of any one amino acid can occur through one or more amino acid transporters. At least five amino acid transporters are present in the basolateral membrane (see Table 36-1). Three amino acid transport

processes on the basolateral membrane mediate amino acid exit from the cell into the blood and thus complete the process of protein assimilation. Two other amino acid transporters mediate uptake from the blood for the purposes of cell nutrition. The three Na+-independent amino acid transport systems appear to mediate amino acid movement out of the epithelial cell into blood. One of these, system y+ (SLC7A1), is also present on the apical membrane. The two Na+-dependent processes facilitate their movement into the epithelial cell. Indeed, these two Na+-dependent transporters resemble those that are also present in nonpolar cells. In general, the amino acids incorporated into protein within villous cells are derived more from those that enter across the apical membrane than from those that enter across the basolateral membrane. In contrast, epithelial cells in the intestinal crypt derive almost all their amino acids for protein synthesis from the circulation; crypt cells do not take up amino acids across their apical membrane.

LIPID DIGESTION Natural lipids are organic compounds of biological origin that are sparingly soluble in water Lipids in the diet are derived from animals or plants and are composed of carbon, hydrogen, and a smaller amount of oxygen. Some lipids also contain small but functionally important amounts of nitrogen and phosphorus (Fig. 45-11). Lipids are typified by their preferential solubility in organic solvents, compared with water. A widely used indicator of the lipidic nature of a compound is its octanol-water partition coefficient, which for most lipids is between 104 and 107. The biological fate of lipids depends critically on their chemical structure as well as on their interactions with

Defects in Apical Amino Acid Transport: Hartnup Disease and Cystinuria

H

artnup disease and cystinuria are hereditary disorders of amino acid transport across the apical membrane. These autosomal recessive disorders are associated with both small intestine and renal tubule abnormalities (see Chapter 36 for the box on hyperaminoacidurias) in the absorption of neutral amino acids in the case of Hartnup disease and of cationic (i.e., basic) amino acids and cystine in the case of cystinuria. The clinical signs of Hartnup disease are most evident in children and include the skin changes of pellagra, cerebellar ataxia, and psychiatric abnormalities. In Hartnup disease, the absorption of neutral amino acids by system B0 (SLC6A19) in the small intestine is markedly reduced, whereas that of cationic amino acids is intact (Fig. 45-9). The principal manifestation of cystinuria is the formation of kidney stones. In cystinuria, the absorption of cationic amino acids by system b0+ (SLC7A9/SLC3A1 dimer) is abnormal—as a result of mutations in SLC7A9 or SLC3A1—but absorption of neutral amino acids is normal.

Because neither of these diseases involves the oligopeptide cotransporter, the absorption of oligopeptides containing either neutral or cationic amino acids is normal in both diseases. Only 10% of patients with Hartnup disease have clinical evidence of protein deficiency (i.e., pellagra) commonly associated with defects in protein or amino acid absorption. The lack of evidence of protein deficiency is a consequence of the presence of more than one transport system for different amino acids, as well as a separate transporter for oligopeptides. Thus, oligopeptides containing neutral amino acids are absorbed normally in Hartnup disease, and oligopeptides with cationic amino acids are absorbed normally in cystinuria. These two genetic diseases also emphasize the existence of amino acid transport mechanisms on the basolateral membrane that are distinct and separate from the apical amino acid transporters. Thus, in both Hartnup disease and cystinuria, oligopeptides are transported normally across the apical membrane and are hydrolyzed to amino acids in the cytosol, and the resulting neutral and cationic amino acids are readily transported out of the cell across the basolateral membrane.

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Section VII • The Gastrointestinal System

A

HARTNUP DISEASE

B

CYSTINURIA

L-Phenylalanine

L-Alanine

L-Arginine or cystine

L-Arginine or cystine

Amino acid transporter Oligopeptide cotransporter

L-PhenylalanylL-Leucine L-ArginylL-Leucine

L-PhenylalanylL-Leucine

Oligopeptide cotransporter

L-ArginylL-Leucine

Normal subjects Hartnup disease Amino acid absorption

Substrate

Amino acid transporter

Normal subjects Cystinuric patients Amino acid absorption

L-Phe

L-Arg

L-Ala

L-PhenylalanylL-Leucine

L-Arg

L-ArginylL-Leucine

Figure 45-9 Genetic disorders of apical amino acid transport. A, In Hartnup disease, an autosomal recessive disorder, the apical system B0 (SLC6A19) is defective. As a result, the absorption of neutral amino acids such as l-phenylalanine, is reduced. (However, the absorption of l-cystine (i.e., Cys-S-S-Cys) and cationic (i.e., basic) amino acids (e.g., l-arginine) remains intact.) The enterocyte can absorb l-phenylalanine normally if the amino acid is present in the form of the dipeptide l-phenylalanyl-l-leucine, inasmuch as the oligopeptide cotransporter PepT1 is normal. B, In cystinuria, an autosomal recessive disorder, the apical system b0+ (SLC7A9/SLC3A1 dimer) is defective. As a result, the absorption of l-cystine (i.e., Cys-S-S-Cys) and cationic (i.e., basic) amino acids (e.g., l-arginine) is reduced. However, the absorption of amino acids that use system B0 (e.g., l-Ala) is normal. The enterocyte can absorb l-arginine normally if the amino acid is present in the form of the dipeptide l-arginyl-l-leucine.

Basolateral Amino Acid Transport Defects: Lysinuric Protein Intolerance

L

ysinuric protein intolerance is a rare autosomal recessive disorder of amino acid transport across the basolateral membrane (Fig. 45-10). Evidence indicates impaired cationic amino acid transport and symptoms of malnutrition. It appears that the defect is in system y+L, which is located solely on the basolateral membrane. System y+L has two subtypes, y+LAT1 (SLC7A7/SLC3A2 dimer) and y+LAT2 (SLC7A6/SLC3A2 dimer). Mutations in the SLC7A7 gene (subtype y+LAT1) cause the disease lysinuric protein intolerance. Cationic amino acids are absorbed normally across the apical membrane in these patients. Unlike in Hartnup disease or cystinuria, in which the enterocytes can absorb the amino acid normally if it is presented as an oligopeptide, in lysinuric protein intolerance the enterocytes cannot absorb the amino acid regardless of whether the amino acid is “free” or is part of an oligopeptide. These observations are best explained by hypothesizing that the patients hydrolyze intracellular oligopeptides properly but have a defect in the transport of cationic amino acids across the basolateral membrane. This defect is present not only in the small intestine, but also in hepatocytes and kidney cells, and perhaps in nonepithelial cells as well.

Lumen

H

Epithelium

Interstitial space

+

Lysine-XX Na+

Lysine-XX PepT1

SLC7A7 defective

Lysine SLC3A2

Lysine

Figure 45-10 A genetic disorder of basolateral amino acid transport, lysinuric protein intolerance is an autosomal recessive defect in which the Na+-independent y+L amino acid transporter on the apical and basolateral membranes is defective. However, the absence of apical y+L (SLC7A6/SLC3A2 or SLC7A7/SLC3A2 dimers) does not present a problem because Na+-dependent amino acid transporters can take up lysine, and PepT1 can take up lysine-containing oligopeptides (lysine-XX). However, no other mechanism exists for moving lysine out of the enterocyte across the basolateral membrane.

Chapter 45 • Nutrient Digestion and Absorption

FATTY ACID

B

OH

GLYCEROL

H O

H

H

C

C

C

C H

TRIACYLGLYCEROL

G

H

H

H

H

H

C

C

C

C

C

C

C

C

C

O

O

O

O

O

OH

C

O C

O C

C

O C

CH2

CH2

CH3

CH3

LYSOPHOSPHATIDYLCHOLINE

CH3 H3C

N

+

H3C

O

O

O

C CH2

O

CH2 O

C CH2

CHOLESTEROL

C

O

CH2

CH2

CH2

CH3

CH3

CH3

I

CHOLESTERYL ESTER

J

H

OH O

CHOLIC ACID

HO

CH3

OH

+

N

CH3

CH3

CH3

OH HO

P

O

CH

O C

CH2

CH3

CH CH2

CH2

CH2

CH2

OH O

CH3

CH3 CH3

–

O

CH2

O

CH3

OH O

O

–

O CH

O

H

H

CH2

O

CH2

H

CH2

H

CH2

CH2

P

H

CH3

CH3

CH2

O

sn2-MONOACYLGLYCEROL

H

CH3

PHOSPHATIDYLCHOLINE

E

H

CH2

F

DIACYLGLYCEROL

H

H

OH OH OH

D

H

CH2 H3C

CH2

CH2 O

C

H

CH2

C

O

C

O

A

OH

CH CH3 CH3

CH3 CH3 CH CH2 CH2 CH3

CH3

CH2

CH3 H3C

CH CH3

Figure 45-11 A to J, Chemical formulas of some common lipids. The example in A is stearic acid, a fully saturated fatty acid with 18 carbon atoms. B shows glycerol, a trihydroxy alcohol, with hydroxyl groups in positions sn1-, sn2-, and sn3-. In C, the left sn1- and center sn2- fatty acids are palmitic acid, a fully saturated fatty acid with 16 carbon atoms. The rightmost sn3- fatty acid is palmitoleic acid, which is also a 16-carbon structure, but with a double bond between carbons 9 and 10. In F, the left sn1- fatty acid is palmitic acid (16 carbons, fully saturated), and the right sn2- fatty acid is palmitoleic acid (16 carbons, double bond between carbons 9 and 10). In I, the example is the result of esterifying cholesterol and palmitic acid (16 carbons, fully saturated).

water and other lipids in aqueous body fluids (e.g., intestinal contents and bile). Thus, lipids have been classified according to their physicochemical interactions with water. Lipids may be either nonpolar and completely insoluble in water (e.g., cholesteryl esters and carotene) or polar and amphi-

philic, that is, having both polar (hydrophilic) and nonpolar (hydrophobic) groups. Added in small amounts, polar lipids form stable or unstable monolayers on the surface of water (see Fig. 2-1C), whereas in bulk their physicochemical behavior varies from insolubility (as is the case with triacyl-

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Section VII • The Gastrointestinal System

glycerols [TAGs] and cholesterol) to the formation of various macroaggregates, such as liquid crystals and micelles. Lesssoluble lipids are incorporated into the macroaggregates of the more-polar lipids and are thus stably maintained in aqueous solutions. The term fat is generally used to refer to TAG—formerly called triglyceride—but it is also used loosely to refer to lipids in general. Dietary lipids are predominantly TAGs, but food also contains membrane lipids, vitamins, and chemicals from the environment Typical adult Western diets contain ~140 g of fat (providing ~55% of the energy), which is more than the recommended intake of less than 30% of total dietary calories ( tauroursodeoxycholate > glycocholate). The electrical charge of the side chain is an important determinant of canalicular transport inasmuch as only negatively charged bile salts are effectively excreted. Secretion of bile salts occurs against a significant cell-to-canaliculus concentration gradient, which may range from 1 : 100 to 1 : 1000. Mutations in the BSEP gene can, in children, cause a form of progressive intrahepatic cholestasis that is characterized by extremely low bile acid concentrations in the bile. Organic Anions

Organic anions that are not bile salts move from the cytoplasm of the hepatocyte to the canalicular lumen through MRP2, the ABCC2 member of the ABC protein family (see Table 5-6) (Fig. 46-5D). MRP2 is electrogenic, ATP dependent, and has a broad substrate specificity—particularly for divalent, amphipathic, phase II conjugates with glutathione, glucuronide, glucuronate, and sulfates. Its substrates include bilirubin diglucuronide, sulfated bile acids, glucuronidated bile acids, and several xenobiotics. In general, transported substrates must have a hydrophobic core and at least two negative charges separated by a specific distance. MRP2 is critical for the transport of GSH conjugates across the canalicular membrane into bile. Although MRP2 has a low affinity for GSH, functional studies suggest that other mechanisms for GSH transport exist. Animal models of defective MRP2 exhibit hyperbilirubinemia, which corresponds phenotypically to the DubinJohnson syndrome in humans. Another canalicular efflux pump for sulfated conjugates is the human ABC protein ABCG2, which transports estrone-3-sulfate (see Fig. 55-10) and dehydroepiandrosterone sulfate (see Fig. 54-5)—breakdown products of sex steroids. Other anions, such as HCO−3 and SO2− 4 , are excreted by anion exchangers.

Organic Cations

Biliary excretion of organic cations is poorly understood. With the exception of transport that is mediated by the MDR proteins such as BSEP (discussed earlier), the hepatic MDR proteins belong to the ABC family of transporters (see Table 5-6). MDR1 (ABCB1) is present in the canalicular membrane, where it mediates the excretion of some organic cations into the bile canaliculus (Fig. 46-7). The nomenclature of the MDRs is especially confusing because different and conflicting numbering systems have been used for different species; we use the human numbering system. MDR1 secretes bulky organic cations, including xenobiotics, cytotoxins, anticancer drugs, and other drugs (e.g., colchicine, quinidine, verapamil, cyclosporine). Other organic cations appear to be secreted into the canaliculus by a transport process driven by a pH gradient (Fig. 46-7). The presence of an electroneutral H-organic cation exchanger has been demonstrated at the canalicular membrane. However, the importance of this process is uncertain because major H+ gradients probably do not exist in the bile canaliculus. In some cases, it appears that organic cations passively move across the apical membrane into the canaliculus, where they are sequestered by biliary micelles.

Biliary Lipids Phospholipid is a major component of bile. MDR3 (ABCB4) is a “flippase” that promotes the active translocation of phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane. Bile salts then extract the PC from the outer leaflet so that the PC becomes a component of bile, where it participates in micelle formation. Indeed, in humans with an inherited deficiency of MDR3, progressive liver disease develops, characterized by extremely low concentrations of phospholipids in the bile. Bile is also the main pathway for elimination of cholesterol. A heterodimer composed of the “half ” ABC transporters ABCG5 and ABCG8 is located on the canalicular membrane. This transporter is responsible for the secretion of cholesterol into bile. Although the mechanism is uncertain, the ABCG5/ABCG8 complex may form a channel for cholesterol translocation or alternatively may undergo a conformational change following ATP hydrolysis, thus flipping a cholesterol molecule into the outer membrane leaflet in a configuration favoring release into the canalicular lumen. Mutations in the genes encoding either of the two ABC monomers lead to sitosterolemia, a disorder associated with defective secretion into the bile of dietary sterols, increased intestinal absorption of plant and dietary sterols, hypercholesterolemia, and early-onset atherosclerosis.

Hepatocytes take up proteins across their basolateral membrane both by specific receptor-mediated endocytosis and by nonspecific fluid-phase endocytosis The hepatocyte takes up macromolecules, such as plasma proteins, from the blood plasma through endocytosis, transports these molecules across the cytoplasm, and then secretes them into the bile through exocytosis. Three forms of endocytosis have been identified in the basolateral (sinusoidal) membrane: fluid-phase endocytosis, adsorptive endocytosis, and receptor-mediated endocytosis. Fluid-phase endocytosis involves the uptake of a small amount of extracellular fluid, with its solutes, and is a result of the constitutive process of membrane invagination and internalization (see Chapter 2). The process is nondiscriminatory and inefficient. Adsorptive endocytosis involves nonspecific binding of the protein to the plasma membrane before endocytosis, and it results in more efficient protein uptake. Receptor-mediated endocytosis is quantitatively the most important mechanism for the uptake of macromolecules (see Chapter 2). After endocytosis, the receptor recycles to the plasma membrane, and the ligand may be excreted directly into bile by exocytosis or delivered to lysosomes for degradation. Receptor-mediated endocytosis is involved in the hepatic removal from the blood of proteins such as insulin, polymeric immunoglobulin A (IgA), asialoglycoproteins, and epidermal growth factor.

BILE FORMATION The secretion of canalicular bile is active and isotonic The formation of bile occurs in three discrete steps. First, the hepatocytes actively secrete bile into the bile canaliculi.

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Section VII • The Gastrointestinal System

Second, intrahepatic and extrahepatic bile ducts not only transport this bile but also secrete into it a watery, HCO−3 rich fluid. These first two steps may produce ~900 mL/day of so-called hepatic bile (Table 46-2). Third, between meals, approximately half the hepatic bile—perhaps 450 mL/day— is diverted to the gallbladder, which stores the bile and isosmotically removes salts and water. The result is that the gallbladder concentrates the key remaining solutes in bile fluid—bile salts, bilirubin, cholesterol, and lecithin—by 10to 20-fold. The 500 mL/day of bile that reaches the duodenum through the ampulla of Vater is thus a mixture of relatively “dilute” hepatic bile and “concentrated” gallbladder bile. The first step in bile formation cannot be ultrafiltration because the hydrostatic pressure in the canaliculi is significantly higher than the sinusoidal perfusion pressure. This situation is in marked contrast to glomerular filtration by the kidney (see Chapter 33), which relies predominantly on passive hydrostatic forces for producing the fluid in Bowman’s space. Instead, bile formation is an active process. It is sensitive to changes in temperature and to metabolic inhibitors. Bile formation by hepatocytes requires the active, energy-dependent secretion of inorganic and organic solutes into the canalicular lumen, followed by the passive movement of water. This movement of water through the tight junctions between hepatocytes carries with it other solutes by solvent drag (see Chapter 19). Canalicular bile is an isosmotic fluid; thus, the intercellular junctions allow the passage

Table 46-2

Composition of Bile

Parameter

Hepatic Bile

Gallbladder Bile

pH

7.5

Na+ (mM)

141-165

K+ (mM)

2.7-6.7

14

Ca2+ (mM)

1.2-3.2

15

Cl− (mM)

77-117

31

HCO−3 (mM)

12-55

19

Total phosphorus (g/L)

0.15

Bile acids (g/L)

3-45

32

Total fatty acids (g/L)

2.7

24

Bilirubin (g/L)

1-2

3

Phospholipids (g/L)

1.4-8.1

Cholesterol (g/L)

1-3.2

6.3

Proteins (g/L)

2-20

4.5

6.0 220

1.4

34

Data from Boyer JL: In Andreoli TE, Hoffman JF, Fanestil DD, Schultz SG (eds): Physiology of Membrane Disorders. New York: Plenum, 1986.

of water and small ions. The canalicular membrane expresses the water channel aquaporin 8 (AQP8). Under basal conditions, AQP8 is predominantly localized to intracellular vesicles but redistributes to the canalicular domain with stimulation by the secretagogue cAMP, thereby increasing apical water permeability. Thus, water transport into the bile canaliculus follows both paracellular and transcellular pathways. Further down the biliary tree (i.e., ducts and gallbladder), where the pore size of paracellular junctions is significantly smaller, solvent drag is not as important. Organic solutes do not readily enter bile distal to the canaliculi. Major organic molecules in bile include bile acids, cholesterol, and phospholipids Bile has two important functions: (1) bile provides the sole excretory route for many solutes that are not excreted by the kidney, and (2) secreted bile salts and acids are required for normal lipid digestion and absorption (see Chapter 44). Both hepatic bile and gallbladder bile are complex secretions that are isosmotic with plasma (~300 mosmol/kg) and consist of water, inorganic electrolytes, and a variety of organic solutes, including bilirubin, cholesterol, fatty acids, and phospholipid (Table 46-2). The predominant cation in bile is Na+, and the major inorganic anions are Cl− and HCO−3 . Solutes whose presence in bile is functionally important include micelle-forming bile acids, phospholipids, and IgA. Bile acids promote dietary lipid absorption through their micelle-forming properties (see Chapter 45). As shown in Figure 45-9, hepatocytes synthesize the so-called primary bile acids—cholic acid and chenodeoxycholic acid—from cholesterol. Indeed, biliary excretion of cholesterol and conversion of cholesterol to bile acids are the principal routes of cholesterol excretion and catabolism, thus making bile formation pivotal for total body cholesterol balance. The first step in this conversion is catalyzed by cholesterol 7αhydroxylase (CYP7a1), a specific cytochrome P-450 enzyme located in the SER. As we see later, secondary bile acids are the products of bacterial dehydroxylation in the terminal ileum and colon. After being absorbed and returning to the liver (enterohepatic circulation, discussed later), these secondary bile acids may also undergo conjugation. Figure 46-9 shows typical examples of conjugation reactions. Phospholipids in bile help to solubilize cholesterol as well as diminish the cytotoxic effects of other bile acids on hepatocytes and bile duct cells. IgA inhibits bacterial growth in bile. Excretory or waste products found in bile include cholesterol, bile pigments, trace minerals, plant sterols, lipophilic drugs and metabolites, antigen-antibody complexes, and oxidized glutathione. Bile is also the excretory route for compounds that do not readily enter the renal glomerular filtrate, either because they are associated with proteins such as albumin or because they are associated with formed elements in blood. Although these compounds are generally lipophilic, they also include the heavy metals. Some bile acids (e.g., the trihydroxy bile acid cholic acid) are only partly bound to serum albumin and may therefore enter the glomerular filtrate. However, they are actively reabsorbed by the renal tubule. In health, bile acids are virtually absent from the urine.

Chapter 46 • Hepatobiliary Function

CHOLESTEROL

PRIMARY BILE ACIDS OH

SECONDARY BILE ACIDS OH

O C

12

OH

O

Bacteria

C

7α-dehydroxylase

3 7α-hydroxylase

OH H

NH

CH2

COO–

Conjugation

7 OH

HO

HO

BILE SALTS

pKa~3.7

HO

Cholic acid

Deoxycholic acid

O C

OH

Glycine

O

Bacteria C

7α-dehydroxylase

OH H

NH

CH2

CH2

SO3–

Conjugation HO

OH

pKa~1.5

HO

Lithocholic acid

Chenodeoxycholic acid

Taurine

Figure 46-9 Synthesis of bile acids. The liver converts cholesterol to the primary bile acids—cholic acid and chenodeoxycholic acid—in a series of 14 reactions occurring in four different cellular organelles. The first reaction is the 7α-hydroxylation of cholesterol. In addition, the action of bacteria in the terminal ileum and colon may dehydroxylate bile acids, thus yielding the secondary bile acids deoxycholic acid and lithocholic acid. The hepatocytes conjugate most of the primary bile acids to small molecules such as glycine and taurine (not shown) before secreting them into the bile. In addition, those secondary bile acids that return to the liver through the enterohepatic circulation may also be conjugated to glycine or taurine, as shown in the figure. The liver may also conjugate some primary and secondary bile acids to sulfate or glucuronate (not shown).

Canalicular bile flow has a constant component driven by the secretion of small organic molecules and a component driven by the secretion of bile acids Total bile flow is the sum of the bile flow from hepatocytes into the canaliculi (canalicular flow) and the additional flow from cholangiocytes into the bile ducts (ductular flow). In most species, the rate of canalicular bile secretion (i.e., milliliters per minute) increases more or less linearly with the rate of bile acid secretion (i.e., moles per minute). Canalicular bile flow is the sum of two components (Fig. 46-10): (1) a “constant” component that is independent of bile acid secretion (bile acid–independent flow) and (2) a rising component that increases linearly with bile acid secretion (bile acid–dependent flow). In humans, most of the canalicular bile flow is bile acid dependent. If we now add the ductular secretion, which is also “constant,” we have the total bile flow in Figure 46-10. We discuss the canalicular secretion in the remainder of this section and ductular secretion in the following section. Bile Acid–Independent Flow in the Canaliculi

The secretion of organic compounds probably provides the major driving force for bile acid–independent flow. For example, glutathione, present in bile in high concentrations, may generate a potent osmotic driving force for canalicular bile formation.

Bile Acid–Dependent Flow in the Canaliculi

The negatively charged bile salts in bile are in a micellar form and

l Tota

bile

flow

n retio sec e l i b Bile-acid ular alic dependent Can flow

Bile flow

Bile-acid independent flow

Ductular secretion

Canalicular bile secretion

Bile-acid excretion rate

Figure 46-10

Components of bile flow.

are—in a sense—large polyanions. Thus, they are effectively out of solution and have a low osmotic activity coefficient. However, the positively charged counter ions accompanying these micellar bile acids are still in aqueous solution and may thus represent the predominant osmotic driving force for water movement in bile acid–dependent flow. If one infuses an animal with a nonphysiological bile acid that does not form micelles or one that forms micelles only at a rather high concentration, the osmotic activity will be higher, and thus the exogenous bile acid will be more effective in producing bile acid–dependent flow. In other words, the slope of the blue bile acid–dependent line in Figure 46-10 would be steeper than for physiological bile acids.

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Section VII • The Gastrointestinal System

Bile flow does not always correlate with the osmotic activity of the bile acid. In some cases, bile acids increase electrolyte and water flux by other mechanisms, such as by stimulating Na+-coupled cotransport mechanisms or by modulating the activity of other solute transporters. For example, the bile acid ursodeoxycholic acid produces a substantial increase in bile flow by markedly stimulating biliary HCO−3 excretion. Bile acids in the lumen may also stimulate the secretion of other solutes by trapping them in the lumen. These solutes include bilirubin and other organic anions, as well as lipids such as cholesterol and phospholipids. The mixed micelles formed by the bile acids apparently sequester these other solutes, thus lowering their effective luminal concentration and favoring their entry. Therefore, excretion of cholesterol and phospholipid is negligible when bile acid output is low, but it increases and approaches maximum values as bile acid output increases.

Lumen

Na+

+

As discussed in the previous section, biliary epithelial cells, or cholangiocytes, are the second major source of the fluid in hepatic bile. Experimentally, one can isolate cholangiocytes from normal liver or from the liver of experimental animals in which ductular hyperplasia has been induced by ligating the bile duct. These cholangiocytes have numerous transporters (Fig. 46-11), including the apical Cl-HCO3 exchanger AE2, 6 of the 11 known human aquaporins (AQPs), and several apical Cl− channels, including the cystic fibrosis transmembrane regulator (CFTR). In a mechanism that may be similar to that in pancreatic duct cells, the ClHCO3 exchanger, in parallel with the Cl− channels for Cl− recycling, can secrete an HCO−3 -rich fluid (see Chapter 42). AQP1, CFTR, and AE2 co-localize to intracellular vesicles in cholangiocytes; secretory agonists cause all three to co-redistribute to the apical membrane. A complex network of hormones, mainly acting through cAMP, regulates cholangiocyte secretory function. Secretin receptors (see Chapter 42) are present on the basolateral membranes of cholangiocytes, a finding that explains why secretin produces water-rich choleresis—that is, bile rich in HCO−3 (i.e., alkaline) but diluted in bile acids. Similarly, the hormones glucagon (see Chapter 50) and vasoactive intestinal peptide (VIP; see Chapter 43) also produce HCO−3 -rich choleresis at the level of the ducts. These hormones raise [cAMP]i and thus stimulate apical Cl− channels and the ClHCO3 exchanger. A Ca2+-activated Cl− channel is also present in the apical membrane. Cholangiocytes are also capable of reabsorbing fluid and electrolytes, as suggested by the adaptation that occurs after removal of the gallbladder (i.e., cholecystectomy). Bile found within the common bile duct of cholecystectomized, fasting animals is similar in composition to the concentrated bile typically found in the gallbladder. Thus, the ducts have partially taken over the function of the gallbladder (see later). The hormone somatostatin inhibits bile flow by lowering [cAMP]i, an effect opposite that of secretin. This inhibition may be caused by enhancing fluid reabsorption by bile ducts

Interstitial space Na+

Cholangiocyte

Cl –

– 2 HCO3

HCO3–

CO2

CA

CO2 H2O

OH–

Na+ H+ Cl –

Na+

Other Cl– channels

K+ K+

Cl –

Secretin stimulates the cholangiocytes of ductules and ducts to secrete a watery, HCO-3-rich fluid

H2O

Cystic fibrosis transmembrane regulator

[cAMP]

Secretin Glucagon VIP

Na+

+

H2O

Somatostatin (inhibitory)

Figure 46-11 Secretion of an HCO−3-rich fluid by cholangiocytes. The apical step of HCO−3 secretion by the duct cell is mediated by a Cl-HCO3 exchanger. The Cl− recycles back to the lumen through Cl− channels, such as CFTR. The basolateral step of HCO−3 secretion probably is mediated in part by the uptake of HCO3− through an electrogenic Na/HCO3 cotransporter. The uptake of CO2, combined with the extrusion of H+ through an Na-H exchanger and an H+ pump, generates the rest of the HCO−3 through carbonic anhydrase (CA). Secretin, glucagon, VIP, and gastrin-releasing peptide (GRP) all are choleretics. Somatostatin either enhances fluid absorption or inhibits secretion.

or by inhibiting ductular secretion of the HCO−3 -rich fluid discussed earlier. Solutes reabsorbed from bile by cholangiocytes can be returned to the hepatocyte for repeat secretion. As shown earlier in Figure 46-2, the intralobular bile ducts are endowed with a rich peribiliary vascular plexus that is supplied by the hepatic artery. The blood draining this plexus finds its way into the hepatic sinusoids. This plexus is analogous to the capillaries of the gut, which, through the portal vein, also find their way into the hepatic sinusoids. Thus, some solutes, such as the hydrophilic bile acid ursodeoxycholic acid, may be absorbed by the cholangiocytes from bile and returned to the hepatocytes for repeat secretion, thus inducing significant choleresis. The gallbladder stores and concentrates bile and delivers it to the duodenum during a meal The gallbladder is not an essential structure of bile secretion, but it does serve to concentrate bile acids up to 10- or even 20-fold during interdigestive periods. Tonic contraction of

Chapter 46 • Hepatobiliary Function

the sphincter of Oddi facilitates gallbladder filling by maintaining a positive pressure within the common bile duct. As we noted earlier, up to 50% of hepatic bile—or ~450 mL/ day—is diverted to the gallbladder during fasting. The remaining ~450 mL/day passes directly into the duodenum. Periods of gallbladder filling between meals are interrupted by brief periods of partial emptying of concentrated bile and probably aspiration of dilute hepatic bile in a process analogous to the function of a bellows. Bile salts and certain other components of bile are concentrated up to 20-fold within the gallbladder lumen because they are left behind during the isotonic reabsorption of NaCl and NaHCO3 by the leaky gallbladder epithelium (Fig. 46-12). The apical step of NaCl uptake and transport is electroneutral and is mediated by parallel Na-H and ClHCO3 exchangers. At the basolateral membrane, Na+ exits through the Na-K pump, whereas Cl− most likely exits by Cl− channels. Both water and HCO−3 move passively from lumen to blood through the tight junctions, which are rather leaky. Water can also move through the cell. The net transport is isotonic, which leaves behind gallbladder bile that is also isotonic but has a higher concentration of bile salts, K+, and Ca2+. Net fluid and electrolyte transport across the gallbladder epithelium is under hormonal regulation. Both VIP (released from neurons innervating the gallbladder) and serotonin inhibit net fluid and electrolyte absorption. Conversely, α-adrenergic blockade of neuronal VIP release increases fluid absorption. Although the gallbladder reabsorbs NaCl by parallel NaH and Cl-HCO3 exchange at the apical membrane, Na-H exchange outstrips Cl-HCO3 exchange; the end result is net secretion of H+ ions. This action neutralizes the HCO−3 and acidifies the bile. The H+ secreted by the gallbladder protonates the intraluminal contents. This action greatly increases the solubility of calcium salts in bile and reduces the likelihood of calcium salt precipitation and gallstone formation. Lumen

Epithelial cell

Interstitial space

K+

Na+

K+ 2 K+

H+ HCO3– Cl– H2O

3 Na Cl–

+

Na

+

Cl– H2O

Common pigment gallstones contain one or more of several calcium salts, including carbonate, bilirubinate, phosphate, and fatty acids. The solubility of each of these compounds is significantly increased by the acidification of bile. Mucus secretion by gallbladder epithelial cells results in the formation of a polymeric gel that protects the apical surface of the gallbladder epithelium from the potentially toxic effects of bile salts. However, excessive mucin synthesis can be deleterious. For example, in animal models of cholesterol cholelithiasis (i.e., formation of gallstones made of cholesterol), a marked increase in mucin release precedes crystal and stone formation. The relative tone of the gallbladder and sphincter of Oddi determines whether bile secreted by the liver flows from the common hepatic duct into the gallbladder or into the duodenum Bile exiting the liver and flowing down the common hepatic duct reaches a bifurcation that permits flow either into the cystic duct and then into the gallbladder or into the common bile duct, through the sphincter of Oddi, and into the duodenum (Fig. 46-4). The extent to which bile takes either path depends on the relative resistance of the two pathways. The sphincter of Oddi—which also controls the flow of pancreatic secretions into the duodenum—corresponds functionally to a short (4- to 6-mm) zone within the wall of the duodenum. The basal pressure within the lumen of the duct at the level of the sphincter is 5 to 10 mm Hg. The pressure in the lumen of the resting common bile duct is also 5 to 10 mm Hg, compared with a pressure of ~0 mm Hg inside the duodenum. The basal contraction of the sphincter prevents reflux of the duodenal contents into the common bile duct. In its basal state, the sphincter exhibits high-pressure, phasic contractions several times per minute. These contractions are primarily peristaltic and directed in antegrade fashion to provide a motive force toward the duodenum. Thus, the sphincter of Oddi acts principally as an adjustable occluding mechanism and a regulator of bile flow. Both hormonal and cholinergic mechanisms appear to be involved in gallbladder emptying. Dietary lipid stimulates the release of cholecystokinin (CCK) from duodenal I cells (see Chapter 44). This CCK not only stimulates pancreatic secretion but also causes smooth muscle contraction and evacuation of the gallbladder. The coordinated response to CCK also includes relaxation of the sphincter of Oddi, thus enhancing bile flow into the duodenum.

ENTEROHEPATIC CIRCULATION OF BILE ACIDS Figure 46-12 Isotonic fluid reabsorption by the gallbladder epithelium. The gallbladder epithelium performs the isotonic absorption of NaCl. The apical step is parallel Na-H exchange and Cl-HCO3 exchange. Because Na-H exchange is somewhat faster, net secretion of acid into the lumen occurs. The basolateral step of NaCl absorption is mediated by the Na-K pump and by Cl− channels. K+ channels provide a route for basolateral K+ recycling. Water follows passively through the tight junctions and through the basolateral membrane.

The enterohepatic circulation of bile acids is a loop consisting of secretion by the liver, reabsorption by the intestine, and return to the liver in portal blood for repeat secretion into bile Bile acids are important for promoting the absorption of dietary lipids in the intestine. The quantity of bile acid that

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Cholestasis

T

he term cholestasis refers to the suppression of bile secretion. Biliary constituents may therefore be retained within the hepatocyte and regurgitated into the systemic circulation. Cholestasis causes three major groups of negative effects: first, regurgitation of bile components (bile acids, bilirubin) into the systemic circulation gives rise to the symptoms of jaundice and pruritus (itching). Second, cholestasis damages hepatocytes, as evidenced by the release of liver enzymes (e.g., alkaline phosphatase) into the plasma. Third, because the bile acids do not arrive in the duodenum, lipid digestion and absorption may be impaired. Many acute and chronic liver diseases produce cholestasis by mechanically obstructing the extrahepatic bile ducts or by impairing bile flow at the level of the hepatocytes or intrahepatic bile ducts. The mechanisms underlying the obstructive and functional forms of cholestasis are complex and have not been completely defined. Experimental models of cholestasis have produced multiple abnormalities: (1) altered plasma membrane composition and fluidity; (2) inhibition of membrane proteins, including the Na-K pump; (3) reduced expression of genes encoding transporters for bile acids and other organic anions; (4) increased permeability of the paracellular pathway, with backdiffusion of biliary solutes into the plasma; (5) altered function of microfilaments, with decreased contractions of bile canaliculi; and (6) loss of the polarized distribution of some plasma membrane proteins. Cholestatic conditions, such as bile duct obstruction, markedly increase the basolateral expression of MRP3—which normally is expressed only minimally. This induction of MRP3 allows the efflux of bile acids and other cholephilic anions from the hepatocyte into sinusoidal blood.

the liver normally secretes in a day varies with the number of meals and the fat content of these meals, but it typically ranges between 12 and 36 g. The liver’s basal rate of synthesis of bile acids from cholesterol (Fig. 46-9) is only ~600 mg/day in healthy humans, sufficient to replace the equivalent losses of bile acid in the feces. Obviously, the gastrointestinal tract must have an extremely efficient mechanism for recycling the bile acids secreted by the liver (Fig. 46-13). This recycling, known as the enterohepatic circulation, occurs as the terminal ileum and colon reabsorb bile acids and return them to the liver in the portal blood. The total pool of bile acids in the gastrointestinal tract is ~3 g. This pool must recirculate ~ 4 to 12 times per day, or as many as 5 or more times for a single fat-rich meal. If reabsorption of bile acids is defective, as can happen after resection of the ileum, de novo synthesis of bile acids by the liver can be as high as 4 to 6 g/day. The intestinal conservation of bile acids is extremely efficient and is mediated both by active apical absorption in the terminal ileum and by passive absorption throughout the intestinal tract Most of the bile secreted into the duodenum is in the conjugated form. Very little of these bile salts are reabsorbed

into the intestinal tract until they reach the terminal ileum, an arrangement that allows the bile salts to remain at high levels throughout most of the small intestine, where they can participate in lipid digestion and absorption (see Chapter 44). However, the enterohepatic circulation must eventually reclaim 95% or more of these secreted bile salts. Some of the absorption of bile acids by the intestines is passive and occurs along the entire small intestine and colon. Nevertheless, the major component of bile acid absorption is active and occurs only in the terminal ileum (Fig. 46-13). Passive absorption of bile acids occurs along the entire small intestine and colon (Fig. 46-13), but it is less intensive than active absorption. The mechanism of bile acid uptake across the apical membrane may consist of either ionic or nonionic diffusion. Nonionic diffusion—or passive diffusion of the protonated or neutral form of the bile acid—is 10-fold greater than ionic diffusion. The extent of nonionic diffusion for a given bile acid depends on the concentration of its neutral, protonated form, which is maximized when the luminal pH is low and the pK of the bile acid is high. At the normal intestinal pH of 5.5 to 6.5, few of the taurineconjugated bile salts are protonated, a small amount of the glycine-conjugated bile salts are protonated, and ~50% of unconjugated bile acids are protonated. Thus, the unconjugated bile acids are in the best position to be reabsorbed by nonionic diffusion, followed by the glycine-conjugated bile acids and then finally by the taurine-conjugated bile acids. Among these unconjugated bile acids, more lipophilic bile acids, such as chenodeoxycholate and deoxycholate, diffuse more readily through the apical membrane than do hydrophilic bile acids such as cholic acid. Nonionic diffusion also depends on the total concentration of the bile acid (i.e., neutral plus charged form), which, in turn, depends on the maximum solubilizing capacity of bile salt micelles for that bile acid. Active absorption of bile acids in the intestine is restricted to the terminal ileum. This active process preferentially absorbs the negatively charged conjugated bile salts—the form not well absorbed by the passive mechanisms. Active uptake of bile salts involves saturation kinetics, competitive inhibition, and a requirement for Na+ (Fig. 46-13). The Na+dependent transporter responsible for the apical step of active absorption is known as the apical Na+/bile salt transporter ASBT (SLC10A2), a close relative of the hepatocyte transporter NTCP (Fig. 46-5C). Once bile salts have entered ileal enterocytes across the apical membrane, they exit across the basolateral membrane via a heteromeric organic solute transporter (Osta/Ostb). Because the most polar bile salts are poorly absorbed by nonionic diffusion, it is not surprising that the ASBT in the apical membrane of the enterocytes of the terminal ileum has the highest affinity and maximal transport rates for these salts. For example, the ASBT is primarily responsible for absorbing the ionized, taurine-conjugated bile salts in the ileum. Conversely, the ASBT in the ileum is relatively poor at absorbing the more lipophilic bile acids, which tend to be absorbed passively in the upper intestine. On their entry into portal blood, the bile acids are predominantly bound to albumin and, to a lesser extent, lipoproteins. The liver removes or clears these bile acids from portal blood by the transport mechanisms

Chapter 46 • Hepatobiliary Function

Unconjugated BA– Synthesis of ~600 mg/day of “primary bile acids”

BA-Z– Conjugated

BA–

Bile salts

Z BA-Z–

LIVER

BA-Z–

Bile storage H.BA GALLBLADDER

SMALL INTESTINE

Passive absorption H.BA

H+ + BA– –

BA-Z

H+ Bacteria in the terminal ileum and the colon deconjugate bile salts (BA-Z–) to form bile acids (H.BA) and also dehydroxylate primary bile acids to form secondary bile acids. TERMINAL ILEUM Deoxycholic acid

+ BA– H.BA Passive absorption

BA-Z– Enterocyte in terminal ileum

H.BA

Lithocholic acid

Na+

COLON

BA-Z– H + BA–

CECUM

H.BA

Conjugated bile acid

ASBT BA-Z–

BA-Z–

OSTα-OSTβ

Passive absorption

~600 mg bile acids lost daily in feces

Figure 46-13 Enterohepatic circulation of bile acids. The bile acids that the liver delivers to the duodenum in the bile are primarily conjugated to taurine or glycine (BA-Z−). Most bile acids are reabsorbed as conjugated bile salts (BA-Z−) in the terminal ileum through an Na+-coupled cotransporter (ASBT). Also in the terminal ileum and colon, bacteria deconjugate a small amount of these bile salts to form unconjugated bile acids (H · BA ↔ H+ + BA−), thereby allowing H · BA to be passively absorbed by nonionic diffusion. In addition, bacteria in the terminal ileum and colon dehydroxylate primary bile acids to form secondary bile acids (see Fig. 46-9). Some of these are passively absorbed, and the rest are excreted in the feces. The absorbed bile acids return to the liver through the portal blood and are then taken up into the hepatocyte for secretion again.

outlined earlier in Figure 46-5C. Hepatic clearance of bile acids is often expressed as the percentage of bile acids removed during a single pass through the liver. The hepatic extraction of bile acids is related to bile acid structure and the degree of albumin binding. It is greatest for hydrophilic bile acids and is least for protein-bound, hydrophobic bile acids. The small fraction of bile acids that escapes active or passive absorption in the small intestine is subject to bacterial modification in the colon. This bacterial modification takes two forms. First, the bacteria deconjugate the bile. Second, the bacteria perform a 7α-dehydroxylation reaction

with the formation of secondary bile acids. These secondary bile acids include deoxycholate and lithocholate (Fig. 46-9). The deconjugated secondary bile acids may then be either absorbed passively in the colon or excreted in the feces; their fate depends on their physicochemical properties and their binding to luminal contents. Up to one third of the deoxycholate formed in the colon may be reabsorbed by nonionic diffusion. Lithocholate, which is relatively insoluble, is absorbed to a much lesser extent. The secondary bile acids formed by colonic bacteria and recycled back to the liver may undergo biotransformation through conjugation to glycine and taurine.

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Gallstones

M

ost gallstones (~80%) consist mainly of cholesterol. Thus, cholelithiasis may be regarded as a disturbance of bile secretion and cholesterol elimination. When cholesterol and phospholipids are secreted together into the bile, they form unilamellar bilayered vesicles. These vesicles become incorporated into mixed micelles that form because of the amphiphilic properties of bile acids. Micellation allows cholesterol to remain in solution in its passage through the biliary tree. However, if the concentration of bile acids is insufficient to maintain all the cholesterol in the form of mixed micelles, the excess cholesterol is left behind as vesicles in the aqueous phase. These cholesterol-enriched vesicles are relatively unstable and are prone to aggregate and form large multilamellar vesicles, from which cholesterol crystals nucleate. Growth of crystals may result in the formation of gallstones. An excess of biliary cholesterol in relation to the amount of phospholipids and bile acids can result from hypersecretion of cholesterol, inadequate secretion of bile acids, or both. Cholelithiasis may be further promoted by other factors, such as gallbladder mucin and other nonmucous glycoproteins, as well as by stasis of bile in the gallbladder.

Thus, the enterohepatic circulation of bile acids is driven by two mechanical pumps: (1) the motor activity of the gallbladder and (2) peristalsis of the intestines to propel the bile acids to the terminal ileum and colon. It is also driven by two chemical pumps: (1) energy-dependent transporters located in the terminal ileum and (2) energy-dependent transporters in the hepatocyte. The bile acid receptor FXR, a member of the nuclear receptor family, controls multiple components of the enterohepatic circulation of bile acids. Primary bile acids are potent agonists of FXR, which transcriptionally regulates several genes involved in bile acid homeostasis. Four examples of negative feedback by activated FXR are as follows: (1) FXR inhibits the expression of cholesterol 7α-hydroxylase (Fig. 46-9), the rate-limiting enzyme for bile acid synthesis; (2) FXR induces the expression of an inhibitory transcription factor—the small heterodimer partner (SHP)—which controls the activity of another nuclear receptor, the liver receptor homologue-1 (LRH-1), which is required for CYP7a1 expression; (3) FXR upregulates BSEP (increasing bile acid secretion; Fig. 46-5C) and downregulates NTCP (decreasing bile acid uptake; Fig. 46-5C) by SHP-dependent mechanisms; and (4) FXR, through SHP, downregulates ASBT and thereby reduces ileal bile acid uptake. Thus, FXR coordinates bile acid synthesis and transport by the liver and intestine.

THE LIVER AS A METABOLIC ORGAN The liver is a metabolically active and highly aerobic organ. It receives ~28% of the total blood flow and extracts ~20% of the oxygen used by the body. The liver is responsible for the synthesis and degradation of carbohydrates, proteins, and lipids. The small molecules that are products of diges-

tion are efficiently sorted in the liver for metabolism, storage, or distribution to extrahepatic tissues for energy. The liver provides energy to other tissues mainly by exporting two substrates that are critical for oxidization in the peripheral tissues, glucose and ketone bodies (e.g., acetoacetate). The liver can serve as either a source or a sink for glucose The liver is one of the key organs that maintain blood glucose concentrations within a narrow range, in a dynamic process involving endogenous glucose production and glucose utilization. The fasting blood [glucose] is normally 4 to 5 mM. Between meals, when levels of insulin are relatively low and levels of glucagon are high (see Chapter 50), the liver serves as a source of plasma glucose, both by synthesizing glucose and by generating it from the breakdown of glycogen. The de novo synthesis of glucose, or gluconeogenesis (see Fig. 51-12), is one of the liver’s most important functions; it is essential for maintaining a normal plasma concentration of glucose, which is the primary energy source for most tissues (see Chapter 57). Glucose is synthesized in the lumen of the ER, principally from amino acids and lactate. Dietary fructose and galactose are also largely converted to glucose. Glucose exits the ER by facilitated diffusion (mediated by GLUT7) and then passes into the blood through another facilitated diffusion mechanism (GLUT2), which has a low affinity and high capacity and is located in the hepatocyte’s basolateral membrane. The second way in which the liver delivers glucose to blood plasma is by glycogenolysis. Stored glycogen may account for as much as 7% to 10% of the total weight of the liver. Glycogenolysis in the liver yields glucose as its major product, whereas glycogen breakdown in muscle produces lactic acid (see Chapter 59). After a meal, when levels of insulin are relatively high, the liver does just the opposite: it acts as a sink for glucose by taking it up from the portal blood and either breaking it down to pyruvate or using it to synthesize glycogen (see Fig. 50-8). Glucose oxidation has two phases. In the anaerobic phase, glucose is broken down to pyruvic acid (glycolysis). In the aerobic phase, pyruvic acid is completely oxidized to H2O and CO2 through the citric acid cycle. The liver also consumes glucose by using it for glycogen synthesis. Carbohydrate that is not stored as glycogen or oxidized is metabolized to fat. All the aforementioned processes are regulated by hormones such as insulin and glucagon (see Chapter 50), which enable rapid responses to changes in the metabolic requirements of the body. The liver synthesizes a variety of important plasma proteins (e.g., albumin, coagulation factors, and carriage proteins) and metabolizes dietary amino acids Protein Synthesis One of the major functions of the liver is to produce a wide array of proteins for export to the blood plasma (Table 46-3). These products include major plasma proteins that are important for maintaining the colloid

Chapter 46 • Hepatobiliary Function

Table 46-3

Proteins Made by the Liver for Export

Major Plasma Proteins

Albumin α1-Fetoprotein Plasma fibronectin (an α2-glycoprotein) C-reactive protein α2-Microglobulin Various other globulins Factors Involved in Hemostasis/Fibrinolysis

Coagulation: fibrinogen and all others except factor VIII Inhibitors of coagulation: α1-antitrypsin and antithrombin III, α2-macroglobulin, protein S, protein C Fibrinolysis: plasminogen Inhibitors of fibrinolysis: α2-antiplasmin Complement C3 Carriage Proteins (Binding Proteins)

Ceruloplasmin Corticosteroid-binding globulin (CBG; also called transcortin) GH-binding protein (low-affinity form) Haptoglobin Hemopexin IGF-binding proteins Retinol-binding protein Sex hormone–binding globulin (SHBG) Thyroid-binding globulin (TBG) Transferrin Transthyretin Vitamin D–binding protein Prohormones

Angiotensinogen Apolipoproteins

Apo Apo Apo Apo Apo Apo Apo

A-I A-II A-IV B-100 C-II D E

GH, growth hormone; IGF, insulin-like growth factor.

osmotic pressure of plasma (see Chapter 19). Other products include factors involved in hemostasis (blood clotting) and fibrinolysis (breakdown of blood clots), carriage proteins that bind and transport hormones and other substances in the blood, prohormones, and lipoproteins (Table 46-4). The liver synthesizes plasma proteins at a maximum rate of 15 to 50 g/day. Amino Acid Uptake

A major role of the liver is to take up and metabolize dietary amino acids that are absorbed by the gastrointestinal tract (see Chapter 44) and are transported to the liver in portal blood. These amino acids are taken up by both Na+-dependent and Na+-independent transporters that are identical to some of the amino acid transporters in the kidney, small intestine, and other tissues (see Table 35-1). An unusual feature of the liver is that, with few exceptions, the same amino acid transporter may be located on

both the basolateral and apical membranes. For example, Na+-dependent glutamate uptake by the excitatory amino acid transporters SLC1A1 (EAAT3) and SLC1A2 (EAAT2) occurs primarily at the apical membrane, but dexamethasone (corticosteroid) treatment can induce their expression at the basolateral membrane. In general, hepatic amino acid transporters are highly regulated at the transcriptional and post-translational levels. Amino Acid Metabolism Under physiological conditions, total and individual plasma concentrations of amino acids are tightly regulated. The liver controls the availability of amino acids in the systemic blood, activating ureagenesis after a high protein meal and repressing it during fasting or low protein intake. Unlike glucose, which can be stored, amino acids must either be used immediately (e.g., for the synthesis of proteins) or broken down. The breakdown of α-amino acids occurs by deamination to α-keto acids and NH+4 (Fig. 46-14). The α-keto acids (“carbon skeleton”), depending on the structure of the parent amino acid, are metabolized to pyruvate, various intermediates of the citric acid cycle (see Fig. 57-9), acetyl coenzyme A (acetyl CoA), or acetoacetyl CoA. The liver detoxifies ~95% of the NH+4 through a series of reactions known as the urea cycle (Fig. 46-14); the liver can also use NH+4—together with glutamate—to generate glutamine. Individual deficiencies in each of the enzymes involved in the urea cycle have been described and result in life-threatening hyperammonemia. The urea generated by the urea cycle exits the hepatocyte through a urea channel, which is, in fact, AQP9. The urea then enters the blood and is ultimately excreted by the kidneys (see Chapter 37). The glutamine synthesized by the liver also enters the blood. Some of this glutamine is metabolized by the kidney to yield glutamate and NH+4, which is exported in the urine (see Chapter 37). The liver is also the main site for the synthesis and secretion of glutathione. GSH is critical for detoxification (in conjugation reactions in the liver) and for protection against oxidative stress in multiple organs. Thus, erythrocytes that have low levels of GSH are more prone to hemolysis. Because more than 90% of the GSH in the circulation is synthesized in the liver, GSH efflux across the basolateral membrane from the hepatocyte to the sinusoid is important. Bidirectional transport of glutathione across the basolateral membrane may occur in part by one of the OATPs as well as MRP4 by cotransport with bile acids. In addition, MRP2 exports some conjugated GSH across the canalicular membrane into bile, as stated earlier, and an unidentified transporter can similarly export smaller amounts of unconjugated GSH.

The liver obtains dietary triglycerides and cholesterol by taking up remnant chylomicrons through receptor-mediated endocytosis As discussed in Chapter 44, enterocytes in the small intestine process fatty acids consumed as dietary triglycerides and secrete them into the lymph primarily in the form of extremely large proteolipid aggregates called chylomicrons (Fig. 46-15). These chylomicrons—made up of triglycerides (80% to 90%), phospholipids, cholesterol, and several apo-

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Table 46-4

Major Classes of Lipoproteins Chylomicrons

VLDLs

IDLs

LDLs

HDLs

Density (g/cm3)

600 kDa), and it accounts for approximately half of the protein content of the thyroid gland. It has relatively few tyrosyl residues (~100/molecule of thyroglobulin), and only a few of these (15 mg/dL) and severe hyperparathyroidism. The condition is life threatening and is characterized by markedly elevated plasma [Ca2+], neuronal malfunction, demineralization of bone, and calcification of soft tissues. These infants die unless the inappropriately regulated parathyroid glands are removed. As in the parathyroid gland, the distal convoluted tubule of the kidney has abundant plasma membrane Ca2+ receptors. Investigators have suggested that serum Ca2+ binds to this renal Ca2+ receptor and inhibits Ca2+ reabsorption (see Chapter 36). Thus, with a mutated receptor, renal Ca2+ reabsorption may not be inhibited until plasma [Ca2+] rises to abnormally high levels. The result would be the increased Ca2+ reabsorption and hypocalciuria characteristic of FHH. The discovery of CaSR led to the development of a CaSR agonist that mimics Ca2+ (a calcimetic). This drug has now come into clinical use for treating patients with parathyroid cancer or hyperparathyroidism secondary to chronic renal disease. Calcimetics decrease the secretion of PTH and secondarily decrease plasma [Ca2+].

Thus, PTH-induced phosphaturia diminishes the Ca/PO ion product and prevents precipitation when Ca2+ mobilization is needed. The body tightly regulates plasma [Ca2+] but allows plasma levels of PO43− to vary rather widely. In addition to affecting PO43− reabsorption, PTH causes a decrease in proximal tubule reabsorption of HCO43− (see Chapter 39) and of several amino acids. These actions appear to play a relatively minor role in whole-body acid-base and nitrogen metabolism, respectively.

Chapter 52 • The Parathyroid Glands and Vitamin D

Stimulation of the Last Step of Synthesis of 1,25Dihydroxyvitamin D A third important renal action of

PTH is to stimulate the 1-hydroxylation of 25-hydroxyvitamin D in the mitochondria of the proximal tubule. The resulting 1,25-dihydroxyvitamin D is the most biologically active metabolite of dietary or endogenously produced vitamin D. Its synthesis by the kidney is highly regulated, and PTH is the primary stimulus to increase 1-hydroxylation. Hypophosphatemia, either spontaneous or induced by the phosphaturic action of PTH, also promotes the production of 1,25-dihydroxyvitamin D. As discussed later, the 1,25-dihydroxyvitamin D formed in the proximal tubule has three major actions: (1) enhancement of renal Ca2+ reabsorption, (2) enhancement of Ca2+ absorption by the small intestine, and (3) modulation of the movement of Ca2+ and PO43− in and out of bone. In bone, PTH can promote net resorption or net deposition The second major target tissue for PTH is bone, in which PTH promotes both bone resorption and bone synthesis. Bone Resorption by Indirect Stimulation of Osteoclasts The net effect of persistent increases of PTH

on bone is to stimulate bone resorption, thus increasing plasma [Ca2+]. Osteoblasts express abundant surface receptors for PTH; osteoclasts do not. Because osteoclasts lack PTH receptors, PTH by itself cannot regulate the coupling between osteoblasts and osteoclasts. Rather, it appears that PTH acts on osteoblasts and osteoclast precursors to induce the production of several cytokines that increase both the number and the activity of bone-resorbing osteoclasts. Precisely which cytokines are involved in the physiological signaling of osteoclasts by PTH-stimulated osteoblasts in vivo is not clear. PTH causes osteoblasts to release agents such as M-CSF and stimulates the expression of RANK ligand (i.e., osteoprotegerin ligand), actions that promote the development of osteoclasts (Fig. 52-4). In addition, PTH and vitamin D stimulate osteoblasts to release interleukin 6 (IL-6), which stimulates existing osteoclasts to resorb bone (Fig. 52-5). One of the initial clues that cytokines are important mediators of osteoclastic bone resorption came from observations on patients with multiple myeloma—a malignancy of plasma cells, which are of B-lymphocyte lineage. The tumor cells produce several proteins that activate osteoclasts and enhance bone resorption. These proteins were initially called “osteoclast-activating factors.” We now know that certain lymphocyte-derived proteins strongly activate osteoclastic bone resorption; these proteins include lymphotoxin, IL-1, and TNF-α. Bone Resorption by Reduction in Bone Matrix

PTH also changes the behavior of osteoblasts in a manner that can promote net loss of bone matrix. For example, PTH inhibits collagen synthesis by osteoblasts and also promotes the production of proteases that digest bone matrix. Digestion of matrix is important because osteoclasts do not easily reabsorb bone mineral if the bone has an overlying layer of unmineralized osteoid.

Bone Deposition Whereas persistent increases in PTH favor net resorption, intermittent increases in plasma [PTH] have predominantly bone synthetic effects. PTH can promote bone synthesis by two mechanisms. First, PTH promotes bone synthesis directly by activating Ca2+ channels in osteocytes, a process that leads to a net transfer of Ca2+ from bone fluid to the osteocyte. The osteocyte then transfers this Ca2+ through gap junctions to the osteoblasts at the bone surface. This process is called osteocytic osteolysis. The osteoblasts then pump this Ca2+ into the extracellular matrix, thus contributing to mineralization. Second, PTH stimulates bone synthesis indirectly in that osteoclastic bone resorption leads to the release of growth factors such as insulin-like growth factor 1 (IGF-1), IGF-2, and transforming growth factor β. The PTH–1-34 peptide is now available as a pharmacological agent for the treatment of osteoporosis. Clinical data show marked increases in bone density—particularly within the axial skeleton—in response to injections of PTH–1-34 once or twice daily. The effects on trabecular bone are striking, with less positive responses seen in cortical bone—particularly in the limbs.

VITAMIN D The active form of vitamin D is its 1,25-dihydroxy metabolite By the 1920s, investigators recognized that dietary deficiency of a fat-soluble vitamin was responsible for the childhood disease rickets. This disorder is characterized clinically by hypocalcemia and multiple skeletal abnormalities. Dietary replacement of vitamin D corrects this disorder and has led to the practice of adding vitamin D to milk, bread, and other products. This practice has greatly reduced the prevalence of this previously common disorder. Our understanding of the involvement of vitamin D in the regulation of plasma [Ca2+] and skeletal physiology has been clarified only over the past 2 decades. Vitamin D exists in the body in two forms, vitamin D3 and vitamin D2 (Fig. 52-9). Vitamin D3 can be synthesized from the 7-dehydrocholesterol that is present in the skin, provided sufficient ultraviolet light is absorbed. This observation explains why nutritional rickets had been a much more prevalent problem in northern countries, where clothing covers much of the skin and where individuals remain indoors much more of the year. Vitamin D3 is also available from several natural sources, including cod and halibut liver, eggs, and fortified milk. Vitamin D2 is obtained only from the diet, largely from vegetables. Vitamins D3 (Fig. 52-9A) and vitamin D2 (Fig. 52-9B) differ only in the side chains of ring D. The side chain in vitamin D3 (cholecalciferol) is characteristic of cholesterol, whereas that of vitamin D2 (ergocalciferol) is characteristic of plant sterols. Vitamin D (i.e., either D2 or D3) is fat soluble but water insoluble. Its absorption from the intestine depends on its solubilization by bile salts (see Chapter 45). In the circulation, vitamin D is found either solubilized with chylomicrons (see Chapter 46) or associated with a plasma binding

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Section VIII • The Endocrine System

A

METABOLISM OF VITAMIN D3 25

H

D

C

7–Dehydrocholesterol

1

A

B

HO Skin

UV light

21

22 20 17

18 11 9

12

26 23

24

25

D 16

13

C 14

15

8

Cholecalciferol (Vitamin D3)

7

6

H

27

19

4 3

HO

5

A

CH2 10 1

2

Liver

25

OH

25–Hydroxycholecalciferol (25–OHD3) CH2 HO Kidney

25

OH

1,25–(OH)2D3

CH2 1

HO B

OH

VITAMIN D2

H

Ergocalciferol (Vitamin D2) CH2 A HO

Figure 52-9

Forms of vitamin D.

protein. Most of the body stores of vitamin D are located in body fat. The body’s pools of vitamin D are large, and only 1% to 2% of the body’s vitamin D is turned over each day. Therefore, several years of very low dietary intake (as well as diminished endogenous synthesis) is required before the endogenous pools are depleted and deficiency develops. The principal active form of vitamin D is not vitamin D2 or D3, but rather a dihydroxylated metabolite of either. Hydroxylation of vitamin D proceeds in two steps (Fig. 52-9A). When circulating levels of 25-hydroxyvitamin D are low, adipocytes release vitamin D into the blood plasma. A cytochrome P-450 mixed-function oxidase, principally in the liver, creates the first hydroxyl group at carbon 25. The 25-hydroxylation of vitamin D does not appear to be highly regulated, but rather it depends on the availability of vitamin D2 or D3. The second hydroxylation reaction occurs in the renal proximal tubule under the tight control of PTH, vitamin D itself, and PO43−. PTH stimulates this 1hydroxylation, whereas PO43− and 1,25-dihydroxyvitamin D (the reaction product) both inhibit the process (Fig. 52-8). In addition to vitamins D2 and D3 and their respective 25-hydroxy and 1,25-dihydroxy metabolites, more than 15 other metabolites of vitamin D have been identified in plasma. However, the specific physiological function of these metabolites, if any, is unclear. Although considered a vitamin because of its dietary requirement, vitamin D can also be considered a hormone, both because it is endogenously synthesized and because even the fraction that arises from the diet must be metabolized to a biologically active form. Vitamin D and its metabolites, like the steroid hormones, circulate bound to a globulin binding protein, in this case a 52-kDa vitamin D–binding protein. This binding protein appears particularly important for the carriage in the blood of vitamins D2 and D3, which are less soluble than their hydroxylated metabolites. Vitamin D and its metabolites arrive at target tissues and, once in the cytosol, associate with the VDR, a transcription factor that is in the family of nuclear receptors (see Chapter 3). Like the thyroid hormone receptor (see Table 4-2), VDR forms a heterodimer with RXR. The VDR specifically recognizes the 1,25-dihydroxyvitamin D with an affinity that is three orders of magnitude higher than that for 25-hydroxyvitamin D. However, because the circulating concentration of 25-hydroxyvitamin D is ∼1000fold higher than that of 1,25-dihydroxyvitamin D, both species probably contribute to the biological actions of the hormone. The biological actions of 1,25-dihydroxyvitamin D appear to be expressed principally, but not exclusively, through regulation of the transcription of a variety of proteins. The VDR/RXR complex associates with a regulatory site in the promoter region of the genes coding for certain vitamin D– regulated proteins. Thus, the occupied VDR alters the synthesis of these vitamin D–dependent proteins. An example is PTH, which stimulates the formation of 1,25-dihydroxyvitamin D. The 5′ regulatory region of the PTH gene has a VDR consensus sequence; when occupied by the VDR complex, this element diminishes transcription of the PTH gene.

Chapter 52 • The Parathyroid Glands and Vitamin D

Vitamin D, by acting on the small intestine and kidney, raises plasma [Ca2+] and thus promotes bone mineralization The actions of vitamin D can be grouped into two categories: actions on classic target tissues involved in regulating body mineral and skeletal homeostasis and a more general action that regulates cell growth. The actions of vitamin D on the small intestine, bone, and kidney serve to prevent any abnormal decline or rise in plasma [Ca2+]. Small Intestine

In the duodenum, 1,25-dihydroxyvitamin D increases the production of several proteins that enhance Ca2+ absorption. Figure 52-10A summarizes the intestinal absorption of Ca2+ (see Chapter 45), which moves from the intestinal lumen to the blood by both paracellular and transcellular routes. In the paracellular route, which occurs throughout the small intestine, Ca2+ moves passively from the lumen to the blood; 1,25-dihydroxyvitamin D does not regulate this pathway. The transcellular route, which occurs only in the duodenum, involves three steps. First, Ca2+ enters the cell across the apical membrane through Ca2+ channels and possibly endocytosis. Second, the entering Ca2+ binds to several high-affinity binding proteins, particularly calbindin. These proteins, together with the exchangeable Ca2+ pools in the endoplasmic reticulum and mitochondria, effectively buffer the cytosolic Ca2+ and maintain a favorable gradient for Ca2+ entry across the apical membrane of the enterocyte. Thus, the intestinal cell solves the problem of absorbing relatively large amounts of Ca2+ while keeping its free, cytosolic [Ca2+] low. Third, the enterocyte extrudes Ca2+ across the basolateral membrane by means of both a Ca2+ pump and an Na-Ca exchanger. Vitamin D promotes Ca2+ absorption primarily by genomic effects that involve induction of the synthesis of epithelial Ca2+ channels and pumps and Ca2+-binding proteins, as well as other proteins (e.g., alkaline phosphatase). Although these actions probably facilitate Ca2+ transport by the intestine, not all steps involved in the action of vitamin D to enhance transcellular Ca2+ transport have been well defined experimentally. The effect of PTH to stimulate intestinal Ca2+ absorption is thought to be entirely indirect and mediated by increasing the renal formation of 1,25dihydroxyvitamin D (Fig. 52-8), which then enhances Ca2+ absorption. Vitamin D also stimulates PO43− absorption by the small intestine (Fig. 52-10B). The initial step is mediated by the NaPi cotransporter (see Chapter 36) and appears to be rate limiting for transepithelial transport and subsequent delivery of PO43− to the circulation. 1,25-Dihydroxyvitamin D stimulates the synthesis of this transport protein and thus promotes PO43− entry into the mucosal cell.

Rickets and Osteomalacia

D

eficiency of vitamin D in children produces the disease rickets, in which bone has abnormal amounts of unmineralized osteoid. Both cortical bone and trabecular bone are involved. The lack of mineralization diminishes bone rigidity and leads to a characteristic bowing of the long bones of the legs. In adults, vitamin D deficiency produces a disorder called osteomalacia. Microscopically, the bone looks very much the same in adult and childhood vitamin D deficiency. However, because the longitudinal growth of the long bones has been completed in adults, bowing of weight-bearing bones does not occur. Instead, the increased unmineralized osteoid content of bone causes a decline in bone strength. Affected individuals are more prone to the development of bone fractures. These fractures may be very small and difficult to see radiographically. As more and more of the bone surface is covered by osteoid and as recruitment of new osteoclasts is diminished, osteoclastic bone resorption is impaired, and hypocalcemia develops. Hypocalcemia causes nerves to become more sensitive to depolarization. In sensory nerves, this effect leads to sensations of numbness, tingling, or burning; in motor nerves, it leads to increased spontaneous contractions, or tetany. Whereas rickets and osteomalacia are very uncommon in developed countries because of vitamin D supplementation, milder degrees of vitamin D deficiency are increasingly recognized, particularly in the elderly population, in whom milk consumption and sunlight exposure are frequently inadequate. The resulting fall in plasma [Ca2+] can lead to mild, secondary hyperparathyroidism. Such continuous elevations of PTH can lead to further bone resorption and worsening osteoporosis. Rickets or osteomalacia also can occur with impaired ability of the kidney to 1-hydroxylate the 25-hydroxyvitamin D previously synthesized in the liver. An acquired version is seen in many patients with chronic renal failure, in which the activity of 1α-hydroxylase is reduced. The genetic form of the 1α-hydroxylase deficiency is a rare autosomal recessive disorder. Either form is called vitamin D–dependent rickets because it can be successfully treated with either 1,25-dihydroxyvitamin D or higher doses of dietary vitamin D2 or vitamin D3 (∼10- to 100-fold) than the 400 U/day used to prevent nutritional rickets.

and even high doses of vitamin D cannot correct this effect. In addition, as in the intestine, vitamin D promotes PO43− reabsorption in the kidney. The effects of vitamin D on PO43− reabsorption, like its effects on Ca2+, are less dramatic than those of PTH. Finally, 1,25-dihydroxyvitamin D directly inhibits the 1-hydroxylation of vitamin D.

Kidney

In the kidney, vitamin D appears to act synergistically with PTH to enhance Ca2+ reabsorption in the distal convoluted tubule (see Chapter 36). High-affinity Ca2+binding proteins, similar to those found in the intestinal mucosa, have been specifically localized to this region of the kidney. It appears that PTH is a more potent regulator of Ca2+ reabsorption than vitamin D is (Fig. 52-8). Indeed, parathyroidectomy increases the fractional excretion of Ca2+,

Bone

The actions of vitamin D on bone are complex and are the result of both indirect and direct actions. The overall effect of vitamin D replacement in animals with dietaryinduced vitamin D deficiency is to increase the flux of Ca2+ into bone. However, as we see later, these major effects of vitamin D on bone are indirect: the action of vitamin D on both the small intestine and the kidneys makes more Ca2+

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Section VIII • The Endocrine System

A INTESTINAL Ca Intestinal lumen

2+

ABSORPTION

Epithelial cell

Interstitial space

Blood

1,25-dehydroxyvitamin D [Ca2+] 100 nM

3 Na

+

2+

Free [Ca ] =~1 mM

Free [Ca2+] =1.2 mM

2+

Ca

mRNA Nucleus

Transcellular Protein synthesis Calbindin

Ca2+ 2+

Ca

Ca2+

B

H+

Calbindin Ca2+Calbindin Ca2+

Paracellular

INTESTINAL PHOSPHATE ABSORPTION

Intestinal lumen

Epithelial cell

Interstitial space

Blood

Nucleus 2 K+

mRNA 2 Na+

3 Na+ Protein synthesis

2– HPO4

H2PO4–

NaPi

2–

HPO4 or H2PO4–

?

Figure 52-10 Intestinal absorption of Ca2+ and PO43−. A, The small intestine absorbs Ca2+ by two mechanisms. The passive, paracellular absorption of Ca2+ occurs throughout the small intestine. This pathway predominates but is not under the control of vitamin D. The second mechanism—the active, transcellular absorption of Ca2+—occurs only in the duodenum. Ca2+ enters the cell across the apical membrane through a channel. Inside the cell, the Ca2+ is buffered by binding proteins, such as calbindin, and is also taken up into intracellular organelles, such as the endoplasmic reticulum. The enterocyte then extrudes Ca2+ across the basolateral membrane through a Ca2+ pump and an Na-Ca exchanger. Thus, the net effect is Ca2+ absorption. The active form of vitamin D—25-dihydroxyvitamin D—stimulates all three steps of transcellular Ca2+ absorption. B, Pi enters the enterocyte across the apical membrane through an Na/Pi (NaPi) cotransporter. Once inside the cell, the Pi is extruded across the basolateral membrane. Thus, the net effect is Pi absorption.

Chapter 52 • The Parathyroid Glands and Vitamin D

available to mineralize previously unmineralized osteoid. The direct effect of vitamin D on bone is to mobilize Ca2+ out of bone. Both osteoblasts and osteoclast precursor cells have VDRs. In response to vitamin D, osteoblasts produce certain proteins, including alkaline phosphatase, collagenase, and plasminogen activator. In addition, as noted earlier (Fig. 52-4), vitamin D and PTH promote the development of osteoclasts from precursor cells. Thus, because vitamin D directly increases the number of mature osteoclasts, supplying vitamin D to bone obtained from vitamin D–deficient animals in in vitro experiments mobilizes Ca2+ from bone into the medium. Additional evidence that vitamin D directly promotes bone resorption comes from experiments on rachitic animals who are maintained on a Ca2+-deficient diet. Treating these animals with vitamin D causes plasma [Ca2+] to rise, an indication of net bone resorption. At the same time, however, the elevated plasma [Ca2+] promotes the mineralization of previously unmineralized osteoid—at the expense of bone resorption from other sites. The direct effects of vitamin D on bone, which are to mobilize Ca2+, seem to be contrary to the overall effect of vitamin D on bone, which is to promote mineralization. These observations, as well as others, have led to the hypothesis, now generally accepted, that the antirachitic action of vitamin D is largely indirect. By enhancing the absorption of Ca2+ and PO43− from the intestine and by enhancing the reabsorption of Ca2+ and PO43− from the renal tubules, vitamin D raises the concentrations of both Ca2+ and PO43− in the blood and extracellular fluid. This increase in the Ca/PO ion product results in net bone mineralization. These indi-

rect effects overshadow the direct effect of vitamin D to increase bone mobilization. Ca2+ ingestion lowers levels of PTH and 1,25-dihydroxyvitamin D, whereas PO43ingestion raises levels of both PTH and 1,25-dihydroxyvitamin D Consider a situation in which a subject ingests a meal containing Ca2+. The rise in plasma [Ca2+] inhibits PTH secretion. The decline in PTH causes a decrease in the resorption of Ca2+ and phosphorus from bone, thus limiting the postprandial increase in plasma Ca2+ and PO43− levels. In addition, the decrease in PTH diminishes Ca2+ reabsorption in the kidney and thus facilitates a calciuric response. If dietary Ca2+ intake remains high, the lower PTH will result in decreased 1-hydroxylation of 25-hydroxyvitamin D, which will eventually diminish the fractional absorption of Ca2+ from the GI tract. If dietary Ca2+ intake is deficient, the body will attempt to restore Ca2+ toward normal by increasing plasma [PTH]. This response will help to mobilize Ca2+ from bone, to promote renal Ca2+ retention, and over time, to increase the level of 1,25-dihydroxyvitamin D, which will enhance gut absorption of Ca2+. If one ingests phosphorus much in excess of Ca2+ (e.g., after drinking several colas), the rise in plasma [PO43−] will lower plasma [Ca2+] because the increased plasma Ca/PO ion product will promote the deposition of mineral in bone. The resultant decrease in plasma [Ca2+] will, in turn, increase

Osteoporosis

A

pproximately 25 million Americans, mostly elderly women, are afflicted with osteoporosis, and between 1 and 2 million of these individuals experience a fracture related to osteoporosis every year. The cost in economic and human terms is immense. Hip fractures are responsible for much of the morbidity associated with osteoporosis, but even more concerning is the observation that as many as 20% of women with osteoporotic hip fractures die within 1 year of their fracture. The major risk factor for osteoporosis is the declining estrogen levels in aging women. Rarely, other endocrine disorders such as hyperthyroidism, hyperparathyroidism, and Cushing disease (hypercortisolism) are responsible. Other risk factors include inadequate dietary Ca2+ intake, alcoholism, cigarette smoking, and a sedentary lifestyle. Strategies to prevent the development of osteoporosis begin in the premenopausal years. High Ca2+ intake and a consistent program of weight-bearing exercises are widely recommended. Pharmacological agents are now available for preventing or at least retarding the development of osteoporosis or for treating the disease once it has become established. These agents can be broadly classified into two groups: antiresorptive drugs and agents that are able to stimulate bone formation. Among the former, estrogen is by far the most widely used therapy. It is most effective when started at the onset of meno-

pause, although it may offer benefits even in patients who are 20 or more years past menopause. Calcitonin is generally offered to women who cannot or are unwilling to take estrogen. However, it is expensive and must be given parenterally; an intranasal spray is also available. Another class of drugs, the bisphosphonates, is becoming popular. These drugs are powerful inhibitors of bone resorption, but some of the first agents of this class have also been found to impair mineralization. The newer bisphosphonates can be safely given in doses that decrease bone resorption without affecting mineralization. Among the drugs that can stimulate bone formation, vitamin D—often given as 1,25-dihydroxyvitamin D (calcitriol)—is combined with Ca2+ therapy to increase the fractional absorption of Ca2+ and to stimulate the activity of osteoblasts. PTH, recently available as an injectable treatment for osteoporosis, potently stimulates osteoblast formation and increases bone mass. PTH also appears to decrease the rate of vertebral fractures. Calcitriol and the bisphosphonates have also been used successfully to treat Paget disease of bone, a disorder characterized by localized regions of bone resorption and reactive sclerosis. The level of bone turnover can be extremely high. Although it remains asymptomatic in many individuals, the disease can cause pain, deformity, fractures, and vertigo and hearing loss if bony overgrowth occurs in the region of the eighth cranial nerve. The cause of Paget disease is not known.

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Section VIII • The Endocrine System

PTH secretion. This rise in PTH will provoke phosphaturia that will act to restore plasma [PO43−] toward normal while Ca2+ and PO43− are mobilized from bone by the action of PTH. Over longer periods, the action of PTH to modulate the 1-hydroxylation of 25-hydroxyvitamin D plays an increasingly important role in defending the plasma [Ca2+] by increasing intestinal Ca2+ absorption.

CALCITONIN AND OTHER HORMONES Calcitonin inhibits osteoclasts, but its effects are transitory Calcitonin is a 32–amino acid peptide hormone made by the clear or C cells of the thyroid gland. C cells (also called parafollicular cells) are derived from neural crest cells of the fifth branchial pouch, which in humans migrate into the evolving thyroid gland. Although it is located within the thyroid, calcitonin’s major, if not sole, biological action relates to the regulation of mineral metabolism and bone turnover. The incidental nature of its relationship with the major functions of the thyroid is emphasized by the finding that in many nonhuman species, C cells are found in a body called the ultimobranchial gland and not in the thyroid at all. Calcitonin is synthesized in the secretory pathway (see Chapter 2) by post-translational processing of a large procalcitonin. As illustrated in Figure 52-11, alternative splicing of the calcitonin gene product gives rise to several biologically active peptides. In the C cells, calcitonin is the only peptide made in biologically significant amounts. Within the central nervous system, calcitonin gene–related peptide (CGRP) is the principal gene product, and it appears to act as a neurotransmitter in peptidergic neurons (see Table 13-1). Calcitonin is stored in secretory vesicles within the C cells, and its release is triggered by raising the extracellular [Ca2+] to levels higher than normal. Conversely, lowering the extracellular [Ca2+] diminishes calcitonin secretion. The threshold [Ca2+] for enhancing calcitonin secretion is in the midphysiological range. In principle, this secretory profile would leave calcitonin well poised to regulate body Ca2+ homeostasis. The precise role for calcitonin in body Ca2+ homeostasis has been difficult to define. This difficulty was first apparent from the simple clinical observation that after complete thyroidectomy with removal of all calcitonin-secreting tissue, plasma [Ca2+] remains normal (provided the parathyroid glands are not injured). Conversely, patients with a rare calcitonin-secreting tumor of the C cells frequently have plasma calcitonin concentrations that are 50 to 100 times normal, yet they maintain normal plasma levels of Ca2+, vitamin D, and PTH. Nevertheless, several lines of evidence suggest that calcitonin does have biologically important actions. First, although calcitonin appears to have a minimal role in the minute-to-minute regulation of plasma [Ca2+] in humans, it does serve an important role in many nonmammalian species. This role is particularly clear for teleost fish. Faced with the relatively high [Ca2+] in sea water (and therefore in food), calcitonin, secreted in response to a rise in plasma [Ca2+], decreases bone resorp-

tion, thus returning the plasma [Ca2+] toward normal. Salmon calcitonin, which differs from human calcitonin in 14 of its 32 amino acid residues, is roughly 10-fold more potent on a molar basis in inhibiting osteoclast function than is the human hormone. The second line of evidence that calcitonin may have biologically important actions is the presence of calcitonin receptors. Like PTH receptors, the calcitonin receptor is a G protein–coupled receptor that, depending on the target cell, may activate either adenylyl cyclase or phospholipase C (see Chapter 3). Within bone, the osteoclast—which lacks PTH receptors—appears to be the principal target of calcitonin. Indeed, the presence of calcitonin receptors may be one of the most reliable methods of identifying osteoclasts. In the osteoclast, calcitonin appears to work by raising [cAMP]i and then presumably acting through one or more protein kinases. Calcitonin inhibits the resorptive activity of the osteoclast and slows the rate of bone turnover. It also appears to diminish osteocytic osteolysis, and this action— together with calcitonin’s effect on the osteoclast—is responsible for the hypocalcemic effect after the short-term administration of pharmacological doses of calcitonin. The hypocalcemic action of calcitonin is particularly effective in circumstances in which bone turnover is accelerated, as occurs in rapidly growing young animals and in human patients with hyperparathyroidism. The antiosteoclastic activity of calcitonin is also useful in treating Paget disease of bone (see the box titled Osteoporosis). However, within hours of exposure of osteoclasts to high concentrations of calcitonin, the antiresorptive action of calcitonin begins to wane. This “escape” from the hypocalcemic effect of calcitonin has limited the use of calcitonin in the clinical treatment of hypercalcemia. The transitory nature of the action of calcitonin appears partly to result from rapid downregulation of calcitonin receptors. In the kidney, calcitonin, like PTH, causes mild phosphaturia by inhibiting proximal tubule PO43− transport. Calcitonin also causes mild natriuresis and calciuresis. These actions may contribute to the acute hypocalcemic and hypophosphatemic actions of calcitonin. However, these renal effects are of short duration and do not appear to be important in the overall renal handling of Ca2+, PO43−, or Na+. Sex steroid hormones promote bone deposition, whereas glucocorticoids promote resorption Although PTH and 1,25-dihydroxyvitamin D are the principal hormones involved in modulating bone turnover, other hormones participate in this process. For example, the sex steroids testosterone and estradiol are needed for maintaining normal bone mass in male and female subjects, respectively. The decline in estradiol that occurs postmenopausally exposes women to the risk of osteoporosis, that is, a decreased mass of both cortical and trabecular bone caused by a decrease in bone matrix (see the box titled Osteoporosis). Osteoporosis is less common in men because their skeletal mass tends to be greater throughout adult life and because testosterone levels in men decline slowly as they age, unlike the abrupt menopausal decline of estradiol in women. Glucocorticoids also modulate bone mass. This action is most evident in circumstances of glucocorticoid excess,

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Chapter 52 • The Parathyroid Glands and Vitamin D

A

5´

Primary transcript(s)

B

C

D

Calcitonin CCP CGRP

3´

RNA processing

Mature mRNA 5´

A or B

C

Thyroid “C” cells D Calcitonin CCP

3´

5´

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Brain D

C

mRNA translation C

N

CGRP

Proteolytic processing

Mature peptides

C

Common region + 4AA

Common region + 6AA

N-terminal peptide (83 amino acids)

3´

mRNA translation

Calcitonin CCP

N

CGRP

N

+ Calcitonin N— S

Proteolytic processing N-terminal peptide (81 amino acids)

N

+ CGRP

—S

—Amide

+ CCP

+ C-terminal peptide

Figure 52-11 Synthesis of calcitonin and CGRP. A common primary RNA transcript gives rise to both calcitonin and CGRP. In the thyroid gland, C cells produce a mature mRNA that they translate to procalcitonin. They then process this precursor to produce an N-terminal peptide, calcitonin (a 32–amino acid peptide), and CCP. In the brain, neurons produce a different mature mRNA and a different “pro” hormone. They process the peptide to produce an N-terminal peptide, CGRP, and a C-terminal peptide.

which leads to osteoporosis, as suggested by the effects of glucocorticoids on the production of osteoprotegerin and osteoprotegerin ligand. The precise cellular mechanisms that mediate the action of androgens, estrogens, or glucocorticoids on bone have not been well defined. Despite the loss of bone that occurs with androgen or estrogen deficiency or glucocorticoid excess, in each case, the coupling of bone synthesis to degradation is qualitatively preserved. Synthesis of new bone continues to occur at sites of previous bone resorption, and no excess of unmineralized osteoid is present. Presumably, the decline in bone mass reflects a quantitative shift whereby the amount of new bone formed at any site is less than what was resorbed. Because this shift occurs at multiple sites, the result is a decline in overall bone mass.

PTH-related peptide, encoded by a gene that is entirely distinct from that for PTH, can cause hypercalcemia in certain malignancies Unlike PTH, which is synthesized exclusively by the parathyroid gland, a peptide called PTHrP appears to be made in many different normal and malignant tissues. The PTH 1R receptor in kidney and bone recognizes PTHrP with an affinity similar to that for intact PTH. PTHrP mimics each of the actions of PTH on kidney and bone. Thus, when present in sufficient concentrations, PTHrP causes hypercalcemia. PTHrP exists in three alternatively spliced isoforms of a single gene product. The gene encoding PTHrP is completely distinct from that for PTH. The similar actions of PTHrP and PTH arise from homology within the first 13 amino acids of PTHrP with native PTH. Only weak homol-

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Section VIII • The Endocrine System

ogy is seen between amino acids 14 and 34 (three amino acids are identical) and essentially no homology beyond amino acid 34. This situation is an unusual example of mimicry among peptides that are structurally quite diverse. The normal physiological function of PTHrP is still largely undefined. The lactating breast secretes PTHrP, and this hormone is present in very high concentrations in milk. PTHrP may promote the mobilization of Ca2+ from maternal bone during milk production. In nonlactating humans, the plasma PTHrP concentration is very low, and PTHrP does not appear to be involved in the day-to-day regulation of plasma [Ca2+]. It appears likely that under most circumstances, PTHrP acts in a paracrine or autocrine, rather than an endocrine, regulatory fashion. Many tumors are capable of manufacturing and secreting PTHrP, among them the following: squamous cell tumors of the lung, head, and neck; renal and bladder carcinomas; adenocarcinomas; and lymphomas. Patients with any of these tumors are subject to severe hypercalcemia of fairly abrupt onset. REFERENCES Books and Reviews Bringhurst FR, Demay MB, Kronenberg HM: Hormones and disorders of mineral metabolism. In Wilson JD, Foster DW, Kro-

nenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed, pp 1155-1209. Philadelphia: WB Saunders, 1998. DeLuca HF: The transformation of a vitamin into a hormone: The vitamin D story. Harvey Lect 1979-1980; 75:333-379. Habener JF, Rosenblatt M, Potts JT Jr: Parathyroid hormone: Biochemical aspects of biosynthesis, secretion, action, and metabolism. Physiol Rev 1984; 64:985-1053. Jones G, Strugnell SA, DeLuca HD: Current understanding of the molecular actions of vitamin D. Physiol Rev 1998; 78:1193-1231. Murer H, Forster I, Hilfiker H, et al: Cellular/molecular control of renal Na/Pi-cotransport. Kidney Int Suppl 1998; 65:2-10. Stein GS, Lian JB, Stein JL, et al: Transcriptional control of osteoblast growth and differentiation. Physiol Rev 1996; 76:593-629. Journal Articles Broadus AE, Mangin M, Ikeda K, et al: Humoral hypercalcemia of cancer: Identification of a novel parathyroid hormone–like peptide. N Engl J Med 1988; 319:556-563. Brown EM, Gamba G, Riccardi D, et al: Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 1993; 366:575-580. Burgess TL, Qian Y, Kaufman S, et al: The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 1999; 145:527-538.

SECTION

IX

T H E R E P R O D U CT I V E SYST E M Chapter 53

• Sexual Differentiation ...... 1113

Chapter 54 • The Male Reproductive System ...... 1128 Chapter 55

Chapter 56

• The Female Reproductive System ...... 1146

• Fertilization, Pregnancy, and Lactation ...... 1170

Chapter 57 • Fetal and Neonatal Physiology ...... 1193

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CHAPTER

53

S E X U A L D I F F E R E N T I AT I O N Ervin E. Jones

One of nature’s primary goals is perpetuation of the species. All living organisms must reproduce in some manner. Nature also favors those species that are able to produce diversification among members, an attribute critical to species survival as the nature of environmental (and other) stresses changes through time. One solution to this problem is sexual differentiation, that is, the evolution of two sexually dissimilar individuals belonging to the same species, one male and one female, and each equipped with its own specific attributes necessary for its particular contribution to the process of procreation. Each sex produces its own type of sex cell (gamete), and the union of male and female gametes generates species-specific progeny. In addition, mechanisms, some simple, some complex, have evolved to ensure the proximity and union of the sex cells (syngamy). Thus, within each species, the relevant sexual characteristics of each partner have adapted differently to achieve the most efficient union of these progenitor cells. These differences between the sexes of one species are called sexual dimorphism. For example, oviparous species such as frogs release their eggs into a liquid medium only when they are in relative proximity to sperm. As effective as this approach is, it also typifies the wastefulness of reproduction among higher species inasmuch as most gametes go unfertilized. Even among species that normally reproduce sexually, sexual dimorphism is not universal. For example, monoecious (i.e., hermaphroditic) species, such as cestodes and nematodes, have the capacity to produce both sperm and eggs. By definition, the ability to produce just one kind of gamete depends on sexually dimorphic differentiation. Throughout evolution, conservation and expression of genes involved in the perpetuation of a species have clearly followed a process of adaptation, which is an advantageous change in structure or function of an organ or tissue to meet the challenges of new conditions. Higher mammals normally have a single pair of sex chromosomes that are morphologically distinguishable from other chromosomes, the autosomes. Each of the sex chromosomes carries genetic information that determines the primary and secondary sexual characteristics of an individual, that is, whether the individual functions and appears as male or female. It has also become abundantly clear that genes determine gender,

sexual expression, and as a result, mechanisms and patterns of reproduction. The functional and spatial organization of all organ systems during development is genetically determined. Thus, the sex of the gonad is genetically programmed: Will a female gonad (ovary) or a male gonad (testis) develop? Although germ cells of the early embryonic gonad are totipotent, these cells develop into female gametes (ova) if the gonad becomes an ovary, but they develop into male gametes (sperm) if the gonad becomes a testis. These two anatomically and functionally distinct gonads determine either “maleness” or “femaleness” and dictate the development of both primary and secondary sexual characteristics. Endocrine and paracrine modulators that are specific for either the ovary or the testis are primarily responsible for female or male sexual differentiation and behavior and therefore the individual’s role in procreation.

GENETIC ASPECTS OF SEXUAL DIFFERENTIATION Meiosis, which occurs only in germ cells, gives rise to male and female gametes Mitosis is the only kind of cell division that occurs in somatic cells. Mitosis results in the formation of two identical daughter cells (Fig. 53-1A), each having the same number of chromosomes (i.e., 46 in humans) and same DNA content as the original cell. Mitosis is a continuum consisting of five phases: prophase, prometaphase, metaphase, anaphase, and telophase. One reason for the genetic identity of the two daughter cells is that no exchange of genetic material occurs between homologous chromosomes, so sister chromatids (i.e., the two copies of the same DNA on a chromosome) are identical. A second reason for the genetic identity is that the sister chromatids of each chromosome split, one going to each daughter cell during anaphase of the single mitotic division. Meiosis occurs only in germ cells. After having undergone several mitotic divisions, the germ cells (spermatogonia in males and oogonia in females)—still with a complement

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Section IX • The Reproductive System

A

MITOSIS

B

MALE MEIOSIS

C

Maternal homologue

Replication of DNA

Paternal homologue

Replication of DNA Prophase

Lining up of individual duplicated chromosomes on the spindle

Lining up of homologous pairs of duplicated chromosomes on the spindle Metaphase I Anaphase I Haploid number of duplicated chromosomes.

Cell division I

Secondary spermatocytes (2N DNA)

Separation of chromatids

Diploid oogonium (2N DNA)

Primary oocyte (4N DNA) Prophase I Primary oocyte– arrested (4N DNA)

Metaphase I Anaphase I Haploid number of duplicated chromosomes.

Cell division I 1st polar body

Telophase I Secondary oocyte (2N DNA)

Separation of chromatids

Anaphase II

Anaphase Meiotic division II

Separation of homologous, duplicated chromosomes

Telophase I

Metaphase Separation of chromatids

Pairing and recombination of homologous chromosomes

Prophase I

Lining up of homologous pairs of duplicated chromosomes on the spindle Separation of homologous, duplicated chromosomes

Diploid number of duplicated chromosomes.

Replication of DNA

Primary spermatocyte (4N DNA)

Pairing and recombination of homologous chromosomes

Meiotic division I

Cell division

Maternal homologue

Diploid spermatogonium (2N DNA)

Paternal homologue

FEMALE MEIOSIS

Cell division II

Anaphase II

Cell division II

Spermatids (1N DNA)

2nd polar body

Mature oocyte (1N DNA)

Figure 53-1 Mitosis and meiosis. A, In mitosis, the two daughter cells are genetically identical to the mother cell. B, In male meiosis, the four daughter cells are haploid. Cell division I produces both recombination (i.e., crossing over of genetic material between homologous chromosomes) and the reduction to the haploid number of chromosomes. Cell division II separates the chromatids of each chromosome, just as in mitosis. C, Female meiosis is similar to male meiosis. A major difference is that instead of producing four mature gametes, it produces only one mature gamete and two polar bodies.

of 2N DNA (N = 23)—undergo two meiotic divisions in both males (Fig. 53-1B) and females (Fig. 53-1C) to reduce the number of chromosomes from the diploid number (2N = 46) to the haploid number (N = 23). Because of this halving of the diploid number of chromosomes, meiosis is often referred to as a reduction division. Meiosis is a continuum composed of two phases: the homologous chromosomes separate during meiosis I, and the chromatids separate during meiosis II. At the start of meiosis I, the chromosomes duplicate so that the cells have 23 pairs of duplicated chromosomes (i.e., each chromosome has two chromatids)—or 4N DNA. During prophase of the first meiotic division, homol-

ogous pairs of chromosomes—22 pairs of autosomal chromosomes (autosomes) in addition to a pair of sex chromosomes—exchange genetic material. This genetic exchange is the phenomenon of crossing over that is responsible for the recombination of genetic material between maternal and paternal chromosomes. At the completion of meiosis I, the daughter cells have a haploid number (23) of duplicated, crossed-over chromosomes—or 2N DNA. During meiosis II, no additional duplication of DNA takes place. The chromatids simply separate so that each daughter receives a haploid number of unduplicated chromosomes— 1 N DNA. A major difference between male and female

Chapter 53 • Sexual Differentiation

gametogenesis is that one spermatogonium yields four spermatids (Fig. 53-1B), whereas one oogonium yields one mature oocyte and two polar bodies (Fig. 53-1C). The details emerge for spermatogenesis in Chapters 54 and 55. When two haploid gametes fuse, a mature spermatozoon from the father and a mature oocyte from the mother, a new individual is formed, a diploid zygote—2 N DNA. When an X- or Y-bearing sperm fertilizes an oocyte, it establishes the zygote’s genetic sex The sex chromosomes that the parents contribute to the offspring determine the genotypic sex of that individual. The genotypic sex determines the gonadal sex, which, in turn, determines the phenotypic sex that becomes fully established at puberty. Thus, sex-determining mechanisms established at fertilization direct all later ontogenetic processes (processes that lead to the development of an organism) involved in male-female differentiation. The process of fusion of a sperm and an ovum is referred to as fertilization, which is discussed in Chapter 55. Fusion of a sperm and egg—two haploid germ cells—results in a zygote, which is a diploid cell containing 46 chromosomes (Fig. 53-2), 22 pairs of somatic chromosomes (autosomes) and a single pair of sex chromosomes. In the female, these sex chromosomes are both X chromosomes, whereas males have one X and one Y chromosome.

1

2

3

4

5

6

Differentiation of the indifferent gonad into an ovary requires two intact X chromosomes

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22 X

X

If female

X

When the karyotypes of normal females and males are compared, two differences are apparent. First, among the 23 pairs of chromosomes in the female, 8 pairs—including the 2 X chromosomes—are of similar size, whereas males have only 71/2 such pairs. Second, instead of a second X chromosome, males have a Y chromosome that is small and acrocentric (i.e., the centromere is located at one end of the chromosome); this chromosome is the only such chromosome that is not present in the female. In the offspring, 23 of the chromosomes—including 1 of the sex chromosomes—are from the mother, and 23— including the other sex chromosome—come from the father. Thus, the potential offspring has a unique complement of chromosomes differing from those of both the mother and father. The ovum provided by the mother (XX) always provides an X chromosome. Because the male is the heterogenetic (XY) sex, half the spermatozoa are X bearing, whereas the other half are Y bearing. Thus, the type of sperm that fertilizes the ovum determines the sex of the zygote. Xbearing sperm produce XX zygotes that develop into females with a 46,XX karyotype, whereas Y-bearing sperm produce XY zygotes that develop into males with a 46,XY karyotype. The genetic sex of an individual is therefore determined at the time of fertilization. The Y chromosome appears to be the fundamental determinant of sexual development. When a Y chromosome is present, the individual develops as a male; when the Y chromosome is absent, the individual develops as a female. In embryos with abnormal sex chromosome complexes, the number of X chromosomes is apparently of little significance.

Y

If male

Figure 53-2 Normal human karyotype. The normal human has 22 pairs of autosomal chromosomes (autosomes) as well as a pair of sex chromosomes. Females have two X chromosomes, whereas males have one X and one Y chromosome.

The primary sex organs of an individual are the gonads. Gene complexes on sex chromosomes determine whether the indifferent gonad differentiates into a testis or an ovary. As discussed later, the Y chromosome exerts a powerful testis-determining effect on the indifferent gonad. The primary sex cords differentiate into seminiferous tubules under the influence of the Y chromosome. In the absence of a Y chromosome, the indifferent gonad develops into an ovary. The differentiated gonads, in turn, determine the sexual differentiation of the genital ducts and external genitalia. The indifferent gonad is composed of an outer cortex and an inner medulla. In embryos with an XX sex chromosome complement, the cortex develops into an ovary, and the medulla regresses. Conversely, in embryos with an XY chromosome complex, the medulla differentiates into a testis, and the cortex regresses. Loss of a sex chromosome causes abnormal gonadal differentiation or gonadal dysgenesis. Loss of one of the X chromosomes of the XX pair results in an individual with an XO sex chromosome constitution and ovarian dysgenesis (see the box titled Gonadal Dysgenesis). Thus, two X chromosomes are necessary for normal ovarian development. In an XO individual, the gonads appear only as streaks on the pelvic sidewall in the adult. Because these streak gonads of XO individuals may contain germ cells, germ cell migration apparently can occur during development. The absence of only some genetic material from one

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Gonadal Dysgenesis

T

he best known example of gonadal dysgenesis is a syndrome referred to as Turner syndrome, a disorder of the female sex characterized by short stature, primary amenorrhea, sexual infantilism, and certain other congenital abnormalities. The cells in these individuals have a total number of 45 chromosomes and a normal karyotype, except they lack a second sex chromosome. The karyotype is 45,XO. Examination of the gonads of individuals with Turner syndrome reveals so-called streak gonads, which are firm, flat, glistening streaks lying below the fallopian tubes. These glands generally do not show evidence of either germinal or secretory elements but, instead, are largely composed of connective tissue arranged in whorls suggestive of ovarian stroma. Individuals with Turner syndrome have normal female differentiation of both the internal and external genitalia, although these genitalia are usually small and immature for the patient’s age. Partial deletion of the X chromosome may also result in the full Turner phenotype, particularly if the entire short arm of the X chromosome is missing. The so-called ring chromosome is an example of an abnormality of the second sex chromosome. A ring chromosome is a small round or oval chromosome that often appears as a single black dot without a central hole. It forms as a result of a deletion and subsequent joining of the two free ends of the chromosome. Formation of a ring chromosome is, in effect, a deletion of the X chromosome and produces the same characteristics as gonadal dysgenesis. The aforementioned defects result from disordered meiosis. A central genetic lesion is an abnormality of the second sex chromosome in some or all of the cells of the person. In at least half of affected individuals, this abnormality appears to be total absence of the second X chromosome. In others, the lesion is structural, as shown by the presence of ring chromosomes that have lost some genetic material. In at least a third of cases, these lesions appear as parts of a mosaicism; that is, some of the germ cells carry the aberrant or absent chromosome, whereas the rest are normal.

X chromosome in an XX individual—for example, as may occur as a result of breakage or deletion—may also cause abnormal sexual differentiation. The testis-determining gene is located on the Y chromosome Investigators have clearly established that a Y chromosome (Fig. 53-3A), with rare exception (see later), is necessary for normal testicular development. Thus, it stands to reason that the gene that determines organogenesis of the testis is normally located on the Y chromosome. This so-called testisdetermining factor (TDF) has been mapped to the short arm of the Y chromosome and, indeed, turns out to be a single gene called SRY (for Sex-determining Region Y). The SRY gene encodes a transcription factor that belongs to the high-mobility group (HMG) superfamily of transcription

A

THE Y CHROMOSOME

B TRANSLOCATION OF TDF REGION TO THE X CHROMOSOME

SRY/TDF 11.3 11.2 p11.1

TDF

TDF

q11.1

TDF Y

11.21

X

Y

X

Equal recombination 11.22

11.23

TDF 12

Y TDF Y

Y chromosome

X X Unequal recombination

Figure 53-3 The location of the testis-determining region of the Y chromosome and an example of translocation. A, The Y chromosome is much smaller than the X chromosome. Giemsa staining of the chromosome results in alternating light and dark bands, some of which are shown here. The short or p arm of the Y chromosome is located above the centromere, whereas the long or q arm is located below it. The numbers to the left of the chromosome indicate the position of bands. The TDF is the SRY gene. B, Crossing-over events between normal X and Y chromosomes of the father can generate an X chromatid that contains a substantial portion of the TDF region and a Y chromatid that lacks its TDF. The figure shows both an equal and an unequal recombination event. If a sperm cell bearing an X chromosome with a translocated TDF fertilizes an ovum, the result is a male with a 46,XX karyotype, because one of the X chromosomes contains the TDF. Conversely, if the sperm cell carries a Y chromosome lacking its TDF, the result can be a 46,XY individual that appears to be female.

factors. The family to which SRY belongs is evolutionarily ancient. One portion of SRY, the 80–amino acid HMG box, which actually binds to the DNA—is highly conserved among members of the family. Rarely, the TDF may also be found translocated on other chromosomes. One example is an XX male (Fig. 53-3B), an individual whose sex chromosome complement is XX but whose phenotype is male. During normal male meiosis, human X and Y chromosomes pair and recombine at the distal end of their short arms. It appears that most XX males arise as a result of an aberrant exchange of genetic material between X and Y chromosomes in the father; in such cases, the TDF is transferred from a Y chromatid to an X chromatid. If the sperm cell that fertilizes the ovum contains such an X chromosome with a TDF, the resultant individual will be an XX male.

Chapter 53 • Sexual Differentiation

Endocrine and paracrine messengers modulate phenotypic differentiation Just as an individual’s genes determine whether the indifferent gonad develops into an ovary or a testis, so does the sex of the gonad dictate the gonad’s endocrine and paracrine functions. Normally, chemical messengers—both endocrine and paracrine—produced by the gonad determine the primary and secondary sexual phenotypes of the individual. However, if the gonads fail to produce the proper messengers, if other organs (e.g., the adrenal glands) produce abnormal levels of sex steroids, or if the mother is exposed to chemical agents (e.g., synthetic progestins, testosterone) during pregnancy, sexual development of the fetus may deviate from that programmed by the genotype. Therefore, genetic determination of sexual differentiation is not irrevocable; numerous internal and external influences during development may modify or completely reverse the phenotype of the individual, whatever the genotypic sex. An abnormal chemical environment can affect sexual differentiation at the level of either the genital ducts or the development of secondary sex characteristics. Higher vertebrates, including humans, have evolved highly elaborate systems of glands and ducts for transporting gametes. This system of glands and conduits collectively comprises the accessory sex organs. Together with the gonads, these accessory sex organs constitute the primary sex characteristics. The gonads produce and secrete hormones that condition and develop these accessory sex organs and, to a large extent, influence phenotypic sexual differentiation; that is, they induce either “maleness” or “femaleness” and influence the psychobiological phenomena involved in sex behavior. Secondary sex characteristics are external specializations that are not essential for the production and movement of gametes; instead, they are primarily concerned with sex behavior and with the birth and nutrition of offspring. Examples include the development of pubic hair and breasts. Not only do the sex steroids produced by the gonads affect the accessory sex organs, but they also modulate the physiological state of the secondary sex characteristics toward “maleness” in the case of the testes and “femaleness” in the case of the ovaries.

DIFFERENTIATION OF THE GONADS After migration of germ cells from the yolk sac, the primordial gonad develops into either a testis or an ovary The primordial germ cells do not originate in the gonad; instead, they migrate to the gonad from the yolk sac along the mesentery of the hindgut at about the fifth week of embryo development (Fig. 53-4A, B). The primordial germ cells of humans are first found in the endodermal epithelium of the yolk sac in the vicinity of the allantoic stalk, and from there the germ cells migrate into the adjoining mesenchyme. They eventually take up their position embedded in the gonadal ridges. Gonadal development fails to progress normally in the absence of germ cells. Thus, any event that

Discordance Between Genotype and Gonadal Phenotype

A

group of individuals has been reported to have no recognizable Y chromosome but do have testes. Some of these individuals are 46,XX and are true hermaphrodites; that is, they possess both male and female sex organs. Other patients have mixed gonadal dysgenesis—a testis in addition to a streak ovary—and a 45,XO karyotype. Some are pseudohermaphrodites; that is, affected individuals have only one type of gonadal tissue, but morphological characteristics of both sexes. All these patterns can result from mosaicisms (e.g., 46,XY/46,XX) or from translocation of the SRY gene (Fig. 53-3B)—which normally resides on the Y chromosome—to either an X chromosome or an autosome. A “normal” testis in the absence of a Y chromosome has never been reported. Another group of individuals with a sex chromosome complex of 46,XY has pure gonadal dysgenesis—streak gonads, but no somatic features of XO. In the past, investigators assumed that these individuals possessed an abnormal Y chromosome. Perhaps the SRY gene is absent, or its expression is somehow blocked.

interferes with germ cell migration may cause abnormal gonadal differentiation. The gonad forms from a portion of the coelomic epithelium, the underlying mesenchyme, and the primordial germ cells that migrate from the yolk sac. At 5 weeks’ development, a thickened area of coelomic epithelium develops on the medial aspect of the urogenital ridge as a result of proliferation of both the coelomic epithelium and cells of the underlying mesenchyme. This prominence, which forms on the medial aspect of the mesonephros, is known as the gonadal ridge (Fig. 53-4B, C). Migration of the primordial germ cells to the gonadal ridge establishes the anlagen for the primordial gonad. The primordial gonad at this early stage of development consists of both a peripheral cortex and a central medulla (Fig. 534C) and has the capacity to develop into either an ovary or a testis. As discussed later, the cortex and medulla have different fates in the male and female. The germ cells themselves seem to direct the sexual development of the gonad. An embryo with an XY chromosome complement undergoes development of the medullary portion of the gonad to become a testis, and the cortex regresses. Conversely, XX germ cells appear to stimulate development of the cortex of the early gonad to become an ovary, and the medulla regresses. Development of the Primitive Testis

In male embryos, primordial germ cells migrate from the cortex of the gonad, in which they were originally embedded, into the primitive sex cords of the medulla (Fig. 53-4D). The primitive sex cords become hollowed out and develop into the seminiferous tubules. The primordial germ cells give rise to spermatogonia, the first cells in the pathway to mature sperm (see Chapter 54). The sex cords give rise to the Sertoli cells.

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A

EARLY EMBRYO (FIFTH WEEK)

B

MIGRATION OF GERM CELLS TO GONADAL RIDGES Gonadal ridge

Yolk sac

Hindgut

Heart

Gonadal ridge

Foregut Hindgut Primordial germ cells

Allantois

C

INDIFFERENT OR PRIMORDIAL GONAD Sympathetic ganglion

Aorta

Suprarenal cortex

Suprarenal medulla D

DEVELOPING TESTIS

Mesonephros Rete testis Medulla

E DEVELOPING OVARY

Rudimentary tunica albuginea

Mesonephros

Mesonephric tubule

Migrating primordial germ cells

Mesonephric (wolffian) duct Cortex

Cortex

Medulla

Tunica albuginea

Primitive sex cords hollow out and form seminiferous tubules.

Cortex

Secondary sex cords

Paramesonephric ¨ (mullerian) duct

Medulla

Urogenital ridge Gonadal Hindgut ridge

Urogenital Primary mesentery sex cords Coelom

Figure 53-4 The early gonad and germ cell migration. A, The primordial germ cells originate in the endodermal endothelium of the yolk sac. B, The primordial germ cells migrate along the mesentery of the hindgut and reach the region of the urogenital ridge called the gonadal ridge. C, The indifferent gonad consists of an outer cortex and an inner medulla. D, The testis develops from the medulla of the indifferent gonad; the cortex regresses. E, The ovary develops from the cortex of the indifferent gonad; the medulla regresses.

The rete testis is a system of thin, interconnected tubules that develop in the dorsal part of the gonad; they drain the seminiferous tubules. The contents of the rete testis flow into the efferent ductules, which—as discussed later—develop from the adjoining tubules of the mesonephros. These tubular structures establish a pathway from the male gonad to the mesonephric duct, which—as also discussed later— evolves into the outlet for sperm. The cortex of the primordial gonad is a thin epithelial layer covering the coelomic surface of the testis.

Development of the Primitive Ovary

In female embryos, the medulla of the gonad regresses, the primary sex cords are resorbed, and the interior of the gonad is filled with a loose mesenchyme that is highly permeated by blood vessels. However, the cortex greatly increases in thickness, and the primordial germ cells remain embedded within it (Fig. 53-4E). Masses of cortical cells are split up on the inner surface of the cortex into groups and strands of cells, or secondary sex cords, surrounding one or several primordial germ cells, or oogonia, during growth of the gonad. These

Chapter 53 • Sexual Differentiation

germ cells become primary oocytes that enter the initial stages of oogenesis. The embryonic gonad determines the development of the internal genitalia and the external sexual phenotype As discussed in the next section of this chapter, several products of the developing male or female gonad have profound effects on differentiation of the internal sex ducts, as well as on development of the external genitalia. Thus, just as genetic sex determines the gonadal phenotype, so also products of the gonad primarily determine the sexual phenotype. Androgens produced by the developing testis cause development of the mesonephric or wolffian ducts. The paramesonephric or müllerian ducts degenerate in the male under the influence of antimüllerian hormone (AMH). In the female embryo, the müllerian ducts develop, whereas the wolffian ducts degenerate. In the absence of a functioning testis, the left and right müllerian ducts develop according to the female phenotype, that is, as the fallopian tubes (oviducts), the uterus, and the upper third of the vagina (see the next section). Just as the absence of male hormones or androgens causes the internal genital ducts to follow a female pattern of differentiation, so also the absence of androgens causes the external genital development to be female. Conversely, testosterone and dihydrotestosterone (DHT) cause masculinization of the external genitalia (see the later section on differentiation of the external genitalia).

DIFFERENTIATION OF THE INTERNAL GENITAL DUCTS The genital ducts are an essential part of the genital organs and are the means by which the sex cells—ova and spermatozoa—are transported to a location where fertilization occurs. As discussed in the previous section, embryos of both sexes have a double set of genital ducts (Fig. 53-5A): the mesonephric or wolffian ducts, which in males develop into the vas deferens and other structures; and the paramesonephric or müllerian ducts, which in females become the oviducts, uterus, and upper third of the vagina. During mammalian development, three sets of kidneys develop, two of which are transient. The pronephric kidney, which develops first, is so rudimentary that it never functions. However, the duct that connects the pronephric kidney to the urogenital sinus—the pronephric duct—eventually serves the same purpose for the second kidney, the mesonephric kidney or mesonephros, as it develops embryologically. Unlike the pronephric kidney, the mesonephros functions transiently as a kidney. It has glomeruli and renal tubules; these tubules empty into the mesonephric duct (Fig. 53-5A), which, in turn, carries fluid to the urogenital sinus. As discussed later, the mesonephros and its mesonephric duct will—depending on the sex of the developing embryo— either degenerate or develop into other reproductive structures. In addition to the mesonephric ducts, a second pair of genital ducts, the paramesonephric or müllerian ducts, will develop as invaginations of the coelomic epithelium on the lateral aspects of the mesonephros. These parameso-

nephric ducts run caudally and parallel to the mesonephric ducts. In the caudal region, they cross ventral to the mesonephric ducts and fuse to form a cylindrical structure, the uterovaginal canal. The third or metanephric kidney becomes the permanent mammalian kidney. Its excretory duct is the ureter. In males, the mesonephros becomes the epididymis, and the mesonephric (wolffian) ducts become the vas deferens, seminal vesicles, and ejaculatory duct During development, the mesonephros ceases to be an excretory organ in both sexes. The only part that remains functional is the portion—in males—that develops into the most proximal end of the epididymis, the efferent ductules. As the mesonephros degenerates, persisting mesonephric tubules develop into many parallel efferent ductules that connect the upstream rete testis to the head of the epididymis, which serves as a reservoir for sperm. The mesonephric ducts develop into the channels through which the spermatozoa exit the testes (Fig. 53-5B). The most proximal portion of the mesonephric duct becomes the head, the body, and the tail of the epididymis. The tail of the epididymis connects to the vas deferens, which also arises from the wolffian duct. A lateral outgrowth from the distal end of the mesonephric duct forms the seminal vesicle. The portion of the mesonephric duct between the seminal vesicle and the point where the mesonephric duct joins the urethra becomes the ejaculatory duct. At about the level where the ejaculatory duct joins with the urethra, multiple outgrowths of the urethra grow into the underlying mesenchyme and form the prostate gland. The mesenchyme of the prostate gives rise to the stroma of the prostate, whereas the prostatic glands develop from endodermal cells of the prostatic urethra. In females, the paramesonephric (müllerian) ducts become the fallopian tubes, the uterus, and the upper third of the vagina In female embryos, both the mesonephros and the wolffian (mesonephric) ducts degenerate. The müllerian ducts establish three functional regions (Fig. 53-5C). The cranial portions of the müllerian ducts remain separate and give rise to the fallopian tubes. The upper end of the duct gains a fringe, which will become the fimbria, by adding a series of minor pits or müllerian tunnels. The midportions of the left and right müllerian ducts fuse and give rise to the fundus and corpus of the uterus. The most distal portions of the bilateral müllerian ducts had previously fused as the uterovaginal primordium. The cranial portion of this common tube gives rise to the cervix and remains the longest portion of the uterus until puberty. The caudal portion of this common tube becomes the upper third of the vagina. In males, development of the wolffian ducts requires testosterone As already noted, the developing embryo has two precursor duct systems (Fig. 53-6A). In a normal male embryo (Fig. 53-6B), the wolffian ducts develop, whereas the müllerian

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A

SEXUALLY INDIFFERENT DUCT SYSTEM Gonads

Paramesonephric ¨ (mullerian) duct

Mesonephros Mesonephric (wolffian) duct Urogenital sinus (developing bladder)

Metanephros

Uterovaginal primordium

Ureter

B

C

MALE DEVELOPMENT

Epididymis

FEMALE DEVELOPMENT

Testes

Wolffian duct (vas deferens)

Ovaries

Metanephric kidneys

Urinary bladder

Ureters

Urinary bladder

Degenerated ¨ mullerian duct

Seminal vesicle

Degenerated wolffian duct

¨ Mullerian duct (oviduct) Uterus

Ejaculatory duct

Prostate

Urethra

Vagina

Urethra

Figure 53-5 Transformation of the genital ducts. A, At the time the gonad is still indifferent, it is closely associated with the mesonephros, as well as the excretory duct (mesonephric or wolffian duct) that leads from the mesonephros to the urogenital sinus. Parallel to the wolffian ducts are the paramesonephric or müllerian ducts, which merge caudally to form the uterovaginal primordium. B, In males, the mesonephros develops into the epididymis. The wolffian duct develops into the vas deferens, seminal vesicles, and ejaculatory duct. The müllerian ducts degenerate. C, In females, the mesonephros and the wolffian (mesonephric) ducts degenerate. The paramesonephric or müllerian ducts develop into the fallopian tubes, the uterus, the cervix, and the upper one third of the vagina.

ducts regress. In a normal female embryo (Fig. 53-6C), the müllerian ducts develop, whereas the wolffian ducts regress. It appears that maturation of one of these systems and degeneration of the other depend on local factors produced by the developing gonad. A classic series of experiments performed by Alfred Jost in 1953 revealed that masculine genital development requires factors produced by fetal testicular tissue. The experimental approach was to castrate rabbit fetuses at various stages of development and allow the pregnancies to continue. Castrating a male fetus before maturation of the wolffian ducts caused the müllerian ducts to persist (i.e., fail to regress) and

induced the development of female internal and external genitalia (Fig. 53-6D). However, castrating female fetuses at a comparable stage in development had no appreciable effect, and müllerian development continued along normal female lines (Fig. 53-6E). Thus, although normal male development requires the testes, development of the fallopian tubes and uterus does not require the ovaries. Unilateral removal of the testis resulted in female duct development on the same (ipsilateral) side as the castration, but virilization of the external genitalia proceeded normally (Fig. 53-6F). Removing both testes—and administering testosterone—resulted in essentially normal development of

Chapter 53 • Sexual Differentiation

A

UNDIFFERENTIATED

B

NORMAL MALE

F

MALE: UNILATERAL EARLY CASTRATE

C NORMAL FEMALE

D

MALE: EARLY CASTRATE

H

TESTOSTERONETREATED FEMALE

Wolffian ducts Müllerian ducts

E

FEMALE: EARLY CASTRATE

G

MALE: EARLY CASTRATE AND TESTOSTERONE

Figure 53-6 Jost experiments. A, Very early in development, both the wolffian (mesonephric) and the müllerian (paramesonephric) ducts are present in parallel. B, The wolffian duct develops into the vas deferens, the seminal vesicles, and the ejaculatory duct. The müllerian ducts degenerate. C, The paramesonephric or müllerian ducts develop into the fallopian tubes, the uterus, the cervix, and the upper one third of the vagina. The wolffian (mesonephric) ducts degenerate. D, Bilateral removal of the testes deprives the embryo of both AMH (also known as MIS) and testosterone, which are both testicular products. As a result of the absence of AMH, the müllerian ducts follow the female pattern of development. In the absence of testosterone, the wolffian ducts degenerate. Thus, the genetically male fetus develops female internal and external genitalia. E, After bilateral removal of the ovaries, müllerian development continues along normal female lines. Thus, the ovary is not required for female duct development. F, Unilateral removal of the testis results in female duct development on the same (ipsilateral) side as the castration. Duct development follows the male pattern on the side with the remaining testis. Virilization of the external genitalia proceeds normally. G, In the absence of both testes, administering testosterone preserves development of the wolffian ducts. However, because of the absence of AMH—which is a product of the testis—no müllerian regression occurs. H, In the presence of both ovaries, the testosterone promotes development of the wolffian ducts. Because there are no testes—and therefore no AMH—the müllerian ducts develop normally.

the wolffian ducts, but no müllerian regression was seen (Fig. 53-6G). Thus, although testosterone can support wolffian development, it is unable to cause müllerian regression. It became clear that a testicular product other than testosterone is necessary for regression of the müllerian ducts. Thus, one would predict that treating a normal female with testosterone would lead to preservation of the wolffian ducts, as well as the müllerian ducts. This pattern of dual ducts is indeed observed (Fig. 53-6H).

In males, antimüllerian hormone causes regression of the müllerian ducts After Jost, other investigators performed experiments indicating that the Sertoli cells of the testis produce a nonsteroid macromolecule—AMH or müllerian-inhibiting substance (MIS)—that causes müllerian degeneration in the male fetus. AMH, a growth-inhibitory glycoprotein, is a member of the transforming growth factor β (TGF-β) superfamily of glycoproteins involved in the regulation of growth and dif-

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Section IX • The Reproductive System

ferentiation (see Chapter 3). Besides TGF-β, this gene superfamily includes the inhibins and activins (see Chapter 55). The proteins produced by this gene family are all synthesized as dimeric precursors and undergo post-translational processing for activation. AMH is glycosylated and is secreted as a 140-kDa dimer consisting of two identical disulfidelinked subunits. The antimitogenic activity and müllerian duct bioactivity of AMH reside primarily in its C-terminal domain. The human AMH gene is located on chromosome 19, and AMH is one of the earliest sexually dimorphic genes expressed during development. The transcription factor SRY, which represents the TDF, may be involved in initiating the transcription of AMH. The sequential timing of SRY and AMH expression is consistent with activation of AMH by SRY, a series of events that may control sexual dimorphism. Although the exact mechanism of AMH action has not been completely clarified, it is thought to involve receptormediated dephosphorylation. AMH appears to act directly on mesenchymal cells of the müllerian duct and, indirectly through the mesenchyme, on müllerian duct epithelial cells. AMH binding has been localized to the mesenchymal cells surrounding the müllerian duct and to the developing oocytes in preantral follicles. During embryogenesis in males, AMH—which is secreted by the Sertoli cells in the testis—causes involution of the müllerian ducts, whereas testosterone—which is secreted by the Leydig cells of the testis—stimulates differentiation of the wolffian ducts. In females, the müllerian ducts differentiate spontaneously in the absence of AMH, and the wolffian ducts involute spontaneously in the absence of testosterone.

DIFFERENTIATION OF THE EXTERNAL GENITALIA The urogenital sinus develops into the urinary bladder, the urethra, and, in females, the vestibule of the vagina Early in embryologic development, a tubular structure called the cloaca is the common termination of the urogenital and gastrointestinal systems (Fig. 53-7A). The cloacal membrane separates the cloaca from the amniotic fluid. Eventually, a wedge of mesenchymal tissue separates the cloaca into a dorsal and a ventral cavity (Fig. 53-7B). The dorsal cavity is the rectum. The ventral compartment is the urogenital sinus. Both the wolffian and the müllerian ducts empty into this urogenital sinus (Fig. 53-5A). The urogenital sinus can be divided into three regions: the vesicle part, the pelvic part, and the phallic part. In the male (Fig. 53-7C), the vesicle part becomes the urinary bladder, the pelvic part becomes the prostatic part of the urethra, and the phallic part becomes the initial portion of the penile urethra. In the female (Fig. 53-7D), the vesicle part of the urogenital sinus also develops into the urinary bladder. The pelvic part becomes the entire female urethra. The phallic portion of the urogenital sinus develops into the vestibule of the

vagina; into this vestibule empty the urethra, the vagina, and the ducts of the greater vestibular glands of Bartholin. As noted earlier, fusion of the caudal portion of the müllerian ducts produces the uterovaginal primordium. As this primordium contacts the dorsal wall of the urogenital sinus, it induces the development of paired sinovaginal bulbs, which grow into the urogenital sinus and then fuse to form a solid core of tissue called the vaginal plate. This plate grows caudally to the phallic portion of the urogenital sinus. Resorption of the center of the vaginal plate creates the vaginal lumen. The remaining cells of the vaginal plate appear to form the vaginal epithelium. During early fetal development, a thin membrane, the hymen, separates the lumen of the vagina from the cavity of the urogenital sinus. Usually, the hymen partially opens during the prenatal period. Occasionally, the hymenal membrane persists completely, does not allow escape of the menstrual effluvium at menarche, and gives rise to a condition known clinically as hematocolpos. In the male, the vagina disappears when the müllerian ducts are resorbed. However, remnants of the vagina sometimes persist as a prostatic utricle. The external genitalia of both sexes develop from common anlagen Although anatomically separate precursors give rise to the internal genitalia, common anlagen give rise to the external genitalia of the two sexes (Fig. 53-8A). Knowledge of the common origins of the external genitalia during normal development facilitates understanding of the ambiguities of abnormal sexual development. The genital tubercle (Fig. 53-8B) develops during the fourth week on the ventral side of the cloacal membrane. As a result of elongation of the genital tubercle, a phallus develops in both sexes. The genital tubercle of the primitive embryo develops into the glans penis in the male (Fig. 538C) and the clitoris in the female (Fig. 53-8D). Until about the end of the first trimester of pregnancy, the external genitalia of males and females are anatomically difficult to distinguish. The phallus undergoes rapid growth in the female initially, but its growth slows, and in the absence of androgens, the phallus becomes the relatively small clitoris in the female. The paired urogenital folds give rise to the ventral aspect of the penis in the male (Fig. 53-8C) and the labia minora in the female (Fig. 53-8D). After formation of the urogenital opening, a groove—the urethral groove—forms on the ventral side of the phallus; this groove is continuous with the urogenital opening. The bilateral urogenital folds fuse over the urethral groove to form an enclosed spongy urethra; the line of fusion is the penile raphe. As the urogenital folds fuse to form the ventral covering of the penis, they do so in a posterior-to-anterior direction, thus displacing the urethral orifice to the tip of the penis. Elongation of the genital tubercle and fusion of the genital folds occur at the 12th to the 14th week of gestation. However, in the female, the urogenital folds normally remain separate as the labia minora. In the male, the genital or labioscrotal swellings fuse to give rise to the scrotum. In females, however, the labioscrotal swellings fuse anteriorly to give rise to the mons pubis and

Chapter 53 • Sexual Differentiation A

PARTITIONING OF CLOACA Allantois

Vesicle part

Genital tubercle

Pelvic part Phallic part

Urogenital sinus

Rectum B

INDIFFERENT UROGENITAL SINUS Mesonephric duct Mesonephros Metanephros Urinary bladder Urorectal septum Ureter

Rectum

C

MALE DEVELOPMENT

Regressed müllerian duct

D

FEMALE DEVELOPMENT

Bladder

Bladder

Vaginal plate

Vaginal plate Rectum

Rectum

Prostatic utricle

Perineal urethra

Uterus

Vagina

Figure 53-7 Differentiation of the urogenital sinus. A, The urorectal septum begins to separate the rectum (dorsal) from the urogenital sinus (ventral). The urogenital sinus is divided into a vesicle (i.e., urinary bladder) part, a pelvic part, and a phallic part. The common space into which the rectum and urogenital sinus empty—the cloaca—is closed by the cloacal membrane. B, At this stage, the rectum and the urogenital sinus are fully separated. The urogenital membrane separates the urogenital sinus from the outside of the embryo. C, The male has a common opening for the reproductive and urinary tracts. The prostatic utricle, which is the male homologue of the vagina, empties into the prostatic urethra. D, A solid core of tissue called the vaginal plate grows caudally from the posterior wall of the urogenital sinus. The lumen of the vagina forms as the center of this plate resorbs. Thus, the female has separate openings for the urinary and reproductive systems.

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A

Figure 53-8 Development of the external genitalia. A, Genital folds and genital swellings surround the cloacal membrane. B, Early in the fourth week of development—in both sexes—the genital tubercle begins to enlarge to form the phallus. C, In males, the genital tubercle becomes the glans penis. The urogenital folds fuse to form the shaft of the penis. The labioscrotal swellings become the scrotum. D, In females, the genital tubercle becomes the clitoris. The urogenital folds remain separate as the labia minora. The labioscrotal swellings become the labia majora where they remain unfused. Ventrally, the labioscrotal swellings fuse to form the mons pubis. Dorsally they fuse to form the posterior labial commissure.

EARLY INDIFFERENT STAGE

Genital fold Genital swelling B

LATE INDIFFERENT STAGE

Cloacal membrane

Genital tubercle

posteriorly to form the posterior labial commissure. The unfused labioscrotal swellings give rise to the labia majora.

C

MALE DEVELOPMENT

D FEMALE DEVELOPMENT

ENDOCRINE AND PARACRINE CONTROL MECHANISMS IN SEXUAL DIFFERENTIATION

Glans Fused urogenital folds Urethral groove

The SRY gene triggers development of the testis, which makes the androgens and AMH necessary for male sexual differentiation

Anus

I already noted that the female pattern of sexual differentiation occurs in the absence of testes. In fact, the embryo follows the female pattern even in the absence of all gonadal tissue. It thus appears that the male pattern of sexual differentiation is directed by endocrine and paracrine control mechanisms. Therefore, I successively examine the control of testicular development, the development of the male internal system of genital ducts, and the development of the male urogenital sinus and external genitalia. Testicular development proceeds in the presence of TDF—the gene product of the SRY gene—before 9 weeks of gestation. If TDF is not present or if TDF is present only after the critical window of 9 weeks has passed, an ovary will develop instead of a testis. Further male-pattern sexual differentiation depends on the presence of three hormones, testosterone, DHT, and AMH. The testis directly produces both testosterone and AMH. Peripheral tissues convert testosterone to DHT.

Prepuce

Testosterone Production

Fused labioscrotal swellings Perineum Anus

Glans penis Glans clitoridis Urethral groove Labium minus Scrotum Labium majus Posterior labial commissure

Mons pubis Urethral orifice Clitoris Body of penis Scrotum Urethral orifice Vestibule of the vagina Hymen Posterior labial commissure Scrotal raphe (line of fusion of labioscrotal swellings)

The primary sex steroid produced by both the fetal and the postnatal testis is testosterone. The testes also produce DHT and estradiol, although in lesser amounts. The Leydig cells are the source of sex steroid production in the testes. The Leydig cells differentiate from mesenchymal tissue that surround the testicular cords. This tissue makes up more than half the testicular volume by 60 days of gestation. The early increase in the number of Leydig cells and secretion of testosterone in humans could depend on either maternal human chorionic gonadotropin (hCG) or fetal luteinizing hormone (LH). The human testis has its greatest abundance of side-chain–cleavage enzyme—which catalyzes the first committed step in steroid synthesis (see Fig. 50-2)—at 14 to 15 weeks of gestation and low values by 26 weeks of gestation. Because hCG follows a similar temporal pattern, it may be hCG that supports early testosterone

Chapter 53 • Sexual Differentiation

production. Late regulation of testosterone production by fetal LH is supported by the finding that the testes of anencephalic fetuses (see the box in Chapter 10 on abnormalities of neural tube closure) at term have few Leydig cells. The Androgen Receptor

Androgens diffuse into target cells and act by binding to androgen receptors, which are present in genital tissues. In the absence of adequate androgen production or functioning androgen receptors, sexual ambiguity occurs. The androgen receptor functions as a homodimer (AR/AR) and is a member of the family of nuclear receptors (see Chapter 3). The AR/AR receptor complex is a transcription factor that binds to hormoneresponse elements on DNA located 5′ from the genes controlled by the androgens (see Table 4-2). Interaction between the receptor-steroid complex and nuclear chromatin causes increased transcription of structural genes, the appearance of mRNA, and subsequent translation and production of new proteins. Congenital absence of the androgen receptor, or the production of abnormal androgen receptor, leads to a syndrome known as testicular feminization (see the box on Impaired Androgen Action in Target Tissues). Dihydrotestosterone Formation In certain target tissues, cytoplasmic 5α-reductase converts testosterone to DHT (see Fig. 54-5), which binds to the same androgen receptor as does testosterone. However, DHT binds to the androgen receptor with an affinity that is ∼100-fold greater than the binding of testosterone to the androgen receptor. Moreover, the DHT-receptor complex binds to chromatin more tightly than does the testosterone-receptor complex. Antimüllerian Hormone

As noted earlier, the Sertoli cells of the testis produce AMH, also known as MIS. AMH is a homodimer of two monomeric glycoprotein subunits that are linked by disulfide bonds.

Androgens direct the male pattern of sexual differentiation of the internal ducts, the urogenital sinus, and the external genitalia Androgens play two major roles in male phenotypic differentiation: (1) they trigger conversion of the wolffian ducts to the male ejaculatory system, and (2) they direct the differentiation of the urogenital sinus and external genitalia. The wolffian phase of male sexual differentiation is regulated by testosterone itself and does not require conversion of testosterone to DHT. In contrast, virilization of the urogenital sinus, the prostate, the penile urethra, and the external genitalia during embryogenesis requires DHT, as does sexual maturation at puberty. Differentiation of the Duct System

After formation of the testicular cords, the Sertoli cells produce AMH, which causes the müllerian ducts to regress. The cranial end of the müllerian duct becomes the vestigial appendix testis at the superior pole of the testis. Shortly after the initiation of AMH production, the fetal Leydig cells begin producing testosterone. The embryonic mesenchyme contains androgen receptors and is the first site of androgen action during for-

Congenital Adrenal Hyperplasia

A

mbiguous genitalia in genotypic females may result from disorders of adrenal function. Several forms of congenital adrenal hyperplasia have been described, including the deficiency of several enzymes involved in steroid synthesis (see Fig. 50-2): the side chain– cleavage enzyme, 17α-hydroxylase, 21α-hydroxylase, 11β-hydroxylase, and 3β-hydroxysteroid dehydrogenase. Deficiencies in 21α-hydroxylase, 11β-hydroxylase, and 3βhydroxysteroid dehydrogenase all lead to virilization in females—and thus ambiguous genitalia—as a result of the hypersecretion of adrenal androgens. 21a-Hydroxylase deficiency, by far the most common, accounts for ∼95% of cases. Some of the consequences of this deficiency are discussed in the box on 21α-hydroxylase deficiency in Chapter 50. As summarized in Figure 50-2, 21α-hydroxylase deficiency reduces the conversion of progesterone to 11-deoxycorticosterone—which goes on to form aldosterone—and also reduces the conversion of 17α-hydroxyprogesterone to 11-deoxycortisol—which is the precursor of cortisol. As a result, adrenal steroid precursors are shunted into androgen pathways. In female infants, the result is sometimes called the adrenogenital syndrome. The external genitalia are difficult to distinguish from male genitalia on visual inspection. The clitoris is enlarged and resembles a penis, and the labioscrotal folds are enlarged and fused and resemble a scrotum. The genitalia thus have a male phenotype in an otherwise normal female infant.

mation of the male urogenital tract. The Sertoli cells also produce a substance referred to as androgen-binding protein (ABP). It is possible that ABP binds and maintains a high concentration of testosterone locally. These high local levels of testosterone stimulate growth and differentiation of the medulla of the gonad into the rete testes, as well as differentiation of the wolffian ducts into the epididymis, the vas deferens, the seminal vesicles, and the ejaculatory duct. Testosterone also promotes development of the prostate from a series of endodermal buds located at the proximal aspect of the urethra. Cells of the wolffian ducts lack 5α-reductase and therefore cannot convert testosterone to DHT. Thus, the internal male ducts respond to testosterone per se and do not require the conversion of testosterone to DHT. In the absence of testosterone, the wolffian system remains rudimentary, and normal male internal ductal development does not occur. Differentiation of the Urogenital Sinus and External Genitalia The cells of the urogenital sinus and external

genitalia, unlike those of the wolffian duct, contain 5αreductase and are thus capable of converting testosterone to DHT. Indeed, conversion of testosterone to DHT is required for normal male development of the external genitalia. Congenital absence of 5α-reductase (see the box titled Impaired Androgen Action in Target Tissues) is associated with normal development of the wolffian duct system but impaired virilization of the external genitalia.

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At ∼9 weeks’ gestation, soon after virilization of the internal genital ducts of the male, development of the external genitalia commences. It is completed by 13 weeks of gestation. In the presence of high intracellular concentrations of DHT, the genital tubercle, the bipotential predecessor to either a clitoris or a penis, elongates to become the glans penis, the corpus spongiosum, and the two corpora cavernosa. Formation of the penis and scrotum is complete by ∼13 weeks, and even extremely high concentrations of testosterone after this time fail to cause midline fusion of the urethral groove or scrotum, although the clitoris will enlarge. The urogenital sinus gives rise to the prostate and the bulbourethral glands, also under the influence of DHT. In the absence of androgen secretion by the fetal testis— or abnormal extragonadal sources—the indifferent external genitalia remain unfused and follow the female pattern of differentiation.

Androgen Dependence of Testicular Descent

I

n preparation for descent, the testes enlarge. In addition, the mesonephric kidneys and wolffian (mesonephric) ducts atrophy. This process frees the testes for their future move down the posterior abdominal wall and across the abdomen to the deep inguinal rings. Testicular descent occurs in three phases during the last two thirds of gestation. During the first stage of testicular descent, rapid growth of the abdominopelvic region causes relative movement of the testes down to the inguinal region (Fig. 53-9A). The role of the gubernaculum—the ligament attaching the inferior part of the testes to the lower segment of the labioscrotal fold—is uncertain. However, the gubernaculum shortens and appears to guide the testis to its place of ultimate functional residence in the scrotum. The second stage of testicular descent is herniation of the abdominal wall adjacent to the gubernaculum (Fig. 53-9B). This herniation, which occurs as a result of increasing abdominal pressure, forms the processus vaginalis; the processus vaginalis then folds around the gubernaculum and creates the inguinal canal. In the third stage, the gubernaculum increases to the approximate diameter of the testis. As its proximal portion degenerates, the gubernaculum draws the testis into the scrotum through the processus vaginalis (Fig. 53-9C). The testes usually complete their descent by the seventh month of gestation; ∼97.5% of full-term infants and 79% of premature infants have fully descended testes at birth. At 9 months of age, only 0.8% of male infants have undescended testes. The incidence of undescended testes in young men is 0.2%. Testicular descent is an androgen-dependent process, and development of the structures involved in testicular descent depends on testosterone. Thus, in testosteronedeficient states caused by inadequate secretion or disordered androgen action, the testes of genetic males often fail to descend. This abnormality can be seen in individuals with both 5α-reductase deficiency and complete androgen resistance (i.e., testicular feminization syndrome).

Androgens and estrogens influence sexual differentiation of the brain Anatomically sexually dimorphic nuclei have been identified in the diencephalons of rodents and lower primates. Gonadal steroids influence the development of these sexually dimorphic nuclei. Androgens do not act directly on the hypothalamus and other areas of the brain having to do with sex behavior and control of gonadotropin secretion. Rather, aromatase—which catalyzes the formation of estrone and estradiol (see Fig. 55-10)—converts androgens to estrogens in the brain. Thus, androgens in the brain serve as prohormones for estrogens. Therefore, estrogens are derived from androgens that appear to masculinize sexually dimorphic nuclei directly in the brain. It is not clear why, in females, estrogens do not masculinize the brain. Gonadal steroids affect sex behavior in both males and females. In rodents, lordosis behavior in females and mounting behavior in males are examples of sex behavior. An example of functional sexual dimorphism in the human brain is the manner in which gonadotropin is released. Gonadotropin release has been described as cyclic in the female and tonic in the male inasmuch as females have midcycle cyclic release of gonadotropin before ovulation, whereas males seem to have a continuous tonic pattern of gonadotropin release. Although controversy continues over the role of prenatal virilization in the determination of sexual dimorphism, sex steroids clearly have an impact on sexual behavior and sexual reference in humans. The appearance of secondary sex characteristics at puberty completes sexual differentiation and development Although at birth humans have the primary and secondary sex organs necessary for procreation, final sexual maturity occurs only at puberty. Profound alterations in hormone

Impaired Androgen Action in Target Tissues

A

s already discussed, in the absence of androgens, male embryos follow a typically female pattern of sexual development. However, such a female developmental pattern can occur even if testosterone levels are normal or elevated. Any defect in the mechanisms by which androgens act on target tissues—in genotypic males—may lead to a syndrome of male pseudohermaphroditism. Affected individuals have a normal male karyotype (46,XY) and unambiguous male gonads but ambiguous external genitalia, or they may phenotypically appear as female. In principle, impaired androgen action could result from a deficiency of the enzyme that converts testosterone to DHT in target tissues, absent androgen receptors, qualitatively abnormal receptors, a quantitative deficiency in receptor levels, or postreceptor defects. The two major forms that have been identified clinically are defects in the conversion of testosterone to DHT (5α-reductase deficiency) and androgen receptor defects.

Chapter 53 • Sexual Differentiation

A

MOVEMENT OF TESTES TO THE INGUINAL REGION Testis

B

HERNIATION OF THE ABDOMINAL WALL

C

DESCENT OF TESTES INTO THE SCROTUM

Ductus deferens Developing pubis Stalk of processus vaginalis

Rectum Gubernaculum

Labioscrotal fold Processus vaginalis

Figure 53-9

Processus vaginalis

Testicular descent.

secretion during the peripubertal period cause changes in the primary and secondary sex organs. The events occurring in puberty are discussed in more detail for both males (see Chapter 54) and females (see Chapter 55). Female

The vagina reflects the effects of estrogens on the vaginal mucosa. The uterus and cervix enlarge, and their secretory functions increase under the influence of estrogen. The uterine glands increase in number and length, and the endometrium and stroma proliferate in response to estrogen secretion. The cervical glands produce increasing quantities of cervical mucus, which serves to lubricate the vaginal vault. The mucous membranes of the female urogenital tract are made of stratified squamous epithelium; these membranes respond to hormones, particularly estrogens. Estrogen levels increase and cause increased epithelial proliferation with the formation of successive intermediate and superficial layers. The cells of the vaginal mucosa are transformed into superficial cells, and the thickness of the vaginal mucosa increases. Development of the breasts occurs under the influence of a complex of hormones. Progesterone is primarily responsible for development of the alveoli, which are analogous to the acini of other exocrine glands. Estrogen is the primary stimulus for development of the duct system that connects the alveoli to the exterior. Insulin, growth hormone, glucocorticoids, and thyroxine contribute to breast development, but they are incapable of causing breast growth by themselves. Lactation is discussed in Chapter 56.

Male

Gubernaculum

The penis undergoes rapid growth under the influence of testosterone secreted by the testes. The testes also increase dramatically in size in response to increasing androgen secretion at puberty.

REFERENCES Books and Reviews Donahoe PK, Budzik GP, Trelstad R, et al: Müllerian-inhibiting substance: An update. Recent Prog Horm Res 1982; 38:279. Grumbach MM, Conte FA: Disorders of sex differentiation. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed, pp 1303-1425. Philadelphia: WB Saunders, 1998. Haqq CM, Donahoe PK: Regulation of sexual dimorphism in mammals. Physiol Rev 1998; 78:1-33. Jost A, Vigier B, Prepin J, Perchellet JP: Studies on sex differentiation in mammals. Recent Prog Horm Res 1973; 29:1-41. Lee MM, Donahoe PK: Müllerian inhibiting substance: A gonadal hormone with multiple functions. Endocr Rev 1993; 14:152-164. Naftolin F, Ryan KJ, Davie KJ, et al: The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res 1975; 31:295-319. Rebar RW: Normal and abnormal sexual differentiation and pubertal development. In Moore TR, Reiter RC, Rebar RW, Baker VV (eds): Gynecology and Obstetrics: A Longitudinal Approach, pp 97-146. New York: Churchill Livingstone, 1993. Journal Articles Griffin JE, Wilson JD: The syndromes of androgen resistance. N Engl J Med 1980; 302:198-209. Judd HL, Hamilton CR, Barlow JJ, et al: Androgen and gonadotropin dynamics in testicular feminization syndrome. J Clin Endocrinol Metab 1972; 34:229-234. New MI, Dupont B, Pang S, et al: An update of congenital adrenal hyperplasia. Recent Prog Horm Res 1981; 37:105-181. Sinclair AH, Berta P, Palmer MS, et al: A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990; 346:240-244. Turner HH: A syndrome of infantilism, congenital webbed neck, and cubitus valgus. Endocrinology 1938; 23:566-574. Wilkins L: Masculinization of the female fetus due to the use of orally given progestins. JAMA 1960; 172:1028-1032.

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54

T H E M A L E R E P R O D U CT I V E SYST E M Ervin E. Jones

The male reproductive system (Fig. 54-1A) consists of two essential elements: the gonads and the complex array of glands and conduits that constitute the sex accessories. The gonads in males are the testes, and they are responsible for the production of gametes, the haploid cells (spermatozoa) necessary for sexual reproduction. The gonads also synthesize and secrete the hormones that are necessary for functional conditioning of the sex organs, control of gonadotropin secretion, and modulation of sexual behavior. The testis (Fig. 54-1B, C) is largely composed of seminiferous tubules and the interstitial cells of Leydig, located in the spaces between the tubules. The seminiferous tubules are lined by seminiferous epithelium, which rests on the inner surface of a basement membrane (Fig. 54-1D). The basement membrane is supported by a thin lamina propria externa. The sex accessories in the male include the paired epididymides, the vas deferens, the seminal vesicles, and the ejaculatory ducts. Also included among the sex accessories are the prostate, the bulbourethral glands (Cowper’s glands), the urethra, and the penis. The primary role of the male sex accessory glands and ducts is to store and transport spermatozoa to the exterior at the proper time, thus enabling spermatozoa to come in contact with and fertilize female gametes.

PUBERTY Puberty occurs in five defined stages During the final month of fetal life, the testes descend (see the box titled Androgen Dependence of Testicular Descent in Chapter 53) into an integumentary pouch called the scrotum. The inguinal canals through which the testes descend are sealed off shortly after birth. Because the internal temperature of the testicle must be closely regulated for optimum function, localization of the testes within the scrotum appears to be a necessary adaptation for testicular function. Aberrant retention of the testes in the abdominal cavity (cryptorchidism) causes marked damage to the seminiferous tubules and diminished testicular function.

1128

Puberty is the transition between the juvenile and adult states, during which time the individual develops secondary sexual characteristics, experiences the adolescent growth spurt, and achieves the ability to procreate. The range of onset of normal male puberty extends from 9 to 14 years. Boys complete pubertal development within 2 to 41/2 years. In a normal boy, the first sign of puberty (stage 2) is enlargement of the testes to greater than 2.5 cm. Testicular enlargement is mainly a result of growth of the seminiferous tubules, but enlargement of the Leydig cells contributes as well. Androgens from the testes are the driving force behind secondary sexual development, although adrenal androgens play a role in normal puberty. The Tanner method of describing the stages of pubertal development is widely accepted. Genital development and growth of pubic hair are best described separately, as indicated by the two columns in Table 54-1. Thus, it is possible for an adolescent boy to be at genital stage 3, pubic hair stage 2. Testicular size is generally determined by using a ruler or calipers. It is expected that a length greater than 2.5 cm is compatible with the onset of normal pubertal development. The testicular volume index is defined as the sum of the length times width product for the left and right testes. An orchidometer allows direct comparison of the patient’s testes with an oval of measured volume. A popular method uses the Prader orchidometer, a set of solid or hollow ovals encompassing the range from infancy to adulthood (1 to 25 mL). The volumes of the testes are then recorded; a volume of 3 mL closely correlates with the onset of pubertal development. Spermarchy, or the first appearance of spermatozoa in early morning urine, occurs at a mean age of ∼13.4 years and corresponds to genital stages 3 to 4 and pubic hair stages 2 to 4. The pubertal spurt, a marked increase in growth rate (total body size), occurs late in puberty in boys, at genital stages 3 to 4. The acceleration of growth appears to be partly a result of increased secretion of growth hormone at puberty and partially a result of testosterone production. Boys experience, on average, 28 cm of growth during the pubertal spurt. The 10-cm mean difference in adult stature between men and women is the result of a greater pubertal growth spurt in boys and to greater height at the onset of peak height

Chapter 54 • The Male Reproductive System

A

B

SAGITTAL SECTION OF THE MALE PELVIS Ductus (vas) deferens

Urinary bladder Ureter

Urinary bladder

Ampulla of ductus deferens

Pubis Corpus cavernosum

Seminal vesicle

Corpus spongiosum Penis

Ejaculatory duct

Penile urethra Scrotum

Bulbourethral gland

Prostatic Bulbous urethra urethra

Testis (left)

Prostate

Ejaculatory duct (initial part)

Bulbourethral (Cowper’s) glands

Seminal vesicle

Prostate gland Anus

URINARY BLADDER (DORSAL VIEW)

Efferent ductules (ductuli efferentes)

C TESTIS Ductus (vas) deferens

Glans penis

Epididymis (head) Rete testis

Epididymis (body)

Epididymis

Tunica albuginea Seminiferous tubules Testicular lobules Septum

Epididymis (tail) D

Figure 54-1 The anatomy of the male internal genitalia and accessory sex organs. A, The two major elements of the male sexual anatomy are the gonads (i.e., testes) and the sex accessories (i.e., epididymis, vas deferens, seminal vesicles, ejaculatory duct, prostate, bulbourethral or Cowper’s glands, urethra, and penis). The urethra can be subdivided into the prostatic urethra, the bulbous urethra, and the penile urethra. B, The vas deferens expands into an ampulla before coursing across the rear of the urinary bladder and merging with the outflow from the seminal vesicle. The merger forms the ejaculatory duct. The left and right ejaculatory ducts penetrate the prostate gland and open into the prostatic urethra. C, The spermatozoa form in the seminiferous tubules and then flow into the rete testis and from there into the efferent ductules, the epididymis, and the vas deferens. D, The seminiferous tubule is an epithelium formed by the Sertoli cells, with interspersed germ cells. The most immature germ cells (the spermatogonia) are near the periphery of the tubule, whereas the mature germ cells (the spermatozoa) are near the lumen of the tubule. The Leydig cells are interstitial cells that lie between the tubules.

Testis

CROSS SECTION OF SEMINIFEROUS TUBULE

Mature sperm Lumen

Spermatogonium

Sertoli cell

Basal lamina surrounding seminiferous tubule

Leydig cell

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Table 54-1

Stages in Male Puberty

Stage

Genital Development

Pubic Hair

1

Preadolescent: the penis, scrotum, and testes are the same size—relative to body size—as in a young child

Preadolescent: no pubic hair is present, only vellus hair, as on the abdomen

2

Scrotum and testes are enlarged

Pubic hair is sparse, mainly at the base of the penis

3

Penis is enlarged, predominantly in length; scrotum and testes are further enlarged

Pubic hair is darker, coarser, and curlier and spreads above the pubis

4

Penis is further enlarged in length and also in diameter; scrotum and testes are further enlarged

Pubic hair is of the adult type, but covers an area smaller than in most adults

5

Adult pattern

Adult pattern

velocity in boys versus girls. Before puberty, boys and girls have the same mean body mass, skeletal mass, and body fat. However, men have 150% of the average woman’s lean and skeletal body mass, and women have 200% of the body fat of men. Men have twice the number of muscle cells that women have and 1.5 times the muscle mass. Androgens determine male secondary sexual characteristics The male sex steroids, which are known as androgens, affect nearly every tissue in the body, including the brain. The development of both the external and the internal genitalia depends on male sex hormones (see Chapter 53). Androgens stimulate adult maturation of the external genitalia and accessory sexual organs, including the penis, the scrotum, the prostate, and the seminal vesicles. Androgens also determine the male secondary sexual characteristics, which include deepening of the voice, as well as evolving male patterns of hair growth. The effects on the voice are a result of androgen-dependent effects on the size of the larynx, as well as the length and thickness of the vocal cords. In boys, the length of the vocal cords increases by ∼50% during puberty, whereas girls have little increase in vocal cord length. The surfaces of the human body that bear secondary sexual hair include the face (particularly the upper lip, chin, and the sideburn areas), the axilla, and the pubic region. Temporal hair recession and male-pattern balding are also androgendependent phenomena. Muscle development and growth are androgendependent processes Androgens have anabolic effects, including stimulation of linear body growth, nitrogen retention, and muscular development in adolescent boys and in men. The biological effects of testosterone and its metabolites have been classified according to their tissue sites of action. Effects that relate to growth of the male reproductive tract or development of secondary sexual characteristics are referred to as androgenic, whereas the growth-promoting effects on somatic tissue are called anabolic. These androgenic and anabolic effects are two independent biological actions of the same

class of steroids. Experimental evidence, however, indicates that these responses are organ specific and that the molecular mechanisms that initiate androgenic responses are the same as those that stimulate anabolic activity.

HYPOTHALAMIC-PITUITARY-GONADAL AXIS AND CONTROL OF MALE SEXUAL FUNCTION The male hypothalamic-pituitary-gonadal axis (Fig. 54-2) controls two primary functions: (1) production of male gametes (spermatogenesis) in the seminiferous tubules and (2) androgen biosynthesis in the Leydig cells in the testes. The hypothalamus produces gonadotropin-releasing hormone (GnRH), which stimulates the gonadotrophs in the anterior pituitary to secrete the two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). As discussed in Chapter 55, the names of these hormones reflect their function in the female reproductive system. LH and FSH control, respectively, the Leydig and Sertoli cells of the testes. The hypothalamus secretes GnRH, which acts on gonadotrophs in the anterior pituitary GnRH, which is synthesized by small-bodied peptidergic neurons in the hypothalamus, stimulates the synthesis, storage, and secretion of gonadotropins by gonadotroph cells in the anterior pituitary. The hypothalamic-pituitaryportal system (see Chapter 47) describes the route by which GnRH and other releasing hormones emanating from the hypothalamus reach the anterior pituitary gland. The neurons that synthesize, store, and release GnRH are dispersed throughout the hypothalamus but are principally located in the arcuate nucleus and preoptic area. Studies involving both rats and primates showed that sites other than the hypothalamus (e.g., the limbic system) of GnRH production can also participate in the control of sex behavior. Neuronal systems originating from other areas of the brain impinge on the hypothalamic GnRHreleasing neurons and thus form a functional neuronal network.

Chapter 54 • The Male Reproductive System

CNS Behavioral effects

Hypothalamus

GnRH

Anterior pituitary LH

FSH

Leydig cell Growth factors

Sertoli cell Inhibin

Androgens

Aromatization

Peripheral aromatization

Androgen-binding protein

Spermatogenesis

Estrogens

Figure 54-2 The hypothalamic-pituitary-testicular axis. Smallbodied neurons in the arcuate nucleus and preoptic area of the hypothalamus secrete GnRH, a decapeptide that reaches the gonadotrophs in the anterior pituitary through the long portal veins. Stimulation by GnRH causes the gonadotrophs to synthesize and release FSH and LH. The LH binds to receptors on the Leydig cells, thus stimulating the transcription of several proteins involved in the biosynthesis of testosterone. FSH binds to receptors on the basolateral membrane of the Sertoli cells, thereby stimulating gene transcription and protein synthesis. These proteins include ABP, aromatase, growth factors, and inhibin. Negative feedback on the hypothalamicpituitary-testicular axis occurs by two routes. First, testosterone inhibits the pulsatile release of GnRH by the hypothalamic neurons and the release of LH by the gonadotrophs in the anterior pituitary. Second, inhibin inhibits the release of FSH by the gonadotrophs in the anterior pituitary.

GnRH is a decapeptide hormone synthesized by aforementioned hypothalamic neurons in the secretory pathway (see Chapter 2). Like many other peptide hormones, GnRH is synthesized as a prohormone—69 amino acids long in this case—from which the mature GnRH is generated by enzymatic cleavage. The synthesis of GnRH is discussed in more detail in Chapter 55. The neurons release GnRH into the extracellular space, to be carried to the anterior pituitary through the long portal vessels. GnRH stimulates the release of both FSH and LH from the gonadotroph cells of the anterior pituitary. FSH and LH are the primary gonadotropins; in males, they stimulate testicular function. The cell surface of the pituitary gonadotrophs is the site of high-affinity membrane receptors for GnRH. These receptors are coupled to the G protein Gαq, which activates phospholipase C (PLC; see Chapter 3). PLC acts on membrane phosphoinositides to liberate inositol 1,4,5-triphosphate (IP3), which triggers Ca2+ release from internal stores, and diacylglycerol (DAG), which stimulates protein kinase C. The results are the synthesis and release of both LH and FSH from the gonadotrophs. Because secretion of GnRH into the portal system is pulsatile, secretion of both LH and FSH by the gonadotrophs is also episodic. The frequency of pulsatile LH discharge in men is ∼8 to 14 pulses over a 24-hour period. FSH pulses are not as prominent as LH pulses, both because of their lower amplitude and because of the longer half-life of FSH in the circulation. Although pulsatile GnRH discharge elicits a corresponding pulsatile release of LH and FSH, continuous administration of GnRH—or intermittent administration of high doses of GnRH analogues—suppresses the release of gonadotropins. As described in the box titled Therapeutic Uses of GnRH in Chapter 55, the mechanism is inhibition of the replenishment of GnRH receptors so that insufficient receptors are available for GnRH function. A clinical application of this principle is in prostatic cancer, in which the administration of GnRH analogues lowers LH and FSH levels and thereby reduces testosterone production (i.e., chemical castration). Products of the testes, particularly sex steroids and inhibin (see later), exert negative feedback control on hypothalamic and anterior pituitary function. Neural elements in the arcuate nucleus respond to sex steroids. Sex steroids alter the frequency and amplitude of the LH secretory pulses in both men and women. Androgens also exert powerful influences on higher brain function, as evidenced by alterations in sex behavior. Under the control of GnRH, gonadotrophs in the anterior pituitary secrete LH and FSH LH and FSH, which are secreted by the gonadotrophs of the anterior pituitary, are the primary regulators of testicular function. LH and FSH are members of the same family of hormones as human chorionic gonadotropin (hCG; see Chapter 56) and thyroid-stimulating hormone (TSH; see Chapter 49). All these glycoprotein hormones are composed of two polypeptide chains designated α and β. Both subunits, α and β, are required for full biological activity. The α subunits of LH and FSH, as well as the α subunits of hCG and TSH, are identical. In humans, the common α subunit

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has 92 amino acids and a molecular weight of ∼20 kDa. The β subunits differ among these four hormones and thus confer specific functional and immunologic characteristics to the intact molecules. Each of the unique β subunits of FSH and LH is 115 amino acids in length. The β subunits of LH and hCG are identical, except the β subunit of hCG has an additional 24 amino acids and additional glycosylation sites at the C terminus. hCG is secreted by the placenta, and some reports have described that small amounts of this substance are made in the testes, pituitary gland, and other nonplacental tissue. The biological activities of LH and hCG are very similar. Indeed, in most clinical uses (e.g., in an attempt to initiate spermatogenesis in oligospermic men), hCG is substituted for LH because hCG is much more readily available. The specific gonadotropin and the relative proportions of each gonadotropin released from the anterior pituitary depend on the developmental age, as well as the existing hormonal milieu. The pituitary gland of the male fetus contains functional gonadotrophs by the end of the first trimester of gestation. Thereafter, gonadotropin secretion rises rapidly and then plateaus. Gonadotropin secretion begins to decline in utero during late fetal life and increases again during the early postnatal period. Male primates release LH in response to GnRH administration at 1 to 3 months of age, a finding indicative of functional competence of the anterior pituitary gland. Also during this time, a short-lived postnatal surge of LH and testosterone secretion occurs in males. Although the cause of this short-lived surge of gonadotropins remains to be understood, it is clearly independent of sex steroids. The sensitivity of the gonadotrophs to stimulation subsequently diminishes, and the system remains quiescent until just before puberty. Release of FSH is greater than that of LH during the prepubertal period, a pattern that is reversed after puberty. GnRH preferentially triggers LH release in men. This preferential release of LH may reflect maturation of the testes, which secrete inhibin, a specific inhibitor of FSH secretion at the level of the anterior pituitary gland. Increased sensitivity of the pituitary to increasing gonadal steroid production may also be responsible for the diminished secretion of FSH. Luteinizing hormone stimulates the Leydig (interstitial) cells of the testis to produce testosterone LH derives its name from effects observed in the female, that is, from the ability to stimulate luteal function. The comparable substance in the male was originally referred to as interstitial cell–stimulating hormone (ICSH). Subsequently, investigators realized that LH and ICSH are the same substance, and the common name became LH. Testosterone production decreases in males after hypophysectomy. This observation led to our current understanding that LH secreted by the anterior pituitary gland is essential for testosterone production by the testis. The interstitial cells of the testis, the Leydig cells, are the primary source of testosterone production in the male. Leydig cells synthesize

androgens from cholesterol by using a series of enzymes that are part of the steroid biosynthetic pathways (see later). LH binds to specific high-affinity cell surface receptors on the plasma membrane of Leydig cells (Fig. 54-3). Binding of LH to this G protein–coupled receptor on the Leydig cell stimulates membrane-bound adenylyl cyclase (see Chapter 3), which catalyzes the formation of cAMP and thus activates protein kinase A (PKA). Activated PKA modulates gene transcription (see Chapter 4) and increases the synthesis of enzymes and other proteins necessary for the biosynthesis of testosterone. Two of these other proteins are the sterolcarrier protein (SCP-2) and the steroidogenic acute regulatory protein or (StAR or STARD1). SCP is a 13.5-kDa protein that appears to transport cholesterol from the plasma membrane or organellar membranes to other organellar membranes, including the outer mitochondrial membrane. StAR belongs to a large family of proteins that contain a ∼210-residue START domain and are involved in lipid trafficking and metabolism. The 37-kDa pro-StAR protein—the precursor to StAR—may participate in ferrying cholesterol from the endoplasmic reticulum (ER) to the outer mitochondrial membrane. The 30-kDa mature StAR protein resides in the mitochondrial intermembrane space (see Fig. 58-10) and extracts cholesterol from the mitochondrial outer membrane, ferries it across the space to the mitochondrial inner membrane, and then deposits the cholesterol in the mitochondrial inner membrane where the cytochrome P-450 side-chain–cleavage (SCC) (P-450scc) enzyme is located. As discussed later, the P-450scc–mediated conversion of cholesterol to pregnenolone is the rate-limiting step in steroidogenesis—including testosterone synthesis. Thus, the net effect of LH on Leydig cells is to stimulate testosterone synthesis. Follicle-stimulating hormone stimulates the sertoli cells to synthesize certain products needed by both the Leydig cells and the developing spermatogonia The Sertoli cells seem to be the primary testicular site of FSH action (Fig. 54-3), as clearly shown by experiments involving suppression of LH secretion. FSH also regulates Leydig cell physiology through effects on the Sertoli cells. The early biochemical events after FSH binding are similar to those described for LH on the Leydig cell. Thus, binding of FSH to a G protein–coupled receptor initiates a series of reactions involving stimulation of adenylyl cyclase, increase in [cAMP]i, stimulation of PKA, transcription of specific genes, and increased protein synthesis. Several proteins are synthesized in response to FSH. Some are important for steroid action: 1. FSH leads to the synthesis of androgen-binding protein (ABP), which is secreted into the luminal space of the seminiferous tubule, near the developing sperm cells. ABP helps to keep local testosterone levels high (see Chapter 53). 2. FSH causes the synthesis of a P-450 aromatase (P-450arom; see Chapter 55). Inside the Sertoli cells, this enzyme converts testosterone, which diffuses from the Leydig cells to the Sertoli cells, into estradiol.

Chapter 54 • The Male Reproductive System

AC cAMP

To bloodstream

Gs

FSH

Capillary

Adenylyl cyclase

LH

FSH receptor Gs cAMP

AC

PKA Sertoli cell

PKA Growth factors Other products New protein synthesis

New protein synthesis Inhibins

Estradiol ABP

Cholesterol Aromatase Enzymes Testosterone Testosterone Leydig cell

Spermatogonium

Extracellular space

Lumen

Figure 54-3 Physiology of the Leydig and Sertoli cells. The Leydig cell (left) has receptors for LH. The binding of LH increases testosterone synthesis. The Sertoli cell (right) has receptors for FSH. (Useful mnemonics: “L” for LH and Leydig, “S” for FSH and Sertoli.) FSH promotes the synthesis of ABP, aromatase, growth factors, and inhibin. Crosstalk occurs between the Leydig cells and the Sertoli cells. The Leydig cells make testosterone, which acts on the Sertoli cells. Conversely, the Sertoli cells convert some of this testosterone to estradiol (because of the presence of aromatase), which can act on the Leydig cells. The Sertoli cells also generate growth factors that act on the Leydig cells.

3. FSH leads to the production of certain growth factors and other products by the Sertoli cells that support sperm cells and spermatogenesis. These substances significantly increase the number of spermatogonia, spermatocytes, and spermatids in the testis. Therefore, it appears that the stimulatory effect of FSH on spermatogenesis is not a direct action of FSH on the spermatogonia; instead, stimulation of spermatogenesis occurs through the action of FSH on the Sertoli cells. FSH may also increase the fertility potential of sperm; it appears that this effect of FSH results from stimulation of motility, rather than from an increase in the absolute number of sperm. 4. FSH causes the Sertoli cells to synthesize inhibins. The inhibins are members of the so-called transforming growth factor β (TGF-β) gene family, which also includes the activins and antimüllerian hormone (see Chapter 53). Inhibins are glycoprotein heterodimers consisting of one α and one β subunit that are covalently linked. The granulosa cells in the ovary and the Sertoli cells in the testis are the primary sources of inhibin in humans, other primates, and the lower vertebrates. I discuss the biology of inhibins and activins in more detail in Chapter 55. Inhibins are secreted into the seminiferous tubule fluid and into the interstitial fluid of the testicle. Inhibins have both paracrine and endocrine actions. Locally, the inhibins are some of the growth factors secreted by the Sertoli cells that are thought to act on the Leydig cells. More importantly, inhibins in the male play an important feedback role in the hypothalamic-pituitary-testicular axis (see later).

The Leydig cells and the Sertoli cells engage in crosstalk. For example, the Leydig cells make testosterone, which acts on the Sertoli cells. In the rat, β endorphin produced by the fetal Leydig cells binds to opiate receptors in the Sertoli cells and inhibits their multiplication. Synthesis of β endorphins could represent a local feedback mechanism by which the Leydig cells modulate the Sertoli cell numbers. Conversely, the Sertoli cells also have an effect on the Leydig cells. For example, the Sertoli cells convert testosterone—manufactured by the Leydig cells—to estradiol, which then acts on the Leydig cells. In addition, FSH acting on the Sertoli cells produces growth factors that may increase the number of LH receptors on the Leydig cells during development and may thus result in an increase in steroidogenesis (i.e., an increase in testosterone production). What, then, is required for optimal spermatogenesis to occur? It appears that two testicular cell types (the Leydig cells and the Sertoli cells) are required, as well as two gonadotropins (LH and FSH) and one androgen (testosterone). First, LH and the Leydig cells are required to produce testosterone. Thus, LH, or rather its substitute hCG, is used therapeutically to initiate spermatogenesis in azoospermic or oligospermic men. Second, FSH and the Sertoli cells are important for the nursing of developing sperm cells and for the production of inhibin and growth factors, which affect the Leydig cells. Thus, FSH plays a primary role in regulating development of the appropriate number of the Leydig cells such that adequate testosterone levels are available for spermatogenesis. During early puberty in boys, both FSH and LH levels increase while, simultaneously, the Leydig cells

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Section IX • The Reproductive System

Plasma testosterone (ng/mL)

Adult 6

Fertilization Birth

Senescence

Puberty

5 4 3 2 1 0

3

6

Months

9

1

10

20

can synthesize cholesterol de novo from acetyl coenzyme A or can take it up as low-density lipoproteins from the extracellular fluid by receptor-mediated endocytosis (see Chapter 1). The two sources appear to be equally important in humans. The Leydig cell uses a series of five enzymes to convert cholesterol to testosterone. Three of these enzymes are P-450 enzymes (see Table 50-2). As summarized in Figure 54-5, because 3β-hydroxysteroid dehydrogenase (3β-HSD) can oxidize the A ring of four intermediates, testosterone synthesis from cholesterol can take four pathways. The following is the “preferred” pathway:

60

Years

Figure 54-4 Plasma testosterone versus age in male humans. (Data from Griffin JE, et al: In Bondy PK, Rosenberg LE: Metabolic Control and Disease. Philadelphia: WB Saunders, 1980; and Winter JS, Hughes IA, Reyes FI, Faiman C: J Clin Endocrinol Metab 1976; 42:679-686.)

proliferate and plasma levels of testosterone increase (Fig. 54-4). The hypothalamic-pituitary-testicular axis is under reciprocal inhibition by testicular hormones and inhibin The hypothalamic-pituitary-testicular axis not only generates testosterone and inhibin but also receives negative feedback from these substances (Fig. 54-2). Normal circulating levels of both testosterone and estradiol exert inhibitory effects on LH secretion in males. Testosterone inhibits the pulsatile release of LH, presumably by inhibiting the pulsatile release of GnRH by the hypothalamus. Testosterone also appears to have negative feedback action on LH secretion at the level of the pituitary gonadotrophs. A testicular hormone also feeds back on FSH secretion. Evidence for negative feedback by a testicular substance on FSH secretion is that plasma FSH concentrations increase in proportion to the loss of germinal elements in the testis. The FSH-inhibiting substance—inhibin—is a nonsteroid present in both the testis and cultures of Sertoli cells. Thus, FSH specifically stimulates the Sertoli cells to produce inhibin, and inhibin “inhibits” FSH secretion. The preponderance of evidence indicates that inhibin diminishes FSH secretion by acting at the level of the anterior pituitary gland (not at the level of the hypothalamus).

TESTOSTERONE The Leydig cells of the testis synthesize and secrete testosterone Cholesterol is the obligate precursor for androgens, as well as for other steroids produced by the testis. The Leydig cell

1. The pathway for testosterone synthesis begins in the mitochondria, where P-450scc (also called 20,22-desmolase) removes the long side chain (carbons 22 to 27) from the carbon at position 20 of the cholesterol molecule (27 carbon atoms). The rate-limiting step in the biosynthesis of testosterone, as for other steroid hormones, is the conversion of cholesterol to pregnenolone. LH stimulates this reaction and is the primary regulator of the overall rate of testosterone synthesis by the Leydig cell. LH appears to promote pregnenolone synthesis in two ways. First, it increases the affinity of the enzyme for cholesterol. Second, LH has long-term action in which it increases steroidogenesis in the testis by stimulating synthesis of the SCC enzyme. 2. The product of the SCC-catalyzed reaction is pregnenolone (21 carbon atoms). In the smooth ER (SER), 17αhydroxylase (P-450c17) then adds a hydroxyl group at position 17 to form 17a-hydroxypregnenolone. 3. In the SER, the 17,20-desmolase (a different activity of the same P-450c17 whose 17α-hydroxylase activity catalyzes the previous step) removes the side chain from carbon 17 of 17α-hydroxypregnenolone. That side chain begins with carbon 20. The result is a 19-carbon steroid called dehydroepiandrosterone (DHEA). 4. In the SER of the Leydig cell, a 17β-hydroxysteroid dehydrogenase (17β-HSD, which is not a P-450 enzyme) converts the ketone at position 17 to a hydroxyl group to form androstenediol. 5. Finally, 3β-HSD (not a P-450 enzyme) oxidizes the hydroxyl group at position 3 of the A ring to a ketone to form testosterone. In addition, the testis can also use 5α-reductase, which is located in the SER, to convert testosterone to dihydrotestosterone (DHT). However, extratesticular tissue is responsible for most of the production of DHT. The conversion of testosterone to DHT is especially important in certain testosterone target cells (see Chapter 53). The Leydig cells of the testes make ∼95% of the circulating testosterone. Although testosterone is the major secretory product, the testis also secretes pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, androsterone, and DHT. Androstenedione is of major importance because it serves as a precursor for extraglandular estrogen formation.

Acetate

Chapter 54 • The Male Reproductive System

Cholesterol (27-carbon compound) 21 20 22 24 25 26 12 18 17 23 16 27 13

11 1

10

A

8

B

14

15

7

5

HO

D

C

19 9

2

4

6

MITOCHONDRIA Side chain cleavage enzyme Pregnenolone Progesterone SER

CH3

CH3

O

C

C

CH3

CH3 CH3 A

D

C

A

B

SER 17 α-Hydroxylase

SER 17 α-Hydroxylase

17 α-Hydroxypregnenolone

CH3

C

C

O

A

B

A

HO

OH

D

C

CH3

Same protein.

O

CH3

OH

D

C

CH3

17 α-Hydroxyprogesterone

CH3 CH3

B

O

SER 17,20-Desmolase

SER 17,20-Desmolase

C

CH3 A

D

B

SER 17β-Hydroxysteroid dehydrogenase Androstenediol ANDROGENS (19 carbons)

CH3 C

CH3 A

OH D

A

SER 17β-Hydroxysteroid dehydrogenase

OH CH3 C

CH3

A

ESTROGENS (18 carbons)

C

B

A

OH CH3 D

B

HO

SER 5 α-Reductase

OH CH3

OH D

CH3 A

B H

Estradiol (E2)

D

Dihydrotestosterone

C

SER 17β-Hydroxysteroid dehydrogenase

Testosterone

SER 5 α-Reductase

CH3

B

HO

O

CH3

D

C A

B

A

Androsterone

O

O

B

HO

CH3

D

C

CH3

Estrone (E1)

O

Aromatase

HO

3β-Hydroxysteroid dehydrogenase

O CH3

CH3

SER

Androstenedione

Dehydroepiandrosterone (DHEA)

HO

B

O

HO

(21 carbons)

D

C

CH3

O

O

C

Mainly in testosterone target cells.

D

B H

Figure 54-5 Biosynthesis of testosterone. This scheme summarizes the synthesis of the androgens from cholesterol. The individual enzymes are shown in the horizontal and vertical boxes; they are located in either the SER or the mitochondria. The SCC enzyme that produces pregnenolone is also known as 20,22 desmolase. The chemical groups modified by each enzyme are highlighted in the reaction product. Four possible pathways from pregnenolone to testosterone are recognized; the preferred pathway in the human testis appears to be the one along the left edge of the figure to androstenediol, followed by oxidation of the A ring to testosterone. Some of these pathways are shared in the biosynthesis of the glucocorticoids and mineralocorticoids (see Fig. 50-2) as well as estrogens (see Fig. 55-9).

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Table 54-2

Androgen Production and Turnover

Steroid

Testosterone Androstenedione Dihydrotestosterone

Blood Production Rate: Hormone Delivered to the Blood (mg/day)

Plasma Concentration (mg/L)

6500

6.5

2000-6000

1.5

300

0.5

Other organs—such as adipose tissue, skin, and the adrenal cortex—also produce testosterone and other androgens In men between the ages of 25 and 70 years, the rate of testosterone production remains relatively constant (Table 542). Figure 54-4 summarizes the changes in plasma testosterone levels as a function of age in male humans. Several tissues besides the testes—including adipose tissue, brain, muscle, skin, and adrenal cortex—produce testosterone and several other androgens. These substances may be synthesized de novo or produced by peripheral conversion of precursors. Moreover, the peripheral organs and tissues may convert sex steroids to less active forms (Fig. 545). Notable sites of extragonadal conversion include the skin and adipose tissue. Androstenedione is converted to testosterone in peripheral tissues. In this case, androstenedione is the precursor for the hormone testosterone. Testosterone can be converted to estradiol or DHT or go “backward” by reversible interconversion to androstenedione. Thus, a potent hormone such as testosterone may also serve as a precursor for a weaker hormone (androstenedione), a hormone with different activities (estradiol), or a more potent hormone having similar activities (DHT). This last example may be illustrated by the effects of DHT on hair follicles, sebaceous glands, and the sex accessories. The adrenal gland (see Chapter 50) is another source of androgen production in both males and females. Normal human adrenal glands synthesize and secrete the androgens DHEA, conjugated DHEA sulfate, and androstenedione. Essentially, all the DHEA in male plasma is of adrenal origin. However, less than 1% of the total testosterone in plasma is derived from DHEA. As summarized in Table 54-2, the plasma concentration of androstenedione in males is only ∼25% that of testosterone. About 20% of androstenedione is generated by peripheral metabolism of other steroids. Although the adrenal gland contributes significantly to the total androgen milieu in males, it does not appear to have significant effects on stimulation and growth of the male accessory organs. Testosterone acts on target organs by binding to a nuclear receptor Most testosterone in the circulation is bound to specific binding proteins. About 45% of plasma testosterone is bound to sex hormone–binding globulin (SHBG)—also called testosterone-binding globulin (TeBG), whereas ∼55% is bound to serum albumin and corticosteroid-binding globulin

(CBG) (see Chapter 50). A small fraction (∼2%) of the total circulating testosterone circulates free, or unbound, in plasma. The free form of testosterone enters the cell by passive diffusion and subsequently exerts biological actions or undergoes metabolism by other organs such as the prostate, liver, and intestines (see the next section). The quantity of testosterone entering a cell is determined by the plasma concentration and by the intracellular milieu of enzymes and binding proteins. Once it diffuses into the cell, testosterone either binds to a high-affinity androgen receptor in the nucleus or is converted to DHT, which also binds to the androgen receptor. The androgen receptor functions as a homodimer (AR/AR) and is a member of the family of nuclear receptors (see Table 4-2) that includes receptors for glucocorticoids, mineralocorticoids, progestins, estrogens, vitamin D, thyroid hormone, and retinoic acid. The gene coding for the androgen receptor is located on the X chromosome. The androgen receptor is a protein with a molecular weight of ∼110 kDa. The androgen-AR complex is a transcription factor that binds to hormone response elements on DNA located 5′ from the genes that the androgens control. Interaction between the androgen-AR complex and nuclear chromatin causes marked increases in transcription, ultimately leading to the synthesis of specific proteins. As a result of these synthetic processes, specific cell functions ensue, including growth and development. The presence of the androgen receptor in a cell or tissue determines whether that tissue can respond to androgens. Whether the active compound in any tissue is DHT or testosterone depends on the presence or absence in that tissue of the microsomal enzyme 5a-reductase, which converts testosterone to DHT. The biological activity of DHT is 30 to 50 times higher than that of testosterone. Some tissues, including the brain, aromatize testosterone to estradiol, and thus the action of this metabolite occurs through the estrogen receptor. Some of the effects of androgens may be nongenomic. For example, androgens may stimulate hepatic microsomal protein synthesis by a mechanism independent of binding to the androgen receptor. Other evidence indicates that the action of androgens on the prostate gland may occur through

Testosterone and the Aging Man

F

or a long time, the abrupt hormonal alterations that signal the dramatic changes of female menopause were believed to have no correlate in men. We now know that men do experience a gradual decline in their serum testosterone levels (Fig. 54-4) and that this decline is closely correlated with many of the changes that accompany aging: decreases in bone formation, muscle mass, growth of facial hair, appetite, and libido. The blood hematocrit also decreases. Testosterone replacement can reverse many of these changes by restoring muscle and bone mass and correcting the anemia. Although the levels of both total and free testosterone decline with age, levels of LH are frequently not elevated. This finding is believed to indicate that some degree of hypothalamic-pituitary dysfunction accompanies aging.

Chapter 54 • The Male Reproductive System

the adenylyl cyclase/PKA system (see Chapter 4) and could result in gene activation under some circumstances. Metabolism of testosterone occurs primarily in the liver and prostate Only small amounts of testosterone enter the urine without metabolism; this urinary testosterone represents less than 2% of the daily testosterone production. The large remaining balance of testosterone and other androgens is converted in the liver to 17-ketosteroids and in the prostate to DHT. The degradation products of testosterone are primarily excreted in the urine as water-soluble conjugates of either sulfuric acid or glucuronic acid. These conjugated testosterone metabolites are also excreted in the feces.

BIOLOGY OF SPERMATOGENESIS AND SEMEN Spermatogenesis includes the mitotic divisions of spermatogonia, the meiotic divisions of spermatocytes to haploid spermatids, and maturation to spermatozoa Mature spermatozoa are derived from germ cells through a series of complex transformations. When seminiferous tubules are viewed in cross section (Fig. 54-1D), the least mature cells are located adjacent to the basement membrane, whereas the most differentiated germ cells are located nearest the lumen. As discussed in Chapter 53, the primordial germ cells migrate into the gonad during embryogenesis; these cells become immature germ cells, or spermatogonia (Fig. 54-6). Beginning at puberty and continuing thereafter throughout life, these spermatogonia, which lie next to the basement membrane of the stratified epithelium lining the seminiferous tubules, divide mitotically (Fig. 54-7). The spermatogonia have the normal diploid complement of 46 chromosomes (2N): 22 pairs of autosomal chromosomes plus 1 X and 1 Y chromosome. Some of the spermatogonia enter into their first meiotic division and become primary spermatocytes. At the prophase of this first meiotic division, the chromosomes undergo crossing over (see Fig. 53-1). At this stage, each cell has a duplicated set of 46 chromosomes (4N): 22 pairs of duplicated autosomal chromosomes, a duplicated X chromosome, and a duplicated Y chromosome. After completing this first meiotic division, the daughter cells become secondary spermatocytes, which have a haploid number of duplicated chromosomes (2N): 22 duplicated autosomal chromosomes and either a duplicated X or a duplicated Y chromosome. These secondary spermatocytes enter their second meiotic division almost immediately. This division results in smaller cells called spermatids, which have a haploid number of unduplicated chromosomes (1N). Spermatids form the inner layer of the epithelium and are found in rather discrete aggregates inasmuch as the cells derived from a single spermatogonium tend to remain together— with cytoplasm linked in a syncytium—and differentiate synchronously. Spermatids transform into spermatozoa in

a process called spermiogenesis, which involves cytoplasmic reduction and differentiation of the tail pieces. Thus, as maturation progresses, developing male gametes decrease in volume. Conversely, maturation leads to an increase in cell number, with each primary spermatocyte producing four spermatozoa, two with an X chromosome and two with a Y chromosome. As additional generations of spermatogonia mature, the advanced cells are displaced toward the lumen of the tubule. Groups of spermatogonia at comparable stages of development undergo mitosis simultaneously. Transformation of spermatogonia into functional spermatozoa requires ∼74 days. Each stage of spermatogenesis has a specific duration. In humans, the life span of the germ cells is 16 to 18 days for spermatogonia, 23 days for primary spermatocytes, 1 day for secondary spermatocytes, and ∼23 days for spermatids. The rate of spermatogenesis is constant and cannot be accelerated by hormones such as gonadotropins or androgens. Germ cells must move forward in their differentiation; if the environment is unfavorable and makes it impossible for them to pursue their differentiation at the normal rate, they degenerate and are eliminated. The most reliable expression of the sperm production rate is the daily number of sperm cells produced per gram of testicular parenchyma. In 20-year-old men, the production rate is ∼6.5 million sperm per gram per day. The rate falls progressively with age and averages ∼3.8 million sperm per gram per day in men 50 to 90 years old. This decrease is probably related to the high rate of degeneration of germ cells during meiotic prophase. Among fertile men, those aged 51 to 90 years exhibit a significant decrease in the percentage of morphologically normal and motile spermatozoa. In summary, three processes occur concurrently in the seminiferous epithelium: (1) an increase in the number of cells by mitosis, (2) a reduction in the number of chromosomes by meiosis, and (3) the production of mature sperm from spermatids by spermiogenesis. Thus, spermatogenesis is a regular, ordered, sequential process resulting in the production of mature male gametes. It is instructive to consider how spermatogenesis in the male differs from oogenesis in the female. In fact, the two processes differ in each of the three steps just noted: (1) in the female, the mitotic proliferation of germ cells takes place entirely before birth, whereas in the male, spermatogonia proliferate only after puberty and then throughout life; (2) the meiotic divisions of a primary oocyte in the female produce only one mature ovum, whereas in the male, the meiotic divisions of a primary spermatocyte produce four mature spermatozoa; and (3) in the female, the second meiotic division is completed only on fertilization (see Chapter 56) and thus no further development of the cell takes place after the completion of meiosis, whereas in the male, the products of meiosis (the spermatids) undergo substantial further differentiation to produce mature spermatozoa. The Sertoli cells support spermatogenesis The Sertoli cells are generally regarded as support or nurse cells for the spermatids (Fig. 54-7). The Sertoli cells are

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Primordial germ cell (2N DNA) Enters gonad Spermatogonium (2N DNA)

Many rounds of mitosis

Mitosis

(4N DNA)

Spermatogonia (2N DNA)

Spermatogonia (2N DNA)

Primary spermatocytes (diploid 4N DNA)

Meiosis I

Secondary spermatocytes (haploid 2N DNA) Meiosis II

Spermatids (haploid 1N DNA)

Mature sperm (Spermatozoa)

Figure 54-6 Spermatogenesis. Early during embryogenesis, the primordial germ cells migrate to the gonad, where they become spermatogonia. Beginning at puberty, the spermatogonia undergo many rounds of mitotic division. Some of these spermatogonia (2N DNA) enter the first meiotic division, at which time they are referred to as primary spermatocytes. During prophase, each primary spermatocyte has a full complement of duplicated chromosomes (4N DNA). Each primary spermatocyte divides into two secondary spermatocytes, each with a haploid number of duplicated chromosomes (2N DNA). The secondary spermatocyte enters the second meiotic division, producing two spermatids, each of which has a haploid number of unduplicated chromosomes (1N DNA). Further maturation of the spermatids yields the spermatozoa (mature sperm). One primary spermatocyte yields four spermatozoa.

large, polyhedral cells extending from the basement membrane toward the lumen of the seminiferous tubule. Spermatids are located adjacent to the lumen of the seminiferous tubules during the early stages of spermiogenesis and are surrounded by processes of Sertoli cell cytoplasm. Tight

junctions connect the adjacent Sertoli cells, to forming a blood-testis barrier—analogous to the blood-brain barrier (see Chapter 11)—that presumably provides a protective environment for developing germ cells. In addition, gap junctions between the Sertoli cells and developing sperma-

Chapter 54 • The Male Reproductive System

Fibroblast Basal compartment

Basal lamina Spermatogonium Tight junction

Adluminal compartment

Nucleus of Sertoli cell Primary spermatocyte Secondary spermatocyte Early spermatid Sertoli cells Late spermiogenesis

The Sertoli Cell–Only Syndrome

I

nvestigators have described a group of normally virilized men whose testes are small bilaterally and whose ejaculates contain no sperm cells (azoospermia). The seminiferous tubules of these men are lined by the Sertoli cells, but the tubules show a complete absence of germ cells. The Sertoli cell–only syndrome (or germinal cell aplasia) accounts for 10% to 30% of male infertility secondary to azoospermia and can either be caused by a single-gene defect or be acquired (e.g., as a result of orchitis, alcoholism, toxic agents). The Leydig cell function is usually preserved. Plasma testosterone and LH levels are usually normal, whereas FSH levels are often, but not always, elevated. It is not entirely clear why FSH levels are elevated in these men. This elevation may result from the absence of germ cells or from suboptimal secretion of inhibin by the Sertoli cells, inasmuch as inhibin is a powerful inhibitor of FSH secretion at the level of the anterior pituitary gland. Segments of Sertoli cell–only tubules may be observed in conditions such as orchitis or exposure to other agents that are toxic to the gonads. However, these individuals generally have functional spermatogenesis in the other seminiferous tubules.

Lumen of seminiferous tubule

Figure 54-7 Interaction of the Sertoli cells and sperm. This figure is an idealized high-magnification view of a portion of the wall of a seminiferous tubule (see Fig. 54-1C). A single Sertoli cell spans from the basal lamina to the lumen of the seminiferous tubule. The adjacent Sertoli cells are connected by tight junctions and surround developing germ cells. From the basal lamina to the lumen of the tubule, gradual maturation of the germ cells occurs.

tozoa may represent a mechanism for transferring material between these two types of cells. Release of the spermatozoa from the Sertoli cell has been called spermiation. Spermatids progressively move toward the lumen of the tubule and eventually lose all contact with the Sertoli cell after spermiation. Sperm maturation occurs in the epididymis The seminiferous tubules open into a network of tubules, the rete testes, which serve as a reservoir for sperm. The rete testes are connected to the epididymis through the efferent ductules (see Chapter 53), which are located near the superior pole of the testicle. The epididymis is a highly convoluted single long duct, 4 to 5 m in total length, on the

posterior aspect of the testis. The epididymis can be divided anatomically into three regions: the head (the segment closest to the testis), the body, and the tail. Spermatozoa are essentially immotile on completion of spermiogenesis. Thus, transfer of spermatozoa from the seminiferous tubule to the rete testes is passive. Secretions flow from the testes through the epididymis, with assistance by ciliary action of the luminal epithelium and contractility of the smooth muscle elements of the efferent duct wall. Thus, sperm transport through this ductal system is also primarily passive. As noted earlier, ∼74 days is required to produce spermatozoa, ∼50 days of which is spent in the seminiferous tubule. After leaving the testes, sperm take 12 to 26 days to travel through the epididymis and appear in the ejaculate. The epididymal transit time for men between the ages of 20 and 80 years does not differ significantly. Sperm are stored in the epididymis, where they undergo a process of maturation before they are capable of progressive motility and fertilization (Table 54-3). Spermatozoa released at ejaculation are fully motile and capable of fertilization, whereas spermatozoa obtained directly from the testis are functionally immature insofar as they cannot penetrate an ovum. However, these immature spermatozoa can fertilize if they are injected into an ovum. During maturation in the epididymis, spermatozoa undergo changes in motility, metabolism, and morphology. Spermatozoa derived from the head (caput) of the epididymis (Fig. 54-1C) are often unable to fertilize ova, whereas larger proportions of spermatozoa captured from the body (corpus) are fertile. Spermatozoa obtained from the tail (cauda) of the epididymis, or from the vas deferens, are almost always capable of fertilization. The epididymis empties into the vas deferens, which is responsible for the movement of sperm along the tract. The

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Table 54-3

Sperm Maturation in the Epididymis

Table 54-4

Normal Values for Semen

Progressive increase in forward motility

Parameter

Value

Increased ability to fertilize

Volume

2-6 mL

Maturation of acrosome

Viscosity

Liquefaction in 1 hr

Molecular reorganization of the plasma membrane: Lipids (stabilization of plasma membrane) Proteins (shedding as well as acquisition of new proteins)

pH

7-8

Count

≥20 million/mL

Ability to bind to zona pellucida

Motility

≥50%

Acquisition of receptors for proteins of the zona pellucida

Morphology

60% normal

Increased disulfide bonds between cysteine residues in sperm nucleoproteins Topographic regionalization of glycosidic residues Accumulation of mannosylated residues on the periacrosomal plasma membrane Decreased cytoplasm and cell volume

vas deferens contains well-developed muscle layers that facilitate sperm movement. The vas deferens passes through the inguinal canal, traverses the ureter, and continues medially to the posterior and inferior aspect of the urinary bladder, where it is joined by the duct arising from the seminal vesicle; together, they form the ejaculatory duct. The ejaculatory duct enters the prostatic portion of the urethra after passing through the prostate. Sperm are stored in the epididymis as well as in the proximal end of the vas deferens. All these accessory structures depend on androgens secreted by the testis for full functional development. The accessory male sex glands—the seminal vesicles, prostate, and bulbourethral glands— produce the seminal plasma Only 10% of the volume of semen (i.e., seminal fluid) is sperm cells. The normal concentration of sperm cells is greater than 20 million/mL, and the typical ejaculate volume is greater than 2 mL. The typical ejaculate content varies between 150 and 600 million spermatozoa. Aside from the sperm cells, the remainder of the semen (i.e., 90%) is seminal plasma, the extracellular fluid of semen (Table 54-4). Very little seminal plasma accompanies the spermatozoa as they move through the testes and epididymis. The seminal plasma originates primarily from the accessory glands (the seminal vesicles, prostate gland, and the bulbourethral glands). The seminal vesicles contribute ∼70% of the volume of semen. Aside from the sperm, the remaining ∼20% represents epididymal fluids, as well as secretions of the prostate gland and bulbourethral glands. However, the composition of the fluid exiting the urethral meatus during ejaculation is not uniform. The first fluid to exit is a mixture of prostatic secretions and spermatozoa with epididymal fluid. Subsequent emissions are composed of mainly secretions derived from the seminal vesicle. The

first portion of the ejaculate contains the highest density of sperm; it also usually contains a higher percentage of motile sperm cells. The seminal plasma is isotonic. The pH in the lumen of the epididymis is relatively acidic (6.5 to 6.8) as the result of H+ secretion by clear cells that are analogous to intercalated cells in the nephron. Addition of the relatively alkaline secretions of the seminal vesicles raises the final pH of seminal plasma to between 7.3 and 7.7. The quiescence of epididymal sperm is not well correlated with pH. Spermatozoa generally tolerate alkalinity better than acidity. A pH near neutrality or slightly higher is optimal for the motility and survival of sperm cells in humans and in other species as well. Seminal plasma contains a plethora of sugars and ions. Fructose and citric acid are contributed to the seminal plasma by the accessory glands, and their concentrations vary with the volume of semen ejaculated. The fructose is produced in the seminal vesicles. In a man with oligospermia (i.e., a low daily sperm output) and a low ejaculate volume (recall that more than half of the ejaculate comes from the seminal vesicles), the absence of fructose suggests obstruction or atresia of the seminal vesicles. Ascorbic acid and traces of B vitamins are also found in human seminal plasma. The prostate gland releases a factor—which contains sugars, sulfate, and a vitamin E derivative—that acts to prevent the clumping of sperm heads. In addition, human semen also contains high concentrations of choline and spermine, although their roles remain to be clarified. Seminal plasma is also rich in Ca2+, Na+, Mg2+, K+, Cl−, and phosphate. Concentrations of Zn2+ and Ca2+ are higher in semen than in any other fluid and most other tissues. Calcium ions stimulate the motility of immature epididymal spermatozoa, but they inhibit the motility of spermatozoa in ejaculates obtained from humans. It appears that the diminished response of sperm to Ca2+ and the acquisition of progressive motility are functions of epididymal maturation. Semen also contains low-molecular-weight polypeptides and proteins. The free amino acids probably arise from the breakdown of protein after the semen is ejaculated. The amino acids may protect spermatozoa by binding heavy metals, which may be toxic, or by preventing the agglutination of proteins. Human semen coagulates immediately after ejaculation. Coagulation is followed by liquefaction, which is apparently caused by proteolytic enzymes, which are contained in

Chapter 54 • The Male Reproductive System

Congenital and Acquired Ductal Obstruction

genetic constitution, social contacts, and the age at which hormones exert their effects. In this section, I describe the neurophysiology of the male sex act.

G

The sympathetic and parasympathetic divisions of the autonomic nervous system control the male genital system

enital duct obstruction may be congenital and may result from ductal absence or structural abnormality, or it may be acquired as a result of stricture, infection, or vasectomy. Genital duct obstruction is found in ∼7% of infertile men. An uncommon cause of male infertility is congenital absence of the vas deferens, which accounts for as many as 50% of cases of congenital ductal obstruction. These patients generally have azoospermic ejaculates with low volume. Congenital absence of the vas deferens is common in male patients with cystic fibrosis (CF) and is sometimes the only manifestation of CF. Epididymal abnormalities range from the presence of an incomplete epididymis to the presence of only small portions of the epididymis; in addition, the seminal vesicles are often absent. Spermatogenesis is thought to be normal inasmuch as testicular biopsy specimens demonstrate germ cells in several stages of development. Obstruction of the epididymis may also occur as a result of gonococcal or tuberculous epididymitis. Smallpox and filariasis are common causes of ductal obstruction in areas where these diseases are endemic. Inspissated secretions may occlude the epididymis in men with Young syndrome or CF. Elective vasectomy, a simple surgical procedure in which a small segment of the vas deferens is removed to ensure male infertility, is currently the leading cause of ductal obstruction. Azoospermia in men with normal testes is the hallmark of genital duct obstruction. However, when specimens of testicles from men who have had vasectomies are examined microscopically, interstitial fibrosis has been found in as many as 20% of cases. This group exhibits low fertility after elective reversal of vasectomy. When the seminiferous tubules are examined, increased thickness of the tubule wall, an increase in cross-sectional tubular area, and decreased numbers of the Sertoli cells are usually noted. Testosterone and gonadotropin levels are normal in most patients with ductal obstruction.

prostatic secretions. Prostatic secretion is rich in acid phosphatase. The natural substrate for acid phosphatase is phosphorylcholine, which is contributed by the seminal vesicles. Hyaluronidase is also present in human semen, although its functional role remains to be clarified. Hyaluronidase is not a product of the accessory glands; rather, it is contained within the sperm cell cytoplasm and is rapidly released into the seminal plasma. Hyaluronidase may perform a role in facilitating penetration of the oocyte by the sperm cell because of the ability of hyaluronidase to depolymerize hyaluronic acid.

MALE SEX ACT Sex steroids influence the central nervous system, even in utero, and play important roles in determining and regulating complex patterns of sexual behavior. However, reproductive behavior is extraordinarily complex and is influenced by numerous factors other than sex steroids, such as one’s

The testes, epididymis, male accessory glands, and erectile tissue of the penis receive dual innervation from the sympathetic and parasympathetic branches of the autonomic nervous system (ANS). The penis also receives both somatic efferent (i.e., motor) and afferent (i.e., sensory) innervation through the pudendal nerve (S2 through S4). Sympathetic Division of the ANS

As described in Chapter 15, the preganglionic sympathetic neurons originate in the thoracolumbar segments of the spinal cord (T1 through T12, L1 through L3; see Fig. 14-4). For the lower portion of the sympathetic chain (T5 and lower), the preganglionic fibers may pass through the paravertebral sympathetic trunk and then pass through splanchnic nerves to a series of prevertebral plexuses and ganglia (see later). Once within one of these plexuses or ganglia, the preganglionic fiber may either (1) synapse with the postganglionic fiber or (2) pass on to a more caudal plexus or ganglion without synapsing. The sympathetic efferent (motor) nerve fibers that are supplied to the male sex organs emanate from five primary prevertebral nerve plexuses (Fig. 54-8): the celiac, superior mesenteric, inferior mesenteric, superior hypogastric, and inferior hypogastric or pelvic plexuses. The celiac plexus is of interest in a discussion of male sex organs only because preganglionic sympathetic fibers pass through this plexus on their way to more caudal plexuses. The superior mesenteric plexus lies on the ventral aspect of the aorta. Preganglionic fibers from the celiac plexus pass through the superior mesenteric plexus on their way to more caudal plexuses. Most of the preganglionic sympathetic fibers pass from the superior mesenteric plexus to the inferior mesenteric plexus, although some of the nerves pass directly to the hypogastric plexus. The superior hypogastric plexus is a network of nerves located distal to the bifurcation of the aorta. The inferior hypogastric or pelvic plexus receives sympathetic supply from the hypogastric nerve. In addition to these five plexuses, two other small ganglia are of interest. The spermatic ganglion is located near the origin of the testicular artery from the aorta. The spermatic ganglion receives fibers directly from the lumbar sympathetic nerves and from branches of several other ganglia. The hypogastric (or pelvic) ganglion is located at the junction of the hypogastric and pelvic nerve trunks. Parasympathetic Division of the ANS

The preganglionic parasympathetic neurons relating to the male reproductive system originate in the sacral segments of the spinal cord (S2 through S4; see Fig. 14-4). These fibers pass through the pelvic nerve to the pelvic plexus, where they synapse with the postganglionic parasympathetic neurons.

Visceral Afferents Sensory fibers are present in all the nerve tracts described (see Fig. 14-2). These fibers travel (1) with the pelvic nerves to the dorsal root of the spinal cord,

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A

SYMPATHETIC INNERVATION

Celiac ganglion

B Celiac plexus

Aorta

Superior mesenteric ganglion

Superior mesenteric plexus

Right aorticorenal ganglion

Left aorticorenal ganglion

Spermatic ganglion

Inferior mesenteric ganglion

SAGITTAL SECTION SHOWING INNERVATION OF MALE GENITAL SYSTEM

Superior hypogastric plexus

Pelvic nerves (pelvic parasympathetics)

Right and left inferior hypogastric nerves

Renal artery

Rectum

Spermatic artery and plexus

Bladder Small and large cavernous nerves

Inferior mesenteric plexus

Pelvic plexus Pudendal nerve

Aorta

Aortic plexus

Dorsal nerve of penis

Superior hypogastric plexus Hypogastric nerve

Inferior hypogastric (pelvic) plexus Hypogastric (pelvic) ganglion

Figure 54-8 Innervation of the male genital system. A, The sympathetic innervation of the male genital system involves a series of prevertebral nerve plexuses and ganglia. B, Three motor pathways as well as a sensory pathway are involved in erection: (1) parasympathetic innervation: preganglionic parasympathetic fibers arise from the sacral spinal cord and from the pelvic nerve, and they synapse in the pelvic plexus; the postganglionic parasympathetic fibers follow the cavernous nerve to the penile corpora and vasculature; (2) sympathetic innervation: preganglionic sympathetic fibers exit the thoracolumbar cord and synapse in one of several prevertebral ganglia; postganglionic fibers reach the genitalia through the hypogastric nerve, the pelvic plexus, and the cavernous nerves; and (3) somatic innervation: somatic (i.e., not autonomic) motor fibers originate in the sacral spinal cord and form the motor branch of the pudendal nerve; the fibers innervate the striated penile muscles. In addition to these three motor pathways, there is also an afferent pathway from the penis. The dorsal nerve of the penis is the main terminus of the sensory pudendal nerve and is the sole identifiable root for tactile sensory information from the penis.

Chapter 54 • The Male Reproductive System

(2) with the sacral nerves to the sympathetic trunk and then rising in the sympathetic trunk to the spinal cord, or (3) with the hypogastric nerve and ascending to more rostral prevertebral plexuses and then to the spinal cord. The principal functions of motor innervation to the male accessory glands include control of smooth muscle contraction, vascular tone, and epithelial secretory activity. Erection is primarily under parasympathetic control The two corpora cavernosa and the corpus spongiosum are usually coordinated in their erection (i.e., tumescence) and detumescence. However, they may act independently inasmuch as their vascular and neuroeffector systems are relatively independent. During erection, relaxation of the smooth muscles of the corpora allows increased inflow of blood to fill the corporal interstices and results in an increase in volume and rigidity. Vascular actions of the smooth muscles of the corpora and the perineal striated muscles are coordinated. For example, contraction of the striated muscles overlying the vascular reservoirs of the penile bulb increases the pressure of the blood in the corpora and promotes increased rigidity. The three major efferent (i.e., motor) pathways for the regulation of penile erection are parasympathetic (pelvic nerve), sympathetic (hypogastric nerve), and somatic (pudendal nerve). Parasympathetic Innervation

The first and most important pathway for erection is the parasympathetic division of the ANS. These fibers derive from the lumbar and sacral portions of the spinal cord and travel through the pelvic nerve, the pelvic plexus, and the cavernous nerve to the penile corpora and vasculature (Fig. 54-8). This pathway is almost entirely parasympathetic, but apparently it also carries some sympathetic fibers (see later). The parasympathetic activity results in vasodilatation of the penile blood vessels, thus increasing blood flow to the cavernous tissue and engorging the organ with blood. In erectile tissue, parasympathetic postganglionic terminals release acetylcholine (ACh) and nitric oxide (NO), similar to the system discussed in Chapter 14 (see Fig. 14-11). First, ACh may bind to M3 muscarinic receptors on endothelial cells. Through Gαq, these receptors would then lead to stimulation of PLC, increased [Ca2+]i, activation of NO synthase, and local release of NO (see Chapter 3). Second, the nerve terminals may also directly release NO. Regardless of the source of NO, this gas diffuses to the vascular smooth muscle cell, where it stimulates guanylyl cyclase to generate cGMP, which, in turn, causes vasodilation (see Chapter 23). See the box titled Erectile Dysfunction.

decrease in this sympathetic tone allows relaxation of the corpora and thus contributes to tumescence. Somatic Innervation The third pathway is the motor branch of the pudendal nerve. It has primarily somatic (i.e., not autonomic) fibers, originates in the sacral spinal cord, and innervates the striated penile muscles. Contraction of the striated ischiocavernosus muscle during the final phase of erection increases pressure inside the corpora cavernosa to values that are even higher than systemic arterial pressure. Contraction of the striated bulbospongiosus muscle increases engorgement of the corpus spongiosum, and thus the glans penis, by pumping blood up from the penile bulb underlying this muscle. Humans are apparently less dependent on their striated penile muscle for achieving and maintaining erection. However, these muscles are active during ejaculation and contribute to the force of seminal expulsion. Postganglionic neurons release other so-called nonadrenergic, noncholinergic neurotransmitters (see Chapter 23)—including NO—that also contribute to the erectile process. Afferent Innervation

The penis also has an afferent pathway. The dorsal nerve of the penis is the main terminus of the sensory pudendal nerve and is the sole identifiable root for tactile sensory information from the penis. Emission is primarily under sympathetic control

The term seminal emission refers to movement of the ejaculate into the prostatic or proximal part of the urethra. Under some conditions, seminal fluid escapes episodically or continuously from the penile urethra; this action is also referred to as emission. Emission is the result of peristaltic contractions of the ampullary portion of the vas deferens, the seminal vesicles, and the prostatic smooth muscles. These actions are accompanied by constriction of the internal sphincter of the bladder, which is under sympathetic control (see Chapter 33), thus preventing retrograde ejaculation of sperm into the urinary bladder (see the box on Ejaculatory Dysfunction: Retrograde Ejaculation). The rhythmic contractions involved in emission result from contraction of smooth muscle. In contrast to other visceral organ systems, the smooth muscle cells of the male ducts and accessory glands fail to establish close contact with one another and show limited electrotonic coupling. In the male accessory glands, individual smooth muscle cells are directly innervated and have only limited spontaneous activity (i.e., multiunit smooth muscle; see Chapter 9). This combination allows a fast, powerful, and coordinated response to neural stimulation. Motor Activity of the Duct System

Sympathetic Innervation

The second pathway, which is thought to be entirely sympathetic, exits the thoracolumbar spinal cord. The preganglionic fibers then course through the least splanchnic nerve, the sympathetic chain, and the inferior mesenteric ganglion. The postganglionic fibers reach the genitalia through the hypogastric nerve, the pelvic plexus, and the cavernous nerves (see earlier). Tonic sympathetic activity contributes to penile flaccidity. During erection, a

A gradation between two forms of smooth muscle activity occurs along the male duct system. The efferent ducts and proximal regions of the epididymis are sparsely innervated, but they display spontaneous contractions that can be increased through adrenergic agents acting on α-adrenergic receptors. In contrast, the distal end of the epididymis and the vas deferens are normally quiescent until neural stimulation is received during the ejaculatory process. Contraction of the smooth muscle of the distal epididymis, vas deferens, and accessory sex

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Erectile Dysfunction

S

ildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) are reasonably well tolerated oral medications used to treat erectile dysfunction. Men with erectile dysfunction experience significant improvement in rigidity and duration of erections after treatment with these medications. As indicated in the text, the smooth muscle tone of the human corpus cavernosum is regulated by the synthesis and release of NO, which raises [cGMP]i in vascular smooth muscle cells, thereby relaxing the smooth muscle and leading to vasodilatation and erection. Breakdown of cGMP by cGMP-specific phosphodiesterase type 5 limits the degree of vasodilation and, in the case of the penis, limits erection. Sildenafil, vardenafil, and tadalafil are highly selective, high-affinity inhibitors of cGMP-specific phosphodiesterase type 5 and thereby raise [cGMP]i in smooth muscle and improve erection in men with erectile dysfunction. The new medications are attractive because they have established efficacy that benefit most men with insufficient erection. These medications stimulate erection only during sexual arousal and thus have a rather natural effect. They can be taken as little as 1 hour before planned sexual activity. One of the side effects of sildenafil is “blue vision,” a consequence of the effect of inhibiting cGMP-specific phosphodiesterase in the retina. In individuals taking other vasodilators, sildenafil can lead to sudden death. In women, sildenafil may improve sexual function by increasing blood flow to the accessory secretory glands (see Chapter 55).

glands occurs in response to stimulation of the sympathetic fibers in the hypogastric nerve and release of norepinephrine. Indeed, an intravenous injection of epinephrine or norepinephrine can induce seminal emission, whereas selective chemical sympathectomy or an adrenergic antagonist can inhibit seminal emission. The role of parasympathetic innervation to the musculature of these ducts and accessory glands in the male is not entirely clear. Parasympathetic fibers may be preferentially involved in basal muscular activity during erection (i.e., before ejaculation) and during urination. Secretory Activity of the Accessory Glands

The effect of autonomic innervation on the secretory activity of the epithelia of the male accessory glands has been studied extensively. Electrical stimulation of the pelvic nerves (parasympathetic) induces copious secretions. The secretory rate depends on the frequency of stimulation and can be blocked with atropine, a competitive inhibitor of muscarinic ACh receptors. Cholinergic drugs induce the formation of copious amounts of secretions when these drugs are administered systemically. Secretions from the bulbourethral glands also contribute to the ejaculate. The bulbourethral glands do not store secretions but produce them during coitus. The secretory activity of the bulbourethral glands also appears to be under cholinergic control inasmuch as administration of atropine causes marked inhibition of secretion from these glands.

Ejaculatory Dysfunction: Retrograde Ejaculation

A

s noted in the text, emission is normally accompanied by constriction of the internal urethral sphincter. Retrograde ejaculation occurs when this sphincter fails to constrict. As a result, the semen enters the urinary bladder rather than passing down the urethra. Retrograde ejaculation should be suspected in patients who report absent or small-volume ejaculation after orgasm. The presence of more than 15 sperm per high-power field in urine specimens obtained after ejaculation confirms the presence of retrograde ejaculation. Lack of emission or retrograde ejaculation may result from any process that interferes with innervation of the vas deferens and bladder neck. Several medical illnesses, such as diabetes mellitus (which can cause peripheral neuropathy) and multiple sclerosis, or the use of pharmaceutical agents that interfere with sympathetic tone can lead to retrograde ejaculation. Retrograde ejaculation may also occur as a result of nerve damage associated with certain surgical procedures, including bladder neck surgery, transurethral resection of the prostate, colorectal surgery, and retroperitoneal lymph node dissection. Retrograde ejaculation from causes other than surgery involving the bladder neck may be treated with pharmacological therapy. Sympathomimetic drugs such as phentolamine (an α-adrenergic agonist), ephedrine (which enhances norepinephrine release), and imipramine (which inhibits norepinephrine re-uptake by presynaptic terminals) may promote normal (i.e., anterograde) ejaculation by increasing the tone of the vas deferens (propelling the seminal fluid) and the internal sphincter (preventing retrograde movement).

Control of the motor activity of the ducts and of the secretory activity of the accessory glands is complex and involves both the sympathetic and the parasympathetic divisions of the ANS. The central nervous system initiates and coordinates all these activities. Ejaculation is under control of a spinal reflex As discussed, seminal emission transports semen to the proximal (posterior) part of the urethra. Ejaculation is the forceful expulsion of this semen from the urethra. Ejaculation is normally a reflex reaction triggered by the entry of semen from the prostatic urethra into the bulbous urethra. Thus, emission sets the stage for ejaculation. The ejaculatory process is a spinal cord reflex, although it is also under considerable cerebral control. The afferent (i.e., sensory) impulses reach the sacral spinal cord (S2 through S4) and trigger efferent activity in the somatic motor neurons that travel through the pudendal nerve. The resulting rhythmic contractions of the striated muscles of the perineal area— including the muscles of the pelvic floor, as well as the ischiocavernosus and bulbospongiosus muscles—forcefully propel the semen through the urethra through the external meatus. In addition, spasmodic contractions of the muscles of the hips and the anal sphincter generally accompany ejaculation.

Chapter 54 • The Male Reproductive System

Neuronal Lesions Affecting Erection and Ejaculation

E

rectile dysfunction is often associated with disorders of the central and peripheral nervous systems. Spinal cord disease and peripheral neuropathies are of particular interest, and spinal cord injuries have been studied in some detail. Erectile capacity is usually preserved in men with lesions of the premotor neurons (neurons that project from the brain to the spinal cord; Table 54-5). In these men, reflexogenic erections occur in 90% to 100% of cases, whereas psychogenic erections do not occur because the pathways from the brain are blocked. Ejaculation is more significantly impaired in upper than in lower motor neuron lesions, presumably because of loss of the psychogenic component. A clinically important feature of the spinal segmentation of nerve roots for generating erection (i.e., thoracolumbar and lumbosacral) is that spinal or peripheral nerve damage may affect only one of the effector systems. Because the lumbosa-

Table 54-5 Effects of Neural Lesions on Erection and Ejaculation Lesion

Reflexogenic Erection

Psychogenic Erection

Effect on Ejaculation

Upper motor neuron

Present

Absent

Significantly impaired

Lower motor neuron

Absent

Present

Less impaired

Orgasm is a term best restricted to the culmination of sexual excitation, as generally applied to both men and women. Orgasm is the cognitive correlation of ejaculation in the male human. Although orgasm, the pleasurable sensation that accompanies ejaculation, is not well understood, clearly, it is as much a central phenomenon as it is a peripheral one. REFERENCES Books and Reviews Ackland JF, Schwartz NB, Mayo KE, Dodson RE: Nonsteroidal signals originating in the gonads. Physiol Rev 1992; 72:731-787. Andersson K-E, Wagner G: Physiology of penile erection. Physiol Rev 1995; 75:191-236.

cral system also carries most of the penile afferents, erection in response to penile stimulation (reflexogenic) is most affected by damage to the lower spinal cord or the nerves that project there. Evidence from men with spinal injuries in the T10 through T12 region has implicated the sympathetic thoracolumbar pathway in mediating erections resulting from sexual stimuli received through the cranial nerves or generated within the brain as memories, fantasies, or dreams. In men with lower motor neuron lesions, reflexogenic erections are absent. However, psychogenic erections still occur in most men with incomplete lesions and in about one fourth of men with complete lesions. It remains uncertain whether this sympathetic pathway is normally the principal route for psychogenic erections or whether it just assumes the role when lumbosacral parasympathetic pathways are damaged.

Griffin JE, et al: The testis. In Bondy PK, Rosenberg LE: Metabolic Control and Disease. Philadelphia: WB Saunders, 1980. Hecht NB: Molecular mechanisms of male germ cell differentiation. Bioessays 1998; 20:555-561. Mather JP, Moore A, Li RH: Activins, inhibins, and follistatins: Further thoughts on a growing family of regulators. Proc Soc Exp Biol Med 1997; 215:209-222. Skinner MK: Cell-cell interaction in the testis. Endocr Rev 1991; 12:45-77. Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed. Philadelphia: WB Saunders, 1998. Journal Articles Beitins IZ, Padmanabhan V, Kasa-Vubu J, et al: Serum bioactive follicle-stimulating hormone concentrations from prepuberty to adulthood: A cross-sectional study. J Clin Endocrinol Metab 1990; 71:1022-1027. Carter AJ, Ballard SA, Naylor AM: Effect of the selective phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog. J Urol 1998; 160:242-246. Koraitim M, Schafer W, Melchior H, Lutzeyer W: Dynamic activity of bladder neck and external sphincter in ejaculation. Urology 1977; 10:130-132. Ludwig DG: The effect of androgen on spermatogenesis. Endocrinology 1950; 46:453-481. Reiter EO, Beitins IZ, Ostrea TR, Gutai JP: Bioassayable luteinizing hormone during childhood and adolescence and in patients with delayed pubertal development. J Clin Endocrinol Metab 1982; 54:155-161. Winter JS, Hughes IA, Reyes FI, Faiman C: Pituitary-gonadal relations in infancy: 2. Patterns of serum gonadal steroid concentrations in man from birth to two years of age. J Clin Endocrinol Metab 1976; 42:679-686.

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CHAPTER

55

T H E F E M A L E R E P R O D U CT I V E SYST E M Ervin E. Jones

REPRODUCTIVE FUNCTION IN THE FEMALE HUMAN Reproductive function in female humans is controlled by hormones that emanate from the hypothalamic-pituitarygonadal axis (see Chapter 47). The release of a mature ovum from an ovary, known as ovulation, is the dominant event of the menstrual cycle. Whereas ovulation in some mammals is triggered by mating, ovulation in the female human is spontaneous and is regulated by cyclic functional interactions among signals coming from the hypothalamus, the anterior pituitary, and the ovaries. Although many aspects of female reproduction are cyclic, maturation and demise (i.e., atresia) of the functional units of the ovaries—the ovarian follicles— are continuous processes that occur throughout reproductive life. The ovaries are not the only female organs that undergo rhythmic changes. Alterations in cervical and uterine function are controlled by changes in the circulating concentrations of ovarian hormones, that is, the estrogens and progestins. For example, the uterine lining or endometrium thickens under the influence of ovarian hormones and deteriorates and sloughs at the end of the cycle when ovarian estrogen and progestin secretion diminishes. Menstruation reflects this periodic shedding of the endometrium. Menstrual cycles are generally repetitive unless they are interrupted by pregnancy or terminated by menopause. All the cyclic physiological changes prepare the female reproductive tract for sperm and ovum transport, fertilization, implantation, and pregnancy. Female reproductive organs include the ovaries and accessory sex organs The ovaries lie on the sides of the pelvic cavity (Fig. 55-1A). A layer of mesothelial cells covers the surface of the ovary. The ovary itself consists of an inner medulla and an outer zone, or cortex, that surrounds the medulla except at the hilar area. The cortex of the ovary in a mature woman contains developing follicles and corpora lutea in various stages of development (Fig. 55-1B). These elements are interspersed throughout the stroma, which includes connective tissue, interstitial cells, and blood vessels. The medulla comprises large blood vessels and other stromal elements.

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The female accessory sex organs include the fallopian tubes, the uterus, the vagina, and the external genitalia. The fallopian tube provides a pathway for the transport of ova from the ovary to the uterus. The distal end of the fallopian tube expands as the infundibulum, which ends in multiple fimbriae. The infundibulum is lined with epithelial cells that have cilia that beat toward the uterus. The activity of these cilia and the contractions of the wall of the fallopian tube, particularly around the time of ovulation, facilitate transport of the ovum. The uterus is a complex, pear-shaped, muscular organ that is suspended by a series of supporting ligaments. It is composed of a fundus, a corpus, and a narrow caudal portion called the cervix. The external surface of the uterus is covered by serosa, whereas the interior, or endometrium, of the uterus consists of complex glandular tissue and stroma. The uterus is continuous with the vagina through the cervical canal. The cervix is composed of dense fibrous connective tissue and muscle cells. The cervical glands lining the cervical canal produce a sugar-rich secretion, the viscosity of which is conditioned by estrogen and progesterone. The human vagina is ∼10 cm in length and is a single, expandable tube. The vagina is lined by stratified epithelium and is surrounded by a thin muscular layer. During development, the lower end of the vagina is covered by the membranous hymen, which is partially perforated during fetal life. In some instances, the hymen remains continuous. The external genitalia include the clitoris, the labia majora, and the labia minora, as well as the accessory secretory glands (including the glands of Bartholin), which open into the vestibule. The clitoris is an erectile organ, which is homologous to the penis (see Chapter 53) and mirrors the cavernous ends of the glans penis.

PUBERTY Puberty marks the transition to cyclic, adult reproductive function Puberty is the transition from a noncyclic, relatively quiescent reproductive endocrine system to a state of cyclic reproductive function that allows procreation. Puberty is the transition between the juvenile state and adulthood during

Chapter 55 • The Female Reproductive System

A

OBLIQUE VIEW OF THE INTERNAL FEMALE SEX ORGANS

Fallopian tube

Fundus of uterus

Intramural portion of fallopian tube

Ovary

Ampulla of fallopian tube Isthmus of fallopian tube Ovary

Endometrium Myometrium Round ligament Infundibulum of fallopian tube

Broad ligament

Fimbriae Hilus Corpus (body) of uterus

Mesovarium Proper ovarian ligament Bladder Cervix of uterus Vagina

B

CROSS SECTION THROUGH AN OVARY

Primary follicles

Primordial follicle

Corpus albicans

Secondary follicle Tertiary follicle

Corpus luteum

Hilus

Blood vessels

Ruptured follicle

Graafian follicle

Discharged oocyte

Figure 55-1

Corona radiata

The anatomy of the female internal genitalia and accessory sex organs.

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Section IX • The Reproductive System

Table 55-1

Stages in Female Puberty

Stage

Breast Development

Pubic Hair

1

Preadolescent: only papillae are elevated

Preadolescent: no pubic hair is present, only vellus hair, as on the abdomen

2

Breasts and papillae are both elevated, and the diameter of the areolae increases

Pubic hair is sparse, mainly along the labia majora

3

The breasts and areolae further enlarge

Pubic hair is darker, coarser, and curlier and spreads over the pubis

4

The areolae and papillae project out beyond the level of the expanding breast tissue

The pubic hair is of adult type, but covers an area smaller than in most adults

5

With further enlargement of the breast, the areolae are now on the same level as the rest of the breast; only the papillae project; adult pattern

Adult pattern

which time secondary sexual characteristics appear, the adolescent growth spurt occurs, and the ability to procreate is achieved. Table 55-1 summarizes the stages of puberty in the female. Puberty in girls involves the beginning of menstrual cycles (menarche), breast development (thelarche), and an increase in adrenal androgen secretion (adrenarche). The precise cause of the onset of puberty is not completely understood, although multiple intrinsic and extrinsic factors play a role. Genetic factors are major determinants of pubertal onset. Other factors, such as nutrition, geographic location, and exposure to light, also play a role. Over the last century, the age of girls at menarche in the United States and Europe has gradually decreased. Although the reason that menarche now occurs at a younger age remains incompletely understood, it is probably because of improved nutritional status. However, better nutritional status alone cannot completely explain the decreased age of pubertal onset. Distance from the equator and lower altitudes are associated with early onset of puberty. A loose correlation is also seen between the onset of menarche in the mother and the onset of menarche in the daughter. The onset of puberty is also related to body composition and to fat deposition. Severe obesity and heavy exercise delay puberty. Gonadotropin levels are low during childhood As shown in Figure 55-2A, a surge in the levels of the pituitary gonadotropins, luteinizing hormone (LH) and folliclestimulating hormone (FSH), occurs during intra-uterine life. A second peak takes place in the immediate postnatal period. However, gonadotropin levels tend to decrease at ∼4 months of age; thereafter, they decline further and remain low until just before puberty. Gonadotropin levels are lowest between 6 and 8 years of age. Although the reason for low gonadotropin secretion by the pituitary in childhood remains unknown, it was once thought to result from feedback inhibition by high levels of gonadal steroids. However, an experiment of nature has revealed that such is not the case. Indeed, girls with gonadal dysgenesis, like physiologically normal girls, have low levels of LH and FSH, even though their

ovaries produce low levels of steroids. Thus, it is likely that the low levels of gonadotropins in the prepubertal period do not reflect high levels of steroids, but rather a high sensitivity to feedback inhibition of the hypothalamic-pituitary system by these steroids. I discuss this feedback mechanism in the next section. During puberty, gonadotropin-releasing hormone secretion becomes pulsatile, and the sensitivity of the gonadotrophs to feedback inhibition by estrogens decreases As shown by the insets to Figure 55-2A, one of the earliest events of puberty is the onset of pulsatile gonadotropin secretion from the pituitary during rapid eye movement (REM) sleep; this pulsatile gonadotropin secretion reflects the pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The development of secondary sexual characteristics follows the onset of sleepassociated pulsatility. With maturation, these pulses occur throughout the day. It is not understood why pulsatile behavior should occur initially only during REM sleep. The precipitating event that is responsible for initiating pulsatile GnRH release is also unknown, although it may reflect the maturation of hypothalamic neurons. Once a pulsatile pattern of gonadotropin secretion is established, it continues throughout reproductive life into menopause. The increased pulsatility of GnRH release eventually leads to a marked increase in plasma LH levels—the LH surge that marks the initiation of the first menstrual cycle. During early pubescence, the LH surges do not occur in a regular pattern, so menstrual cycles are generally irregular. As the reproductive system matures, the LH surges gradually come at regular intervals, and cyclic reproductive function becomes firmly established. The appearance of GnRH pulsatility early in puberty is associated with decreased sensitivity of the hypothalamicpituitary system to circulating sex steroids. In young girls, even low levels of sex steroids are sufficient to feed back on the hypothalamic-pituitary system and to block the release

Chapter 55 • The Female Reproductive System

A

PATTERNS OF GONADOTROPIN LEVELS THROUGHOUT THE LIFE OF A FEMALE Day Night

LH secretion patterns Fetus

Childhood

Puberty

Reproductive years

Menopause

Infancy

FSH

100 Plasma gonadotropins (mU/mL) 70

monthly surges LH

10

rth Bi d 3r d 2n t 1s

10–14 yr

6 mo

50 yr

Trimesters

B

C AGE DEPENDENCE OF FEEDBACK SENSITIVITY

NEGATIVE FEEDBACK OF ESTROGENS ON GONADOTROPIN RELEASE In an adult, much higher levels occur. Child

Adolescent

Adult Relative sensitivity of gonadotropin release to negative feedback by sex steroids

Relative gonadotropin release In a child, even a low level of steroid blocks the release of gonadotropin.

High sensitivity

Concentration of sex steroids

Low sensitivity

Fetus

Infancy and childhood

Puberty Adult

Figure 55-2 Gonadotropin function during life. A, The levels of both LH and FSH peak during fetal life and again during early infancy, before falling to low levels throughout the rest of childhood. At the onset of puberty, LH and FSH levels slowly rise and then begin to oscillate at regular monthly intervals. At menopause, gonadotropin levels rise to very high levels. The four insets show daily changes in gonadotropin levels. B, This is a highly schematic plot of how estrogen levels negatively feed back on gonadotropin secretion by the gonadotroph cells of the anterior pituitary. In childhood, even very low estrogen levels are sufficient to suppress gonadotropin output fully. In adolescence, higher levels of estrogens are required. In the adult woman, estrogens must be at very high levels to suppress gonadotropin release. C, This is a plot—versus age—of the midpoints of curves such as those in B.

of gonadotropins (Fig. 55-2B). As a girl goes through puberty, the levels of steroids required to block gonadotropin release progressively become higher and higher. At about the same time, the levels of sex steroids also rise. Eventually, a situation is reached in which the monthly oscillations in sex steroid levels produce the full range of feedback inhibition of gonadotropin release. Thus, during maturation, the sensitivity of the hypothalamic-pituitary system to inhibition by sex steroids falls to reach the low level that is characteristic

of the adult (Fig. 55-2C). As discussed later, in addition to the negative feedback of sex steroids on gonadotropin release, positive feedback also occurs near the midpoint of the menstrual cycle. During puberty, basal levels of LH and FSH increase (Fig. 55-2A). Concentrations of androgens and estrogens also increase many-fold as a result of gonadal stimulation by FSH and LH. The LH surge that occurs at midcycle is thus superimposed on an already high basal level of circulating LH.

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Section IX • The Reproductive System

Ovarian cycle

Follicular phase

Endometrial cycle

Menses 0

2

Luteal phase

Proliferative phase 4

6

8

10

12

Secretory phase 14 Days

16

18

20

22

24

26

28–0

Figure 55-3 The ovarian and endometrial cycles. The menstrual cycle comprises parallel ovarian and endometrial cycles. The follicular phase of the ovarian cycle and the menses start on day 0. In this idealized example, ovulation occurs on day 14, and the entire cycle lasts 28 days.

HYPOTHALAMIC-PITUITARY-GONADAL AXIS AND CONTROL OF THE FEMALE MENSTRUAL RHYTHM The menstrual cycle includes both the ovarian and endometrial cycles The menstrual cycle actually involves cyclic changes in two organs: the ovary and the uterus (Fig. 55-3). The ovarian cycle includes the follicular phase and the luteal phase, separated by ovulation. The endometrial cycle includes the menstrual, the proliferative, and the secretory phases. Although menstrual cycles are generally regular during the reproductive years, the length of the menstrual cycle may be highly variable because of disturbances in neuroendocrine function. The mean menstrual cycle is 28 days long, but considerable variation occurs during both the early reproductive years and the premenopausal period. Irregular menses during adolescence and the premenopausal period occur primarily because of the increased frequency of anovulatory cycles. The first phase of the ovarian cycle is the follicular phase—during which FSH stimulates a follicle to complete its development (i.e., folliculogenesis). The follicular phase begins with the initiation of menstruation and averages ∼14 days in length. The duration of the follicular phase is the most variable of the cycle. During folliculogenesis, the granulosa cells of the follicles increase production of the estrogen estradiol, which stimulates the endometrium to undergo rapid and continuous growth and maturation. This period is the proliferative phase of the endometrial cycle. A rapid rise in ovarian estradiol secretion eventually triggers a surge in LH, which causes ovulation. After releasing its ovum, the follicle transforms into a corpus luteum, which is why the second half of the ovarian cycle is called the luteal phase. The luteal cells produce progesterone and estrogen, which stimulate further endometrial growth and development. This period is the secretory phase of the endometrial cycle. For unknown reasons, the corpus luteum rapidly diminishes its production of estrogens and progestins, thereby resulting in a catastrophic degeneration of the endometrium that leads to menstrual bleeding. This period is the menstrual phase of the endometrial cycle. The hypothalamic-pituitary-ovarian axis drives the menstrual cycle Neurons in the hypothalamus synthesize, store, and release GnRH. Long portal vessels carry the GnRH to the anterior

pituitary, where the hormone binds to receptors on the surface of gonadotrophs. The results are the synthesis and release of both FSH and LH from the gonadotrophs. These trophic hormones, LH and FSH, stimulate the ovary to synthesize and secrete the sex steroids estrogens and progestins. The ovaries also produce peptides called inhibins and activins. Together, these ovarian steroids and peptides exert both negative and positive feedback on both the hypothalamus and the anterior pituitary. This complex interaction is unique among the endocrine systems of the body inasmuch as it generates a monthly pattern of hormone fluctuations. Because the cyclic secretion of estrogens and progestins primarily controls endometrial maturation, menstruation reflects these cyclic changes in hormone secretion. Neurons in the hypothalamus release GnRH in a pulsatile fashion At the rostral end of the hypothalamic-pituitary-ovarian axis (Fig. 55-4), neurons in the arcuate nucleus and the preoptic area of the hypothalamus synthesize GnRH. They transport GnRH to their nerve terminals for storage and subsequent release. As discussed later, each of the aforementioned two groups of neurons is responsible for a very different kind of rhythm of GnRH secretion. Axons of the GnRH neurons project directly to the median eminence, the extreme basal portion of the hypothalamus, and terminate near portal vessels. These vessels carry GnRH to the gonadotrophs in the anterior pituitary. The gene encoding GnRH is located on chromosome 9 (Fig. 55-5). The mature mRNA for GnRH encodes a preprohormone composed of 92 amino acids. After removing the 23–amino acid signal sequence (residues −23 to −1), the neuron produces a prohormone (residues 1 to 69). Cleavage of this prohormone yields the decapeptide GnRH (residues 1 to 10), a 56–amino acid peptide (residues 14 to 69) referred to as GnRH-associated peptide (GAP), and three amino acids that link the two. The neuron transports both GnRH and GAP down the axon for secretion into the portal circulation. The importance of GAP is unknown, but it may inhibit prolactin secretion. GnRH is present in the hypothalamus at 14 to 16 weeks’ gestation, and its target, the gonadotropin-containing cells (gonadotrophs), are present in the anterior pituitary gland as early as 10 weeks’ gestation. The hypothalamic-pituitary system is functionally competent by ∼23 weeks’ gestation, at which time fetal tissues release GnRH. The GnRH neurons do not release GnRH continuously, but rather in rhythmic pulses (Fig. 55-6). GnRH is released

Chapter 55 • The Female Reproductive System

CNS

( / )

Hypothalamus

( / ) Figure 55-4 Hypothalamic-pituitary-ovarian axis. Smallbodied neurons in the arcuate nucleus and the preoptic area of the hypothalamus secrete GnRH, a decapeptide that reaches the gonadotrophs in the anterior pituitary through the long portal veins. GnRH binds to a G-proteincoupled receptor on the gonadotroph membrane, triggering the IP3/DAG pathway, raising [Ca2+]i and phosphorylation. Stimulation causes the gonadotrophs to synthesize and release two gonadotropins—FSH and LH—that are stored in secretory granules. Both FSH and LH are glycoprotein heterodimers comprising common α subunits and unique β subunits. The LH binds to receptors on theca cells, thus stimulating Gαs, which, in turn, activates adenylyl cyclase. The resultant rise in [cAMP]i stimulates protein kinase A (PKA), which increases the transcription of several proteins involved in the biosynthesis of progestins and androgens. The androgens enter granulosa cells, which convert the androgens to estrogens. The dashed arrow indicates that the granulosa cells also have LH receptors. FSH binds to receptors on the basolateral membrane of granulosa cells, also activating PKA, thereby stimulating gene transcription and synthesis of the relevant enzymes (e.g., aromatase), activins, and inhibins. Negative feedback on the hypothalamic-pituitary-ovarian axis occurs by several routes. The activins and inhibins act only on the anterior pituitary. The estrogens and progestins act on both the anterior pituitary and on the hypothalamic neurons, by exerting both positive and negative feedback controls. CNS, central nervous system.

GnRH

( / ) Anterior pituitary

( / ) LH

56 AA

14

GnRH-associated peptide

Inhibins

Androgens Activins

Progestins

Estrogens

Negative feedback Positive feedback

69 C

Glu His Trp Ser Tyr Gly Leu Arg Pro Gly

1

Granulosa cell

Theca cell

Processing sites –23 –1 10 AA N Signal peptide GnRH

FSH

10

Figure 55-5 Map of the gonadotropin-releasing-hormone gene. The mature mRNA encodes a preprohormone with 92 amino acids. Removal of the 23–amino acid signal sequence yields the 69–amino acid prohormone. Cleavage of this prohormone yields GnRH.

in bursts into the portal vessels about once per hour, thereby intermittently stimulating the gonadotrophs in the anterior pituitary. Because the half-life of GnRH in blood is only 2 to 4 minutes, these hourly bursts of GnRH cause clearly discernible oscillations in portal plasma GnRH levels that result in hourly surges in release of the gonadotropins LH and FSH. Early in the follicular phase of the cycle, when the gonadotrophs are not very GnRH sensitive, each burst of GnRH elicits only a small rise in LH (Fig. 55-6A). Later in

Reproductive tract

the follicular phase, when the gonadotrophs in the anterior pituitary become much more sensitive to the GnRH in the portal blood, each burst of GnRH triggers a much larger release of LH (Fig. 55-6B). Although the mechanisms controlling the hourly pulses of GnRH remain unclear, the pulse generator for GnRH is thought to be located in the arcuate nucleus of the medial basal hypothalamus, where one group of GnRH neurons resides. In rodents, bursts of nerve impulses from neurons in these nuclei correspond in time with the pulsatile release of GnRH from the hypothalamus and with the episodic release of LH from the anterior pituitary. These data suggest that a built-in system within the hypothalamus controls the pulsatile discharge of GnRH from nerve terminals. The pulse-generating mechanism is key to control of cyclic reproductive function and to regulation of the menstrual cycle. The frequency of GnRH release, and thus LH release, determines the specific response of the gonad. Pulses spaced 60 to 90 minutes apart upregulate the gonadotrophs’ GnRH receptors and thus stimulate the release of gonadotropins. However, continuous administration of GnRH (or an analogue) causes downregulation of the gonadotrophs’ GnRH

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Section IX • The Reproductive System

A

EARLY FOLLICULAR PHASE GnRH pulses

B LATE FOLLICULAR PHASE GnRH pulses

120

120

Plasma 80 LH (mU/mL)

Plasma 80 LH (mU/mL) LH

40

Therapeutic Uses of GnRH

C

LH

40 0

0 0

1

2 3 4 Time (hr)

5

0

1

2 3 4 Time (hr)

5

High levels of estradiol, typical of the late follicular phase, enhance the sensitivity of the gonadotrophs to GnRH.

Figure 55-6 Pulsatile release of GnRH and pulsatile secretion of LH. (Data from Wang CF, Lasley BL, Lein A, Yen SS: J Clin Endocrinol Metab 1976; 42:718-728.)

receptors and thus suppresses gonadotropin release and gonadal function (see the box titled Therapeutic Uses of GnRH). In addition to the hourly rhythm of GnRH secretion, orchestrated by the arcuate nucleus, a monthly rhythm of GnRH secretion also occurs—in rhesus monkeys. A massive increase in GnRH secretion at midcycle is, in part, responsible for the LH surge, which, in turn, leads to ovulation. Which neurons produce the massive surge in GnRH that leads to the LH surge? These are not the GnRH neurons in the arcuate nucleus but, rather, those in the preoptic area. The preoptic GnRH neurons have inhibitory γaminobutyric acid (GABA) receptors, whereas the arcuate GnRH neurons have inhibitory opioid receptors. Later in this chapter, I discuss how these two sets of GnRH neurons may underlie the negative and positive feedback produced by estrogens. GnRH stimulates gonadotrophs in the anterior pituitary to secrete FSH and LH, which stimulate ovarian cells to secrete estrogens and progestins GnRH enters the anterior pituitary through the portal system and binds to GnRH receptors on the surface of the gonadotroph, thus initiating a series of cellular events that result in the synthesis and secretion of gonadotropins (Fig. 55-7). GnRH binds to a G protein–linked receptor coupled to Gαq. The result is activation of phospholipase C (PLC), which, in turn, hydrolyzes phosphatidylinositol 4,5biphosphonate (PIP2) to inositol 1,4,5-triphosphate (IP3), and diacylglycerol (DAG) (see Chapter 3). Both IP3 and DAG are second messengers. Release of Ca2+ from the endo-

ontinuous administration of GnRH leads to downregulation (suppression) of gonadotropin secretion, whereas pulsatile release of GnRH causes upregulation (stimulation) of FSH and LH secretion. Clinical problems requiring upregulation of gonadotropin secretion, which leads to stimulation of the gonads, are therefore best treated by a pulsatile mode of GnRH administration. In contrast, when the patient requires gonadal inhibition, a continuous mode of administration is necessary. An example of a disease requiring pulsatile GnRH administration is Kallmann syndrome. Disordered migration of GnRH cells during embryologic development causes Kallmann syndrome, which in adults results in hypogonadotropic hypogonadism and anosmia (loss of sense of smell). Normally, primordial GnRH cells originate in the nasal placode during embryologic development. These primitive cells then migrate through the forebrain to the diencephalon, where they become specific neuronal groups within the medial basal hypothalamus and preoptic area. In certain individuals, both male and female, proper migration of GnRH cells fails to occur. The cause of Kallmann syndrome was confirmed in humans when researchers studied a fetus at 19 weeks’ gestation that had complete deletion of the X-linked Kallmann locus. The GnRH cells were found along their known migration route, but not in the brain. Girls and women with Kallmann syndrome generally have amenorrhea (no menstrual cycles). However, the pituitary and gonads of these individuals can function properly when appropriately stimulated. Thus, women treated with exogenous gonadotropins or GnRH analogues—pulsatile administration with a programmed infusion pump—can have normal folliculogenesis, ovulation, and pregnancy. An example of a disease requiring continuous GnRH administration to downregulate gonadal function is endometriosis. Endometriosis is a common condition caused by the aberrant presence of endometrial tissue outside the uterine cavity. This tissue responds to estrogens during the menstrual cycle and is a source of pain and other problems, including infertility. In patients with endometriosis, continuous administration of GnRH analogue inhibits replenishment of the receptor for GnRH in the gonadotrophs in the anterior pituitary. As a result, insufficient numbers of GnRH receptors are available for optimum GnRH action, thereby diminishing gonadotropin secretion and producing relative hypoestrogenism. Because estrogen stimulates the endometrium, continuous administration of GnRH or GnRH analogues causes involution and diminution of endometriotic tissue. Leiomyomas (smooth muscle tumors) of the uterus (also called a uterine fibroid) are also estrogen dependent. When estrogen levels are decreased, the proliferation of these lesions is decreased. Therefore, leiomyomas of the uterus can also be effectively treated by continuous administration of GnRH analogues.

plasmic reticulum by IP3 causes an increase in [Ca2+]i. This Ca2+ induces the Ca2+ channels at the cell membrane to open and allows an influx of extracellular Ca2+ that sustains the elevated [Ca2+]i. The rise in [Ca2+]i triggers exocytosis and gonadotropin release.

Chapter 55 • The Female Reproductive System

8 2+ The [Ca ]i triggers exocytosis and release of gonadotropin.

1 Binding of GnRH to G-protein– linked receptor activates the PLC pathway and the release of Ca2+ from internal stores. Ca2+

From hypothalamus GnRH

Gq

2 The activated PLC also leads to the formation of DAG…

PLC 7 Ca2+ released from internal stores activates Ca2+ channels leading to sustained [Ca2+]i.

2+

Ca

PKC

3 …which stimulates PKC. PKC

Gonadotroph cell Follicular phase: estrogen

6 Gonadotropins are synthesized, dimerized, and glycosylated in the secretory pathway, regulated by the rhythm of GnRH.

α β-FSH β-LH

Luteal phase: estrogen and progesterone Inhibin (from ovary)

Activin (from ovary) 5 LH and FSH are αβ dimers. The α subunits are identical. The β subunit determines specificity.

Figure 55-7

4 PKC phosphorylates targets that indirectly stimulate gene transcription.

Gonadotropin secretion. PKC, protein kinase C.

In addition to the IP3 pathway, GnRH also acts through the DAG pathway. The DAG formed by PLC stimulates protein kinase C, which indirectly leads to increases in gene transcription. The net effect is an increase in synthesis of the gonadotropins FSH and LH. In addition, GnRH increases mRNA levels for certain immediate early response genes (e.g., c-Fos, c-Jun, and JunB). The GnRH receptor is internalized and partially degraded in the lysosomes. However, a portion of the GnRH receptor is shuttled back to the cell surface. Return of the GnRH receptor to the cell membrane is referred to as receptor replenishment and is related to the upregulation of receptor activity discussed earlier. The mechanism through which GnRH receptor replenishment occurs remains unclear. FSH and LH are in the same family as thyroid-stimulating hormone (TSH; see Chapter 49) and human chorionic gonadotropin (hCG; see Chapter 56). All four are glycoprotein hormones with α and β chains. The α chains of all four of these hormones are identical; in humans, they have 92 amino acids and a molecular weight of ∼20 kDa. The β chains of FSH and LH are unique and confer the specificity of the hormones. The rhythm of GnRH secretion influences the relative rates of expression for genes encoding the syn-

thesis of the α, βFSH, and βLH subunits of FSH and LH. GnRH pulsatility also determines the dimerization of the α and βFSH subunits, or α and βLH, as well as their glycosylation. Differential secretion of FSH and LH is also affected by several other hormonal mediators, including ovarian steroids, inhibins, and activins. I discuss the role of these agents in the section on feedback control of the hypothalamicpituitary-ovarian axis. Thus, depending on the specific hormonal milieu produced by different physiological circumstances, the gonadotroph produces and secretes the α and β subunits of FSH and LH at different rates. The secretion of LH and FSH is further modulated by neuropeptides, amino acids such as aspartate, neuropeptide Y, corticotropin-releasing hormone (CRH), and endogenous opioids. Before ovulation, the LH and FSH secreted by the gonadotrophs act on cells of the developing follicle. The theca cells of the follicle have LH receptors, whereas the granulosa cells have both LH and FSH receptors. Both LH and FSH are required for estrogen production because neither the theca cell nor the granulosa cell can carry out all the required steps. After ovulation, LH acts on the cells of the corpus luteum; recall that after ovulation, the cells of the follicle give rise to the corpus luteum.

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LH and FSH bind to specific receptors on the surface of their target cells. Both the LH and the FSH receptors are coupled through Gαs to adenylyl cyclase (see Chapter 3), which catalyzes the conversion of ATP to cAMP. cAMP stimulates protein kinase A, which not only stimulates the enzymes involved in steroid biosynthesis but also induces the synthesis of certain proteins and increases cell division. Among the proteins whose synthesis is promoted by gonadotropins is the low-density lipoprotein (LDL) receptor required for cholesterol uptake and the aromatase required for estrogen synthesis.

THE INHIBIN/ACTIVIN SUBUNITS α βA βB

INHIBINS α

ACTIVINS βA

S S

Ovaries also produce peptide hormones: inhibins, which inhibit FSH secretion, and activins, which activate it The inhibins and the activins are peptides that modulate FSH secretion by the gonadotrophs. The transforming growth factor β (TGF-β) supergene family is a group of molecules that are structurally related and include TGF-β, antimüllerian hormone (AMH; see Chapter 53), the activins, the inhibins, and other glycoproteins. These growth factors modulate growth and differentiation during development. The inhibins and activins are dimers constructed from a related set of building blocks: a glycosylated 20-kDa α subunit and two nonglycosylated 12-kDa β subunits, one called βA and the other called βB (Fig. 55-8). The inhibins are always composed of one α subunit and either a βA or a βB subunit; the α and β subunits are linked by disulfide bridges. The α-βA dimer is called inhibin A, whereas the α-βB dimer is called inhibin B. The activins, however, are composed of two β-type subunits. Thus, three kinds of activins are recognized: βA-βA, βB-βB, and the heterodimer βA-βB. The inhibins are produced by the granulosa cells of the follicle, as well as other tissues, including the pituitary, the brain, the adrenal gland, the kidney, the bone marrow, the corpus luteum, and the placenta. FSH specifically stimulates the granulosa cells to produce inhibins. Also involved in the regulation of inhibin production are certain other factors, including hormones and growth-stimulating factors. Estradiol may stimulate inhibin production through an intraovarian mechanism. Just before ovulation, after the granulosa cells acquire LH receptors, LH also stimulates the production of inhibin by granulosa cells. The biological action of the inhibins is primarily confined to the reproductive system. As discussed later, the inhibins inhibit FSH production by gonadotrophs. The activins are produced in the same tissues as the inhibins, but they stimulate—rather than inhibit—FSH release from pituitary cells. Both the ovarian steroids (estrogens and progestins) and peptides (inhibins and activins) feed back on the hypothalamic-pituitary axis As summarized in Figure 55-4, the ovarian steroids—the estrogens and progestins—exert both negative and positive feedback on the hypothalamic-pituitary axis. Whether the feedback is negative or positive depends on both the concentration of the gonadal steroids and the duration of the exposure to these steroids (i.e., the time in the menstrual cycle).

βA

S S βA

α S S βB Inhibins are always composed of one α subunit and either a βA or a βΒ subunit. The α and β subunits are linked by disulfide bridges.

βB S S βB βA S S βB Activins are also dimers, but are composed of two β-type subunits: βA-βA, βB-βB, and the heterodimer βA-βB.

Figure 55-8 The inhibins and activins. The inhibins and activins are peptide hormones that are made up of a common set of building blocks. For both the inhibins and the activins, disulfide bonds link the two subunits.

In addition, the ovarian peptides—the inhibins and activins—also feed back on the anterior pituitary. Negative Feedback by Ovarian Steroids Throughout most of the menstrual cycle, the estrogens and progestins that are produced by the ovary feed back negatively on both the hypothalamus and the gonadotrophs of the anterior pituitary. The net effect is to reduce the release of both LH and FSH. The estrogens exert negative feedback at both low and high concentrations, whereas the progestins are effective only at high concentrations. Although estrogens inhibit the GnRH neurons in the arcuate nucleus and preoptic area of the hypothalamus, this inhibition is not direct. Rather, the estrogens stimulate interneurons that inhibit the GnRH neurons. In the arcuate nucleus, these inhibitory neurons exert their inhibition through opiates. However, in the preoptic area, the inhibitory neurons exert their inhibitory effect through GABA, a classic inhibitory neurotransmitter (see Chapter 13). Positive Feedback by Ovarian Steroids Although ovarian steroids feed back negatively on the hypothalamicpituitary axis during most of the menstrual cycle, they have the opposite effect at the end of the follicular phase. Levels of estrogen, mainly estradiol, rise gradually during the first half of the follicular phase of the ovarian cycle and then steeply during the second half (Fig. 55-9). After the estradiol

Chapter 55 • The Female Reproductive System

Ovarian cycle

Follicular phase

Luteal phase

Events in the ovary Developing follicle

Corpus luteum Ovulation

80

LH FSH (mU/mL)

LH

Estradiol

FSH Inhibin

Progesterone

60

Estradiol (pg/mL) 40

1000

Progesterone (ng/mL) 10

800

8

600

6

1600

400

4

800

200

2

0

0

Inhibin (ng/mL)

0

20

0

0 Endometrial cycle

2 Menses

4

6

8

10

12

14 Days

Proliferative phase

16

18

20

22

24

26

28–0

Secretory phase

Figure 55-9 Hormonal changes during the menstrual cycle. The menstrual cycle is a cycle of the hypothalamic-pituitary-ovarian axis, as well as a cycle of the targets of the ovarian hormones: the endometrium of the uterus. Therefore, the menstrual cycle includes both an ovarian cycle—which includes the follicular phase, ovulation, and the luteal phase—and an endometrial cycle—which includes the menstrual, the proliferative, and the secretory phases.

levels reach a certain threshold for a minimum of 2 days— and perhaps because of the accelerated rate of estradiol secretion—the hypothalamic-pituitary axis reverses its sensitivity to estrogens; that is, estrogens now feed back positively on the axis. One manifestation of this positive feedback is that estrogens now increase the sensitivity of the gonadotrophs in the anterior pituitary gland to GnRH. As discussed in the next section, this switch to positive feedback promotes the LH surge. Indeed, pituitary cells that are cultured in the absence of estrogen have suboptimal responses to GnRH. Once high levels of estrogens have properly conditioned the gonadotrophs, rising levels of progesterone during the late follicular phase also produce a positive feedback response and thus facilitate the LH surge. Negative Feedback by the Inhibins

The inhibins inhibit FSH secretion by the gonadotrophs of the anterior pituitary (hence the name inhibin) in a classic negative feedback arrangement. The initial action of inhibin appears to be

beyond the Ca2+-mobilization step in FSH secretion. In cultured pituitary cells, even very small amounts of inhibin markedly reduce mRNA levels for both the αLH/FSH and the βFSH subunits. As a result, inhibins suppress FSH secretion. In contrast, inhibins have no effect on the mRNA levels of βLH. In addition to their actions on the anterior pituitary, the inhibins also have the intraovarian effect of decreasing androgen production, which can have secondary effects on intrafollicular estrogen production. Positive Feedback by the Activins Activins promote marked increases in βFSH mRNA and FSH release, with no change in βLH formation. The stimulatory effect of activins on FSH release is independent of GnRH action. Like the inhibins, the activins also have the intraovarian action of stimulating the synthesis of estrogens. Thus, by their actions on both the gonadotrophs and the ovaries, the activins and inhibins regulate the activity of the follicular cells during the menstrual cycle.

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Modulation of gonadotropin secretion by positive and negative ovarian feedback produces the normal menstrual rhythm We already saw in Figure 55-6 that the pulsatile release of GnRH from the hypothalamus, generally occurring every 60 to 90 minutes, triggers a corresponding pulsatile release of LH and FSH from the gonadotrophs of the anterior pituitary. Because the gonadotropins elicit the release of ovarian steroids, and these steroids modulate the hypothalamicpituitary axis, the interaction between the ovarian steroids and gonadotropin release is an example of feedback. This feedback is especially interesting because it is bidirectional in that it elicits negative feedback throughout most of the menstrual cycle but positive feedback immediately before ovulation. Figure 55-9 illustrates the cyclic hormonal changes during the menstrual cycle. The time-averaged records of LH and FSH levels mask their hour-by-hour pulsatility. The follicular phase is characterized by a relatively high frequency of GnRH—and thus LH—pulses. Early in the follicular phase, when levels of estradiol are low but rising, the frequency of LH pulses remains unchanged, but their amplitude gradually increases with time. We see this increase in amplitude in Figure 55-6, in which the early and late follicular phases are compared. Later in the follicular phase of the menstrual cycle, the higher estrogen levels cause both the frequency and the amplitude of the LH pulses to increase gradually. During this time of high estradiol levels, the ovarian steroids are beginning to feed back positively on the hypothalamicpituitary axis. Late in the follicular phase, the net effect of this increased frequency and amplitude of LH and FSH pulses is an increase in their time-averaged circulating levels (Fig. 55-9). The LH surge is an abrupt and dramatic rise in the LH level that occurs around the 13th to 14th day of the follicular phase in the average woman. The LH surge peaks ∼12 hours after its initiation and lasts for ∼48 hours. The peak concentration of LH during the surge is ∼3-fold greater than the concentration before the surge (Fig. 55-9). The LH surge is superimposed on the smaller FSH surge. Positive feedback of estrogens, progestins, and activins on the hypothalamicpituitary axis is involved in the induction of this LH surge. The primary trigger of the gonadotropin surge is a rise in estradiol to very high threshold levels just before the LH surge. The rise in estrogen levels has two effects. First, the accelerated rate of increase in estradiol levels in the preovulatory phase sensitizes the gonadotrophs in the anterior pituitary to GnRH pulses (Fig. 55-6). Second, the increasing estrogen levels also modulate hypothalamic neuronal activity and induce a GnRH surge, presumably through GnRH neurons in the preoptic area of the hypothalamus. Thus, the powerful positive feedback action of estradiol induces the midcycle surge of LH and, to a lesser extent, FSH. Gradually rising levels of the activins—secreted by granulosa cells— also act in a positive feedback manner to contribute to the FSH surge. In addition, gradually increasing levels of LH trigger the preovulatory follicle to increase its secretion of progesterone. These increasing—but still “low”—levels of progesterone also have a positive feedback effect on the

hypothalamic-pituitary axis that is synergistic with the positive feedback effect of the estrogens. Thus, although progesterone is not the primary trigger for the LH surge, it augments the effects of estradiol. The gonadotropin surge causes ovulation and luteinization. The ovarian follicle ruptures, probably because of weakening of the follicular wall, and expels the oocyte and with it the surrounding cumulus and corona cells. This process is known as ovulation, and it is discussed in more detail in Chapter 56. As discussed later, a physiological change—luteinization—in the granulosa cells of the follicle causes these cells to secrete progesterone rather than estradiol. The granulosa and theca cells undergo structural changes that transform them into luteal cells, a process known as luteinization. The pulsatile rhythm of GnRH release and gonadotropin secretion is maintained throughout the gonadotropin surge. As the luteal phase of the menstrual cycle begins, circulating levels of LH and FSH rapidly decrease (Fig. 55-9). This fall-off in gonadotropin levels reflects negative feedback by three ovarian hormones—estradiol, progesterone, and inhibin. Moreover, as gonadotropin levels fall, so do the levels of ovarian steroids. Thus, immediately after ovulation we see more or less concurrent decreases in the levels of both gonadotropins and ovarian hormones. Later, during the luteal phase, the luteal cells of the corpus luteum gradually increase their synthesis of estradiol, progesterone, and inhibin (Fig. 55-9). The rise in concentration of these hormones causes—in typical negative feedback fashion—the continued decrease of gonadotropin levels midway through the luteal phase. One of the mechanisms of this negative feedback is the effect of progesterone on the hypothalamic-pituitary axis. Recall that at the peak of the LH surge, both the frequency and the amplitude of LH pulses are high. Progesterone levels rise, and high levels stimulate inhibitory opioidergic interneurons in the hypothalamus, thus inhibiting the GnRH neurons. This inhibition decreases the frequency of LH pulses, although the amplitude remains rather high. By ∼48 hours before onset of the menses, the pulsatile rhythm of LH secretion has decreased to one pulse every 3 to 4 hours. As a result, circulating levels of LH slowly fall during the luteal phase. During the late luteal phase, the gradual demise of the corpus luteum leads to decreases in the levels of progesterone, estradiol, and inhibin (Fig. 55-9). After the onset of menstruation, the hypothalamicpituitary axis returns to a follicular-phase pattern of LH secretion (i.e., a gradual increase in the frequency of GnRH pulses).

OVARIAN STEROIDS Starting from cholesterol, the ovary synthesizes estradiol, the major estrogen, and progesterone, the major progestin Estrogens in female humans are derived from the ovary and the adrenal gland and from peripheral conversion in adipose tissue. In a nonpregnant woman, estradiol, the primary cir-

Chapter 55 • The Female Reproductive System

Table 55-2

Benefits and Risks of Oral Contraceptives

Oral Contraceptives Decrease the Risk of

Ovarian cancer Endometrial cancer Ovarian retention cysts Ectopic pregnancy Pelvic inflammatory disease Benign breast disease Oral Contraceptives Increase the Risk of

Benign liver tumors Cholelithiasis (gallstones) Hypertension Heart attack Stroke Deep vein thrombosis Pulmonary embolus

culating estrogen, is secreted principally by the ovary. The precursor for the biosynthesis of the ovarian steroids, as it is for all other steroid hormones produced elsewhere in the body, is cholesterol. Cholesterol is a 27-carbon sterol that is both ingested in the diet and synthesized in the liver from acetate (see Chapter 46). Ovarian cells can synthesize their own cholesterol de novo. Alternatively, cholesterol can enter cells in the form of LDL cholesterol and can bind to LDL receptors. As shown in Figure 55-10, a cytochrome P-450 enzyme (see Table 50-2) known as the side-chain–cleavage enzyme (or 20,22-desmolase) catalyzes the conversion of cholesterol to pregnenolone. This reaction is the rate-limiting step in estrogen production. Ovarian cells then convert pregnenolone to progestins and estrogens. The initial steps of estrogen biosynthesis from pregnenolone follow the same steps as synthesis of the two so-called adrenal androgens dehydroepiandrosterone (DHEA) and androstenedione, both of which have 19 carbon atoms. These steps are discussed in connection with both substances (see Figs. 50-2 and 54-5). The Leydig cells in the testis can use either of two pathways to convert these weak androgens to testosterone. Cells in the ovaries are different because, as shown in Figure 55-10, they have an aromatase that can convert androstenedione to estrone and testosterone to estradiol. This aromatization also results in loss of the 19-methyl group (thus, the estrogens have only 18 carbons), as well as conversion of the ketone at position 3 to a hydroxyl in the A ring of the androgen precursor. Once formed, estrone can be converted into the more powerful estrogen estradiol, and vice versa, by 17β-hydroxysteroid dehydrogenase (17β-HSD). Finally, the liver can convert both estradiol and estrone into the weak estrogen estriol. The two major progestins, progesterone and 17ahydroxyprogesterone, are formed even earlier in the biosynthetic pathway than the adrenal androgens. Functionally, progesterone is the more important progestin, and it has higher circulating levels.

Estrogen biosynthesis requires two ovarian cells and two gonadotropins, whereas progestin synthesis requires only a single cell In the follicular phase of the menstrual cycle, the follicle synthesizes estrogens, whereas in the luteal phase, the corpus luteum does the synthesis. A unique aspect of estradiol synthesis is that it requires the contribution of two distinct cell types: the theca and granulosa cells within the follicle and the theca-lutein and granulosa-lutein cells within the corpus luteum (Fig. 55-11). I discuss these cells—as well as development of the follicle and corpus luteum—in the next major section. The superficial theca cells and theca-lutein cells can take up cholesterol and produce the adrenal androgens, but they do not have the aromatase necessary for estrogen production. However, the deeper granulosa cells and granulosalutein cells have the aromatase, but they lack the 17α-hydroxylase and 17,20-desmolase (which are the same protein) necessary for making the adrenal androgens. Another difference between the two cell types is that—in the follicle—the superficial theca cell is near blood vessels and is hence a source of LDL cholesterol. The granulosa cell, conversely, is far from blood vessels and instead is surrounded by LDL-poor follicular fluid. Thus, in the follicular stage, the granulosa cells obtain most of their cholesterol by de novo synthesis. However, after formation of the corpus luteum, the accompanying vascularization makes it possible for the granulosa-lutein cell to take up LDL cholesterol from the blood and to thus synthesize large amounts of progesterone. A final difference between the two cell types is that theca cells have LH receptors, and granulosa cells have both LH and FSH receptors. Because of their unique physiological properties, neither the theca/theca-lutein cells nor the granulosa/granulosalutein cells can make estrogens by themselves. According to the two-cell, two-gonadotropin hypothesis, estrogen synthesis occurs in the following steps: Step 1. LH stimulates the theca cell, through the adenylyl cyclase pathway, to increase its synthesis of LDL receptors and the side-chain–cleavage enzyme. Step 2. Thus stimulated, the theca cell increases its synthesis of androstenedione. Step 3. The androstenedione synthesized in the theca cells freely diffuses to the granulosa cells. Step 4. FSH, also acting through the adenylyl cyclase pathway, stimulates the granulosa cell to produce aromatase. Step 5. The aromatase converts androstenedione to estrone (Fig. 55-10). 17β-HSD then converts the estrone to estradiol. Alternatively, 17β-HSD can first convert the same androstenedione to testosterone, and then the aromatase can convert this product to estradiol. By these pathways, theca-derived androgens are converted to estrogens in the granulosa cell. Step 6. The estradiol diffuses into the blood vessels. At low concentrations, the weak androgens produced by the theca cells are substrates for estrogen synthesis by the

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Figure 55-10 Biosynthesis of the ovarian steroids. This scheme summarizes the synthesis of the progestins and estrogens from cholesterol. The individual enzymes are shown in the horizontal and vertical boxes; these enzymes are located in either the smooth endoplasmic reticulum (SER) or the mitochondria. The side-chain–cleavage enzyme that produces pregnenolone is also known as 20,22-desmolase. The chemical groups modified by each enzyme are highlighted in the reaction product. The ovary differs from the testis in having aromatase, which converts androgens to estrogens. Certain of these pathways are shared in the biosynthesis of the glucocorticoids and mineralocorticoids (see Fig. 50-2) and estrogens (see Fig. 54-5).

Cholesterol (27-carbon compound)

Acetate

21 20 22 24 25 26 17 23 12 18 16 27 13

11 1

19 9

A

10

2

D

C

8

B

14

15

7

5

HO

4

6

MITOCHONDRIA Side-chain–cleavage enzyme Pregnenolone Progesterone

C

O

SER

CH3

CH3

A

D

C

CH3

B

A

PROGESTINS (21 carbons)

D

C

CH3

O

C CH3

CH3

B

O

HO

SER 17 α-Hydroxylase

SER 17 α-Hydroxylase

17 α-Hydroxypregnenolone

17 α-Hydroxyprogesterone

CH3 C

A

OH

B

HO

SER 17,20-Desmolase

CH3 CH3 A

O D

C

CH3

A

B

O

Androstenedione

Estrone (E1)

C

CH3 A

Estriol (E3)

CH3

O CH3 A

B

O D

C

D

OH CH3

Liver

C

B

HO

SER 17β-Hydroxysteroid Dehydrogenase

Aromatase

O

SER 17β-Hydroxysteroid Dehydrogenase

ESTROGENS (18 carbons)

SER 17,20-Desmolase

B

HO

OH D

C

CH3

SER

Dehydroepiandrosterone (DHEA)

3β-Hydroxysteroid Dehydrogenase

CH3

O

C

D

C

Same protein

CH3

O

CH3

ANDROGENS (19 carbons)

1158

A

OH

D

B

HO

SER 17β-Hydroxysteroid Dehydrogenase

er

Liv

Androstenediol

Testosterone

Estradiol (E2)

OH CH3 CH3 A HO

C

OH CH3

D CH3

B A

C

CH3 C

D A

B

O

granulosa cells, in addition to enhancing the aromatase activity of granulosa cells. However, at high concentrations, conversion of androgens to estrogens is diminished. Instead, the weak androgens are preferentially converted by 5αreductase (see Fig. 54-5) to more potent androgens, such as dihydrotestosterone, a substance that cannot be converted to estrogen. Furthermore, these 5α-reduced androgens

OH D

B

HO

inhibit aromatase activity. Thus, the net effect of a highandrogen environment in the follicle is to decrease estrogen production. These androgens also inhibit LH receptor formation on follicular cells. In the luteal phase of the cycle, luteinization of the follicle substantially changes the biochemistry of the theca and granulosa cells. As part of the formation of the corpus

Chapter 55 • The Female Reproductive System

The Birth Control Pill

H

ormonal contraception is the most commonly used method of contraception in the United States; ∼30% of sexually active women take the oral contraceptive pill (OCP). Numerous combination (i.e., estrogen and progestin) oral contraceptives and progestin-only pills are available. The estrogens and progestins used in OCPs have varying potencies. In the United States, two estrogen compounds are approved for oral contraceptive use: ethinyl estradiol and mestranol. The progestins used in OCPs are modified steroids in which the methyl at position 19 (Fig. 55-10) is removed; these progestins include norethindrone, norgestrel, norethynodrel, norethindrone acetate, and ethynodiol diacetate. A new generation of progestins—including gestodene and norgestimate—have reduced androgenic effects. The woman takes the OCP daily for 21 days out of the 28-day cycle; she takes no pill, a placebo, or an iron pill during days 22 to 28. No medication is usually given during this fourth week, to allow withdrawal bleeding to occur. Three regimens of contraceptive steroid administration are used: 1. Monophasic or fixed-combination OCPs. The pills taken for the first 21 days of the cycle are identical. 2. Multiphasic or varying-dose OCPs. The pills contain two or three different amounts of the same estrogen and progestin, the dosages of which vary at specific intervals during the 21-day medication period. Multiphasic OCPs generally maintain a low dose of estrogen throughout the cycle, combined with varying amounts of progestin. The rationale for this type of formulation is that the woman takes a lower total dose of steroid but is not at increased risk of breakthrough endometrial bleeding. 3. Progestin-only OCPs (“minipill”). The woman takes these estrogen-free OCPs daily for 3 weeks of a 4-week cycle. This regimen may be associated with irregular, low-grade, breakthrough endometrial bleeding. The progestin-only OCP is a good option for nursing mothers, as well as women for whom estrogens are contraindicated (e.g., those with thromboembolic disease, cerebral vascular incidents, and hypertension).

luteum, blood vessels invade deep toward the granulosalutein cells. Recall that in the follicle, the granulosa cells had been surrounded by follicular fluid, which is poor in LDL cholesterol. The increased vascularity facilitates the delivery of LDL cholesterol to the granulosa-lutein cells. In addition, LH stimulates the granulosa-lutein cell to take up and process cholesterol—as it does in theca cells. The net effect is the increased progesterone biosynthesis that is characteristic of the midluteal phase. Indeed, the major products of the corpus luteum are progesterone and 17α-hydroxyprogesterone, although the corpus luteum also produces estradiol. As indicated in Figure 55-11, the granulosa-lutein cells cannot make either 17α-hydroxyprogesterone or estradiol directly because these cells lack the protein that has dual activity for 17α-hydroxylase and 17,20-desmolase (Fig. 55-10). Thus, 17α-hydroxyprogesterone synthesis necessi-

Biological Action of Oral Contraceptives The contraceptive effectiveness of OCPs accrues from several actions. Like natural ovarian steroids, contraceptive steroids feed back both directly at the level of the hypothalamus (decreasing secretion of GnRH) and at the level of the gonadotrophs in the anterior pituitary (Fig. 55-4). The net effect is suppressed secretion of the gonadotropins, FSH and LH. The low FSH levels are insufficient to stimulate normal folliculogenesis; the low LH levels obviate the LH surge and therefore inhibit ovulation. However, in the commonly used doses, contraceptive steroids do not completely abolish either gonadotropin secretion or ovarian function. The progestin effect of the OCP causes the cervical mucus to thicken and become viscid and scant. These actions inhibit sperm penetration into the uterus. The progestins also impair the motility of the uterus and oviducts and therefore decrease transport of both ova and sperm to the normal site of fertilization in the distal fallopian tube (see Chapter 56). Progestins also produce changes in the endometrium that are not conducive for implantation of the embryo. These changes include decreased glandular production of glycogen and thus diminished energy for the blastocyst to survive in the uterus. Progestin-only OCPs do not effectively inhibit ovulation, as do the combination pills. However, they do produce the other actions: mucus thickening, reduced motility, and impaired implantation. Because they are inconsistent inhibitors of ovulation, the progestin-only OCPs have a substantially higher failure rate than does the combined type of OCP. Side effects of the compounds in OCPs are those associated with estrogens and progestins and include nausea, edema, headaches, and weight gain. Side effects of progestins include depression, mastodynia, acne, and hirsutism. Many of the side effects associated with the progestin component of the pill are the result of the androgenic actions of the progestins used, particularly the acne and hirsutism. The potential benefits of the newer progestins include decreased androgenic effects, such as increased sex hormone–binding globulin, improved glucose tolerance (see Chapter 51), and increased high-density lipoprotein and decreased LDL cholesterol (see Chapter 46). The clinical impact of these changes remains to be determined. Table 55-2 lists the major benefits and risks of OCPs.

tates that progesterone first moves to the theca-lutein cell (Fig. 55-11), which can convert progesterone to 17αhydroxyprogesterone, as well as androstenedione. Furthermore, estradiol synthesis necessitates that androstenedione from the theca-lutein cell moves to the granulosa-lutein cell for aromatization and formation of estradiol. The principal functions of estrogens are stimulation of cellular proliferation and growth of sex organs and other tissues related to reproduction Most estrogens in blood plasma are bound to carrier proteins, as are testosterone and other steroid hormones. In the case of estradiol, 60% is bound to albumin and 38% to sex hormone–binding globulin (SHBG)—also known as

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LDL Theca cell

LDL

LH

Granulosa cell Cholesterol

Cholesterol

Gs

cAMP

cAMP

AC

Pregnenolone

AC

Pregnenolone LH

These steps only become important during the luteal phase.

Gs

Progesterone

17α-Hydroxylase and 17,20-desmolase activities are absent.

Progesterone

17α-OH Progesterone

Androstenedione 17β-HSD

Androstenedione

Testosterone Aromatase

Aromatase

Estrone

Gs

cAMP

FSH

AC

17β-HSD

Testosterone Estradiol Capillary Aromatase activity is absent.

Figure 55-11 Two-cell, two-gonadotropin model. During the follicular phase, the major product of the follicle is estradiol, whereas during the luteal phase, the major products of the corpus luteum are the progestins, although estradiol synthesis is still substantial. In the follicular phase, LH primes the theca cell to convert cholesterol to androstenedione. Because the theca cell lacks aromatase, it cannot generate estradiol from this androstenedione. Instead, the androstenedione diffuses to the granulosa cell, whose aromatase activity has been stimulated by FSH. The aromatase converts the androstenedione to estradiol. In the luteal phase, the vascularization of the corpus luteum makes low LDL available to the granulosa-lutein cells. Thus, both the theca-lutein and the granulosa-lutein cells can produce progesterone, the major product of the corpus luteum. For production of 17α-hydroxyprogesterone (17α-OH progesterone), some of the progesterone diffuses into the theca-lutein cell, which has the 17α-hydroxylase activity needed for converting the progesterone to 17α-hydroxyprogesterone. The theca-lutein cell can also generate the androstenedione, which diffuses into the granulosa-lutein cell for estradiol synthesis. AC, adenylyl cyclase.

testosterone-binding globulin (TeBG; see Chapter 54). The latter name is doubly a misnomer; not only does TeBG bind estradiol, but also TeBG levels are twice as high in women as they are in men. At least one reason for the higher levels in women is that estrogens (including birth control pills) stimulate the synthesis of SHBG. Only 2% of total plasma estradiol circulates as the free hormone. Because of their lipid solubility, estrogens readily cross cell membranes. Although it was once believed that estrogens bound to cytoplasmic receptors, more recently it became clear that the receptor for estradiol resides in the cell nucleus (see Chapter 3). The estrogen receptor (ER) functions as a homodimer (see Table 4-2). The estrogen–estrogen receptor complex interacts with steroid response elements on chromatin and rapidly induces the transcription of specific genes to produce mRNA. The RNA enters the cytoplasm and increases protein synthesis, which modulates numerous cellular functions. Over the next several hours, DNA synthesis increases, and the mitogenic action of estrogens becomes apparent. Estrogens almost exclusively affect particular target sex organs that have the estrogen receptor. These organs include the uterus and the breasts.

The progestins, particularly progesterone, stimulate glandular secretion in reproductive tissue and promote the maturation of certain estrogen-stimulated tissue. One of the most prominent actions of progesterone, which binds to the dimeric progesterone receptor (PR; see Table 4-2), is the induction of secretory changes in the endometrium. The endometrium must be conditioned by estrogen for progesterone to act effectively. During the latter half of the menstrual cycle, progesterone induces final maturation of the uterine endometrium for reception and implantation of the fertilized ovum.

THE OVARIAN CYCLE: FOLLICULOGENESIS, OVULATION, AND FORMATION OF THE CORPUS LUTEUM Follicles mature in stages from primordial to graafian (or preovulatory) follicles Oocyte maturation—the production of a haploid female gamete capable of fertilization by a sperm—begins in the

Chapter 55 • The Female Reproductive System

fetal ovary. The primordial germ cells migrate from the hind gut to the gonadal ridge. These primordial germ cells develop into oogonia, or immature germ cells, which, in turn, proliferate in the fetal ovary by mitotic division (see Chapter 53). By 6 to 7 weeks of intrauterine life, ∼10,000 oogonia are present. This figure is the result of migration and rapid mitotic division; up until this time, no atresia occurs. By ∼8 weeks’ gestation, ∼600,000 oogonia are present, and they may enter prophase of the first meiosis and become primary oocytes. From this point onward, the number of germ cells is determined by three ongoing processes: mitosis, meiosis, and atresia. By middle fetal life, all the mitotic divisions of the female germ cells have been completed, and the number of germ cells peaks at 6 to 7 million around 20 weeks’ gestation. At this point, oogonia enter their first meiotic division. During prophase of the first meiosis, when the primary oocytes have a duplicated set of 23 chromosomes (4N DNA)—22 duplicated pairs of autosomal chromosomes and 1 pair of duplicated X chromosomes)—crossing over occurs (see Fig. 53-1C). Meiosis arrests in prophase I. This prolonged state of meiotic arrest is known as the dictyotene stage. During the remainder of fetal life and childhood, the number of primary oocytes gradually declines to ∼2 to 2.5 million at birth and to ∼400,000 just before puberty. As we shall see, they remain primary oocytes—arrested in prophase I of meiosis—until just before ovulation, many years later, when meiosis is completed and the first polar body is extruded. Oocyte maturation is complete when the resulting haploid oocyte is capable of fertilization by a sperm. The few primary oocytes that survive are those that are surrounded by flat, spindle-shaped follicular or pregranulosa cells (Fig. 55-12). This oocyte-pregranulosa cell complex is enclosed by a basement lamina. At this stage of development, the primary oocyte with its surrounding single layer of pregranulosa cells is called a primordial follicle. Primordial follicles are 30 to 60 μm in diameter. The first primordial follicle usually appears ∼6 weeks into intrauterine life, and the generation of primordial follicles is complete by ∼6 months after birth. The ovarian follicle is the primary functional unit of the ovary. Throughout reproductive life, some 90% to 95% of all follicles are the primordial (i.e., “nongrowing”) follicles discussed earlier. The growing follicles that are recruited from this pool of primordial follicles undergo a striking series of changes in size, morphology, and physiology. This follicular development, as well as the subsequent ovulation, is central to control of the menstrual cycle. The first step in follicular growth is that a primordial follicle becomes a primary follicle. The primary follicle (Fig. 55-12) forms as the spindle cells of the primordial follicle become cuboidal cells. In addition, the oocyte enlarges. Thus, the primary follicle contains a larger primary oocyte that is surrounded by a single layer of cuboidal granulosa cells. The secondary follicle (Fig. 55-12) contains a primary oocyte surrounded by several layers of cuboidal granulosa cells. The granulosa cells of a primary follicle proliferate and give rise to several layers of cells. In addition, stromal cells differentiate, surround the follicle, and become the theca cells. These theca cells are on the outside of the follicle’s

Primary oocyte (4N DNA)

Pregranulosa cell Primary oocyte (4N DNA) Basement membrane

Primordial follicle

Granulosa cell Zona pellucida Primary oocyte (4N DNA) Basement membrane

Primary follicle

Granulosa cell Zona pellucida Theca cell

Secondary follicle

Primary oocyte (4N DNA) Basement membrane Theca extrerna Theca interna Basement membrane Granulosa cells Antrum Zona pellucida

Early tertiary follicle

Primary oocyte (4N DNA) First meiotic division completed

Antrum

Graafian follicle

Theca externa Theca interna Basement membrane Granulosa cells

Secondary oocyte (2N DNA)

Zona pellucida

First polar body (2N DNA)

Corona radiata Cumulus oophorus

Beginning of second meiotic division, fertilization, and completion of second meiotic division Corona radiata

Ovulated ovum Second polar body

Fertilized oocyte

Figure 55-12

The maturation of the ovarian follicle.

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basement membrane. The oocyte increases in size to a diameter of ∼120 μm. As the developing follicle increases in size, the number of granulosa cells increases to ∼600, and the theca cells show increasing differentiation. The progression to secondary follicles also entails the formation of capillaries and an increase in the vascular supply to developing follicular units. As the increasingly abundant granulosa cells secrete fluid into the center of the follicle, they create a fluid-filled space called the antrum. At this stage, the follicle is now a tertiary follicle (Fig. 55-12), the first of the two antral stages. In contrast to this tertiary follicle, the primordial, primary, and secondary follicles are solid masses of cells that lack an antrum; they are therefore referred to as preantral follicles. In tertiary follicles, gap junctions are located among both theca cells and granulosa cells. In addition, tight junctions and desmosomes exist between adjacent cells. Gap junctions may also exist between the oocyte and the granulosa cells closest to the oocyte and may function as thoroughfares to transport nutrients and information from the granulosa cells to the oocyte and vice versa. The granulosa cells closest to the oocyte also secrete the mucopolysaccharides that form the zona pellucida immediately surrounding the oocyte. As the antrum enlarges, it nearly encircles the oocyte, except for a small mound or cumulus that attaches the oocyte to the rest of the follicle, whose diameter increases to 20 to 33 mm. This preovulatory or graafian follicle (Fig. 55-12) is the second of the two antral stages. The granulosa cells of the tertiary and graafian follicles are of three types: (1) mural granulosa cells, which are the farthest from the center of the follicle, are the most metabolically active, and contain large quantities of LH receptors and enzymes that are necessary for the synthesis of steroids; (2) cumulus granulosa cells are shed with the oocyte at the time of ovulation; and (3) antral granulosa cells, which face the antrum, are left behind within the follicle to become the large luteal cells of the corpus luteum. The capacity of the three types of granulosa cells to generate steroids differs. Cumulus cells contain neither the side-chain–cleavage enzyme (P-450scc) nor aromatase (P-450arom) and therefore cannot generate estrogens. Moreover, cumulus cells respond less to LH and have a low overall LH receptor content. The exact role of the cumulus layer has not been definitively established, although investigators have postulated that the cumulus layer may function as a feeder layer and may provide stem cells that differentiate into other follicular cell types. Both FSH and LH stimulate follicular growth Even in fetal life and childhood, some primordial follicles can develop all the way to the antral stage. However, these follicles all undergo atresia (death of the ovum, followed by collapse of the follicle and scarring) at some stage in their development. At the time of puberty, the increase in levels of gonadotropins and ovarian steroids produces a marked increase in the rate of follicular development. During the luteal phase of each cycle, a cohort of primordial follicles is recruited for further development into graafian follicles, a process that occurs during the follicular phase of the next

cycle. Thus, primordial follicles may remain in a nongrowing state for 50 years before they develop into primary follicles. All along the course of this development, follicular units undergo atresia until only one dominant graafian follicle remains at the time of ovulation. Some controversy exists about the length of this developmental process. Some investigators believe that the entire developmental process takes three to four monthly cycles. However, the predominant view is that a cohort of primary follicles is recruited during the end of one cycle, and one of these follicles develops into the dominant graafian follicle. Primary Follicles

Appropriate structural and functional development of the follicle necessitates that the follicle is exposed to the appropriate sequence of three hormones: FSH, estradiol, and LH. FSH secretion occurs during early fetal life, and FSH can be detected as early as 5 months into gestation (Fig. 55-2). Three pieces of evidence suggest that gonadotropins and estrogens are essential for early follicular growth, that is, for the progression from primordial to primary follicles. First, hypophysectomy (i.e., removal of the pituitary) causes depletion of primordial follicular units in primates. Thus, with the absence of gonadotropins—as well as other pituitary hormones—and a reduction in estrogens, no follicular development takes place in females. Second, patients with resistant ovary syndrome have a normal complement of primordial follicles, but the follicles do not develop beyond the primordial stage. Although these patients have high circulating levels of FSH, they have no receptors for FSH and LH in their ovaries. Thus, FSH or LH must be able to act on the ovary for follicular development to occur. Third, in individuals with 17α-hydroxylase deficiency—and thus low estrogen levels—follicular development does not progress beyond the primordial stage. Circulating levels of LH in these individuals are normal or high. These three lines of evidence suggest that removal of the gonadotropins, the gonadotropin receptors, or the estrogens halts early follicular growth.

Secondary Follicles As primary follicles form secondary follicles, the theca cells proliferate and acquire LH receptors, as well as the ability to synthesize steroids. Moreover, the granulosa cells acquire receptors for FSH, androgens, and estrogens. When the granulosa cells acquire FSH receptors, the follicular unit becomes a functional steroid-producing entity. Tertiary Follicles Formation of the antrum, which leads to the development of a tertiary follicle, also requires gonadotropins. FSH, acting in concert with estrogens, causes the proliferation of granulosa cells after development of the antrum, and as a result, the total number of receptors for FSH is increased. FSH, along with estradiol, also induces the proliferation of LH receptors in granulosa cells. Graafian Follicles

When human granulosa cells from graafian follicles are studied in vitro, the mitotic and steroidsynthesizing characteristics of these cells reflect the hormonal condition of the follicle from which they came. Both FSH and estradiol are needed for mitosis to occur in granulosa cells. FSH, LH, and estradiol are necessary for maximum

Chapter 55 • The Female Reproductive System

progesterone production by granulosa cells. Premature exposure of developing follicles to LH inhibits mitosis, as well as steroidogenesis. Therefore, the follicle must be exposed to the appropriate sequence of hormones (e.g., FSH, followed by estradiol and then LH) for appropriate maturational and functional development. Each month, a cohort of follicles is recruited, one of which achieves dominance The consensus is that the monthly cycle of folliculogenesis actually begins from the primary follicle stage 2 to 3 days before onset of the menses of the previous cycle. At this time, FSH levels begin to increase (Fig. 55-9) because of decreasing inhibin concentrations, thus inducing folliculogenesis, which is completed in the next cycle. Although we do not understand why some primordial follicles—and not others—join a developing cohort of follicles, FSH is thought to be at least partly responsible for continued development of a cohort of follicles each cycle. The number of follicles in a cohort depends on the residual pool of remaining follicles in the ovary. As the cycle continues, only some of the cohort of follicles continue to develop in response to gonadotropin secretion. The other members of the cohort of follicles undergo atresia. The one follicle destined to ovulate is recruited during the early days of the current menstrual cycle and eventually achieves dominance. Although the mechanism of selection of the dominant follicle is not completely understood, it is thought to be caused by estrogen-induced events within the follicles. As estrogen levels rise during the follicular phase of the cycle, the pituitary gradually lowers its secretion of FSH (Fig. 55-9). Rising inhibin levels also feed back on the anterior pituitary to decrease FSH secretion. Peak inhibin levels correlate with the number of follicles present and rise in parallel with circulating estradiol levels. Decreased levels of FSH cause a decline in FSHdependent aromatase activity in granulosa cells. As a result, estrogen production decreases in the less mature follicles. Conversely, estrogen increases the effectiveness of FSH in the more mature follicles by increasing the number of FSH receptors. Although the dominant follicle continues to be dependent on FSH, it has more FSH receptors, a greater rate of granulosa cell proliferation, more FSH-dependent aromatase activity, and more estrogen production than the less dominant follicles. Because the less dominant follicles have less aromatase activity, the androstenedione in the theca cells cannot be converted as readily to estrogens in the granulosa cell. Instead, the androstenedione either builds up or is converted to other androgens. The less dominant follicles consequently undergo atresia under the influence of androgens in their local environment. In contrast, the production of estrogens and inhibins allows the dominant follicle to become prominent and to gain an even greater edge over its competitors. The vascular supply to the thecal layer of the dominant follicle increases rapidly, so that during the late follicular phase, the vasculature of the dominant follicle is several-fold greater than that of other follicles in the cohort. Increased vascularity may allow greater FSH delivery to the dominant follicle and may thus help to maintain dominance of the follicle selected for ovulation.

Estradiol secretion by the dominant follicle triggers the LH surge, which, in turn, signals ovulation Ovulation occurs at the midpoint of every normal menstrual cycle and is triggered by the LH surge, which, in turn, is stimulated by rapidly rising levels of estradiol. Estradiol secretion by the dominant follicle increases rapidly near the end of the late follicular phase (Fig. 55-9). This dramatic rise in circulating estradiol exerts positive feedback on the anterior pituitary and sensitizes it to GnRH. The net effect of a rising estradiol level is induction of the LH surge. The LH surge is generally initiated 24 to 36 hours after peak estradiol secretion is achieved, and ovulation usually occurs ∼36 hours after onset of the LH surge and ∼12 hours after its peak. Thus, it appears that the developing follicle, through its increased estradiol secretion, signals the hypothalamicpituitary system that follicular maturation is complete and that the hypothalamic-pituitary axis can now release a bolus of gonadotropin to induce ovulation. The LH surge appears to terminate in part as a result of rising levels of progesterone, through negative feedback, and in part as a result of loss of the positive feedback that is derived from estradiol. Depletion of gonadotropin stores in the anterior pituitary gland may also contribute to termination of the LH surge. At the time of the LH surge, the primary oocyte (4N DNA), which had been arrested in the prophase of its first meiotic division since fetal life (see Chapter 53), now resumes meiosis and completes its first meiotic division several hours before ovulation. The result of this first meiotic division is a small first polar body, which degenerates or divides to form nonfunctional cells, and a much larger secondary oocyte. Both the first polar body and the secondary oocyte, like secondary spermatocytes (see Chapter 54), have a haploid number of duplicated chromosomes (2N DNA): 22 duplicated somatic chromosomes and 1 duplicated X chromosome. This secondary oocyte begins its second meiotic division, but it becomes arrested in metaphase until the time of fertilization (see Chapter 56). The secondary oocyte is surrounded by the zona pellucida and one or more layers of follicular cells, the corona radiata. Before ovulation, the cumulus oophorus expands under the influence of LH, and eventually a complex consisting of the cumulus, the oocyte, and its surrounding cells breaks free with its “stalk” and floats inside the antrum, surrounded by follicular fluid. Breaking away of the oocyte-cumulus complex is probably facilitated by increased hyaluronidase synthesis that is stimulated by FSH. Release of the oocyte from the follicle—ovulation— follows thinning and weakening of the follicular wall, probably under the influence of both LH and progesterone. Both LH and progesterone enhance the activity of proteolytic enzymes (e.g., collagenase) within the follicle and thus lead to the digestion of connective tissue in the follicular wall. Prostaglandins, particularly those in the E and F series, may also contribute to ovulation, perhaps by triggering the release of lysosomal enzymes that digest the follicular wall. Ultimately, a stigma—or spot—forms on the surface of the dominant follicle. As this stigma balloons out and forms a vesicle, it ruptures, and the oocyte is expelled. Ovulation is apparently facilitated by increased intrafollicular pressure

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and contraction of smooth muscle in the theca as a result of prostaglandin stimulation. The expelled oocyte, with its investment of follicular cells, is picked up by the fimbriae of the fallopian tube (Fig. 55-1) as they move over the surface of the ovary. The oocyte is then transported through the infundibulum into the ampulla by means of ciliary movement of the tubal epithelium, as well as by muscular contractions of the tube. Fertilization, if it occurs, takes place in the ampullary portion of the fallopian tube. The resulting zygote subsequently resides there for ∼72 hours, followed by rapid transport through the isthmus to the uterine cavity, where it floats free for an additional 2 to 3 days before attaching to the endometrium. After ovulation, the theca and granulosa cells of the follicle differentiate into the theca-lutein and granulosa-lutein cells of the highly vascularized corpus luteum After expulsion of the oocyte, the granulosa and theca cells are thrown up into folds that occupy the follicular cavity and form the corpus luteum, a temporary endocrine organ whose major product is progesterone. The corpus luteum is highly vascularized, and surrounding blood vessels penetrate the theca and granulosa layers. Blood accumulates in the resealed antral cavity of the corpus luteum soon after ovulation. Formation of the corpus luteum occurs as a result of transformation of the granulosa and theca cells under the influence of LH (Fig. 55-12). The theca cells at the periphery of the follicle differentiate into stroma and give rise to thecalutein cells—also known as small luteal cells. The granulosa cells, in contrast, enlarge and give rise to granulosa-lutein cells—also known as large luteal cells. Therefore, the mature corpus luteum is composed of two cell types: theca-lutein and granulosa-lutein cells. During the luteal phase of the menstrual cycle, estrogens and progestins inhibit folliculogenesis. Luteal function begins to decrease ∼11 days after ovulation. The mechanisms responsible for luteal regression—or luteolysis—remain open to speculation. One school of thought postulates that withdrawal of trophic support results in demise of the corpus luteum, whereas the second school maintains that local factors induce luteal regression. For example, prostaglandin F2α inhibits luteal function and terminates the life of the corpus luteum. Growth and involution of the corpus luteum produce the rise and fall in progestins and estrogens during the luteal phase of the menstrual cycle Although the corpus luteum produces both estrogen and progesterone, the luteal phase is primarily dominated by progesterone secretion. Estrogen production by the corpus luteum is largely a function of the theca-lutein or the small cells, which also produce androgens. Progestin production in the corpus luteum is primarily a function of the granulosa-lutein or large cells (Fig. 55-11), which also produce estrogens. As shown earlier in Figure 55-9, progesterone production rises before follicular rupture. After ovulation, proges-

terone levels rise sharply and peak in ∼7 days. Progesterone acts locally to inhibit follicular growth during the luteal phase. In addition, progesterone may act centrally by inhibiting gonadotropin secretion. Progestins are also antiestrogens. As a result, progestins acting locally may downregulate ERs and may reduce the effectiveness of estradiol. Therefore, increasing progesterone production may have adverse effects on folliculogenesis. Estradiol levels also rise during the luteal phase (Fig. 55-9) and reflect production by the corpus luteum. The estradiol produced during the luteal phase is necessary for the occurrence of progesterone-induced changes in the endometrium. Unless rescued by hCG (see Chapter 56)—produced by the syncytial trophoblasts of the blastocyst—luteal production of progesterone ceases toward the end of the menstrual cycle. hCG produced by the developing conceptus maintains steroidogenic function of the corpus luteum until approximately the ninth week of gestation, at which time placental function is well established. If not rescued by pregnancy, the hormone-producing cells of the corpus luteum degenerate and leave behind a fibrotic corpus albicans.

THE ENDOMETRIAL CYCLE In the human female fetus, the uterine mucosa is capable of responding to steroid hormones by 20 weeks’ gestation. Indeed, some of the uterine glands begin secreting material by the 22nd week of gestation. Endometrial development in utero apparently occurs in response to estrogens derived from the maternal placenta. At ∼32 weeks’ gestation, glycogen deposition and stromal edema are present in the endometrium. As estrogenic stimulation is withdrawn after delivery, the endometrium regresses, and at ∼4 weeks after birth, the glands are atrophic and lack vascularization. The endometrium remains in this state until puberty. The ovarian hormones drive the morphological and functional changes of the endometrium during the monthly cycle The ovarian steroids—estrogens and progestins—control the cyclic monthly growth and breakdown of the endometrium. The three major phases in the endometrial cycle are the menstrual, proliferative, and secretory phases. The Menstrual Phase

If the oocyte was not fertilized and pregnancy did not occur in the previous cycle, a sudden diminution in estrogen and progesterone secretion will signal the demise of the corpus luteum. As hormonal support of the endometrium is withdrawn, the vascular and glandular integrity of the endometrium degenerates, the tissue breaks down, and menstrual bleeding ensues; this moment is defined as day 1 of the menstrual cycle (Fig. 55-13). After menstruation, all that remains on the inner surface of most of the uterus is a thin layer of nonepithelial stromal cells and some remnant glands. However, epithelial cells remain in the lower uterine segments, as well as regions close to the fallopian tubes.

Chapter 55 • The Female Reproductive System

Ovarian cycle

Follicular phase

Luteal phase

Events in the ovary Developing follicle Menstrual phase

Endometrial cycle

Corpus luteum

Proliferative phase Early

Secretory phase

Advanced

Early

Late (premenstrual)

Middle

Menstrual phase Zona compacta

Approximately 14 days Endometrial changes

Zona spongiosa Functional layer

Zona basalis 0

4

8–9

14

15–18

25

28–0

4

Days 99 Basal body 98 temperature (°F) 97 Ovulation occurs on day 14.

Figure 55-13 The endometrial cycle. The ovarian cycle includes the follicular phase—in which the follicle develops—and the luteal phase—in which the remaining follicular cells develop into the corpus luteum. The endometrial cycle has three parts: the menstrual, the proliferative, and the secretory phases.

The Proliferative Phase After menstruation, the endometrium is restored by about the fifth day of the cycle (Fig. 55-13) as a result of proliferation of the basal stromal cells on the denuded surface of the uterus (the zona basalis), as well as the proliferation of epithelial cells from other parts of the uterus. The stroma gives rise to the connective tissue components of the endometrium. Increased mitotic activity of the stromal and glandular epithelium continues throughout the follicular phase of the cycle and beyond, until ∼3 days after ovulation. Cellular hyperplasia and increased extracellular matrix result in thickening of the endometrium during the late proliferative phase. The thickness of the endometrium increases from ∼0.5 to as much as 5 mm during the proliferative phase. Proliferation and differentiation of the endometrium are stimulated by estrogen that is secreted by the developing follicles. Levels of estrogen rise early in the follicular phase and peak just before ovulation (Fig. 55-9). ER levels in the endometrium also increase during the follicular phase of the menstrual cycle. Levels of endometrial ER are highest during the proliferative phase and decline after ovulation in response to changing levels of progesterone. As already discussed, estradiol binds to a nuclear receptor on the endometrial cell, and the activated receptor interacts with the hormone response elements of specific genes and modulates their transcription rates.

Estrogen is believed to act on the endometrium in part through its effect on the expression of proto-oncogenes (see Chapter 3) that are involved in the expression of certain genes. Part of the effect of estrogens is to induce the synthesis of growth factors such as the insulin-like growth factors (IGFs, also called somatomedins; see Chapter 48), TGFs, and epidermal growth factor (EGF). These autocrine and paracrine mediators are necessary for maturation and growth of the endometrium. Estrogen causes the stromal components of the endometrium to become highly developed. Estrogen also induces the synthesis of progestin receptors in endometrial tissue. Levels of progestin receptors peak at ovulation, when estrogen levels are highest, to prepare the cells for the high progestin levels of the luteal phase of the cycle. Progesterone, in contrast, opposes the action of estrogen on the epithelial cells of the endometrium and functions as an antiestrogen. Although progesterone inhibits epithelial cell proliferation, it promotes proliferation of the endometrial stroma. Progesterone exerts its primary antiestrogen effects by stimulating 17β-HSD and sulfotransferase, enzymes that convert estradiol to weaker compounds. Thus, 17β-HSD may convert the estradiol captured from the extracellular environment to estrone (Fig. 55-10), and sulfotransferase may conjugate estrogens to sulfate and produce derivatives such as estradiol 3-sulfate. Estrone is far less

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active than estradiol, and sulfated estrogens are by themselves biologically inactive. The Secretory Phase During the early luteal phase of the ovarian cycle, progesterone further stimulates the 17β-HSD and sulfation reactions. These antiestrogenic effects halt the proliferative phase of the endometrial cycle. Progesterone also stimulates the glandular components of the endometrium and thus induces secretory changes in the endometrium. The epithelial cells exhibit a marked increase in secretory activity, as indicated by increased amounts of endoplasmic reticulum and mitochondria. These increases in synthetic activity occur in anticipation of the arrival and implantation of the blastocyst. The early secretory phase of the menstrual cycle (Fig. 55-13) is characterized by the development of a network of interdigitating tubes within the nucleolus of the endometrial epithelial cells. These tubes are referred to as the nucleolar channel system, and progesterone apparently stimulates their development. The cytoplasm invaginates near the nucleolar channel system, although it remains unclear whether actual connections exist between the channel system and the cytoplasm. The nucleolar channel system may provide a route of transport for mRNA to the cytoplasm. During the middle to late secretory phase, evidence of increased secretory capacity of the endometrial glands becomes more apparent. Vascularization of the endometrium increases, the glycogen content increases, and the thickness of the endometrium increases to 5 to 6 mm. The endometrial glands become engorged with secretions. They are no longer straight; instead, they become tortuous and achieve maximal secretory activity at approximately day 20 or 21 of the menstrual cycle. The changes in the endometrium are not limited to the glands; they also occur in the stromal cells between the glands. Beginning 9 to 10 days after ovulation, stromal cells that surround the spiral arteries of the uterus enlarge and develop eosinophilic cytoplasm, with a prominent Golgi complex and endoplasmic reticulum. This process is referred to as predecidualization. The rounded decidual cells differentiate from spindle-shaped fibroblast-like stromal cells under the influence of progesterone. As the stromal cells differentiate into decidual cells, their biochemical activity changes, and they form secretory products typical of decidual cells. Laminin, fibronectin, heparin sulfate, and type IV collagen surround matrices of decidualizing cells. Multiple foci of these decidual cells spread throughout the upper layer of the endometrium and form a dense layer called the zona compacta (Fig. 55-13). This spreading is so extensive that the glandular structures of the zona compacta become inconspicuous. Inflammatory cells accumulate around glands and blood vessels. Edema of the midzone of the endometrium distinguishes the compact area from the underlying zona spongiosa, where the endometrial glands become more prominent. Together, the superficial zona compacta and the midlevel zona spongiosa make up the so-called functional layer of the endometrium. This functional layer is the region that proliferates early in the monthly endometrial cycle, that later interacts with the embryo during pregnancy, that is shed after pregnancy, and that is also shed each month during

menstruation. The deepest layer of the endometrium—the zona basalis—is the layer left behind after parturition or menstruation. The cells of the zona basalis give rise to the proliferation at the beginning of the next endometrial cycle. During the late luteal phase of the menstrual cycle, just before the next menstruation, levels of both estrogens and progestins diminish, and these decreased ovarian steroid levels lead to eventual demise of the upper two thirds of the endometrium. During this period, the spiral arteries rhythmically go into spasm and then relax. This period of the cycle is sometimes referred to as the ischemic phase. As cells begin to die, hydrolases are released from lysosomes and cause further breakdown of the endometrium. Prostaglandin production increases as a result of the action of phospholipases liberated from lysosomes. Necrosis of vascular cells leads to microhemorrhage. The average loss of blood, tissues, and serous fluid amounts to ∼30 mL. Menstrual blood does not clot because of the presence of fibrolysins released from necrotic endometrial tissue. The effective implantation window is 3 to 4 days From studies of embryo transfer to recipient mothers in oocyte donation programs (see the box in Chapter 56 on in vitro fertilization), when both the age of the donated embryo and the time of the endometrial cycle of the recipient are known, the period of endometrial receptivity for implantation of the embryo is estimated to extend from as early as day 16 to as late as day 19 of the menstrual cycle. Of course, because implantation must normally follow the ovulation that occurs on day 14 and because fertilization normally occurs within 1 day of ovulation, the effective window is less than 4 days, from day 16 to day 19. In contrast, when embryos are transferred on cycle days 20 through 24, no pregnancies are achieved. Although the mechanisms underlying endometrial receptivity remain unclear, several changes in the endometrium are believed to be associated with increased receptivity of the endometrium for the embryo. The formation of microvilli and pinopods (i.e., protrusions of endometrial cells near gland openings) during the midluteal phase and the secretion of extracellular matrix composed of such materials as glycoproteins, laminin, and fibronectin may provide a surface that facilitates attachment of the embryo (see Chapter 56).

THE FEMALE SEX ACT Female sexual desire—libido—is a complex phenomenon that consists of physical and psychological effects, all modulated by circulating sex steroids. Libido varies during the ovarian cycle, and the frequency of female sexual activity increases around the time of ovulation. There may also be an increase in the rate of initiation of sexual activity by women around the time of ovulation. These changes may, in part, reflect the increased secretion of androgenic steroids that occurs just before and during ovulation secondary to the LH surge.

Chapter 55 • The Female Reproductive System

The female sex response, which is elicited by physical, psychic, and hormonal stimuli, occurs in four distinct phases Although sexual function has a strong physiological basis, it is not possible to separate sexual response from the other emotional and contributing factors involved in sexual relationships. Masters and Johnson published, in their now classic work Human Sexual Response, a discussion of data obtained on the sexual cycles of 700 subjects. Our current understanding of the female sexual response is based on their findings. Masters and Johnson described four stages of the sex response in women: the excitement or seduction phase, plateau, orgasm, and resolution. Following is a brief description of each phase.

Table 55-3

Female Sexual Response Cycle

Excitement

Warmth and erotic feelings Increased sexual tension Deep breathing Increased heart rate Increased blood pressure Generalized vasocongestion Skin flush Breast engorgement

Excitement

The excitement or arousal phase of the female sex response may be initiated by a multitude of internal or external stimuli, including psychological factors, such as thoughts and emotions, and physical factors, such as sight and tactile stimuli. Table 55-3 summarizes the responses of the excitement phase, many of which reflect activity of the parasympathetic division of the autonomic nervous system (ANS). Sexual intensity rises in crescendo fashion. Plateau

The plateau stage is the culmination of the excitement phase as it reaches its peak. It is associated with a marked degree of vasocongestion throughout the body. Orgasm

During orgasm, the sexual tension that has built up in the entire body is released. The climax, or orgasm, is intense and includes a myotonic response throughout the body. Muscle contractions start 2 to 4 seconds after the woman begins to experience orgasm, and they repeat at 0.8-second intervals. The actual number of contractions, as well as their intensity, varies from woman to woman. Some women observed to have orgasmic contractions are not aware that they are having an orgasm. Masters and Johnson suggested that prolonged stimulation during the excitement phase leads to more pronounced orgasmic activity. Whereas the excitement phase is under the influence of the parasympathetic division of the ANS, as is the erection phase in men, orgasm seems to be related to the sympathetic division, as is the emission phase in men (see Chapter 54). Resolution

The last phase of the female sex response is a return of the woman’s physiological state to the preexcitement level. During the resolution phase, the woman generally experiences a feeling of personal satisfaction, wellbeing, and relaxation of sexual desire. A new sexual excitement cycle may be initiated at any time after orgasm without the refractory phase that occurs in men.

Nipple erection (myotonic effect) Engorgement of labia and clitoris Vaginal “sweating” (transudative lubrication) Secretions from Bartholin glands Uterine tenting into pelvis Plateau

Marked vasocongestion “Sex flush” (maculopapular rash on breasts, chest, and epigastrium) Nipple erection Engorgement of the labia Engorgement of lower third of the vagina, with narrowing of diameter Dilation of upper two thirds of vagina Clitoral swelling and erection Vaginal “sweating” Uterine tenting Orgasm

Release of tension Generalized, rhythmic myotonic contractions Contractions of perivaginal muscles and anal sphincter Uterine contractions Resolution

Return to pre-excitement state

Both the sympathetic and the parasympathetic divisions control the female sex response Much of the response in the excitement phase results from stimulation of the parasympathetic fibers of the ANS. In some cases, anticholinergic drugs may interfere with a full

Personal satisfaction and well-being New excitement cycles may be initiated

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response in this stage. Dilatation of blood vessels in the erectile tissues causes engorgement with blood and erection of the clitoris, as well as distention of the peri-introital tissues and subsequent narrowing of the lower third of the vagina. Parasympathetic fibers emanating from the sacral plexus (see Chapter 14) innervate these erectile tissues, just as in men (see Chapter 54). In addition, the parasympathetic system innervates Bartholin’s glands, which empty into the introitus, as well as the vaginal glands. Adequate lubrication is necessary to minimize the friction of intercourse and thus maximize the stimulation to achieve orgasm. Both physical stimulation and psychic stimuli are important for female orgasm. Psychic stimuli are coordinated through the cerebrum, which causes the generalized tension throughout the body, as discussed earlier, and also modulates the autonomic response. The female orgasm is also coordinated through a spinal cord reflex that results in rhythmic contractions of the perineal muscles. The afferent pathways for this spinal cord reflex follow the pudendal nerves, which emanate through sacral segments 2 to 4 and are the primary innervation to the perineum and the female external genitalia. This spinal cord reflex is similar to that observed in men.

The female sex response facilitates sperm transport through the female reproductive tract The spinal reflexes previously discussed may also increase uterine and cervical activity and may thus promote transport of gametes. The cervix dilates during orgasm, thereby facilitating sperm transport into the upper part of the reproductive tract. The release of oxytocin at the time of orgasm stimulates uterine contractility, which also facilitates gamete transport. Although 150 to 600 million sperm cells (see Chapter 54) are normally deposited in the vagina during sexual intercourse, ∼100,000 reach the cavity of the uterus, and only 50 to 100 viable sperm reach the distal fallopian tube where fertilization occurs. Aside from the one or more sperm that will fertilize the ovum (or ova), most sperm degenerate, to be disposed of by the female genital tract. Sperm transport is accomplished by swimming movements of the sperm tail through the mucus of the cervical canal. The sperm reach the ampulla of the fallopian tubes within 5 minutes of ejaculation. Clearly, this rapid rate of transport could not be achieved by the swimming activity of the sperm alone. Therefore, uterine or tubal activity must serve a major role in sperm transport.

MENOPAUSE Menopause, or the climacteric, signals the termination of reproductive function in women. Cyclic reproductive function ceases, menstruation comes to an end, and childbearing is generally no longer possible. Also occurring are significant physiological changes (Table 55-4) that have a major impact on health.

Table 55-4 The Menopausal Syndrome and Physical Changes in Menopause Menopausal Syndrome

Physical Changes in Menopause

Vasomotor instability

Atrophy of the vaginal epithelium

Hot flashes

Changes in vaginal pH

Night sweats

Decrease in vaginal secretions

Mood changes

Decrease in circulation to vagina and uterus

Short-term memory loss

Pelvic relaxation

Sleep disturbances

Loss of vaginal tone

Headaches Loss of libido

Cardiovascular disease Osteoporosis Alzheimer disease

Only a few functioning follicles remain in the ovaries of a menopausal woman Progressive loss of ovarian follicular units occurs throughout life. Approximately 6 to 7 million germ cells are present in the two ovaries of the developing female fetus at 20 weeks’ gestation. At birth, only ∼1 to 2 million follicular units remain in the ovaries. At puberty ∼400,000 remain, a finding again reflecting the continued process of atresia during the prepubertal years. Puberty generally occurs in American girls at ∼12.5 years of age. The average age of menopause in American women is 51.5 years. Thus, it is estimated that more than 400 oocytes are ovulated during the reproductive life of a woman. At menopause and during the ensuing 5 years, the ovary contains only an occasional secondary follicle and a few primary follicles in a prominent stroma. The massive loss of oocytes over the reproductive life of a woman—from 400,000 at puberty to virtually none at menopause—is the result of the rapid, continuous process of atresia during reproductive life. During each cycle, a large cohort (∼10 to 30) of follicles is recruited, but only one follicle reaches dominance and ovulates. The others become atretic. However, even if we multiply the number of follicles in a cohort by the total number of menstrual cycles, we cannot account for all 400,000 of the prepubertal units. Thus, most of the primordial and primary ovarian follicles are lost as a result of atresia during the reproductive life of the individual. During menopause, levels of the ovarian steroids fall, whereas gonadotropin levels rise The loss of functional ovarian follicles is primarily responsible for menopause in primates. Even before the onset of meno-

Chapter 55 • The Female Reproductive System

Hormone Replacement Therapy During Menopause

A

lthough the mean age at menopause is ∼51.5 years, changes in hormone secretion patterns are seen much earlier. Increases in levels of FSH occur as early as 35 years of age. The mechanisms responsible for this change remain to be elucidated. However, it is clear that ovarian function begins to diminish far in advance of a woman’s last menstrual period. The increase in gonadotropin secretion is probably a result of decreased folliculogenesis leading to decreased secretion of sex steroids and inhibin and thus lowered negative feedback on the gonadotrophs during the perimenopausal period. The characteristic changes associated with menopause are primarily the result of low circulating estrogen levels. Estrogen is a very important regulatory hormone in girls and women. In addition to the role of estrogen in reproductive processes, this hormone has profound effects on several other physiological systems (Table 55-4). Hormone replacement therapy is indicated during menopause to alleviate the menopausal syndrome and to prevent or diminish the physical changes that occur as a result of estrogen deficiency. Menopausal hormone replacement therapy consists of estrogen and progestin administration. The reason for administering progestins is that the endometrium is at significant risk of neoplasia from the unopposed actions of estrogens. Thus, progestins are not generally administered to women who have had hysterectomies. Estrogen replacement is very effective against the menopausal syndrome, as well as against osteoporosis and cardiovascular disease. However, because of side effects (e.g., menstruation) and concern about endometrial and breast cancer, compliance with hormone replacement therapy is often compromised. The selective ER modulators (SERMS) comprise a group of structurally dissimilar compounds that interact with ERs. However, these agents act as either estrogen agonists or estrogen antagonists, depending on the target tissue and hormonal status of the individual. The exact mechanisms through which SERMS elicit their effects in specific tissues remain unclear and constitute an area of active research. The estrogen antagonist effects of SERMS may be mediated by classic competition for the ER. SERMS such as tamoxifen and raloxifene have beneficial effects, similar to those of estrogens, on bone and the cardiovascular system, whereas they antagonize estrogen in reproductive tissue. Clearly, the ideal SERM would have all the beneficial effects of estrogen without the negative and potentially dangerous side effects. For example, the perfect SERM would alleviate the menopausal syndrome, protect against cardiovascular and Alzheimer disease, and act as estrogen agonists in certain reproductive tissues and as antagonists in others.

pause, significant hormonal changes occur very early during reproductive life. Because of a gradual decline in the number of follicles, the decreased ovarian production of estrogen reduces the negative feedback to the anterior pituitary and leads to increased levels of FSH. Increased levels of FSH are seen as early as 35 years of age, even though cyclic reproductive function continues. When compared with

younger women, older—but premenopausal—women have diminished estradiol production and decreased luteal function during natural cycles. Diminished inhibin production by the aging ovary may also contribute to the sharp rise in FSH levels that occurs in the perimenopausal period of life. During menopause, estradiol levels are generally less than 30 pg/mL, and progesterone levels are often less than 1 ng/ mL of plasma. Both these values are somewhat less than the lowest levels seen during the menstrual cycle of a younger woman (Fig. 55-9). Ovarian production of androstenedione is minimal during menopause, although androstenedione production by the adrenal cortex remains normal. Because the output of estrogens, progestins, and inhibins from the ovaries falls to very low levels during menopause, negative feedback on the hypothalamic-pituitary-ovarian axis (Fig. 55-4) becomes minimal. As a result, levels of FSH and LH may be higher than those seen during the midcycle surge in premenopausal women—the futile attempt of the axis to stimulate follicular development and production of the female sex steroids. During menopause, the anterior pituitary still secretes FSH and LH in pulses, presumably after cyclic release of GnRH from the hypothalamus (Fig. 55-2A). Although gonadotropins cannot generally stimulate the postmenopausal ovary, it appears that the gonadotrophs in the anterior pituitary can respond to exogenous GnRH. REFERENCES Books and Reviews Adashi EY, Rock JA, Rosenwaks Z (eds): Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott-Raven, 1996. de Kretser DM, Robertson DM, Risbridger GP: Recent advances in the physiology of human inhibin. J Endocrinol Invest 1990; 13:611-624. Dufau ML: The luteinizing hormone receptor. Annu Rev Physiol 1998; 60:461-496. Marshall WA, Tanner JM: Puberty. In Falkner F, Tanner JM (eds): Human Growth, vol 2, pp 171-209. New York: Plenum, 1986. Masters WH, Johnson VE: Human Sexual Response. Boston: Little, Brown, 1966. Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed. Philadelphia: WB Saunders, 1998. Woodruff TK, Mather JP: Inhibin, activin and the female reproductive axis. Annu Rev Physiol 1995; 57:214-244. Journal Articles Erickson GF, Wang C, Hsueh AJW: FSH induction of functional LH receptors in granulosa cells cultured in a chemically defined medium. Nature 1979; 279:336-338. Fiddes JC, Goodman HM: The gene encoding the common alpha subunit of the four human glycoprotein hormones. J Mol Appl Genet 1981; 1:3-18. Ryan KJ: Granulosa-thecal cell interaction in ovarian steroidogenesis. J Steroid Biochem 1979; 11:799-800. Schwanzel-Fukuda M, Pfaff DW: Origin of luteinizing hormonereleasing hormone neurons. Nature 1989; 338:161-164. Veldhuis JD, Dufau ML: Estradiol modulates the pulsatile secretion of bioactive luteinizing hormone in vivo. J Clin Invest 1987; 80:631-638. Wang CF, Lasley BL, Lein A, Yen SS: The functional changes of the pituitary gonadotrophs during the menstrual cycle. J Clin Endocrinol Metab 1976; 42:718-728.

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56 F E R T I L I Z AT I O N , P R E G N A N C Y, A N D L A C TAT I O N Ervin E. Jones

TRANSPORT OF GAMETES AND FERTILIZATION Cilia and smooth muscle transport the egg and sperm within the female genital tract Following ovulation, the fimbriae of the fallopian tube sweep over the ovarian surface and pick up the oocyte—surrounded by its complement of granulosa cells, the cumulus oophorus, and corona radiata (see Chapter 55)—and deposit it in the fallopian tube. Shortly after ovulation, movements of the cilia and the smooth muscle of the fallopian tube propel the oocyte-cumulus complex toward the uterus. A man normally deposits 150 to 600 million sperm into the vagina of a woman at the time of ejaculation. Only 50 to 100 of these sperm actually reach the ampullary portion of the fallopian tube, where fertilization normally occurs. However, the sperm get there very quickly, within ∼5 minutes of ejaculation. The swimming motion of the sperm alone cannot account for such rapid transport. Forceful contractions of the uterus, cervix, and fallopian tubes propel the sperm into the upper reproductive tract during female orgasm. Prostaglandins in the seminal plasma may induce further contractile activity.

The capacitation of the spermatozoa that occurs in the female genital tract enhances the ability of the sperm cell to fertilize the ovum As discussed in Chapter 54, maturation of sperm continues while they are stored in the epididymis. In most species, neither freshly ejaculated sperm cells nor sperm cells that are removed from the epididymis are capable of fertilizing the egg until these cells have undergone further maturation (capacitation) in the female reproductive tract or in the laboratory. Capacitation is a poorly understood physiological process by which spermatozoa acquire the ability to penetrate the zona pellucida of the ovum. The removal or modification of a protective protein coat from the sperm cell membrane appears to be an important molecular event in the process of capacitation.

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In women, sperm do not need to pass through the cervix and uterus to achieve capacitation. Successful pregnancy can occur with gamete intrafallopian transfer (GIFT), in which spermatozoa and oocytes are placed directly into the ampulla of the fallopian tube, and also with direct ultrasound-guided intraperitoneal insemination, in which the sperm are deposited in the peritoneal cavity, near the fimbria. Thus, capacitation of sperm in the reproductive tract is not strictly organ specific. As evidenced by the success of in vitro fertilization and embryo transfer (IVF-ET; see the box titled In Vitro Fertilization and Embryo Transfer), capacitation is feasible even if the sperm does not make contact with the female reproductive tract. Fertilization begins as the sperm cell attaches to the zona pellucida and undergoes the acrosomal reaction, and it ends with the fusion of the male and female pronuclei After ovulation, the egg in the fallopian tube is in a semidormant state. If it remains unfertilized, the ripe egg will remain quiescent for some time and eventually degenerates. In the case of fertilization, the sperm normally comes into contact with the oocyte in the ampullary portion of the tube, usually several hours after ovulation. Fertilization causes the egg to awaken (activation), thus initiating a series of morphological and biochemical events that lead to cell division and differentiation. Fertilization occurs in eight steps: Step 1. The sperm head weaves its way past the follicular cells and attaches to the zona pellucida that surrounds the oocyte (Fig. 56-1). The zona pellucida is composed of three glycoproteins; ZP1 cross-links the filamentous ZP2 and ZP3 into a latticework. Receptors on the plasma membrane of the sperm cell bind to ZP3, thereby initiating a signal transduction cascade. Step 2. As a result of the sperm-ZP3 interaction, the sperm cell undergoes the acrosomal reaction, a prelude to the migration of the sperm cell through the mucus-like zona pellucida. The acrosome is a unique sperm organelle, essentially a large secretory vesicle, that originates from the Golgi complex in the spermatid (see Chapter 54). The

Chapter 56 • Fertilization, Pregnancy, and Lactation

1 The sperm cell weaves past follicular cells and binds to the zona pellucida.

Follicular cells

2 A rise in [Ca2+]i inside the sperm cell triggers the exocytosis of the acrosome (acrosomal reaction), which contains hydrolytic enzymes. 3 Hydrolytic enzymes contained in the acrosomal cap are released. These enzymes locally dissolve the zona pellucida. The whip-like action of the tail pushes the sperm head toward the oocyte membrane.

ZP3

Sperm nucleus

Vitelline envelope Bonds Egg plasma membrane Sperm-binding receptors

Acrosome  Pronucleus

Enzymes released as acrosome breaks down

Cortical vesicle Zona pellucida (jelly coat)

4 With the head of the sperm now lying sideways, microvilli on the oocyte surround the sperm head. The two membranes fuse. The contents of the sperm cell enter the oocyte; the sperm-cell membrane remains behind.

[Ca2+]

Egg cytosol

Vitelline envelope

1st

Female polar 2nd bodies

 Pronucleus

5 A rise in [Ca2+]i inside the oocyte triggers the cortical reaction, in which there is exocytosis of granules that previously lay immediately beneath the plasma membrane. The enzymes released lead to changes in the zonapellucida proteins, causing the zona pellucida to harden, preventing the entry of other sperm cells. 6 The rise in [Ca2+]i inside the oocyte induces the completion of the oocyte's second meiotic division and the formation of the second polar body, which usually lies next to the  Pronucleus first polar body. 7 The head of the sperm enlarges to become the male pronucleus.

Fertilization membrane

8 The male and female pronuclei fuse.

Figure 56-1

Fertilization. The illustration summarizes the eight steps of fertilization. ZP, zona pellucida.

acrosome contains hydrolyzing enzymes that are necessary for the sperm to penetrate the zona pellucida. The acrosome lies in front of and around the anterior two thirds of the sperm nucleus, much like a motorcycle helmet fits over one’s head. During the acrosomal reaction, an increase in [Ca2+]i triggers fusion of the outer acrosomal membrane with the sperm cell’s plasma membrane and results in the exocytosis of most of the acrosomal contents. Step 3. The spermatozoon penetrates the zona pellucida. One mechanism of this penetration is the action of the acrosomal enzymes. Protease inhibitors can block the penetration of spermatozoa through the zona pellucida. The sperm cell also penetrates the zona pellucida by

mechanical action. The sperm head rapidly oscillates about a fulcrum that is situated in the neck region. This rapid, vigorous, rocking action occurs with a frequency of approximately six to eight oscillations per second. The sperm penetrates the zona pellucida at an angle, thus creating a tangential cleavage slit and leaving the sperm head lying sideways against the oocyte membrane. Step 4. The cell membranes of the sperm and the oocyte fuse. Microvilli on the oocyte surface envelop the sperm cell, which probably binds to the oocyte membrane through specific proteins on the surfaces of the two cells. The posterior membrane of the acrosome—which remains part of the sperm cell after the acrosomal reaction—is the first portion of the sperm to fuse with the

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plasma membrane of the egg. The sperm cell per se does not enter the oocyte. Rather, the cytoplasmic portions of the head and tail enter the oocyte and leaving the sperm cell plasma membrane behind, similar to a snake’s crawling out of its skin. Step 5. The oocyte undergoes the cortical reaction. As the spermatozoon penetrates the oocyte’s plasma membrane, it initiates formation of inositol 1,4,5-triphosphate (IP3) and causes Ca2+ release from internal stores (see Chapter 3) and an increase in [Ca2+]i and [Ca2+]i waves. This rise in [Ca2+]i, in turn, triggers the oocyte’s second meiotic division—discussed later—and the cortical reaction. In the cortical reaction, small electron-dense granules that lie just beneath the plasma membrane fuse with the oocyte’s plasma membrane. Exocytosis of these granules releases enzymes that act on glycoproteins in the zona pellucida and cause them to harden. In the process, polysaccharides are liberated from these glycoproteins. From a teleological perspective, the cortical granule reaction prevents polyspermy. Polyspermic embryos are abnormal because they are polyploid. They do not develop beyond the early cleavage stages. Step 6. The oocyte completes its second meiotic division. The oocyte, which had been arrested in the prophase of its first meiotic division since fetal life (see Chapter 53), completed its first meiotic division at the time of the surge of luteinizing hormone (LH), which occurred several hours before ovulation (see Chapter 55). The results were the first polar body and a secondary oocyte with a haploid number of duplicated chromosomes (see Fig. 53-1). Before fertilization, this secondary oocyte had begun a second meiotic division, which was arrested in metaphase. The rise in [Ca2+]i inside the oocyte—which the sperm cell triggers, as noted earlier—causes not only the cortical reaction but also the completion of the oocyte’s second meiotic division. One result is the formation of the second polar body, which contains a haploid number of unduplicated maternal chromosomes. The oocyte extrudes the chromosomes of the second polar body, together with a small amount of ooplasm, into a space immediately below the zona pellucida; the second polar body usually lies close to the first polar body. The nucleus of the oocyte also contains a haploid number of unduplicated chromosomes. As its chromosomes decondense, the nucleus of this mature ovum becomes the female pronucleus. Step 7. The sperm nucleus decondenses and transforms into the male pronucleus, which, like the female pronucleus, contains a haploid number of unduplicated chromosomes (see Fig. 54-6). The cytoplasmic portion of the sperm’s tail degenerates. Step 8. The male and female pronuclei fuse, to form a new cell, the zygote. The mingling of chromosomes (syngamy) can be considered as the end of fertilization and the beginning of embryonic development. Thus, fertilization results in a conceptus that bears 46 chromosomes, 23 from the maternal gamete and 23 from the paternal gamete. As noted in Chapter 53, fertilization of the ovum by a sperm bearing an X chromosome produces a zygote with XX sex chromosomes; this develops into a female. Fertilization with a Y-bearing sperm produces an XY

zygote, which develops into a male. Therefore, chromosomal sex is established at fertilization.

IMPLANTATION OF THE DEVELOPING EMBRYO As discussed, the ovum is fertilized in the ampullary portion of the fallopian tube several hours after ovulation (Fig. 56-2), and the conceptus remains in the fallopian tube for ∼72 hours, during which time it develops to the morula stage (i.e., a mulberry-shaped solid mass of 12 or more cells), receiving nourishment from fallopian tube secretions. During these 3 days, smooth muscle contractions of the isthmus prevent advancement of the conceptus into the uterus while the endometrium is preparing for implantation. The mechanisms by which the ovum is later propelled through the isthmus of the fallopian tube to the uterus probably include beating of the cilia of the tubal epithelium and contraction of the fallopian tube. After the morula rapidly moves through the isthmus to the uterine cavity, it floats freely in the lumen of the uterus and transforms into a blastocyst (Fig. 56-2). A blastocyst is a ball-like structure with a fluid-filled inner cavity. Surrounding this cavity is a thin layer of trophoectoderm cells that forms the trophoblast, which develops into a variety of supporting structures, including the amnion, the yolk sac, and the fetal portion of the placenta. On one side of the cavity, attached to the trophoblast, is an inner cell mass, which develops into the embryo proper. The conceptus floats freely in the uterine cavity for ∼72 hours before it attaches to the endometrium. Thus, implantation of the human blastocyst normally occurs 6 to 7 days following ovulation. Numerous maturational events occur in the conceptus as it travels to the uterus. The embryo must be prepared to draw nutrients from the endometrium on arrival in the uterine cavity, and the endometrium must be prepared to sustain the implantation of the blastocyst. Because of the specific window in time during which implantation can occur, temporal relationships between embryonic and endometrial maturation assume extreme importance. The presence of an embryo leads to decidualization, a completion of the predecidualization of the endometrium that was initiated during the late secretory phase of the endometrial cycle During the middle to late secretory phase of the normal endometrial cycle, the endometrium becomes more vascularized and thickened, and the endometrial glands become tortuous and engorged with secretions. These changes, driven by progesterone from the corpus luteum, peak at ∼7 days after ovulation. Additionally, beginning 9 to 10 days after ovulation, a process known as predecidualization begins near the spiral arteries (see Chapter 55). During predecidualization, stromal cells transform into rounded decidual cells, and these cells spread across the superficial layer of the endometrium to make it more compact (zona compacta) and to separate it from the deeper, more spongy layer (zona spongiosa; see Fig. 55-13). If conception fails to occur, the

Chapter 56 • Fertilization, Pregnancy, and Lactation

Intramural portion Endometrium of fallopian tube Fundus of uterus

Myometrium

Ampulla of fallopian tube

Isthmus of fallopian tube Ovary

Corpus (body) of uterus

Infundibulum of fallopian tube

Hilus

Fimbriae

Mesovarium Proper ovarian ligament Bladder Cervix of uterus Vagina

Transport of the conceptus to the uterus.

secretory activity of the endometrial glands decreases, followed by regression of the glands 8 to 9 days after ovulation, which is ultimately followed by menstruation. When pregnancy occurs, the predecidual changes in the endometrium are sustained and extended, thus completing the process of decidualization. The decidua is the specialized endometrium of pregnancy. Its original name was membrana decidua, a term referring to the membranes of the endometrium that are shed following pregnancy, like the leaves of a deciduous tree. Because the degree of decidualization is considerably greater in conception cycles than in nonconception cycles, it is likely that the blastocyst itself promotes decidualization. Indeed, either the presence of the embryo or a traumatic stimulus that mimics the embryo’s invasion of the endometrium induces changes in the endometrium. The area underneath the implanting embryo becomes the decidua basalis (Fig. 56-3). Other portions of the decidua that become prominent later in pregnancy are the decidua capsularis, which overlies the embryo, and the decidua parietalis, which covers the remainder of the uterine surface. The upper zona compacta layer and the middle zona spongiosa layer of the nonpregnant endometrium are still recognizable in the decidualized endometrium of pregnancy. The glandular epithelium within the zona spongiosa continues its secretory activity during the first trimester. Some of the glands take on a hypersecretory appearance in what has been referred to as the Arias-Stella phenomenon of early pregnancy—named after the pathologist Javier Arias-Stella. Although the decidualized endometrium is most prominent during the first trimester, before the establishment of the definitive placenta, elements of decidualization persist throughout gestation.

Embryo in amniotic sac

Myometrium

Yolk sac Chorionic villi Extracoelomic cavity Endometrium

Figure 56-2

Decidua basalis

Uterine cavity

Decidua capsularis Decidua parietalis Cervical canal

Vagina

Figure 56-3 The three decidual zones during early embryonic development (∼13 to 14 days after fertilization). The figure shows a sagittal section through a pregnant uterus, with the anterior side to the right.

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In Vitro Fertilization and Embryo Transfer

I

VF is a procedure in which an oocyte is or oocytes are removed from a woman and are then fertilized with sperm under laboratory conditions. Early development of the embryo also proceeds under laboratory conditions. Finally, the physician transfers one or more embryos to the uterine cavity, where the embryo will, one hopes, implant and develop. Indications. Indications for IVF-ET include disorders that impair the normal meeting of the sperm and the egg in the distal portion of the fallopian tube. In addition to ovulatory dysfunction, these disorders include tubal occlusion, tubalperitoneal adhesions, endometriosis, or other disease processes of the female peritoneal cavity. In addition, IVF-ET is indicated in some cases of male-factor infertility (abnormalities in male reproductive function) or unexplained infertility. Ovarian stimulation. Because the success rates are less than 100% for each of the stages of IVF-ET, the physician needs several oocytes, all obtained in a single ovarian cycle. However, a woman normally develops a single dominant follicle each cycle (see Chapter 55). Thus, to obtain the multiple oocytes for IVF-ET, the physician must stimulate the development of multiple follicles in the woman by controlled ovarian hyperstimulation. Although this procedure qualitatively mimics the hormonal control of the normal cycle, the high dose of gonadotropins triggers the development of many follicles. The physician administers some combination of FSH and LH or pure FSH preparation, either intramuscularly or subcutaneously. Because exogenous gonadotropins stimulate the ovaries directly, GnRH analogues (see box in Chapter 55 on therapeutic uses of GnRH) are often used to downregulate the hypothalamic-pituitary axis during controlled ovarian stimulation. One usually administers these GnRH analogues before initiating gonadotropin therapy, primarily to prevent a premature LH surge and ovulation. Cycle monitoring. After administering the gonadotropins, the physician monitors the simulated follicular growth in the ovaries with sonographic imaging. Size, number, and serial growth of ovarian follicles may be assessed daily or at other appropriate intervals. Serum estradiol levels provide an additional measure of follicular growth and function. When estradiol levels and follicular growth indicate—by established criteria— appropriate folliculogenesis, the physician simulates a natural LH surge by injecting hCG, which is a close relative of LH (see Chapter 55). However, in this case, the simulated LH surge completes the final maturation of multiple follicles and oocytes. As we already know, ovulation usually occurs 36 to 39 hours following the beginning of the LH surge. Thus, the physician plans oocyte retrieval in such a way to allow maximal follicular maturation, but still to harvest the oocytes before ovulation. Thus, retrievals are scheduled for 34 to 36 hours following the administration of hCG. Oocyte retrieval. The physician retrieves oocytes by aspirating them from individual follicles, under sonographic guid-

Uterine secretions nourish the preimplantation embryo, promote growth, and prepare it for implantation Before the embryo implants in the endometrium and establishes an indirect lifeline between the mother’s blood and its

ance. With the patient under conscious or unconscious sedation, and after applying a local anesthetic to the posterior vaginal wall, the physician inserts a probe, equipped with a needle guide, into the vagina. After inserting a 16- to 18-gauge needle through the vaginal wall, the specialist aspirates the follicular fluid from each mature follicle and collects it in a test tube containing a small amount of culture medium. The eggs are identified in the follicular fluid, are separated from the fluid and other follicular cells, and are then washed and prepared for insemination. This procedure normally yields 8 to 15 oocytes. Insemination. The sperm sample is subjected to numerous washes, followed by column chromatography to separate the sperm cells from the other cells and from debris found in the ejaculate. Each egg is inseminated with 50,000 to 300,000 motile sperm in a drop of culture medium and is incubated overnight. Fertilization can usually be detected by the presence of two pronuclei in the egg cytoplasm after 16 to 20 hours. Fertilization rates generally range from 60% to 85%. Embryo development is allowed to continue in vitro for another 48 to 120 hours until embryos are transferred to the uterus. Among couples whose male partner has very low numbers of motile sperm, high fertilization rates can be achieved using intracytoplasmic sperm injection (ICSI). Micromanipulation techniques are used to inject a sperm cell into the cytoplasm of each egg in vitro. Fertilization rates after ICSI are generally 60% to 70%, or approximately equivalent to conventional insemination in vitro. Embryo transfer. After culturing the cells for 48 to 120 hours, the physician transfers three to four embryos to the uterus at the four- to eight-cell stage (after 2 days) or fewer embryos at the blastocyst stage (after 5 days). Embryos are selected and are loaded into a thin, flexible catheter, which is inserted into the uterine cavity to the desired depth under ultrasonic guidance. The woman usually receives supplemental progesterone to support implantation and pregnancy. In certain cases, the embryos are transferred to the fallopian tube during laparoscopy. This procedure is referred to as tubal embryo transfer (TET). The rationale for this procedure is that the fallopian tube contributes to the early development of the embryo as it travels down the tube to the uterus. Success rates. Implantation rates usually range from 8% to 15% per embryo transferred. In the United States, the mean live birth rate per ET procedure is ∼33%. Success rates in IVFET depend on numerous factors, including age as well as the type and severity of the disease causing infertility. GIFT. In certain cases of infertility, the physician collects the oocytes and sperm cells in much the same way as described earlier for IVF-ET, but directly transfers the gametes to the fallopian tube, where fertilization occurs. GIFT is accomplished using laparoscopic techniques.

own, it must receive its nourishment from uterine secretions. Following conception, the endometrium is primarily controlled by progesterone, which initially comes from the corpus luteum (see Chapter 55). The uterine glandular epithelium synthesizes and secretes several steroid-dependent proteins (Table 56-1) that are thought to be important for

Chapter 56 • Fertilization, Pregnancy, and Lactation

Table 56-1 Endometrial Proteins, Glycoproteins, and Peptides Secreted by the Endometrial Glands During Pregnancy Mucins Prolactin Insulin-like growth factor–binding protein 1 (IGFBP-1) Placental protein 14 (PP14) or glycodelin Pregnancy-associated endometrial α2-globulin (α2-PEG) Endometrial protein 15 Fibronectin Laminin

Table 56-2

Substances Secreted by the Blastocyst

Immunoregulatory Agents

Platelet-activating factor (PAF) Early pregnancy factor Immunosuppressive factor PGE2 Interleukins 1α, 6, and 8 Interferon α Leukemia inhibitory factor Colony-stimulating factor Human leukocyte antigen 6 Fas ligand Metalloproteases (facilitate invasion of trophoblast into the endometrium)

Collagen type IV

Collagenases: digest collagen types I, II, III, VII, and X Gelatinases: two forms, digest collagen type IV and gelatin Stromelysins: digest fibronectin, laminin, and collagen types IV, V, and VII

Heparan sulfate

Serine Proteases (facilitate invasion of trophoblast into the endometrium)

Entactin

Proteoglycan Integrins Albumin β Lipoprotein Relaxin Acidic fibroblast growth factor Basic fibroblast growth factor Pregnancy-associated plasma protein A (PAPP-A) Stress response protein 27 (SRP-27)

Other Factors or Actions

hCG: autocrine growth factor Ovum factor Early pregnancy factor Embryo-derived histamine-releasing factor Plasminogen activator and its inhibitors Insulin-like growth factor 2 (IGF-2): promotes trophoblast invasiveness Estradiol β1 Integrin Fibroblast growth factor (FGF) Transforming growth factor α (TGF-α) Inhibins

CA-125 β Endorphin Leu-enkephalin Diamine oxidase Plasminogen activator (PA) Plasminogen activator inhibitor Renin Progesterone-dependent carbonic anhydrase Lactoferrin

the nourishment, growth, and implantation of the embryo. The endometrium secretes cholesterol, steroids, and various nutrients, including iron and fat-soluble vitamins. It also synthesizes matrix substances, adhesion molecules, and surface receptors for matrix proteins, all of which may be important for implantation. Pinopods appear as small, finger-like protrusions on endometrial cells between day 19 (about the time the embryo would arrive in the uterus) and day 21 (about the time of implantation) of the menstrual cycle; they persist for only 2 to 3 days. Pinopod formation appears to be progesterone

dependent, and it is inhibited by estrogens. Pinopods endocytose macromolecules and uterine fluid and absorb most of the fluid in the lumen of the uterus during the early stages of embryo implantation. By removing uterine luminal fluid, the pinopods may allow the embryo and the uterine epithelium to approximate one another more closely. Because apposition and adhesion of the embryo to the uterus are the first events of implantation, the presence and action of pinopods may determine the extent of the implantation window. The blastocyst secretes substances that facilitate implantation If the blastocyst is to survive, it must avoid rejection by the maternal cellular immune system. It does so by releasing immunosuppressive agents (Table 56-2). The embryo also synthesizes and secretes macromolecules that promote implantation, the development of the placenta, and the maintenance of pregnancy. Both short-range and long-range embryonic signals may be necessary for implantation, although the nature of some of these signals remains enigmatic. One short-range signal from the blastocyst may stimulate local changes in the endo-

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metrium at the time of its apposition to the endometrium. A long-range signal that is secreted by the early blastocyst is human chorionic gonadotropin (hCG), which is closely related to LH (see Chapter 55) and sustains the corpus luteum in the presence of rapidly falling levels of maternal LH. hCG is one of the most important of the factors secreted by the trophoblast of the blastocyst, both before and after implantation. Besides rescuing the corpus luteum, hCG is an autocrine growth factor that promotes trophoblast growth and placental development. hCG levels are high in the area where the trophoblast faces the endometrium. hCG may have a role in the adhesion of the trophoblast to the epithelia of the endometrium, and it also has protease activity. During implantation, the blastocyst apposes itself to the endometrium, adheres to epithelial cells, and then finally breaks through the basement membrane and invades the stroma As noted earlier, the conceptus lies unattached in the uterine cavity for ∼72 hours. About halfway through this period (i.e., 5 to 6 days after ovulation), the morula transforms into the blastocyst (Fig. 56-4A). Before the initiation of implantation, the zona pellucida that surrounds the blastocyst degenerates. This process, known as hatching of the embryo, occurs 6 to 7 days after ovulation. Lytic factors in the endometrial cavity appear to be essential for the dissolution of the zona pellucida. The blastocyst probably also participates in the process of zona lysis and hatching; when an unfertilized egg is placed in the uterus under the same conditions, its zona pellucida remains intact. A factor produced by the blastocyst may activate a lytic factor that is derived from a uterine precursor. Plasmin, produced from plasminogen, is a plausible candidate for this uterine factor, because plasmin exhibits a lytic effect on the zona pellucida in vitro, and inhibitors of plasmin block in vitro hatching of rat blastocysts. Implantation occurs in three stages: (1) apposition, (2) adhesion, and (3) invasion. Apposition

The earliest contact between the blastocyst wall, the trophoectoderm, and the endometrial epithelium is a loose connection called apposition (Fig. 56-4B). Apposition usually occurs in a crypt in the endometrium. From the standpoint of the blastocyst, it appears that apposition occurs at a site where the zona pellucida is ruptured or lysed and where it is possible for the cell membranes of the trophoblast to make direct contact with the cell membranes of the endometrium. Although the preimplantation blastocyst is asymmetric, it seems that the entire trophoectoderm has the potential to interact with the endometrium, and the final correct orientation—with the inner cell mass pointing toward the endometrium—occurs by free rotation of the inner cell mass within the sphere of overlying trophoectoderm cells.

Adhesion

The trophoblast appears to attach to the uterine epithelium through the microvilli of the trophoblast; ligandreceptor interactions are probably involved in adhesion (Fig. 56-4C). The receptors for these ligand-receptor interactions are often members of the integrin family (see Chapter 2)

and can be either on the blastocyst or on the endometrium. Integrins are bifunctional integral membrane proteins; on their intracellular side, they interact with the cytoskeleton, whereas on their extracellular side, they have receptors for matrix proteins such as collagen, laminin, fibronectin, and vitronectin. Therefore, ligand-receptor interactions have two possible orientations. For the first, the extracellular surface of the trophoblast has integrins for binding fibronectins, laminin, and collagen type IV. Thus, during implantation, the trophoblast binds to the laminin that is distributed around the stromal (decidual) cells of the endometrium. Fibronectin, a component of the basement membrane, probably guides the implanting embryo (see later) and is subsequently broken down by the trophoblast. For the second orientation of matrix-integrin interactions, the extracellular surface of the glandular epithelium also expresses integrins on days 20 to 24 of the menstrual cycle, the implantation window (see Chapter 55). The expression of receptors for fibronectin and vitronectin (i.e., integrins) may serve as markers of the endometrial capacity for implantation. Small peptides containing sequences that are homologous to specific sequences of fibronectin block blastocyst attachment and outgrowth on fibronectin. In addition to the integrin-matrix interactions, another important class of ligand-receptor interactions appears to be between heparin or heparan sulfate proteoglycans (see Chapter 2), which are attached to the surface of the blastocyst and surface receptors on the uterine epithelial cell. These endometrial proteoglycan receptors increase as the time of implantation approaches. Any of the foregoing ligand-receptor interactions can lead to cytoskeletal changes. Thus, adhesion of the trophoblast through ligand-receptor interactions may dislodge the uterine epithelial cells from their basal lamina and may thereby facilitate access of the trophoblast to the basal lamina for penetration. Invasion

As the blastocyst attaches to the endometrial epithelium, the trophoblastic cells rapidly proliferate, and the trophoblast differentiates into two layers: an inner cytotrophoblast and an outer syncytiotrophoblast (Fig. 56-4D). The syncytiotrophoblast is a multinucleated mass without cellular boundaries. During implantation, long protrusions from the syncytiotrophoblast extend among the uterine epithelial cells. The protrusions dissociate these endometrial cells by secreting tumor necrosis factor α (TNF-α), which interferes with the expression of cadherins (cell adhesion molecules; see Chapter 2) and β-catenin (an intracellular protein that helps to anchor cadherins to the cytoskeleton). The syncytiotrophoblast protrusions then penetrate the basement membrane of the uterine epithelial cells and ultimately reach the uterine stroma. The trophoblast secretes several autocrine factors, which appear to stimulate invasion of the endometrial epithelium, as well as proteases (Table 56-2). By degrading the extracellular matrix, metalloproteases and serine proteases may control both the proliferation and the invasion of the trophoblast into the endometrium. Around the site of penetration of the syncytiotrophoblast, uterine stromal cells take on a polyhedral shape and become laden with lipids and glycogen. These are the decidual cells

Chapter 56 • Fertilization, Pregnancy, and Lactation

A

B APPOSITION

HATCHING

Endometrial capillary

Degenerating zona pellucida

Endometrial gland

Blastocyst cavity

Endometrial epithelium Inner cell mass Embryonic pole

Inner cell mass

Trophoblast

Trophoblast Blastocyst cavity

Lytic factors break down the zona pellucida.

C

D INVASION

ADHESION

Endometrial capillary

Endometrial stroma Glandular secretion

Endometrial gland Syncytiotrophoblast

Inner cell mass

Uterine cavity

Trophoblast Blastocyst cavity

Amniotic cavity Embryonic epiblast Cytotrophoblast

Exocoelomic cavity

Embryonic hypoblast Exocoelomic membrane

Figure 56-4

Embryo hatching, apposition, adhesion, and invasion.

discussed earlier. The decidual cells degenerate in the region of the invading syncytiotrophoblast and thus provide nutrients to the developing embryo. The blastocyst superficially implants in the zona compacta of the endometrium and eventually becomes completely embedded in the decidua. As the finger-like projections of the syncytiotrophoblast invade the endometrium, they reach the maternal blood supply and represent a primordial form of the chorionic villus of the mature placenta, as discussed in the next section.

PHYSIOLOGY OF THE PLACENTA Eventually, almost all the materials that are necessary for fetal growth and development move from the maternal circulation to the fetal circulation across the placenta, either by passive diffusion or by active transport. Except for CO2, waste products are largely excreted through the amniotic fluid.

At the placenta, the space between the fetus’s chorionic villi and the mother’s endometrial wall contains a continuously renewed pool of extravasated maternal blood Within the syncytium of the invading syncytiotrophoblast, fluid-filled holes called lacunae develop 8 to 9 days after fertilization (Fig. 56-5A). Twelve to 15 days after fertilization, the finger-like projections of the syncytiotrophoblast finally penetrate the endothelial layer of small veins of the endometrium. Later, these projections also penetrate the small spiral arteries. The result is free communication between the lacunae of the syncytiotrophoblast and the lumina of maternal blood vessels (Fig. 56-5B). Within 12 to 15 days after fertilization, some cytotrophoblasts proliferate and invade the syncytiotrophoblast, to form finger-like projections that are the primary chorionic villi.

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A

8 DAYS AFTER FERTILIZATION Endometrium

Lumen of uterus Lacuna Embryonic disk Amniotic cavity

Maternal blood vessels

Cytotrophoblast Syncytiotrophoblast B

12-15 DAYS

Endometrial epithelium Forming body stalk Extraembryonic coelom Lacuna

Yolk sac Forming chorion C

Primary chorionic villus Maternal blood vessels

20 DAYS

Body stalk Tertiary chorionic villus Mesenchyme Extraembryonic coelom

Yolk sac

Amniotic cavity Ectoderm Mesoderm Endoderm Lacuna in contact with maternal blood vessel

Fetal blood vessel

Figure 56-5 Development of the placenta. A, Shortly after the blastocyst has implanted (6 to 7 days after fertilization), the syncytiotrophoblast invades the stroma of the uterus (i.e., the decidua). Within the syncytiotrophoblast are lacunae. B, The invading syncytiotrophoblast breaks through into endometrial veins first, and then later into the arteries, thus creating direct communication between lacunae and maternal vessels. In addition, the proliferation of cytotrophoblasts creates small mounds known as primary chorionic villi. C, The primary chorionic villus continues to grow with the proliferation of cytotrophoblastic cells. In addition, mesenchyme from the extraembryonic coelom invades the villus, to form the secondary chorionic villus. Eventually, these mesenchymal cells form fetal capillaries; at this time, the villus is known as a tertiary chorionic villus. The lacunae also enlarge by merging with one another.

With further development, mesenchymal cells from the extraembryonic mesoderm invade the primary chorionic villi, which now are known as secondary chorionic villi. Eventually, these mesenchymal cells form fetal blood vessels de novo, at which point the villi are known as tertiary chorionic villi (Fig. 56-5C). Continued differentiation and

amplification of the surface area of the fetal tissue protruding into the maternal blood create mature chorionic villi. The outer surface of each villus is lined with a very thin layer of syncytiotrophoblast, which has prominent microvilli (brush border) that face the maternal blood. Under the syncytiotrophoblast lie sparse cytotrophoblasts, mesenchyme, and fetal blood vessels. The lacunae, filled with maternal blood, eventually merge with one another, to create one massive, intercommunicating intervillous space (Fig. 56-6). The fetal villi protruding into this space resemble a thick forest of trees arising from the chorionic plate, which is the analogue of the soil from which the trees sprout. Thus, in the mature placenta, fetal blood is separated from maternal blood only by the fetal capillary endothelium, some mesenchyme and cytotrophoblasts, and a thin layer of syncytiotrophoblast. Maternal Blood Flow

The maternal arterial blood is discharged from ∼120 spiral arteries; these arteries may have multiple openings, not all of which need be open at the same time. Blood enters in pulsatile spurts through the wall of the uterus and moves in discrete streams into the intervillous space toward the chorionic plate (Fig. 56-6). Small lakes of blood near the chorionic plate dissipate the force of the arterial spurts and reduce blood velocity. The maternal blood spreads laterally and then reverses direction and cascades over the closely packed villi. Blood flow slows even more, to allow adequate time for exchange. After bathing the chorionic villi, the maternal blood drains through venous orifices in the basal plate, enters the larger maternal placental veins, and ultimately flows into the uterine and other pelvic veins. No capillaries are present between the maternal arterioles and venules; the intervillous space is the functional capillary. Because the intervillous spaces are very narrow, and the arterial and venous orifices are randomly scattered over the entire base of the placenta, the maternal blood moves efficiently among the chorionic villi and avoids arteriovenous shunts. The spiral arteries are generally perpendicular, and the veins are generally parallel to the uterine wall. Thus, because of both the geometry of the maternal blood vessels and the difference between maternal arterial and venous pressure, the uterine contractions that occur periodically during pregnancy, as well as during delivery, attenuate arterial inflow and completely interrupt venous drainage. Thus, the volume of blood in the intervillous space actually increases, to provide continual, albeit reduced, exchange. The principal factors that regulate the flow of maternal blood in the intervillous space are maternal arterial blood pressure, intra-uterine pressure, and the pattern of uterine contraction. Fetal Blood Flow The fetal blood originates from two umbilical arteries. Unlike systemic arteries after birth, umbilical arteries carry deoxygenated blood. As these umbilical arteries approach the placenta, they branch repeatedly beneath the amnion, penetrate the chorionic plate, and then branch again within the chorionic villi, to form a capillary network. Blood that has obtained a significantly higher O2 and nutrient content returns to the fetus from the placenta through the single umbilical vein.

Chapter 56 • Fertilization, Pregnancy, and Lactation

From

m ot her To m othe r

Myometrium Chorionic villus Maternal vein

Figure 56-6 The mature placenta. With further development beyond that shown in Figure 56-5C, the outer surface of the mature chorionic villus is covered with a thin layer of syncytiotrophoblast. Under this are cytotrophoblasts, mesenchyme, and fetal blood vessels. The lacunae into which the villi project gradually merge into one massive intervillous space. Maternal blood is trapped in this intervillous space, between the endometrium on the maternal side and the villi on the fetal side. In the mature placenta, as shown here, “spiral” arteries from the mother empty directly into the intervillous space, which is drained by maternal veins. The villi look like a thick forest of trees arising from the chorionic plate, which is the analogue of the soil from which the trees sprout.

Maternal artery

Intervillous space (filled with maternal blood)

Fetal arteriole Fetal venule

Mature chorionic villus (covered with syncytiotrophoblast)

Maternal portion of placenta

Chorionic plate Umbilical arteries From fetus To fetus

The amniotic fluid that fills the amniotic cavity serves two important functions. First, it serves as a mechanical buffer and thus protects the fetus from external, physical insults. Second, it serves as a mechanism by which the fetus excretes many waste products. The water in the amniotic fluid turns over at least once a day. After the fetal kidneys mature (10 to 12 weeks), the renal excretions of the fetus are the major source of amniotic fluid production (∼75%); pulmonary secretions account for the rest. Fluid removal occurs through the actions of the fetal gastrointestinal tract (∼55%), amnion (∼30%), and lungs (∼15%).

Umbilical vein From fetus

Table 56-3

Decidual cells (maternal)

Maternal and Fetal Oxygen Levels

Site

PO2 (mm Hg)

Hemoglobin Saturation

Maternal Values

Uterine artery

100

97.5%

30-35

57%-67%

30

57%

Umbilical arteries

23

60.5%

Umbilical vein

30

85.5%

Intervillous space Uterine vein Fetal Values

Gases and other solutes move across the placenta through simple diffusion, facilitated diffusion, secondary active transport, and endocytosis The placenta is the major lifeline between the mother and the fetus. It provides nutrients and O2 to the fetus, and it removes CO2 and certain waste products from the fetus. O2 and CO2 Transport

The maternal blood coming into the intervillous space has a gas composition similar to that of systemic arterial blood: a PO2 of ∼100 mm Hg (Table 56-3), a PCO2 of ∼40 mm Hg, and a pH of 7.40. However, the diffusion of O2 from the maternal blood into the chorionic villi of the fetus causes the PO2 of blood in the intervillous space to fall, so the average PO2 is 30 to 35 mm Hg. Given the O2 dissociation curve of maternal (i.e., adult) hemoglobin (Hb), this PO2 translates to an O2 saturation of ∼65%. The PO2 of blood in the umbilical vein is even less. Despite the relatively low PO2 of the maternal blood in the intervillous space, the fetus does not suffer from a lack of O2. Because fetal Hb has a much higher affinity for O2 than does maternal Hb, the fetal Hb can extract O2 from the maternal Hb (see Chapter 29). Thus, a PO2 of 30 to 35 mm Hg, which yields an Hb saturation of ∼65% in the intervillous space in the mother’s blood, produces an Hb saturation of ∼85% in the umbili-

cal vein of the fetus (Table 56-3), assuming that the O2 fully equilibrates between intervillous and fetal blood. Other mechanisms of ensuring adequate fetal oxygenation include the relatively high cardiac output per unit body weight of the fetus and the increasing O2-carrying capacity of fetal blood late in pregnancy as the Hb concentration rises to a level 50% higher than that of the adult. The transfer of CO2 from the fetus to the mother is driven by a concentration gradient between the blood in the umbilical arteries and that in the intervillous space. Near the end of pregnancy, the PCO2 in the umbilical arteries is ∼48 mm Hg, and the PCO2 in the intervillous space is ∼43 mm Hg, a gradient of ∼5 mm Hg. The fetal blood also has a somewhat lower affinity for CO2 than does maternal blood, thus favoring the transfer of CO2 from the fetus to mother. Other Solutes

Various other solutes besides O2 and CO2 move across the placenta between the mother and the fetus

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and avail themselves of numerous transport mechanisms. Some of these solutes, such as the waste products urea and creatinine, probably move passively from fetus to mother. The lipid-soluble steroid hormones shuttle among the mother, the placenta, and the fetus by simple diffusion. Glucose moves from the mother to the fetus by facilitated diffusion, and amino acids move by secondary active transport (see Chapter 5). The placenta also transports several other essential substances, such as vitamins and minerals, that are needed for fetal growth and development. Many substances are present in the fetal circulation at concentrations higher than in the maternal blood, and they must be actively transported against concentration or electrochemical gradients. The necessary energy (i.e., ATP) is derived from glycolysis and the citric acid cycle, for which the enzymes are present in the human placenta at term. Also present are the enzymes for the pentose phosphate pathway, an alternative pathway for the oxidation of glucose, which provides the NADPH that is necessary for several synthetic pathways that require reducing equivalents in the human placenta at term. The placenta takes up large molecules from the mother through receptor-mediated endocytosis (see Chapter 2). The uptake of substances such as low-density lipoproteins (LDL), transferrin, hormones (e.g., insulin), and antibodies (e.g., immunoglobulin G) increases throughout gestation until just before birth. The placenta makes a variety of peptide hormones, including human chorionic gonadotropin and human chorionic somatomammotropin The placenta plays a key role in steroid synthesis, which is discussed in the next major section. In addition, the placenta manufactures numerous amines, polypeptides (including peptide hormones and neuropeptides), proteins, glycoproteins, and steroids (Table 56-4). Among these peptides are the placental variants of all known releasing hormones, which are produced by the hypothalamus (see Chapter 47). These placental releasing hormones may act in a paracrine fashion, controlling the release of local placental hormones, or they may enter the maternal or fetal circulations. In addition, several proteases are also present in the placenta. Although the placenta synthesizes a wide variety of substances, the significance of many of these substances is not clear. The most important placental peptide hormone is hCG. In the developing blastocyst, and later in the mature placenta, the syncytiotrophoblast cells synthesize hCG, perhaps under the direction of progesterone and estrogens. The placenta also produces two human chorionic somatomammotropins, hCS1 and hCS2, also called human placental lactogen (hPL). hCS1 and hCS2 are polypeptide hormones structurally related to growth hormone (GH) and placentalvariant GH, as well as to prolactin (PRL; see Table 48-1). They play a role in the conversion of glucose to fatty acids and ketones, thus coordinating the fuel economy of the fetoplacental unit. The fetus and placenta use fatty acids and ketones as energy sources and store them as fuels in preparation for the early neonatal period, when a considerable reservoir of energy is necessary for the transition from intra-

Table 56-4

Hormones Made by the Placenta

Peptide Hormones and Neuropeptides

hCG Thyrotropin (thyroid-stimulating hormone [TSH]) Placental-variant growth hormone hCS1 and hCS2, also known as hPL (hPL1 and hPL2) Placental proteins PP12 and PP14 TRH Corticotropin-releasing hormone (CRH) Growth hormone–releasing hormone (GHRH) GnRH Substance P Neurotensin Somatostatin Neuropeptide Y ACTH-related peptide The inhibins Steroid Hormones

Progesterone Estrone Estradiol Estriol

uterine life to life outside the uterus. hCS1 and hCS2 also promote the development of maternal mammary glands during pregnancy. In addition to its secretory functions, the placenta also stores vast amounts of proteins, polypeptides, glycogen, and iron. Many of these stored substances can be used at times of poor maternal nutrition and also during the transition from intrauterine to extrauterine life.

THE MATERNAL-PLACENTAL-FETAL UNIT During pregnancy, progesterone and estrogens rise to levels that are substantially higher than their peaks in a normal cycle Following ovulation during a normal or nonconception cycle, the cells of the ovarian follicle functionally transform into luteal cells, which produce mainly progesterone, but also estrogens (see Chapter 55). However, the corpus luteum has a finite life span, which lasts only ∼12 days before it begins its demise in the presence of declining LH levels. As a consequence of luteal demise, levels of both progesterone and estrogens decline. In contrast, during pregnancy, maternal levels of progesterone and estrogens (estradiol, estrone, estriol) all increase and reach concentrations substantially higher than those achieved during a normal menstrual cycle (Fig. 56-7). These elevated levels are necessary for maintaining pregnancy. For example, progesterone reduces uterine motility and inhibits propagation of contractions. How are these elevated levels of female steroids achieved? Early in the first trimester, hCG that is manufactured by the syncytiotrophoblast rescues the corpus luteum, which is the major source of progesterone and estrogens. This function of the corpus luteum in the

Chapter 56 • Fertilization, Pregnancy, and Lactation

ovary continues well into early pregnancy. However, by itself, the corpus luteum is not adequate to generate the very high steroid levels characteristic of late pregnancy. The developing placenta itself augments its production of progesterone and estrogens, so by 8 weeks of gestation, the placenta has become the major source of these steroids. The placenta continues to 200 100 50 20 10 5 ng/mL

2 1.0 0.5

Progesterone Estradiol (E2) Estriol (E3) Estrone (E1)

0.2 0.1 0.05 -12 -8 -4

0

4 8 12 16 20 24 28 32 36 40 Gestational age (wk)

Figure 56-7 Maternal levels of progesterone and the estrogens just before and during pregnancy. The y-axis scale is logarithmic. The zero point on the x-axis is the time of fertilization. The progesterone spikes near −8 and −4 weeks refer to the two menstrual cycles before the one that resulted in the pregnancy. (Data from Wilson JD, Foster DW, Kronenberg HM, Larsen PR [eds]: Williams Textbook of Endocrinology, 9th ed. Philadelphia: WB Saunders, 1998.)

Table 56-5

produce large quantities of estrogens, progestins, and other hormones throughout gestation. Estriol, which is not important in nonpregnant women, is a major estrogen during pregnancy (Fig. 56-7). After 8 weeks of gestation, the coordinated biosynthetic activity of the maternal-placentalfetal unit maintains high levels of progesterone and estrogens Although it emerges as the major source of progesterone and estrogens (Table 56-5), the placenta cannot synthesize these hormones by itself; it requires the assistance of both mother and fetus. This joint effort in steroid biosynthesis has led to the concept of the maternal-placental-fetal unit. Figure 56-8—which resembles the maps describing the synthesis of glucocorticoids, mineralocorticoids (see Fig. 50-2), male steroids (see Fig. 54-5), and female steroids (see Fig. 55-10, later)—illustrates the pathways used by the maternalplacental-fetal unit to synthesize progesterone and the estrogens. Figure 56-9 summarizes the exchange of synthetic intermediates among the three members of the maternalplacental-fetal unit. Unlike the corpus luteum, which manufactures progesterone, estrone, and estradiol early in pregnancy (see Chapter 55), the placenta is an imperfect endocrine organ. First, the placenta cannot manufacture adequate cholesterol, the precursor for steroid synthesis. Second, the placenta lacks two crucial enzymes that are needed for synthesizing estrone and estradiol. Third, the placenta lacks a third enzyme that is needed to synthesize estriol. The enzymes missing from the placenta are listed in Table 56-5, and they also are indicated with a brown background in Figures 56-8 and 56-9. The maternal-placental-fetal unit overcomes these placental shortcomings in two ways. First, the mother supplies most of the cholesterol as LDL particles (see Chapter 46). With this supply of maternal cholesterol, the placenta can

Roles of the Mother, Placenta, and Fetus in Steroid Biosynthesis Needs

Contributes

Lacks

Progesterone Estrone Estradiol Estriol

LDL cholesterol

Adequate synthetic capacity for progesterone and estrogens

Placenta

3β-Hydroxysteroid dehydrogenase aromatase (P-450arom)

Adequate cholesterol synthesizing capacity 17α-Hydroxylase (P-450c17; needed to synthesize estrone and estradiol) 17,20-Desmolase (P-450c17; needed to synthesize estrone and estradiol) 16α-Hydroxylase (needed to synthesize estriol)

Fetus

17α-Hydroxylase (P450c17; needed to synthesize estrone and estradiol) 17,20-Desmolase (P450c17; needed to synthesize estrone and estradiol) 16α-Hydroxylase (needed to synthesize estriol)

3β-Hydroxysteroid dehydrogenase aromatase (P450arom)

Mother

1181

1182

Section IX • The Reproductive System Cholesterol 21 20 22 2425 26 17 23 16 27 13

Placenta (Fetus lacks)

12 18 11

2

10

A HO

Acetate

Fetus (Placenta lacks)

15

14

8

B

Common to placenta and fetus

7

5

4

D

C

19 9

1

6

Side-chain–cleavage enzyme

Pregnenolone

Progesterone

CH3

CH3 O

C CH3 A

D

C

O

C CH3

CH3

A

D

C

CH3

B

B

O

HO

17α-Hydroxylase

17α-Hydroxylase

17α-Hydroxypregnenolone CH3

CH3

C

C

O

CH3 CH3 A

17α-Hydroxyprogesterone

CH3

OH

D

C

B

A

17,20-Desmolase

16α-Hydroxylase

CH3

D

C

CH3 A

O

B

HO

17,20-Desmolase Androstenedione

CH3

O CH3

O D

C

CH3 A

Estrone (E1)

16α-Hydroxylase

Dehydroepiandrosterone (DHEA)

B

O

3β-Hydroxysteroid Dehydrogenase

HO

OH

D

C

CH3

Same protein.

O

D

C A

B

B

HO

O

16-Hydroxy estrone CH3

OH D

C A

O

B

HO

17β-Hydroxysteroid dehydrogenase

Androstenediol

CH3 A

OH

OH CH3

D

C B

A

D

C

CH3

HO

B

CH3 C

CH3 A

O

CH3

OH

C

CH3

B

A O

CH3 C

OH D

B

HO

16α-Hydroxytestosterone

16α-Hydroxyandrostenedione

D

Estradiol (E2)

A

O

16α-Hydroxy DHEA

HO

Aromatase

CH3

Testosterone

O

OH CH3

OH

D

C

CH3

B

A

Estriol (E3) OH CH3

OH

D

C B A

O HO

Figure 56-8 Synthesis of progesterone and the estrogens by the maternal-placental-fetal unit. Individual enzymes are shown in the horizontal and vertical boxes. See Figures 50-2, 54-5, and 55-9 for cellular localizations of enzymes. Chemical groups modified by each enzyme are highlighted in the reaction products. The fetus lacks 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase (P-450arom), shown on the blue background. Placenta lacks 17α-hydroxylase and 17,20-desmolase activity (contributed by the same protein, P-450c17) and 16α-hydroxylase, shown on the brown background. The blue and brown color coding of enzymes distinguishes fetus from placenta, whereas color coding in previous steroidogenesis figures indicates subcellular localization.

B

D

OH

Chapter 56 • Fertilization, Pregnancy, and Lactation

FETUS ADRENAL LDL cholesterol MOTHER

PLACENTA SCCE

Acetate

LDL cholesterol

Pregnenolone sulfate

Pregnenolone 3β-HSD

Progesterone

17α-Hydroxylase 17,20 Desmolase

Progesterone Sulfatase DHEA

DHEA-S Sulfatase

DHEA-S

3β-HSD 17β-HSD Aromatase

Estradiol Estradiol 17β-HSD

16α-Hydroxylase

17β-HSD Estrone

Estriol

3β-HSD 17β-HSD Aromatase

Estriol

Estrone

LIVER Sulfatase

16α-OH DHEA

16α-OH DHEA-S

Figure 56-9 The interactions of the maternal-placental-fetal unit. The details of the enzymatic reactions are provided in Figure 56-8. SCCE is the side-chain–cleavage enzyme; the S in DHEA-S and 16α-OH DHEA-S represents sulfate. 17β-HSD, 17β-hydroxysteroid dehydrogenase.

generate large amounts of progesterone and can export it to the mother, thus solving the problem of maintaining maternal progesterone levels after the corpus luteum becomes inadequate. Second, the fetal adrenal gland and liver supply the three enzymes lacking in the placenta. The fetal adrenal glands are up to this metabolic task; at term, these glands are as large as those of an adult. The fetus does not synthesize estrogens without assistance, for two reasons. First, it cannot, because the fetus lacks the enzymes that catalyze the last two steps in the production of estrone, the precursor of estradiol. These two enzymes are also necessary to synthesize estriol. The enzymes missing from the fetus are listed in Table 56-5, and they also are indicated with a blue background in Figures 56-8 and 56-9. Second, the fetus should not synthesize estrogens without assistance. If the fetus were to carry out the complete, classic biosynthesis of progesterone and the estrogens, it would expose itself to dangerously high levels of hormones that are needed not by the fetus, but by the mother. The fetus and its placenta use three strategies to extricate themselves from this conundrum. First, because the fetus lacks the two enzymes noted earlier, it never makes anything beyond dehydroepiandrosterone (DHEA) and 16α-hydroxyDHEA (Fig. 56-8). In particular, the fetus cannot make progesterone or any of the three key estrogens. Second, the placenta is a massive sink for the weak androgens that the

fetus does synthesize, thus preventing the masculinization of female fetuses. Third, the fetus conjugates the necessary steroid intermediates to sulfate, which greatly reduces their biological activity (Fig. 56-9). Thus, as pregnenolone moves from the placenta to the fetus, it is sulfated. The products of fetal pregnenolone metabolism are also sulfated (DHEA-S and 16α-hydroxy-DHEA-S) as long as they reside inside the fetus. It is only when DHEA-S and 16α-hydroxy-DHEA-S finally move to the placenta that a sulfatase removes the sulfate groups, and thus the placenta can complete the process of steroidogenesis and can export the hormones to the mother.

RESPONSE OF THE MOTHER TO PREGNANCY The mean duration of pregnancy is ∼266 days (38 weeks) from the time of ovulation or 280 days (40 weeks) from the first day of the last menstrual period. During this time, the mother experiences numerous and profound adaptive changes in her cardiovascular system, fluid volume, respiration, fuel metabolism, and nutrition. These orderly changes reflect the effects of various hormones, as well as the increase in the size of the pregnant uterus.

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Both maternal cardiac output and blood volume increase during pregnancy

Increased levels of progesterone during pregnancy increase alveolar ventilation

The maternal blood volume starts to increase during the first trimester, expands rapidly during the second trimester, rises at a much lower rate during the third trimester, and finally achieves a plateau during the last several weeks of pregnancy. Maternal blood volume may have increased by as much as 45% near term in singleton pregnancies and up to 75% to 100% in twin or triplet pregnancies. The ultimate increase in blood volume results from an increase in the volume of both the plasma and erythrocytes. However, the rise in plasma volume begins earlier and is ultimately greater (∼50%) than the rise in total erythrocyte volume (∼33%). A proposed mechanism for the increase in plasma volume is that elevated progesterone and estrogens cause a vasodilation that decreases peripheral vascular resistance and thus renal perfusion. One mechanism of the vasodilation is refractoriness to the pressor effects of angiotensin II. The renin-angiotensin-aldosterone axis responds by increasing aldosterone, which augments renal reabsorption of salt and water. In addition, pregnancy causes a leftward shift of the relationship between arginine vasopressin (AVP) release and plasma osmolality (see Chapter 41). Immediately after the delivery of the placenta, with the attendant decreases in progesterone and estrogen levels, the mother commences vigorous diuresis. The increase in blood volume is needed to meet the demands of the enlarged pregnant uterus with its greatly hypertrophied vascular system. It also protects mother and fetus against the deleterious effects of impaired venous return in the supine and erect positions, and it safeguards the mother against the adverse effects of the blood loss associated with parturition. Cardiac output increases appreciably during the first trimester of pregnancy (by 35% to 40%), but it increases only slightly during the second and third trimesters (∼45% at term). The increase in cardiac output, which reflects mainly an increase in stroke volume but also heart rate, is highly targeted. Renal blood flow increases 40%. Uterine blood flow rises from just 1% to 15% of cardiac output. Blood flow to the heart (to support increased cardiac output), skin (to increase heat radiation), and breasts (to support mammary development) also increases. However, no change occurs in blood flow to the brain, gut, or skeleton. The increase in cardiac output with physical activity is greater in pregnant women (for most of the pregnancy) than it is in nonpregnant women. Despite the large increase in plasma volume, mean arterial pressure (MAP) usually decreases during midpregnancy and then rises during the third trimester, although it normally remains at or lower than normal. The reason for this initial fall in MAP is a decrease in peripheral vascular resistance, possibly reflecting—in part—the aforementioned vasodilating effects of progesterone and estradiol. Posture has a major effect on cardiac output (see Chapter 25). In late pregnancy, cardiac output is typically higher when the mother is in the lateral recumbent position than when she is in the supine position. In the supine position, the fundus of the enlarged uterus rests on the inferior vena cava near L5, thereby impeding venous return to the heart.

During pregnancy, hormonal and mechanical factors lead to several anatomical changes that have the net effect of increasing alveolar ventilation. The level of the diaphragm rises ∼4 cm, probably reflecting the relaxing effects of progesterone on the diaphragm muscle and fascia. At the same time, the costovertebral angle widens appreciably as the transverse diameter of the thoracic cage increases ∼2 cm. Although these two changes have opposite effects on the residual volume (RV) of air in the lungs (see Chapter 27), the elevation of the diaphragm dominates, thus causing a net decrease in RV and functional residual capacity (FRC). Vital capacity (VC), maximal pulmonary ventilation, and pulmonary compliance do not change appreciably. Total pulmonary resistance falls, thereby facilitating airflow. Because of the increased size of the abdominal contents during pregnancy, the abdominal muscles are less effective in aiding forced expirations. Although pregnancy has little effect on respiratory rate, it increases tidal volume (VT) markedly—by ∼40%— . and thereby increases alveolar ventilation (VA; see Chapter . 30). These increases in VT and VA are some of the earliest physiological changes during pregnancy, beginning 6 weeks after fertilization. They may reflect, at least in part, a direct stimulatory effect of progesterone and, to a lesser extent, estrogen on the medullary respiratory centers. The physio. logical effect of the increased VA during pregnancy is a fall in maternal arterial PCO2, which typically decreases from a value before pregnancy of ∼40 to ∼32 mm Hg, despite the net increase in CO2 production that reflects fetal metabolism. A side effect is mild respiratory alkalosis for which the kidneys compensate by lowering plasma [HCO3−] modestly (see Chapter 28). Pregnancy increases the demand for dietary protein, iron, and folic acid During pregnancy, an additional 30 g of protein will be needed each day to meet the demand of the growing fetus, placenta, uterus, and breasts, as well as the increased maternal blood volume. Most protein should come from animal sources, such as meat, milk, eggs, cheese, poultry, and fish, because these foods furnish amino acids in optimal combinations. Almost any diet that includes iodized salt and adequate caloric intake to support the pregnancy also contains enough minerals, except iron (see Table 45-3). Pregnancy necessitates a net gain of ∼800 mg of circulating iron to support the expanding maternal Hb mass, the placenta, and the fetus. Most of this iron is used during the latter half of pregnancy. A nonpregnant woman of reproductive age needs to absorb ∼1.5 mg/day of iron in a diet that contains 15 to 20 mg/day (see Chapter 45). In contrast, during pregnancy, the average required iron uptake rises to ∼7 mg/day. Very few women have adequate iron stores to supply this amount of iron, and a typical diet seldom contains sufficient iron. Thus, the recommended supplementation of elemental iron is 60 mg/day, taken in the form of a simple ferrous iron salt. Maternal folate requirements increase significantly during pregnancy, in part reflecting an increased demand for pro-

Chapter 56 • Fertilization, Pregnancy, and Lactation

ducing blood cells. This increased demand can lead to lowered plasma folate levels or, in extreme cases, to maternal megaloblastic anemia (see Chapter 45). Folate deficiency may cause neural tube defects in the developing fetus. Because oral supplementation of 400 to 800 μg/day of folic acid produces a vigorous hematologic response in pregnant women with severe megaloblastic anemia, this dose would almost certainly provide very effective prophylaxis. Less than one third of the total maternal weight gain during pregnancy represents the fetus The recommended weight gain during a singleton pregnancy for a woman with a normal ratio of weight to height (i.e., body mass index) is 11.5 to 16 kg. This number is higher for women with a low body mass index. A weight gain of 14 kg would include 5 kg for intrauterine contents—the fetus (3.3 kg), placenta and membranes (0.7 kg), and amniotic fluid (1 kg). The maternal contribution of 9 kg would include increases in the weight of the uterus (0.7 kg), the blood (1.3 kg), and the breasts (2.0 kg), as well as adipose tissue and interstitial fluid (5.0 kg). The interstitial fluid expansion may be partly the result of increased venous pressure created by the large pregnant uterus and, as noted earlier, partly caused by aldosterone-dependent Na+ retention. For a woman whose weight is normal before pregnancy, a weight gain in the recommended range correlates well with a favorable outcome of the pregnancy. Most pregnant women can achieve an adequate weight gain by eating— according to appetite—a diet adequate in calories, protein, minerals, and vitamins. Seldom, if ever, should maternal weight gain be deliberately restricted to less than this level. Failure to gain weight is an ominous sign; birth weight parallels maternal weight, and neonatal mortality rises with low birth weight, particularly for babies weighing less than 2500 g.

PARTURITION Throughout most of pregnancy, the uterus is quiescent. Both progesterone and relaxin may promote this inactivity. Table 56-6

Weak and irregular uterine contractions occur throughout the last month of pregnancy. Eventually, a series of regular, rhythmic, and forceful contractions develops to facilitate thinning and dilation of the cervix—the obstetric definition of labor (Table 56-6). These contractions may last for several hours, a day, or even longer and may eventually result in the expulsion of the fetus, placenta, and membranes. Although not all the factors leading to the initiation of labor are known, endocrine, paracrine, and mechanical stretching of the uterus all play a role. Once labor is initiated, it is sustained by a series of positive feedback mechanisms.

Signals from the placenta or fetus may initiate labor In rabbits, withdrawal of progesterone, made primarily in the placenta, results in prompt evacuation of the uterus; administration of progesterone delays the onset of labor. However, most human studies have failed to provide evidence that progesterone levels fall before the onset of labor. Nonetheless, it appears that progesterone plays an important role in maintaining the length of gestation in primates. Other studies point to the importance of the fetal hypothalamic-pituitary-adrenal axis in the preparation for, or initiation of, parturition. In the fetal lamb, transection of the hypothalamic portal vessels prolongs gestation. In the human, an equivalent disruption of the fetal hypothalamic-pituitary-adrenal axis occurs in anencephalic fetuses, in which the cerebral hemispheres are absent and the rest of the brain is severely malformed. Indeed, gestation is prolonged in human pregnancies with anencephalic fetuses. Infusing adrenocorticotropic hormone (ACTH) into fetal lambs with intact adrenal glands, or directly infusing cortisol, causes premature parturition. Although the theory that cortisol plays a role in initiating parturition remains attractive, several naturally occurring instances of failure of cortisol production in the human fetus do not prolong gestation. As discussed in the next section, prostaglandins appear to play a crucial role in the initiation of labor.

Stages of Labor

Stage

Characteristics

0

Uterine tranquility and refractoriness to contraction

1

Uterine awakening, initiation of parturition, extending to complete cervical dilatation

2

Active labor, from complete cervical dilatation to delivery of the newborn

3

From delivery of the fetus to expulsion of the placenta and final uterine contraction

Physiological Changes

Increase in the number of gap junctions between myometrial cells; increase in the number of OT receptors

Data from Casey ML, MacDonald PC: In Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology. Philadelphia: WB Saunders, 1998.

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Prostaglandins initiate uterine contractions, and both prostaglandins and oxytocin sustain labor Whereas hormones (particularly oxytocin [OT]) and paracrine factors (particularly prostaglandins) play an important role in stimulating the uterine contractions that sustain labor, only the prostaglandins are believed to have a key role in the initiation of labor. Prostaglandins

The uterus, the placenta, and the fetal membranes synthesize and release prostaglandins (see Chapter 3). Prostaglandins from the uterine decidual cells, particularly prostaglandins F2 and E2 (PGF2α and PGE2), act by a paracrine mechanism on uterine smooth muscle cells. OT (see later) stimulates uterine decidual cells to increase their PGF2α synthesis. Arachidonic acid, the precursor of prostaglandins, is present in very high concentrations in the fetal membranes near term. Prostaglandins have three major effects. First, prostaglandins strongly stimulate the contraction of uterine smooth muscle cells. Second, PGF2α potentiates the contractions induced by OT by promoting formation of gap junctions between uterine smooth muscle cells; estradiol also increases the number of gap junctions (see Chapter 9). These gap junctions permit synchronous contraction of the uterine smooth muscle cells, reminiscent of the contraction of the ventricles of the heart. Third, prostaglandins also cause softening, dilatation, and thinning (effacement) of the cervix, which occurs early during labor. This softening is akin to an inflammatory reaction in that it is associated with an invasion by polymorphonuclear leukocytes (e.g., neutrophils). Because of these effects, prostaglandins are used to induce labor and delivery in certain clinical settings. Prostaglandins may physiologically initiate labor. Both PGF2α and PGE2 evoke myometrial contractions at any stage of gestation, regardless of the route of administration. The levels of prostaglandins or their metabolic products naturally increase in the blood and amniotic fluid just before and during labor. Arachidonic acid instilled into the amniotic cavity causes the uterus to contract and to expel its contents. Aspirin, which inhibits the enzyme cyclooxygenase (see Chapter 3), reduces the formation of PGF2α and PGE2, thus inhibiting labor and prolonging gestation. Oxytocin

The nonapeptide OT is closely related to AVP (Fig. 56-10). The two hormones apparently evolved from vasotocin, the single neurohypophyseal hormone in nonmammalian vertebrates. OT and AVP, which both differ from vasotocin by a single amino acid, are synthesized in the cell bodies of the neurons in the supraoptic and paraventricular nuclei of the hypothalamus. Both OT and AVP then move by fast axonal transport to the posterior pituitary gland, where they are stored in the nerve terminals until they are released in response to the appropriate stimuli. Both OT and AVP are closely associated with—and released with— peptides known as neurophysins. Circulating OT binds to Gαq-coupled OT receptors on the plasma membrane of uterine smooth muscle cells; this process triggers the phospholipase C cascade (see Chapter 3). Presumably, formation of IP3 leads to Ca2+ release from

Oxytocin (OT)

Vasopressin (AVP)

N

N

Cys

Cys

Tyr

Tyr

Ile Gln

S S

Phe Gln

DDAVP

Cys Tyr

S S

Phe Gln

Asn

Asn

Asn

Cys

Cys

Cys

Pro

Pro

Pro

Leu

Arg

DArg

Gly

Gly

Gly

Amide

Amide

Amide

S S

Figure 56-10 Comparison of structures of OT and AVP. DDAVP is a synthetic AVP in which the N-terminal Cys is deaminated and lArg at position 8 is replaced with d-Arg (see the box on diabetes insipidus in Chapter 38).

internal stores and to an increase in [Ca2+]i. The rise in [Ca2+]i activates calmodulin, which stimulates myosin light-chain kinase to phosphorylate the regulatory light chain and to cause contraction of uterine smooth muscle cells (see Chapter 9) and increased intrauterine pressure. OT also binds to a receptor on decidual cells, thereby stimulating PGF2α production, as discussed earlier. Estrogen increases the number of OT receptors in the myometrial and decidual tissue of pregnant women. The uterus actually remains insensitive to OT until ∼20 weeks’ gestation, at which time the number of OT receptors increases progressively to 80-fold higher than baseline values by ∼36 weeks’ gestation, plateaus just before labor, then rises again to 200-fold during early labor. The time course of the expression of OT receptors may account for the increase in spontaneous myometrial contractions even in the absence of increased plasma OT levels. Whereas the uterus is sensitive to OT only at the end of pregnancy, it is susceptible to prostaglandins throughout pregnancy. Although the prevailing view is that OT of maternal origin is not involved in initiating labor in humans, maternal OT may help to maintain labor. OT of fetal origin, which moves to the maternal circulation, could be involved in the onset of labor, because fetal plasma OT levels rise during the first stage of labor (Table 56-6). However, infusing OT at pharmacological doses into the circulation of the fetal lamb only stimulates uterine contractions. Therefore, normal levels of fetal OT probably have little influence on labor. Once labor is initiated (stage 1), maternal OT is released in bursts, and the frequency of these bursts increases as labor progresses. The primary stimulus for the release of maternal OT appears to be distention of the cervix; this effect is known as the Ferguson reflex. OT is an important stimulator of myometrial contraction late in labor. During the second stage of labor, OT release may play a synergistic role in the expulsion of the fetus by virtue of its ability to stimulate prostaglandin release. During the third stage of labor, uterine contractions induced by OT are also important for constricting uterine blood vessels at the site where the placenta used to be, thus

Chapter 56 • Fertilization, Pregnancy, and Lactation

promoting hemostasis (i.e., blood coagulation). Basal maternal plasma OT levels are unchanged after delivery. Fetal plasma OT levels are higher after vaginal delivery than after delivery by cesarean section, presumably because the maternal OT triggered by the Ferguson reflex crosses the placenta into the fetus. Relaxin This 48–amino acid polypeptide hormone, structurally related to insulin, is produced by the corpus luteum, the placenta, and the decidua. Relaxin may play a role in keeping the uterus in a quiet state during pregnancy. Production and release of relaxin increase during labor, when relaxin may soften and may thus help to dilate the cervix. Mechanical Factors

Mechanical stretch placed on the uterine muscle may lead to the rhythmic contractions of labor. Thus, the increase in the size of the uterine contents to a critical level may stimulate uterine contractions, thereby leading to initiation of labor.

Positive Feedback Once labor is initiated, several positive feedback loops involving prostaglandins and OT help to sustain it. First, uterine contractions stimulate prostaglandin release, which itself increases the intensity of uterine contractions. Second, uterine activity stretches the cervix, thus stimulating OT release through the Ferguson reflex. Because OT stimulates further uterine contractions, these contractions become self-perpetuating.

Table 56-7 Hormones Affecting the Mammary Gland During Pregnancy and Breast-Feeding Mammogenic Hormones (promote cell proliferation) Lobuloalveolar Growth

Estrogen Growth hormone (IGF-1) Cortisol Prolactin Relaxin? Ductal Growth

Estrogen Growth hormone Cortisol Relaxin Lactogenic Hormones (promote initiation of milk production by alveolar cells)

Prolactin hCS (or hPL) Cortisol Insulin (IGF-1) Thyroid hormones Growth hormone? Withdrawal of estrogens and progesterone Galactokinetic Hormones (promote contraction of myoepithelial cells and thus milk ejection)

OT AVP (1% to 20% as powerful as OT) Galactopoietic Hormones (maintain milk production after it has been established)

Involution of the uterus is primarily the result of a changing endocrine milieu Almost immediately following delivery of the newborn, marked changes occur in the endocrine status of the mother. During pregnancy, many hormones are secreted in massive quantities. Estrogens are mitogenic, causing considerable hypertrophy of the uterine muscle cells during gestation. As the levels of these hormones fall abruptly, stimulation ceases, and uterine smooth muscle cells decrease in size. The vasculature of the uterus regresses, and blood flow to the uterus is significantly curtailed, thus leading to further involution of this organ.

LACTATION The fundamental secretory unit of the breast (Fig. 56-11A) is the alveolus (Fig. 56-11B, C), which is surrounded by contractile myoepithelial cells and adipose cells. These alveoli are organized into lobules, each of which drains into a ductule. Groups of 15 to 20 ductules drain into a duct, which widens at the ampulla—a small reservoir. The lactiferous duct carries the secretions to the outside. Breast development at puberty depends on several hormones, but primarily on the estrogens and progesterone. During pregnancy, gradual increases in levels of PRL and hCS, as well as very high levels of estrogens and progesterone, lead to full development of the breasts.

PRL (primary) Cortisol and other metabolic hormones (permissive) IGF-1, insulin-like growth factor type 1.

As summarized in Table 56-7, hormones affecting the breast are mammogenic (promoting the proliferation of alveolar and duct cells), lactogenic (promoting initiation of milk production by alveolar cells), galactokinetic (promoting contraction of myoepithelial cells, and thus milk ejection), or galactopoietic (maintaining milk production after it has been established). The epithelial alveolar cells of the mammary gland secrete the complex mixture of sugars, proteins, lipids, and other substances that constitute milk Milk is an emulsion of fats in an aqueous solution containing sugar (lactose), proteins (lactalbumin and casein), and several cations (K+, Ca2+, and Na+) and anions (Cl− and phosphate). The composition of human milk differs from that of human colostrum (the thin, yellowish, milk-like substance secreted during the first several days after parturition) and cow’s milk (Table 56-8). Cow’s milk has nearly three times more protein than human milk, almost exclusively a result of its much higher casein concentration. It also has a higher electrolyte content. The difference in composition between human milk and cow’s milk is important because a newborn,

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A

LACTATING BREAST

B

LOBULE

Adipose tissue Secretory lobule Enlarged secretory lobules Elaborated duct system

Alveoli

Lactiferous duct

C

ALVEOLUS

Ductule Milk lipids Milk Basal lamina Ampulla

Alveolar cell Myoepithelial cell Capillary

D

SECRETORY EPITHELIAL CELL 1 Secretory pathway

Alveolar lumen

Exocytosis

2 Transcellular endocytosis/exocytosis

Proteincontaining vesicle

Alveolar cell cytosol

Proteins

Myoepithelial cell

Vesicle containing protein

Milk lipid Nucleus 3 Lipid pathway

Vesicles containing sugars and salts.

Receptormediated endocytosis

Connective tissue

Salts Endoplasmic reticulum

Sugars

4 Transcellular salt and water transport through channels and transporters.

Golgi

Basal lamina

Capillary

5 Paracellular pathway for ions and water.

Figure 56-11 Cross section of the breasts and milk production. A, The breast consists of a series of secretory lobules, which empty into ductules. The ductules from 15 to 20 lobules combine into a duct, which widens at the ampulla—a small reservoir. The lactiferous duct carries the secretions to the outside. B, The lobule is made up of many alveoli, the fundamental secretory units. C, Each alveolus consists of secretory epithelial cells (alveolar cells) that actually secrete the milk, as well as contractile myoepithelial cells, which are, in turn, surrounded by adipose cells. D, The alveolar cell secretes the components of milk through five pathways.

Chapter 56 • Fertilization, Pregnancy, and Lactation

Table 56-8

Composition of Human Colostrum, Human Milk, and Cow’s Milk (per Deciliter of Fluid)

Component

Human Colostrum

Human Milk

Cow’s Milk

Total protein (g)

2.7

0.9

3.3

Casein (% of total protein)

44

44

82

Total fat (g)

2.9

4.5

3.7

Lactose (g)

5.7

7.1

4.8

Caloric content (kcal)

54

70

69

Calcium (mg)

31

33

125

Iron (μg)

10

50

50

Phosphorus (mg)

14

15

96

7-8 × 106

1-2 × 106

—

Cells (macrophages, neutrophils, and lymphocytes)

with his or her delicate gastrointestinal tract, may not tolerate the more concentrated cow’s milk. The epithelial cells in the alveoli of the mammary gland secrete the complex mixture of constituents that make up milk by five major routes (Fig. 56-11D): 1. Secretory pathway. The milk proteins lactalbumin and casein are synthesized in the endoplasmic reticulum and are sorted to the Golgi apparatus (see Chapter 2). Here alveolar cells add Ca2+ and phosphate to the lumen. Lactose synthetase in the lumen of the Golgi catalyzes synthesis of lactose, the major carbohydrate. Lactose synthetase has two components, a galactosyl transferase and lactalbumin, both made in the endoplasmic reticulum. Water enters the secretory vesicle by osmosis. Finally, exocytosis discharges the contents of the vesicle into the lumen of the alveolus. 2. Transcellular endocytosis and exocytosis. The basolateral membrane takes up maternal immunoglobulins by receptor-mediated endocytosis (see Chapter 2). Following transcellular transport of these vesicles to the apical membrane, the cell secretes these immunoglobulins (primarily IgA) by exocytosis. The gastrointestinal tract of the infant takes up these immunoglobulins (see Chapter 45), which are important for conferring immunity before the infant’s own immune system matures. 3. Lipid pathway. Epithelial cells synthesize short-chain fatty acids. However, the longer chain fatty acids (>16 carbons) that predominate in milk originate primarily from the diet or from fat stores. The fatty acids form into lipid droplets and move to the apical membrane. As the apical membrane surrounds the droplets and pinches off, it secretes the milk lipids into the lumen in a membranebound sac. 4. Transcellular salt and water transport. Various transport processes at the apical and basolateral membranes move small electrolytes from the interstitial fluid into the lumen of the alveolus. Water follows an osmotic gradient

generated primarily by lactose (present at a final concentration of ∼200 mM) and, to a lesser extent, by the electrolytes. 5. Paracellular pathway. Salt and water can also move into the lumen of the alveolus through the tight junctions (see Chapter 5). In addition, cells, primarily leukocytes, squeeze between cells and enter the milk. Prolactin is essential for milk production, and suckling is a powerful stimulus for prolactin secretion PRL is a polypeptide hormone that is structurally related to GH, placental-variant GH, and hCS1 and hCS2 (see Table 48-1). Like GH, PRL is made and released in the anterior pituitary; however, lactotrophs rather than somatotrophs, are responsible for PRL synthesis. Another difference is that whereas GH-releasing hormone stimulates somatotrophs to release GH, dopamine (DA) inhibits the release of PRL from lactotrophs. Thus, the removal of inhibition promotes PRL release. The actions of PRL on the mammary glands (Table 56-7) include the promotion of mammary growth (mammogenic effect), the initiation of milk secretion (lactogenic effect), and the maintenance of milk production once it has been established (galactopoietic effect). Although the initiation of lactation requires the coordinated action of several hormones, PRL is the classic lactogenic hormone. Initiating milk production also necessitates the abrupt fall in estrogens and progesterone that accompanies parturition. PRL is also the primary hormone responsible for maintaining milk production once it has been initiated. PRL binds to a tyrosine kinase–associated receptor (see Chapter 3) in the same family of receptors as the GH receptor. PRL receptors, which have equal affinities for GH, are present in tissues such as breast, ovary, and liver. Presumably through pathways initiated by protein phosphorylation at tyrosine residues, PRL stimulates transcription of the genes

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3 Neurons from the spinal cord also stimulate the production and release of oxytocin from the paraventricular and supraoptic nuclei. Oxytocin is released in the posterior pituitary and into the systemic blood, where it then makes its way to the breast and myoepithelial cells.

2 Neurons from the spinal cord inhibit dopamine (DA) release from the arcuate nucleus. The decreased level of DA removes the inhibition that DA normally produces on lactotrophs in the anterior pituitary, leading to prolactin release. Prolactin stimulates milk production in the breast. Spinal cord

4 Preoptic Neurons from the area spinal cord inhibit neurons in the arcuate nucleus and the preoptic area of the hypothalamus, causing a fall in GnRH production. The reduced stimulation of gonadotrophs inhibits the ovarian cycle.

Paraventricular nucleus

Supraoptic nucleus 1 Stimulus from suckling travels from breast, through the spinal cord to the hypothalamus.

From breast

Arcuate nucleus

Hypothalamus

DA

GnRH Anterior lobe of pituitary

Oxytocin

Posterior lobe of pituitary

Gonadotrophs

Prolactin-releasing lactotrophs

Oxytocin To breast Prolactin

Figure 56-12 Effect of suckling on the release of PRL, OT, and GnRH. Suckling has four effects. First, it stimulates sensory nerves, which carry the signal from the breast to the spinal cord, where the nerves synapse with neurons that carry the signal to the brain. Second, in the arcuate nucleus of the hypothalamus, the afferent input from the nipple inhibits neurons that release DA. DA normally travels through the hypothalamicportal system to the anterior pituitary, where it inhibits PRL release by lactotrophs. Thus, inhibition of DA release leads to an increase in PRL release. Third, in the supraoptic and paraventricular nuclei of the hypothalamus, the afferent input from the nipple triggers the production and release of OT in the posterior pituitary. Fourth, in the preoptic area and arcuate nucleus, the afferent input from the nipple inhibits GnRH release. GnRH normally travels through the hypothalamic-portal system to the anterior pituitary, where it stimulates the synthesis and release of FSH and LH. Thus, inhibiting GnRH release curbs FSH and LH release and thereby inhibits the ovarian cycle.

Chapter 56 • Fertilization, Pregnancy, and Lactation

that encode several milk proteins, including lactalbumin and casein. Suckling is the most powerful physiological stimulus for PRL release. Nipple stimulation triggers PRL secretion through an afferent neural pathway through the spinal cord, thereby inhibiting dopaminergic neurons in the median eminence of the hypothalamus (Fig. 56-12). Because DA normally inhibits PRL release from the lactotrophs, it is called a PRL-inhibitory factor (PIF). Thus, because suckling decreases DA delivery through the portal vessels, it relieves the inhibition on the lactotrophs in the anterior pituitary and stimulates bursts of PRL release. Treating women with DA-receptor agonists rapidly inhibits PRL secretion and milk production. Several factors act as PRL-releasing factors (PRFs): thyrotropin-releasing hormone (TRH), angiotensin II, substance P, β endorphin, and AVP. In the rat, suckling stimulates the release of TRH from the hypothalamus. In lactating women, TRH leads to increased milk production. Estradiol modulates PRL release in two ways. First, estradiol increases the sensitivity of the lactotroph to stimulation by TRH. Second, estradiol decreases the sensitivity of the lactotroph to inhibition by DA. During the first 3 weeks of the neonatal period, maternal PRL levels remain tonically elevated. Thereafter, PRL levels decrease to a constant baseline level higher than that observed in women who are not pregnant. If the mother does not nurse her young, PRL levels generally fall to nonpregnant levels after 1 to 2 weeks. If the mother does breast-feed, increased PRL secretion is maintained for as long as suckling continues. Suckling causes episodic increases in PRL secretion with each feeding, thus producing peaks in PRL levels superimposed on the elevated baseline PRL levels. After the infant completes a session of nursing, PRL levels return to their elevated baseline and remain there until the infant nurses again. Oxytocin and psychic stimuli initiate milk ejection (“let-down”) OT, which can promote uterine contraction, also enhances milk ejection by stimulating the contraction of the network of myoepithelial cells surrounding the alveoli and ducts of the breast (galactokinetic effect). Nursing can sometimes cause uterine cramps. During nursing, suckling stimulates nerve endings in the nipple and triggers rapid bursts of OT release (Fig. 56-12). This neurogenic reflex is transmitted through the spinal cord, the midbrain, and the hypothalamus, where it stimulates neurons in the paraventricular and supraoptic nuclei that release OT from their nerve endings in the posterior pituitary. From the posterior pituitary, OT enters the systemic circulation and eventually reaches the myoepithelial cells arranged longitudinally on the lactiferous ducts and around the alveoli in the breast (Fig. 56-11C, D). Because these cells have OT receptors, OT causes them to contract by mechanisms similar to those for the contraction of uterine smooth muscle, described earlier. The result is to promote the release of pre-existing milk after 40 to 60 seconds, a process known as the let-down reflex. In addition to the suckling stimulus, many different psychic stimuli emanating from the infant, as well as neuro-

endocrine factors, also promote OT release. The site or sound of an infant may trigger milk let-down, a phenomenon observed in many mammals. Thus, the posterior pituitary releases OT episodically even in anticipation of suckling. This psychogenic reflex is suppressed when fear, anger, or other stresses are encountered, and the results are inhibition of OT release and suppression of milk outflow. Suckling inhibits the ovarian cycle Lactation generally inhibits cyclic ovulatory function. Suckling likely reduces the release of gonadotropin-releasing hormone (GnRH) by neurons in the arcuate nucleus and the preoptic area of the hypothalamus (Fig. 56-12). Normally, GnRH travels through the portal vessels to the gonadotrophs in the anterior pituitary. Thus, the decreased GnRH release induced by suckling reduces the secretion of follicle-stimulating hormone (FSH) and LH and has a negative effect on ovarian function. As a result, breast-feeding delays ovulation and normal menstrual cycles. However, if the mother continues to nurse her infant for a prolonged period, ovulatory cycles will eventually resume. Suckling intensity and frequency, which decrease with the introduction of supplementary foods to the infant, determine the duration of anovulation and amenorrhea in well-nourished women. In breast-feeding women in Bangladesh, the period of anovulation averages 18 to 24 months. If the mother does not nurse her child after delivery, ovulatory cycles resume, on average, 8 to 10 weeks after delivery, with a range of up to 18 weeks. REFERENCES Books and Reviews Casey ML, MacDonald PC: Endocrine changes of pregnancy. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology. Philadelphia: WB Saunders, 1998. Fuchs A-R: Physiology and endocrinology of lactation. In Gabbe SG, Niebyl JR, Simpson JL (eds): Obstetrics: Normal and Problem Pregnancies, 3rd ed. New York: Churchill Livingstone, 1996, pp 137-157. Lamberts SWJ, Macleod RM: Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 1990; 70:279-318. Moore TR, Reiter RC, Rebar RW, Baker VV (eds): Gynecology and Obstetrics: A Longitudinal Approach. New York: Churchill Livingstone, 1993. Ramsey EM, Eonner MW: Placental Vasculature and Circulation. Philadelphia: WB Saunders, 1980. Stulc J: Placental transfer of inorganic ions and water. Physiol Rev 1997; 77:805-836. Vonderhaar BK, Ziska SE: Hormonal regulation of milk protein gene expression. Annu Rev Physiol 1989; 51:641-652. Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed. Philadelphia: WB Saunders, 1998. Yen SSC, Jaffe RB (eds): Reproductive Endocrinology. Philadelphia: WB Saunders, 1986. Journal Articles Fuch AR, Fuchs F, Husslein P, et al: Oxytocin receptors and human parturition: A dual role for oxytocin in the initiation of labor. Science 1982; 215:1396-1398. Goebelsmann U, Jaffe RB: Oestriol metabolism in pregnant women. Acta Endocrinol 1971; 66:679-693.

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Liggins GC: Initiation of parturition. Br Med Bull 1979; 35:145-150. Perez A, Vela P, Masnick GS, Potter RG: First ovulation after childbirth: The effect of breast feeding. Am J Obstet Gynecol 1972; 114:1041-1047. Tabibzadeh S, Babaknia A: The signals and molecular pathways involved in implantation, a symbiotic interaction between blas-

tocyst and endometrium involving adhesion and tissue invasion. Hum Reprod 1995; 10:1579-1602. Wigglesworth JS: Vascular organization of the human placenta. Nature 1967; 216:1120-1121. Wilkening RB, Meschia G: Fetal oxygen uptake, oxygenation, and acid-base balance as a function of uterine blood flow. Am J Physiol 1983; 244:H749-H755.

CHAPTER

57

F E TA L A N D N E O N ATA L P H Y S I O L O G Y Ervin E. Jones

Growth of the fetus begins soon after fertilization, when the first cell division occurs. Cell division, hypertrophy, and differentiation are highly coordinated events that result in the growth and development of specialized organ systems. The fetus, fetal membranes, and placenta develop and function as a unit throughout pregnancy, and their development is interdependent. The growth trajectory of fetal mass is relatively flat during the first trimester, increases linearly at the beginning of the second trimester, and rises rapidly during the third trimester.

BIOLOGY OF FETAL GROWTH Growth occurs by hyperplasia and hypertrophy The growth of an organ occurs as a result of an increase in cell number (hyperplasia), an increase in cell size (hypertrophy), or both. We can define three sequential phases of growth: (1) pure hyperplasia, (2) hyperplasia and concomitant hypertrophy, and (3) hypertrophy alone. The time courses of the three phases of growth are organ specific. For example, the placenta goes through all three phases of growth, but these phases are compressed because the placental life span is relatively short. Moreover, simple hypertrophy is the primary form of placental growth. Thus, the weight, RNA content, and protein content of the human placenta increase linearly until term, but cell number does not increase during the third trimester. In contrast to placental growth and development, growth of the fetus occurs almost entirely by hyperplasia. Thus, DNA content increases linearly in all fetal organs beginning early in the second trimester. Stimuli that either increase or decrease cell number, cell size, or both may accelerate or retard the growth of the whole fetus or of individual organs. The phase of growth during which the stimulus acts determines the response of the organ. For example, malnutrition occurring during the period of hyperplasia retards cell division and causes a deficiency in cell number. Therefore, adequacy of nutrition early in life may determine the number of cells in any organ. This effect on cell number may be irreversible, even if normal nutrition is restored later. Con-

versely, malnutrition occurring during the period of hypertrophy causes a reduction in cell size. However, this effect can be reversed, and normal cell size can be achieved if adequate nutrition is restored. Thus, reversibility depends on the timing of the stimulus. Genetic factors primarily determine growth during the first half of gestation, and epigenetic factors determine growth during the second half The fertilized egg contains the genetic material that directs cell multiplication and differentiation and guides development of the human phenotype. For specific developmental events to occur at precise times (Table 57-1), a programmed sequence of gene activation and suppression is necessary. Ignoring apoptosis, the fertilized egg must undergo an average of ∼42 divisions to reach newborn size. A fertilized ovum, weighing less than 1 ng, gives rise to a newborn weighing slightly more than 3 kg (an increase of more than 1012 fold). Not only must the total cell number in a term fetus lie within relatively narrow limits, but also the developmental program must trigger cell differentiation after a specified number of cell divisions. After birth, only approximately five additional divisions are necessary for the net increase in mass that is necessary to achieve adult size. Obviously, many tissues (e.g., gastrointestinal tract, skin, blood cells) must continually undergo cell division to replenish cells lost by apoptosis. Although the genetic makeup of the fetus principally determines its growth and development, other influences— both stimulatory and inhibitory—are superimposed on the genetic program. During the first half of pregnancy, the fetus’ own genetic program is the primary determinant of growth, thus constraining patterns of growth. During the second half of pregnancy, the patterns of growth and development are more variable. The four primary epigenetic factors at work during the second half of pregnancy are placental, hormonal, environmental (e.g., maternal nutrition, disease, drugs, altitude), and metabolic (e.g., diabetes). We discuss the first two factors (placental and hormonal) in the next two sections.

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Table 57-1

Chronologic Development of Organs, Systems, and Body Form

Organ

Chronology of Development

Bronchial apparatus and pharyngeal pouches

4th week, ridges and grooves appear over the future neck region

Thyroid gland

4th week, endoderm appears over the floor of the pharynx

Tongue

4th week, primordia appear in the floor of the pharynx

Face

End of 4th week, primordia appear

Palate

Begins in the 5th week

Upper respiratory system

4th week, laryngotracheal groove appears

Digestive system Foregut derivatives Pharynx and its derivatives Lower respiratory tract Esophagus, stomach, proximal duodenum (from stomach to entry of the common bile duct) Liver, biliary tract, gallbladder, pancreas Midgut derivatives Small intestine, except proximal duodenum Cecum, appendix Ascending colon and proximal half of transverse colon Hindgut derivatives Distal half of transverse colon Descending and sigmoid colon Rectum and upper portion of anal canal Part of the urogenital system

4th week, primitive gut forms 4th week, formation of midgut derivatives begins

Kidneys, urinary bladder, urethra

5th week, permanent adult kidney begins to develop

Adrenal glands

5th week, primordia of adrenal glands develop

Gonads, genital ducts, external genitalia

5th week, gonadal ridges form

Heart

3rd week, development of the heart begins

Atria

5th week, the atria are formed

Ventricles

5th week, the ventricles form

Fetal circulation

3rd week, embryonic blood vessels develop

Brain and spinal cord

End of 4th week, primary vesicles form and walls of the neural tube thicken to form the spinal cord

Pituitary

6th week, connection of Rathke’s with oral cavity disappears

Limbs

End of 4th week, limb buds appear

Skull

7th week, paired cartilages begin to fuse to form the cranium

6th week, primordia appear, midgut elongates

End of 7th week, anal canal has formed

Chapter 57 • Fetal and Neonatal Physiology

Table 57-2

Determinants of Birth Weight

Factor

Contribution to Final Birth Weight

Maternal environment

30%

Maternal genotype

20%

Paternal genotype

20%

Fetal genotype (excluding gender)

15%

Fetal gender

2%

Multifactorial (e.g., gestational age at delivery, multiple gestation)

13%

Studies of birth weights in families reveal that both parental and fetal genotypes affect birth weight (Table 57-2). The mother contributes to birth weight both through her influence on the environment that she provides for the fetus (∼30%) and through the genes that she passes on to the fetus (∼20%). The mother’s contribution to the fetal environment includes maternal health and nutritional status, environment, lifestyle, age (e.g., adolescents and older women have infants with lower birth weight), parity, prepregnancy weight and prenatal weight gain, early fat deposition, height, chronic diseases, infection, and stress. The father contributes to birth weight only through the genes that he passes on to his child (∼20%). The unique fetal genotype—the interaction of the alleles provided by the parents (e.g., dominant versus recessive genes) considered apart from the individual contributions of the two parents—contributes ∼15%. The gender of the fetus contributes ∼2%. The remaining ∼13% of the contribution to birth weight is multifactorial and may include variations in such factors as gestational age at delivery and multiple gestation (e.g., twinning). Increases in placental mass parallel periods of rapid fetal growth The placenta plays several important roles in fetal growth and development. In addition to its transport and storage functions, the placenta is involved in numerous biosynthetic activities. These include the synthesis of steroids, such as estrogen and progesterone, and protein hormones, such as human chorionic gonadotropin (hCG) and the human chorionic somatomammotropins (hCSs) (see Chapter 56). Fetal growth closely correlates with placental weight. During periods of rapid fetal growth, placental weight increases. As the placental mass increases, the total surface area of the placental villi (see Chapter 56) increases to sustain gas transport and fetal nutrition. Moreover, maternal blood flow to the uterus and fetal blood flow to the placenta also increase in parallel with the increase in placental mass. Placental growth increases linearly until ∼4 weeks before birth. Intrauterine growth restriction (IUGR; see the box titled Growth Restriction) may occur as a result of decreased pla-

cental reserve caused by any insult. Adequate placental reserve is particularly important during the third trimester, when fetal growth is very rapid. For example, mothers who smoke during pregnancy tend to have small placentas and are at high risk of delivering a low birth weight baby. Insulin, the insulin-like growth factors, and thyroxine stimulate fetal growth Chapter 48 includes a discussion of several hormones— including glucocorticoids, insulin, growth hormone (GH), the insulin-like growth factors (IGFs), and thyroid hormones—that are important for achieving final adult mass. Glucocorticoids and Insulin As its major energy source, the growing fetus uses glucose, which moves across the placenta by facilitated diffusion. Unlike the adult, who uses sophisticated hormonal systems to control blood glucose levels (see Chapter 51), the fetus is passive: the exchange of glucose across the placenta controls fetal blood glucose levels. The fetus normally has little need for gluconeogenesis, and the levels of gluconeogenic enzymes in the fetal liver are low. Glucocorticoids in the fetus promote the storage of glucose as glycogen in the fetal liver, a process that increases greatly during the final month of gestation in preparation for the increased glycolytic activity required during and immediately after delivery. Near term, when fetal glucose metabolism becomes sensitive to insulin, this hormone contributes to the storage of glucose as glycogen, as well as to the uptake and utilization of amino acids, and lipogenesis (see Chapter 51). Transient increases in maternal blood glucose levels after meals are closely mirrored by increases in fetal blood glucose levels. This transient fetal hyperglycemia leads to increased fetal production of insulin. Maternal insulin cannot cross the placenta. In a mother with poorly controlled diabetes (see the box on diabetes mellitus in Chapter 51), sustained maternal hyperglycemia leads to sustained fetal hyperglycemia and therefore fetal hyperinsulinemia. The resulting high levels of fetal insulin, which is a growth factor (see Chapter 48), increase both the size of fetal organs (organomegaly) and fetal body mass (macrosomia). During the last half of the third trimester, fetal weight in poorly controlled diabetic pregnancies generally exceeds that in normal pregnancies. In some cases, large fetal size leads to problems at delivery. Indeed, the frequency of cesarean section is much higher in deliveries of fetuses born to diabetic mothers. Insulin-Like Growth Factors

Postnatally, GH acts by binding to GH receptors, primarily in the liver, and triggering the production of somatomedin or IGF-1. IGF-2 is not so much under the control of GH. The IGF-1 receptor is similar, but not identical, to the insulin receptor and can bind both IGF-1 and IGF-2, as well as insulin (see Chapter 48). In the fetus, both IGF-1 and IGF-2, which are mitogenic peptides, are extremely important for growth. IGF-1 and IGF-2 are present in the fetal circulation from the end of the first trimester, and their levels increase thereafter in both mother and fetus. Birth weight correlates positively with IGF levels. However, both relative levels of the IGFs and control of the IGFs are very different in the fetal stage than they are

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postnatally. First, fetal IGF-2 levels are much higher than IGF-1 levels; IGF-1 and IGF-2 levels resemble those in adults soon after birth. Second, in the fetus, IGF-1 and IGF-2 levels correlate poorly with GH levels. Indeed, it appears that GH may have only a minimal effect on fetal growth. For example, anencephalic fetuses (see Chapter 10 for the box on abnormalities of neural tube closure), which have low GH levels, generally grow normally. Moreover, unlike the adult liver, the fetal liver has relatively few GH receptors.

current with development of the hypothalamic-pituitary portal system. Hypothyroidism has adverse effects on fetal growth, generally reflected as a reduction in the size of organs such as the heart, kidney, liver, muscle, and spleen. Peptide Hormones Peptide hormones secreted by the placenta (see Table 56-4) can act through endocrine, paracrine, and autocrine mechanisms to stimulate growth and differentiation in several organ systems.

Epidermal Growth Factor

The fetus has abundant epidermal growth factor (EGF) receptors (see Chapter 3), and EGF is well known for its mitogenic properties, especially with regard to development of ectodermal and mesodermal structures. However, the fetus has no detectable mRNA encoding EGF. Thus, transforming growth factor α (TGFα), another potent mitogen, which binds to EGF receptors on target cells, may act as a ligand for the EGF receptor.

Thyroid Hormones

The thyroid hormones are obligatory for normal growth and development (see Chapter 49). Before the second trimester, most of the thyroxine (T4) in the fetus is maternal. Fetal production of thyrotropin (thyroid-stimulating hormone [TSH]) and the thyroid hormone T4 begin to increase in the second trimester, con-

Many fetal tissues produce red blood cells early in gestation Early during gestation, production of red blood cells (erythropoiesis) occurs in many tissues not normally thought of as erythropoietic in the adult. Erythropoiesis begins during the third week of fetal development in the yolk sac and placenta. At approximately the fourth week of gestation, the endothelium of blood vessels and the mesenchyme also begin to contribute to the erythrocyte pool, shortly followed by the liver. The bone marrow, spleen, and other lymphoid tissues begin to produce red blood cells only near the end of the first trimester. All these organ systems except bone marrow gradually lose their ability to manufacture blood

Growth Restriction

I

UGR is an abnormality of fetal growth and development. IUGR has been variously defined as a birth weight lower than the 3rd, 5th, or 10th percentile for gestational age or a birth weight that is more than two standard deviations lower than the mean for gestational age. The growth-restricted fetus is at substantial risk of morbidity and mortality. Specific risks include birth asphyxia, neonatal hypoglycemia, hypocalcemia, meconium aspiration, persistent pulmonary hypertension of the newborn, pulmonary hemorrhage, thrombocytopenia, polycythemia, delayed neurologic development, and hypothermia. Ultrasound methods offer objective, reliable means for identifying IUGR. Intrauterine measurements of biparietal diameter (distance between the two parietal eminences of the head) and abdominal circumference predict IUGR in as many as 90% of the cases. The three recognized categories of IUGR are related to the time of onset of the pathologic process, as follows: Type I or symmetrical IUGR refers to the infant with decreased growth potential. Type I IUGR accounts for 20% to 30% of growth-restricted fetuses. The entire fetus is small for gestational age. Length, weight, and abdominal and head circumferences are all less than the 10th percentile for gestational age. Type I IUGR results from growth inhibition during early fetal development (4 to 20 weeks’ gestation), a period referred to as the hyperplastic stage of fetal development. Thus, the pathologic result is fewer cells in the fetus. Causes include intrauterine infections (e.g., rubella, cytomegalovirus), chromosomal disorders, congenital malformations, maternal drug ingestion, and maternal smoking. Of fetuses with severe, early onset of growth retardation, ∼25% have aneuploidy (i.e., abnormal number of chromosomes). Uniformly (or symmetri-

cally) diminished growth of these fetuses may result from inhibition of mitosis during early development. Type II or asymmetric IUGR refers to the infant with restricted growth, most frequently caused by uteroplacental insufficiency. This type accounts for 70% to 80% of growthrestricted fetuses. This type of growth restriction results from an insult that occurs later in gestation than type I IUGR, usually after 28 weeks’ gestation. Late in the second trimester, hypertrophy dominates. A rapid increase in cell size and increases in the formation of fat, muscle, bone, and other tissues occur. Fetuses with type II IUGR have a normal total number of cells, but these cells are smaller than normal. The distinguishing feature of the fetus with asymmetric IUGR is that the fetus has a normal length and head circumference (brain-sparing effect), but abdominal growth slows during the late second and early third trimesters. Redistribution of fetal CO occurs, with increased flow to the brain, heart, and adrenals and decreased glycogen storage and liver mass. This form of IUGR is most often associated with maternal disease such as kidney disease, chronic hypertension, and severe diabetes mellitus, among others. Intermediate IUGR is a combination of types I and II IUGR and accounts for 5% to 10% of all growth-restricted fetuses. It probably occurs during the middle phase of fetal growth (20 to 28 weeks’ gestation), between the hyperplastic and hypertrophic phases. During this middle period, mitotic rate decreases and overall cell size increases progressively. Chronic hypertension, lupus nephritis, or other maternal vascular diseases that are severe and begin early in the second trimester may result in intermediate IUGR, with symmetric growth and no significant brain-sparing effect.

Chapter 57 • Fetal and Neonatal Physiology

cells, and by the third trimester, the bone marrow becomes the dominant source of blood cells. The erythrocytes formed early in gestation are nucleated, but as fetal development progresses, more and more of the circulatory erythrocytes are non-nucleated. The blood volume in the common circulation of the fetoplacental unit increases as the fetus grows. The fraction of total erythrocytes that are reticulocytes (immature, non-nucleated erythrocytes with residual polyribosomes) is high in the young fetus, but it decreases to only ∼5% at term. In the adult, the reticulocyte count is normally less than 1%. The life span of fetal erythrocytes depends on the age of the fetus; in a term fetus, it is ∼80 days, or two thirds that in an adult. The life span of erythrocytes of less mature fetuses is much shorter. The hemoglobin (Hb) content of the fetal blood rises to ∼15 g/dL by midgestation, equivalent to the level in normal men. The Hb concentration of fetal blood at term is higher than the Hb concentration of maternal blood, which may be only ∼12 g/dL. Embryonic Hb with different combinations of α-type and β-type chains (see Table 29-1) is present very early in gestation. A genetic program of development governs the eventual transition to fetal Hb (HbF), which predominates at birth. HbA and a small amount of HbA2 gradually replace HbF during the first 12 months of life, thus culminating in the adult pattern of Hb expression (see Table 29-2). The fetal gastrointestinal and urinary systems excrete products into the amniotic fluid by midpregnancy The fetus imbibes considerable quantities of amniotic fluid by 20 weeks’ gestation. However, not until the final 12 weeks of gestation is fetal gastrointestinal function similar to that of the normal infant at term. The fetal gastrointestinal tract continuously excretes small amounts of meconium into the amniotic fluid. Meconium consists of excretory products from the gastrointestinal mucosa and glands, along with unabsorbed residua from the imbibed amniotic fluid. By the beginning of the second trimester, the fetus also begins to urinate. Fetal urine constitutes ∼75% of amniotic fluid production (see Chapter 56). The fetal renal system does not acquire the capacity to regulate fluid, electrolyte, and acid-base balance until the beginning of the third trimester. Full development of the renal system does not occur until several months following delivery. A surge in protein synthesis, with an increase in muscle mass, is a major factor in the rapid fetal weight gain during the third trimester Fetal tissues constantly synthesize and break down proteins. Protein synthesis predominates throughout gestation, especially during the third trimester, when fetal protein synthesis—primarily in muscle and liver—increases 3- to 4-fold. The number of ribosomes per cell increases throughout gestation and early postnatal life. The efficiency of ribosomes at translating mRNA may also improve during gestation. Substrate availability (i.e., amino acids) and modulation of the synthetic apparatus by endocrine and other factors

play important roles in regulating protein synthesis during gestation. The formation of each peptide bond requires four molecules of ATP, so the energy cost of protein synthesis is 0.86 kcal/g. Protein synthesis comprises 15% to 20% of fetal metabolic expenditure in the third trimester. At equivalent phases of development, fetuses across several species invest similar fractions of total energy in protein synthesis. Because glucose is the major metabolic fuel, a shortfall of oxidized metabolic substrates (e.g., glucose and lactate) has a direct, negative impact on protein synthesis. Increases in skeletal muscle mass account for 25% to 50% of fetal weight gain during the second half of gestation, when the number of muscle cells increases 8-fold and cell volume increases ∼2.6-fold. Although skeletal muscle fibers are not differentiated in the first half of gestation, distinct type I and type II muscle fibers (see Chapter 9) appear in equal amounts between 20 and 26 weeks of gestation. Fetal lipid stores increase rapidly during the third trimester Fetal fat stores account for only 1% of fetal body weight during the first trimester. By the third trimester, as much as 15% of fetal body weight is fat. At birth, humans have more fat than other warm-blooded animals (e.g., the newborn cat has 2%; the guinea pig, 9.5%; the rat, 11%), with the exception of hibernating mammals and migratory birds. Approximately half the increase in body fat reflects increased lipid transport across the placenta, and the other half reflects increased fatty acid (FA) synthesis in the fetal liver. Blood levels of fetal lipids (i.e., triglycerides, FAs, and ketone bodies) remain low before 32 weeks’ gestation. In the last 2 months, the fetus increases its lipid storage as triglycerides in white and brown adipose tissue as well as in liver. During this period, both subcutaneous fat (i.e., white fat) and deep fat (i.e., white and brown) increase exponentially. The stored fat ensures adequate fuel stores for postnatal survival, and it also provides thermal insulation to the newborn. In addition, brown fat is important for thermogenesis in the postnatal period. Several factors are responsible for increased lipid stores in the near-term fetus. Increases in fetal albumin facilitate FA transfer across the placenta. Insulin acts on fetal hepatocytes to stimulate lipogenesis. Insulin also promotes the availability of substrates, including glucose and lactate, which, in turn, increase the synthesis of fat (see Chapter 51).

DEVELOPMENT AND MATURATION OF THE CARDIOPULMONARY SYSTEM Fetal lung development involves repetitive branching of both the bronchial tree and the pulmonary arterial tree The fetal lung begins as an outpouching of the foregut at ∼24 days’ gestation. Several days later, this lung bud branches into two tubular structures, the precursors of the main bronchi. At 4 to 6 weeks’ gestation, the bronchial tree begins

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to branch repetitively. The further maturation of the lungs occurs in four overlapping phases: (1) the pseudoglandular period, (2) the canalicular period, (3) the terminal sac period, and (4) the alveolar period. During the pseudoglandular period (5 to 17 weeks), the lung “airways” resemble branching exocrine glands. The canalicular period is characterized by canalization of the airways (16 to 25 weeks) and is complete when ∼17 generations of airways have formed, including the respiratory bronchioles. Each respiratory bronchiole gives rise to as many as six alveolar ducts, which give rise to the primitive alveoli during the second trimester. The branching of the pulmonary arterial tree parallels, both temporally and spatially, the branching of the bronchial tree. However, at ∼24 weeks’ gestation, considerable interstitial tissue separates the capillaries from the respiratory epithelium. Thus, if the fetus were born at this stage of its development, the premature infant would have a very low diffusing capacity (see Chapter 30), owing to the great distance between the edge of the alveolar lumen and the edge of the capillary lumen. During the terminal sac period (24 weeks’ gestation to birth), the respiratory epithelium thins greatly, and the capillaries push into the alveolar sacs. The potential for gas exchange improves after ∼24 weeks’ gestation, when capillaries proliferate and come into closer proximity to the thin type I alveolar pneumocytes (see Chapter 26). During this period, surfactant synthesis and storage begin (although not extensively) in the differentiated type II cells. In the alveolar period of lung development (late fetal life to 8 years of age), final alveolar growth occurs. Alveolar-like structures are present at ∼32 weeks’ gestation, and at 34 to 36 weeks’ gestation, 10% to 15% of the adult number of alveoli will be present. Alveolar number continues to increase until as late as 8 years of age. An increase in cortisol, in conjunction with other hormones, triggers production of surfactant by type II alveolar pneumocytes in the third trimester Hormones play a major role in controlling fetal lung growth and development in preparation for ex utero function. A key target is surfactant (see Chapter 27), which increases lung compliance. Numerous hormones stimulate surfactant biosynthesis, including glucocorticoids, thyroid hormones, thyrotropin-releasing hormone, and prolactin, as well as growth factors such as EGF. Glucocorticoids in particular play an essential role in stimulating fetal lung maturation by increasing the number of both type II alveolar pneumocytes and lamellar bodies (see Chapter 27) within these cells. Glucocorticoid receptors are probably present in lung tissue at midterm. Fetal cortisol levels rise steadily during the third trimester and surge just before birth. Two thirds of this cortisol is of fetal origin; the rest crosses the placenta from the mother. The predominant phospholipid in surfactant is dipalmitoylphosphatidylcholine (DPPC). Glycogen serves as a primary energy and carbon source for the FAs involved in phospholipid synthesis (Fig. 57-1). The FAs used in the synthesis of surfactant enter the type II cells directly from the bloodstream. The condensation of diacylglycerol with cyto-

Glycogen HCO–3

+

Acetyl CoA carboxylase

Glucose

+

Pi

Pyruvate Acetyl CoA

Malonyl CoA

Fatty acid synthase (7-step/multifunctional)

Palmitate CH3

O

(CH2)14

C

Choline

–

O

Choline kinase

Dihydroxyacetone phosphate or glycerol-3-phosphate

Phosphocholine CTP

(CTP: phosphocholine) cytidyl transferase

PPi OH CH3

CH

O

O

C

O C

CH3

CH2 CH

CH2

O

P

O

–

O O

P

CH3

CH3

O

O

N

CDP-choline –

O

Cytidine H3C CH3

1,2-diacylglycerol (DAG)

(CDP-choline: 1,2-diacylglycerol) phosphocholine transferase

CMP CH3 CH

CH2

P

CH3

CH3

O O

+ N

O–

O CH3

CH

O

O

C

O C

CH2

O

Dipalmitoylphosphatidylcholine (DPPC) or dipalmitoyl lecithin

H3C CH3

Figure 57-1 Synthesis of DPPC. Before birth, cortisol upregulates several enzymes that are important for the synthesis of surfactant, including FA synthase and phosphocholine transferase. CoA, coenzyme A.

Chapter 57 • Fetal and Neonatal Physiology

Respiratory Distress Syndrome

R

espiratory distress syndrome (RDS) affects 10% to 15% of infants born prematurely. In very immature infants, delivered before 30 weeks of gestation, cyanosis, tachypnea, nasal flaring, intercostal and subcostal retractions, the use of accessory musculature, and grunting may be immediately apparent in the delivery room. In more mature preterm infants, these symptoms may evolve over several hours. A chest radiograph reveals atelectasis with air bronchograms (i.e., air-filled bronchi standing out against the white background of collapsed lung tissue). Infants with severe RDS may develop edema and respiratory failure that requires mechanical ventilation. Uncomplicated cases usually resolve spontaneously. Because RDS occurs in premature infants, the course is often confounded by the coexistence of a patent ductus arteriosus. This combination of problems raises the risk for short- and long-term complications, such as alveolar rupture with pneumothorax and pulmonary interstitial emphysema, necrotizing enterocolitis, intraventricular hemorrhage, and bronchopulmonary dysplasia. RDS is caused by a deficiency of pulmonary surfactant. Although prematurity is by far the single most important risk factor for developing RDS, others include male sex, cesarean section, perinatal asphyxia, second twin pregnancy, and maternal diabetes. Surfactant insufficiency can result from abnormalities of surfactant synthesis, secretion, or reutilization. Decreased lung compliance and atelectasis result from both structural immaturity and surfactant deficiency, thus promoting airway collapse. The consequent right-to-left shunting of blood past poorly ventilated alveoli results in hypoxemia, which—at the level of the alveoli—causes capillary damage and leakage of plasma proteins into the alveolar space. These proteins may inactivate surfactant, thus exacerbating the underlying condition. The discovery that a deficiency of surfactant is the underlying problem in infants with RDS led investigators to look for ways of assessing fetal lung maturity and adequacy of surfactant production before delivery, so elective induction or cesarean section could be timed successfully in infants who need to be delivered prematurely. Clinical tests for assessing lung maturity exploit the knowledge that the major surfactant lipids are phosphatidyl cholines (i.e., lecithins) and that phosphatidylglycerol (PG) is also overrepresented (see Chapter 27). A ratio of lecithin to sphingomyelin (L/S ratio) greater than 2.0 in the amniotic fluid is consistent with mature lungs, as is a positive PG assay. The 2000 National Institutes of Health Antenatal Steroid Consensus Conferences recommended antenatal steroid therapy for pregnant women with fetuses between 24 and 34 weeks’ gestational ages who are at risk of preterm delivery within 7 days. This treatment accelerates lung maturation and surfactant production. In the newborn who develops signs of RDS soon after birth, surfactant is instilled into the trachea, preferably within the first hour after delivery. The administration of antenatal steroids and postnatal surfactant has markedly reduced the mortality from RDS and has improved the clinical course described earlier.

sine diphosphate choline ultimately leads to the production of DPPC. At ∼32 weeks’ gestation, increases in cortisol and in the other hormones mentioned previously stimulate several regulatory enzymes, including FA synthase and phosphocholine transferase. Thus, the net effect is vastly increased production of pulmonary surfactant late in gestation. Coincident with increased surfactant synthesis are large increases in lung distensibility and stability on inflation. Fetal respiratory movements begin near the end of the first trimester but wane just before birth Fetal breathing movements have been confirmed in humans by both Doppler ultrasound and tocodynamometer (an external device that records uterine movements) studies, commencing near the end of the first trimester. It appears that hypoxia and tactile stimulation of the fetus promote these breathing movements, which occupy less than half of any 24-hour period. Near term, breathing movements are regular, similar to those found after birth. However, just before labor, fetal breathing decreases. The fetal lung undergoes many changes in preparation for birth. In utero, the alveoli and airways of the fetal lung are filled with a volume of fluid approximating the functional residual capacity (see Chapter 27) of the neonatal lung. The onset of labor is accompanied by increases in catecholamines and arginine vasopressin, which decrease fluid production by the fetal lung and initiate its active reabsorption. The pulmonary circulation absorbs the majority of the fluid, and the pulmonary lymphatics absorb some as well. A small portion of the lung fluid is forced out of the trachea as the fetus passes through the birth canal. The fetal circulation has four unique shunts: the placenta, the ductus venosus, the foramen ovale, and the ductus arteriosus The circulatory system differentiates from the mesoderm of the embryo. The fetal heart begins to beat in the fourth week of gestation. The major difference between the circulatory system of the fetus and that of the adult is the presence of the placenta. The placenta performs for the fetus functions that—at least in part—are performed by four organ systems in extrauterine life: (1) the lungs (gas exchange), (2) the gastrointestinal tract (nutrition), (3) the liver (nutrition, waste removal), and (4) the kidneys (fluid and electrolyte balance, waste removal). Thus, the fetal heart pumps large quantities of blood through the placenta and smaller amounts of blood through the other four organ systems. The key principle governing the unique pattern of blood flow in the fetus is the presence of four shunts, that is, pathways that allow blood to bypass the future postnatal route. These shunts are illustrated in Figure 57-2. Because, to a large extent, the right and left sides of the fetal heart pump in parallel rather than in series, and because the inputs and outputs of these two sides mix, we define the combined cardiac output (CCO) as the sum of the outputs of the right and left ventricles. Figure 57-2A shows the fraction of the CCO that flows through the fetal circulatory system at important checkpoints. Figure 57-2B shows values for PO2

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Fetal Asphyxia

A

ny insult that interferes with the ability of the placenta to exchange O2 and CO2 between the maternal and fetal circulations may lead to fetal asphyxia. Common causes include maternal hypotension, abruptio placentae (i.e., breaking away of a portion of the placenta from the uterine wall), and a prolapsed umbilical cord (i.e., the umbilical cord falls into the birth canal in front of the head or other part of the fetus). The results are low fetal PO2, high PCO2, and acidosis. These effects can decrease myocardial function, lead to lowering of CO and further compromise of O2 delivery to the tissues, and thereby create a vicious cycle. In the brain, asphyxia produces substantial alterations in cerebral intracellular metabolism. As the brain is forced to shift from aerobic to anaerobic metabolism, high-energy phosphate compounds (e.g., ATP) decrease in concentration, and their breakdown products (e.g., ADP and inorganic phosphate) increase. Lactic acid accumulates. Changes in PO2, PCO2, pH, and metabolism can profoundly affect neurotransmitter release and re-uptake and hence concentrations of various ions in neurons, glia, and brain extracellular fluid (see Chapter 11). Asphyxia also may lead to accumulation of prostaglandins and leukotrienes, vasoactive compounds that can dilate microvessels in critical areas of the brain, thus permitting the generation of free radicals that, in turn, lead to cell damage. Fetuses experiencing chronic O2 deficiency in utero are at increased risk of delayed breathing immediately after birth, in part because their energy reserves are already low. Therefore, fetal hypoxia can lead to neonatal hypoxia. The metabolic derangements of asphyxia remain evident for as long as 24 hours after birth.

and the percentage of saturation of HbF at these same checkpoints. The numeric values in Figure 57-2 are reasonable values for a healthy fetus. Placenta The first of these shunts is the placenta itself. Of the CCO late in gestation, ∼69% reaches the thoracic aorta (Fig. 57-2A). Half of the CCO enters the placenta as deoxygenated blood through the paired umbilical arteries, which arise from the two common iliac arteries. This massive blood flow to the placenta not only shunts blood away from the lower trunk, but also lowers effective blood flow to all abdominal viscera, including the kidneys. The umbilical arteries branch repeatedly under the amnion and ultimately form dense capillary networks within the terminal villi (see Chapter 56). The single umbilical vein returns oxygenated blood (which has a PO2 of 30 to 35 mm Hg) back to the fetus from the placenta. This blood enters the ductus venosus, which then merges with the inferior vena cava. Ductus Venosus

This second shunt bypasses the liver, which is largely nonfunctional. The ductus venosus allows blood from the umbilical vein, ∼50% of CCO, to enter the inferior vena cava directly, without ever entering the liver. In addition, some blood from the portal circulation may

enter the ductus venosus. Blood from the ductus venosus then combines with blood from the inferior vena cava, ∼19% of CCO, which drains the lower body and liver. Thus, ∼69% of the CCO (PO2 ≅ 27 mm Hg) enters the right atrium. Foramen Ovale The third major shunt is blood entering the right atrium and then crossing the foramen ovale to enter the left atrium. The foramen ovale is an oval hole in the septum dividing the atria, located in the posterior aspect of the right atrium. Of the 69% of the CCO that enters the right atrium through the inferior vena cava, ∼27% shunts through the foramen ovale directly into the left atrium. This movement represents a right-to-left shunt. Therefore, the left side of the heart receives relatively well oxygenated blood (PO2 ≅ 27 mm Hg) from the inferior vena cava. In addition, the left atrium receives 7% of the CCO as poorly oxygenated blood (PO2 ≅ 20 mm Hg) from the nonfunctional lungs. Thus, the left ventricle pumps a total of 27% + 7% = 34% of the CCO (PO2 ≅ 25 mm Hg). Because this blood enters the aorta upstream from the ductus arteriosus, it primarily flows to the head and forelimbs. Returning now to the right atrium, 69% − 27% = 42% of the CCO entering the right atrium from the inferior vena cava does not shunt through the foramen ovale. This relatively well-oxygenated blood (PO2 ≅ 27 mm Hg) joins the relatively poorly oxygenated 21% of the CCO (PO2 ≅ 17 mm Hg) that enters the right atrium from the superior vena cava and another 3% from the coronary vessels—a total of 24% of CCO. Because of the valve-like nature of the septum surrounding the foramen ovale, none of the incoming blood from the superior vena cava or coronary vessels shunts through the foramen ovale. Rather, it goes through the tricuspid valve to the right ventricle. Thus, the right ventricle receives 42% + 21% + 3% = 66% of the CCO (PO2 = 18 to 22 mm Hg). The PO2 in the fetal right ventricle is somewhat lower than that in the left ventricle. The blood from the right ventricle then enters the trunk of the pulmonary artery. Ductus Arteriosus The fourth major shunt, also a rightto-left shunt, directs blood from the pulmonary artery to the aorta through the ductus arteriosus. The ductus arteriosus contains substantial smooth muscle in its vessel wall. The patency of this vessel is due to active relaxation of this smooth muscle, mediated by prostaglandins, particularly prostaglandin E2 (PGE2). Fetal PGE2 levels are as much as 5-fold higher than adult levels are. Administering prostaglandin inhibitors to an experimental fetal animal causes the ductus arteriosus to vasoconstrict. Although 66% of the CCO enters the pulmonary artery, only 7% of the CCO perfuses the unventilated fetal lungs, reflecting the high resistance of the pulmonary vasculature in the fetus. This high resistance is the result of hypoxic vasoconstriction and acidosis (see Chapter 31), the collapsed state of the airways, and perhaps leukotrienes (particularly leukotriene D4 [LTD4]). The rest of the blood entering the pulmonary artery, 66% − 7% = 59% of the CCO (PO2 ≅ 22 mm Hg), enters the descending aorta through the ductus arteriosus and mixes with the blood from the aortic arch, 10% of CCO, that did not perfuse the head and upper body (PO2 ≅ 25 mm Hg). Thus, the descending aorta receives 59%

Chapter 57 • Fetal and Neonatal Physiology

A

BLOOD FLOW EXPRESSED AS PERCENT OF COMBINED CARDIAC OUTPUT

21% 21%

31%

10% 59%

66% Ventricles

4 Ductus arteriosus 7%

Upper body and brain

3% 66%

34%

69%

Atria Lungs 7%

21% 27%

3%

To show fetal blood supply more clearly, the atria have been shown below the ventricles in this diagram.

42% Foramen ovale 3 Inferior vena cava

69%

27%

Liver 2 Ductus venosus

Aorta

Ductus arteriosus Here, the foramen ovale is open.

Umbilical vein 50%

Portal vein

19% Right atrium

To/from GI tract

Right ventricle

Navel 1 Placenta

50%

Legs

Left ventricle

Legs

Umbilical arteries

Figure 57-2 Fetal circulation. A, This schematic drawing shows the major elements of the fetal circulation. Note that—in the main drawing—the heart is upside down, for the sake of presenting the blood flow as simply as possible. The heart is right side up in the inset. Because the inputs and outputs of the right and left hearts mix, we define the CCO as the sum of the outputs of the right and left ventricles. The percentage of the CCO that passes various checkpoints is represented as a number in a black box. The CCOs of the right ventricle (66%) and the left ventricle (34%) add up to 100%. The fetal circulation has four major shunts: the placenta, the ductus venosus, the foramen ovale, and the ductus arteriosus. Continued

Left atrium

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B

OXYGEN SATURATION / PO2

65% 25

00% = O2 saturation 00 = mm Hg 65% 25 4 Ductus arteriosus

Ventricles Upper body and brain

55% 22

65% 25

Atria Lungs 29% 17

70% 27

70% 27

46% 20

60% 23

Foramen ovale 3 Liver 2 Ductus venosus Umbilical vein

85% 30

Portal vein

29% 17

To/from GI tract

60% 23

Navel 1 Placenta

Umbilical arteries

Legs

Figure 57-2, cont’d B, The schematic drawing is the same as in A, except—at each checkpoint—we show the O2 saturation of HbF against a black background and the PO2 (in mm Hg) against a white background. The relationship between the O2 saturation and PO2 figures is based on the O2 saturation curve for HbF (similar to the curve labeled Hb + CO2 in Fig. 29-7).

Chapter 57 • Fetal and Neonatal Physiology

+ 10% = 69% of the CCO (PO2 ≅ 23 mm Hg). The placenta receives blood with a PO2 of 23 mm Hg, and returns blood to the fetus with a PO2 of 30 to 35 mm Hg.

CARDIOPULMONARY ADJUSTMENTS AT BIRTH As the newborn exits the birth canal, it takes its first breath, which not only expands the lungs, but also triggers a series of changes in the circulatory system. At the same time, the newborn loses its nutritional connection to the mother and apprehends a cold new world. Three major changes in metabolism accompany birth: hypoxia, hypoglycemia, and hypothermia. We discuss the adaptations of the respiratory and cardiovascular systems in this major section and adjustments of other organ systems in the next. Loss of the placental circulation requires the newborn to breathe on its own Although separation of the placenta does not occur until several minutes after birth, vasoconstriction in the umbilical arteries terminates the ability of the placenta to deliver oxygenated blood to the newborn immediately upon birth. Thus, even though the newborn may remain attached to its placenta during the first few moments of life, it is essential that the baby begins to breathe immediately. Umbilical vasoconstriction has two origins. First, stretching the umbilical arteries during delivery stimulates them to constrict. Second, the sudden rise in the systemic arterial PO2 in the newborn also stimulates and maintains vasoconstriction in the umbilical arteries. Birth may also be associated with an “autotransfusion” as blood in the placental circulation preferentially moves into the body of the emerging baby. Because the umbilical veins do not constrict, as do the umbilical arteries, blood flows from placenta to newborn if the newborn is below the level of the placenta, and if the umbilical cord is not clamped. This autotransfusion may constitute 75 to 100 mL, which is a substantial fraction of the newborn’s total blood volume of ∼300 mL. At birth, the newborn must transform its circulatory system from one that supports gas exchange in the placenta to one that supports O2 and CO2 exchange in the lungs. In addition, other circulatory adjustments must occur as the gastrointestinal tract, liver, and kidneys assume their normal roles. As the lungs become functional at birth, the pulmonary and systemic circulations shift from interconnected and parallel systems to separate entities that function in series. Mild hypoxia and hypercapnia, as well as tactile stimuli and cold skin, trigger the first breath The first breath is the defining event for the newborn. Not only does it inflate the lungs, but also—as discussed later—it triggers circulatory changes that convert the fetal pattern of blood flow to the adult pattern. The functional capabilities of the lungs depend on their surface area available for gas exchange, the ability of surfactant to maximize lung compli-

ance, neural mechanisms that control breathing, and the aforementioned circulatory changes. The first breath is normally also the most difficult inspiration of a lifetime. A considerable negative pressure within the intrapleural space is necessary to overcome the effects of surface tension. The infant’s first inspiratory effort requires a transpulmonary pressure (PTP)—the pressure difference between the intrapleural space and alveolar air spaces—of 60 cm H2O to increase the lung volume by ∼40 mL. In contrast, a typical adult only needs to change PTP by ∼2.5 cm H2O during a typical tidal volume of 500 mL (see Chapter 27). The newborn’s first ventilatory effort creates an airwater interface for the first time, opening the alveoli. Breathing becomes far easier once the alveoli are open and the type II alveolar pneumocytes deliver surfactant to the air-water interface. Thus, the second inspiration may require a PTP of only 40 cm H2O. The newborn may not achieve the adult level of relative lung compliance until 1 hour after birth. Very immature neonates, who lack adequate surfactant (see the box titled Respiratory Distress Syndrome), may have difficulty expanding the lungs. The rapid onset of breathing immediately after delivery appears to be induced by a temporary state of hypoxia and hypercapnia. In most normal deliveries, these changes in PO2 and PCO2 result from the partial occlusion of the umbilical cord. Tactile stimulation and decreased skin temperature also promote the onset of breathing. When newborns do not begin to breathe immediately, hypercapnia and hypoxia increase and provide further simulation for the infant to breathe. The peripheral and central chemoreceptors are responsible for sensing the blood gas parameters (i.e., low PO2, high PCO2, and low pH) characteristic of the asphyxia that accompanies birth. In addition, increased sympathetic tone may stimulate breathing at the time of birth by constricting vessels to the peripheral chemoreceptors, thereby lowering the local PO2 in the microenvironment of the glomus cells and mimicking even more severe hypoxia (see Chapter 32). Finally, independent of the initial stimuli that trigger breathing, other central nervous system mechanisms may help to sustain breathing in the newborn. The neonate’s ability to control the blood gas parameters depends on the sensitivity of the lung’s mechanical (i.e., stretch) reflexes, the sensitivity of the central and peripheral chemoreceptors, the gestational and postnatal age, the ability of the respiratory muscles to resist fatigue, and the effects of the sleep state. Sleeping newborn infants, especially premature newborns, tend to have increased respiratory variability from breath to breath. For example, they exhibit periodic breathing, which consists of breaths with intermittent respiratory pauses (generally of a few to several seconds’ duration) and varying tidal volumes. Periodic breathing and increased respiratory variability, including periodic breathing, occur more frequently in rapid eye movement sleep than during quiet sleep, a state characterized by regular breathing. In human adults and in adult experimental animals, periodic breathing may reflect an exaggerated ventilatory response to CO2—which causes arterial PCO2 to fall, thus lowering respiratory drive. However, the mechanisms underlying periodic breathing in the newborn may not be the same.

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At birth, removal of the placental circulation increases systemic vascular resistance, whereas pulmonary expansion decreases pulmonary vascular resistance As noted earlier, the fetal circulation has four unique shunts absent in the adult: the placental circulation, the ductus venosus, the foramen ovale, and the ductus arteriosus. At or around birth, these shunts disappear. In addition, the pulmonary circulation, which received only ∼7% of the CCO in the fetus, now accepts the entire cardiac output (CO). In this and the next three sections, closure of each of these four shunts is discussed. Closure of the Placental Circulation

In the fetus, the placental circulation receives ∼50% of the CCO (Fig. 57-2A). Thus, the placental circulation represents a major parallel path in the systemic circulation and accounts for the low vascular resistance of the fetal systemic circulation. As the placental circulation disappears at birth, the total peripheral resistance doubles. Because blood flow through the descending aorta is essentially unchanged, aortic pressure must increase, thereby causing upstream pressure in the left ventricle to increase as well. Opening of the Pulmonary Circulation

As noted earlier, during fetal life, pulmonary vascular resistance is high as the result of hypoxic vasoconstriction, acidosis, the collapsed state of the airways, and perhaps agents such as LTD4 (see earlier). As a result, only ∼7% of the CCO of the term fetus flows through the lungs, a figure corresponding to ∼11% of the right ventricular output. At birth, expansion of the lungs by itself markedly decreases pulmonary vascular resistance, perhaps by triggering the release of prostaglandin I2 (PGI2 or prostacyclin). In addition, the increase in PO2 and pH that occurs with breathing leads to pulmonary vasodilation. Together, these changes reduce pulmonary vascular resistance more than 5-fold (Fig. 57-3). Because the blood flow through the pulmonary vasculature increases by a slightly smaller factor, pressure in the pulmonary artery decreases. As a result, upstream pressure in the right ventricle also falls.

Birth 2.0 Pulmonary 1.5 vascular resistance 1.0 mm Hg kg mL/min 0.5 0 200 Pulmonary blood flow mL kg min

150 100 50 0 60

Mean pulmonary arterial pressure (mm Hg)

40 20 0 -7

-5

-3

-1 1 2 Time (weeks)

5

7

Figure 57-3 Effect of birth on pulmonary vascular resistance, blood flow, and mean arterial pressure. In the fetus, pulmonary vascular resistance is high, pulmonary blood flow is low, and mean pulmonary arterial pressure is high. At birth, each of these three situations rapidly reverses. The primary event is the fall in resistance, which occurs because of the following: (1) the pulmonary blood vessels are no longer being crushed; (2) breathing causes increased PO2, which, in turn, causes vasodilation; and (3) local prostaglandins cause vasodilation. The reason that pressure falls after birth is that the fall in pulmonary vascular resistance is greater than the rise in blood flow. (Data from Rudolf AM: Congenital Diseases of the Heart: Clinical-Physiological Considerations. Armonk, NY: Futura, 2001.)

Closure of the ductus venosus within the first 3 hours of life forces portal blood to perfuse the liver

At birth, left atrial pressure begins to exceed right atrial pressure, thus causing the foramen ovale to close

During fetal life, a large fraction of the blood in the portal vein bypasses the liver by entering the ductus venosus and merging with blood from the umbilical vein (Fig. 57-2A). Although blood flow through the umbilical vein ceases soon after birth, the majority of the portal blood continues to flow through the ductus venosus. Thus, immediately after birth, portal flow through the liver remains low. Within ∼3 hours after term birth, however, constriction of the vascular smooth muscle within the ductus venosus completely occludes this shunt pathway. As a result, pressure in the portal vein increases markedly, thereby diverting blood into the liver. The mechanisms underlying the contraction of the muscular walls of the ductus venosus remain unknown.

In the fetus, blood from the inferior vena cava moves preferentially from the right atrium across the foramen ovale into the left atrium (Fig. 57-2A). After entering the left ventricle, this well-oxygenated blood moves into the ascending aorta and primarily perfuses the head, neck, and coronary arteries. At birth, the decrease in the pulmonary vascular resistance increases blood flow through the lungs; the results are increased venous return to the left atrium and elevated left atrial pressure. At the same time, the venous return to the right atrium falls from 69% + 21% + 3% = 93% of the CCO of both ventricles in the fetus to 100% of the CO of a single ventricle at birth. Thus, right atrial pressure falls. The net effect is a reversal of the pressure gradient across the atrial septum, pushing the foramen ovale’s “valve”—

Chapter 57 • Fetal and Neonatal Physiology

B A

CLOSURE OF FORAMEN OVALE AND DUCTUS ARTERIOSUS

OXYGEN SATURATION / PO2 IN THE NEWBORN 00% = O2 saturation 00 = mm Hg

The atria have been shown below the ventricles in this diagram. 99% 60

At birth, the ductus arteriosus… …and the foramen ovale both close.

65% 25

99% 60

99% 60

99% 60

Atria

Hepatic veins

65% 25

65% 25

Ventricles

Right atrium

Constricted ductus arteriosus

Lungs 99% 60 Right ventricle

Left ventricle

Left atrium

65% 25

Foramen ovale

Figure 57-4 Changes in the circulation at and around birth. A, Closure of these two shunts establishes separate right and left circulatory systems. As the pressure in the left atrium rises higher than the pressure in the right atrium—owing to the large decrease in pulmonary vascular resistance—the flap of the foramen ovale pushes against the septum, thus preventing blood flow from the left to the right atrium. Eventually, this flap seals shut. As aortic pressure exceeds the pressure of the pulmonary artery, blood flow through the ductus arteriosus reverses. Well-oxygenated aortic blood now flows through the ductus arteriosus. This high PO2 causes vasoconstriction, which functionally closes the ductus arteriosus within a few hours. Falling prostaglandin levels also contribute to the rapid closure. Eventually, the lumen of the ductus becomes anatomically obliterated. B, The elimination of the fetal shunts and the oxygenation of blood in the lungs lead to major increases in the O2 saturation and PO2 in the circulation.

situated on the “left” side of the septum—against the opening of the foramen ovale (Fig. 57-4A). Closing this valve usually prevents what would otherwise be movement of blood from the left to the right atrium of the newborn. The left side of the heart now receives blood only from the lungs. Gradually, a permanent seal forms between the valve and the wall of the septum, a process that can take as little as a few months or as long as a few years. In some newborns, the valve does not completely seal to the septum, thus leaving a remnant potential pathway between the two atria. However, the 15% to 20% of adults with this condition do not have left-to-right shunting, because the valve of the foramen ovale is effective even if it is not completely sealed. However, if right atrial pressure should increase to more than left atrial pressure for some pathologic reason (e.g., pulmonary hypertension), then a right-to-left shunt between the atria would occur. Closure of the ductus arteriosus completes the separation between the pulmonary and systemic circulations During fetal life, blood flows from the pulmonary artery, through the ductus arteriosus, and into the aorta (Fig. 57-2A). Prostaglandins maintain the patency of the ductus

arteriosus during fetal life. Immediately after birth, the ductus arteriosus remains open. However, it now conducts blood in the direction opposite from that of the fetus: from the aorta to the pulmonary artery. This reversal of blood flow is the result of the increased systemic resistance (which elevates aortic pressure) and decreased pulmonary vascular resistance (which lowers pulmonary arterial pressure). Obviously, the open ductus arteriosus during the first few hours of postnatal life constitutes an undesirable left-toright shunt. Fortunately, within a few hours after term birth, the ductus arteriosus closes functionally because its muscular wall constricts (Fig. 57-4A). Usually, all blood flow through the ductus arteriosus ceases within 1 week after birth. Within a month or so, the lumen becomes obliterated anatomically because of thrombosis (i.e., blood clot within the lumen), proliferation of the vessel’s intimal layer, and growth of fibrous tissue. Occasionally, the ductus arteriosus fails to close. The incidence of patent ductus arteriosus is one in several thousand. The relatively rapid functional closure of the ductus arteriosus is primarily the result of the increased PO2 of the blood perfusing this vessel immediately after birth. As the PO2 of blood flowing through the ductus arteriosus rises from 18 to 22 mm Hg in utero to ∼60 mm Hg a few hours after birth, the smooth muscle in the wall of the ductus arteriosus

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contracts. In newborns who are hypoxemic, the low PO2 has three effects: (1) pulmonary vascular resistance and pressure remain high, (2) the ductus arteriosus remains patent, and (3) the patent ductus arteriosus maintains a right-to-left shunt. In these infants, raising the inspired PO2 closes the ductus arteriosus. If these infants are allowed to breathe room air again too quickly, the ductus arteriosus will reopen. Other factors, in addition to a high PO2, contribute to the rapid functional closure of the ductus arteriosus. Shortly after birth, circulating levels of prostaglandins fall quickly, thus relieving the ductus arteriosus of the vasodilating influence of these substances. Preterm infants tend to maintain high circulating prostaglandin levels, a feature that may account for their tendency to patent ductus arteriosus. Treating such infants with indomethacin (a nonsteroidal anti-inflammatory drug that inhibits cyclooxygenase and thereby reduces prostaglandin synthesis) (see Chapter 3 for the box on inhibition of cyclooxygenase isoforms by aspirin) induces closure of the ductus arteriosus, even at a low PO2. Norepinephrine, acetylcholine, and bradykinin also produce constrictor responses. The closure of the ductus arteriosus completes the separation of the right and left circulations initiated with closure of the foramen ovale. Whereas the ventricles functioned in parallel in the fetus, now they function in series in the neonate. As a result, the O2 saturation of the newborn’s Hb is similar to the adult’s (Fig. 57-4B). However, because the O2 saturation curve of HbF is shifted relatively leftward (see Chapter 29), the newborn achieves these O2 saturations at lower PO2 values. In the neonate, the sum of the ventricular outputs of the two ventricles (i.e., twice the CO) is larger than the CCO in the fetus, a result primarily of a marked rise in the output of the left ventricle, which doubles its stroke volume. Compared with the adult, the newborn has a markedly lower systemic vascular resistance and thus can achieve a relatively high blood flow with a relatively low perfusion pressure.

NEONATAL PHYSIOLOGY In humans, the neonatal period is defined as the first 4 weeks of life. The newborn’s ability to survive during this period depends on the adequate development and maturation of various fetal organ systems, as well as on adaptations of these organ systems to extrauterine life. As the newborn loses the nutritional link with the placenta, the infant must now rely on his or her own gastrointestinal tract. Moreover, other functions normally carried out by the placenta are now entrusted to the liver and kidneys. Finally, on exiting its uterine “incubator,” the newborn must stabilize his or her body temperature. Although the newborn is prone to hypothermia, nonshivering thermogenesis in brown fat helps to keep the neonate warm The body loses heat to the environment by radiation, conduction, convection, and evaporation (see Chapter 59). The relative importance of these processes depends on the cir-

cumstances. For instance, at birth, the infant moves from a warm and liquid environment to cool and dry surroundings. Hence, evaporation is the main source of heat loss immediately after delivery. However, even after the newborn’s skin is dry, the infant is at risk for losing body heat by each of the previously discussed mechanisms. The major reasons are as follows: (1) the large skin surface area of the newborn relative to the small body mass (i.e., large surface-to-volume ratio), (2) the limited ability of the newborn to generate heat through muscle contraction (e.g., shivering thermogenesis), (3) the newborn’s poor thermal insulation from the environment by adipose tissue, and (4) the inability of the newborn to adjust his or her own protection (e.g., put on warmer clothes) from the thermal stress of the environment or to modify that environment (e.g., turn up the thermostat). Premature and growth-retarded infants are at an even higher risk for heat loss and hypothermia. Fortunately, the newborn has one important asset for fighting hypothermia: nonshivering thermogenesis, a process that occurs primarily in liver, brain, and brown fat (Fig. 57-5). Cold stress triggers an increase in the levels of epinephrine and TSH. TSH stimulates the release of the thyroid hormones, predominantly T4 (see Chapter 49). Working in parallel, epinephrine activates, particularly in brown fat, the 5′/3′-monodeiodinase responsible for the peripheral conversion of circulating T4 to the far more active triiodothyronine (T3). T3 acts locally in brown fat to uncouple mitochondrial oxidation from phosphorylation and thereby to increase heat production. Brown fat differs from white fat in having a high density of mitochondria; the cytochromes in these mitochondria give the brown fat cells their color. Newborns have particularly high levels of brown fat in the neck and midline of the upper back. In brown fat, the locally generated T3 upregulates a protein called uncoupling protein UCP1, originally called thermogenin. This protein is an H+ channel located in the inner mitochondrial membrane. Normally, intracellular purine nucleotides (e.g., ATP, GDP) inhibit UCP1. However, epinephrine, acting through a cAMP pathway, activates the lipase that liberates FAs from triglycerides. These FAs relieve the inhibition of the H+ channel and increase its conduction of protons. Consequently, the protons generated by electron transport enter the mitochondrion through UCP1, which dissipates the H+ gradient needed by the H+-translocating ATP synthase (see Chapter 5). Thus, the mitochondria in brown fat can produce heat without producing useful energy in the form of ATP. The oxidation of FAs in brown fat generates ∼27 kcal/kg of body weight each day and contributes a large fraction of the neonate’s high metabolic rate. The neonate mobilizes glucose and fatty acids soon after delivery Carbohydrate Metabolism Elimination of the placental circulation at birth means that the newborn now has to forage for his or her own food. However, the newborn may not start suckling for ∼6 hours. During late fetal life, glucocorticoids promote rapid accumulation of glycogen through their action on glycogen synthase. In its first few hours, the

Chapter 57 • Fetal and Neonatal Physiology

COLD STRESS

TSH

Brown fat cell Epinephrine

Thyroid gland

Extracellular space

Epinephrine

Epinephrine receptor

T4

Plasma membrane AC 5´ Monodeiodinase

Cytosol T3

G-protein complex

cAMP

+

Fat lobule

Pi

C cAMP

cAMP

PKA (inactive)

C R

R R

R cAMP

cAMP

C

C

RXR

THR

PKA (active) UCP (H+ channels)

DNA

Triacylglycerol lipase (inactive)

Transcription

Triacylglycerol lipase (active)

TRE

Free fatty acids

Nucleus T3 upregulates uncoupling proteins (UCP), + which are H channels. UCP becomes part of the mitochondrial inner membrane.

Triacylglycerols

Mitochondrion

Brown fat cell cytosol Purine nucleotides such as ATP and GTP normally block UCP. Channel remains closed.

Mitochondrion intermembrane space

Outer membrane

H+-transporting ATPase (ATP synthase) I

III

H+

IV

UCP

Free fatty acids Free fatty acids relieve the block and open the channel. The entering H+ short circuits the H+ gradient that is set up by the electron-transport chain, resulting in the production of heat in the matrix. I

V

III

H+ H+

IV

V

UCP H+

H+

H+

Electron-transport chain Mitochondrial matrix

+

Inner membrane

H+

H+

H+

+

Pi

Pi

HEAT

Figure 57-5 Nonshivering thermogenesis in brown fat. AC, adenylyl cyclase; RXR, retinoid X receptor; THR, thyroid hormone receptor; TRE, thyroid response element.

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neonate uses glycogenolysis to mobilize hepatic glycogen stores, thereby releasing glucose into the bloodstream. The two enzymes needed for breaking down hepatic glycogen, phosphorylase, and glucose-6-phosphatase (G6Pase; see Chapter 58) are present in the fetus but do not become active until soon after birth. The newborn depletes his or her hepatic glycogen stores in the first 12 hours of life. Stores of glycogen in cardiac muscle are 10 times those in the adult, and those in the skeletal muscle are 3 to 5 times those in the adult, but the fetus mainly uses the glycogen stored in these tissues to provide glucose for local use. The net effect is that, during the first day of life, blood glucose levels may decline to 40 to 50 mg/dL, although they soon rise to near adult values once nutrition becomes adequate. Infants born of diabetic mothers run a very high risk of having pathologic hypoglycemia (i.e., 2 days), the liver metabolizes FAs to raise plasma levels of ketone bodies sufficiently to supply much of the brain’s oxidative fuel needs. The second priority for the body is to maintain its protein reserves (e.g., contractile proteins, enzymes, nervous tissue) in times of fasting. The body also has two main priorities for energy repletion after fasting. First, following a meal, liver and muscle replenish their limited glycogen reserves. Once these stores

The period after an overnight fast serves as a useful reference point because it represents the period before the transition from the fasted to the fed state. At this time, the concentrations of insulin, glucagon, and metabolic substrates that were altered by meal ingestion during the preceding day have returned to some baseline. Moreover, the body is in a relative steady state in which the rate of release of endogenous fuels from storage depots closely matches fuel consumption. Requirement for Glucose

After an overnight fast, the decline in circulating insulin leads to a marked decrease in glucose uptake by insulin-sensitive tissues (e.g., muscle) and a shift toward the use by these tissues of FAs mobilized from fat stores. Nevertheless, the average adult continues to metabolize glucose at a rate of 7 to 10 g/hr. Total body stores of free glucose, which exists mostly in the extracellular space, amount to only 15 to 20 g or ∼2 hours’ worth of glucose fuel. However, the useful glucose store is even less if we consider that the plasma [glucose]—normally ∼90 mg/dL (5.0 mM) after an overnight fast—may not fall to less than ∼55 mg/dL (3.0 mM) before brain function becomes abnormal. Thus, maintaining plasma [glucose] in the presence of this ongoing glucose use, particularly by the brain, requires that the body produce glucose at rates sufficient to match its ongoing consumption.

Gluconeogenesis Versus Glycogenolysis

Four to 5 hours after a meal (perhaps longer for a very large meal), a fall in plasma [insulin] and a rise in [glucagon] cause the liver to begin breaking down its stores of glycogen and releasing it as glucose (see Chapter 51). Moreover, both the liver and, to a lesser extent, the kidney generate glucose by gluconeogenesis. The release of glucose by these two organs is possible

Chapter 58 • Metabolism

Cori cycle Glucose-alanine cycle Liver

ECF

GLUT2

RBC Glucose

Lactate

Glycogen

Gly

co

AQP9

Glucose ~5 mM

G1P

ge

no

G6P

lys

is

Glycerol

Insulin independent Brain

Gluconeogenesis

Adipocytes Pyruvate Glu αKG

FA

Glycerol Alanine Lactate

FA

GLUT4 TAG

Muscle Glucose Alanine Gut Glutamine

Gluconeogenic substances: Lactate Alanine Glutamine Fatty acids (FA)

Lactate Ala Gln

Pyruvate

αKG Glu

αKA αAA FA

Glu αAA αKA Protein

Glycerol

Figure 58-13 Overnight fast. αAA, α-amino acid; AQP9, aquaporin 9; ECF, extracellular fluid; αKA, α-keto acid; αKG, α-ketoglutarate.

because they are the only two with significant amounts of G6Pase, which catalyzes the conversion of G6P to glucose. Net hepatic glycogenolysis and gluconeogenesis each contribute ∼50% whole-body glucose production during the first several hours of a fast. Gluconeogenesis: The Cori Cycle

In the first several hours of a fast, the brain consumes glucose at the rate of 4 to 5 g/hr, which is two thirds the rate of hepatic glucose production (∼180 g/day). Obligate anaerobic tissues also metabolize glucose but convert it primarily to lactate and pyruvate. The liver takes up these products and uses gluconeogenesis to regenerate glucose at the expense of energy. The liver releases the glucose for uptake by the glucoserequiring tissues, thus completing the Cori cycle (Fig. 58-13).

Gluconeogenesis: The Glucose-Alanine Cycle

After an overnight fast, the body as a whole is in negative nitrogen balance. Muscle and splanchnic tissues are the principal sites of protein degradation and release of amino acids into the blood. Alanine and glutamine, which are particularly important, represent ∼50% of total amino acid released by muscle, even though these amino acids represent only 10% to 13% of total amino acids in muscle protein. The reason that alanine and glutamine are overrepresented is that muscle synthesizes them (Fig. 58-13). During fasting, breakdown of muscle protein yields amino acids, which subsequently transfer their amino groups to α-ketoglutarate (supplied by

the citric acid cycle) to form glutamate. Glutamine synthase can then add a second amino group to glutamate, thus producing glutamine. Alternatively, alanine aminotransferase can transfer the amino group of glutamate to pyruvate (the product of glucose breakdown), thereby generating alanine and α-ketoglutarate. Both glutamine and alanine enter the blood. The intestine uses some of the glutamine as an oxidative fuel and releases the amino groups into portal blood as either alanine or ammonia. The amino acids taken up by the liver provide carbon for gluconeogenesis. On a molar basis, alanine is the principal amino acid taken up by the liver. In the first several hours of fasting, the liver principally uses alanine for gluconeogenesis. Because the carbon backbone of alanine came from glucose metabolism in muscle, and the liver regenerates glucose from this alanine, the net effect is a glucose-alanine cycle between muscle and liver, analogous to the Cori cycle. In addition to its role in gluconeogenesis, the glucosealanine cycle is critical for nitrogen metabolism, thus providing a nontoxic alternative to ammonia for transferring amino groups—derived from muscle amino acid catabolism—to the liver (Fig. 58-13). The hepatocytes now detoxify the amino groups on alanine and other amino acids by generating urea, which the kidney then excretes (see Figs. 39-6 and 46-14). Another key amino acid in nitrogen metabolism is glutamine, which muscle releases into the blood for uptake by the gut and liver as well as the kidney. The kidney uses the carbon skeleton of glutamine for renal gluconeogenesis and converts the amino group to ammonia, which it excretes

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(see Chapter 39). This ammonia excretion is particularly important in maintaining body acid-base balance during fasting. Combined, alanine and glutamine account for more than 40% of the amino acid carbon used by liver and kidneys in gluconeogenesis. Neither the Cori cycle nor the glucose-alanine cycle in muscle yields new carbon skeletons. Rather, both cycles transfer energy—and the glucose-alanine cycle also transfers nitrogen—between muscle and liver. The energy for hepatic glucose synthesis comes from oxidation of fat in the liver. Lipolysis

Finally, the fall in plasma [insulin] after an overnight fast permits the release of FAs and glycerol from fat stores (see Fig. 51-10). This response appears to be more pronounced in visceral than peripheral fat depots. The decline in [insulin] and the ensuing lipolysis are sufficient to supply FAs to extracerebral tissues (e.g., muscle, heart, liver) for fuel and glycerol to the liver for gluconeogenesis. However, these changes are not sufficient to stimulate the hepatic conversion of FA to ketone bodies. The body never completely suppresses gluconeogenesis. When an individual ingests a meal, gluconeogenic flux provides glucose for hepatic glycogen stores (indirect pathway). During fasting, the liver redirects the gluconeogenic flux to provide glucose for delivery to the circulation. Starvation beyond an overnight fast enhances gluconeogenesis and lipolysis

We have just seen that, during an overnight fast, glycogenolysis and gluconeogenesis contribute about equally to maintain a fasting plasma glucose concentration of ∼90 mg/ dL (5.0 mM). What happens if we extend our fast for 1 or 2 days? Because the glucose utilization rate is 7 to 10 g/hr, if half of this were provided by glycolysis (as is true for an overnight fast), the hepatic glycogen stores of ∼70 g that remain after an overnight fast would be sufficient to last only an additional day. However, in the early stages of starvation, the body compensates by accelerating gluconeogenesis. Orchestrating the metabolic adaptations in the early stages of starvation—increased gluconeogenesis, but also increased proteolysis and lipolysis—are a decline in [insulin] to a level lower than that seen after an overnight fast and a modest increase in portal vein [glucagon]. Insulin deficiency promotes all aspects of the metabolic response, whereas the effect of glucagon is confined to the liver (see Chapter 51). Enhanced Gluconeogenesis

Adaptations in both liver and muscle are responsible for increasing gluconeogenesis (Fig. 58-13). In muscle, acceleration of proteolysis leads to the release of alanine and other glycogenic amino acids, whereas the liver accelerates its conversion of gluconeogenic amino acids into glucose. This enhanced gluconeogenesis, however, is not the result of increased availability of substrates, because plasma levels of alanine and other glycogenic amino acids decline. Instead, fasting upregulates key gluconeogenic enzymes (see Chapter 51) and thus makes gluconeogenesis more efficient. The dependence of gluconeogenesis on proteolysis is reflected by an increase in urinary nitrogen excretion in the early phase of starvation. During the first 24 hours of a fast,

the average 70-kg person excretes 7 to 12 g of elemental nitrogen in the urine, equivalent to 50 to 75 g of protein. Because tissue protein content does not exceed 20% by weight for any tissue, 50 to 75 g of protein translates to 250 to 375 g of lean body mass lost on the first day of a fast. Enhanced Lipolysis

The activation of HSL increases release of FAs and glycerol from TAG stores in adipose tissue and muscle (Fig. 58-13). The increased availability of glycerol provides the liver with an additional substrate for gluconeogenesis, as discussed earlier, that contributes to glucose homeostasis. Moreover, the increased availability of FAs to muscle and other peripheral tissues limits their use of glucose, preserves glucose for the CNS and other obligate glucose-using tissues, and thereby diminishes the demands for gluconeogenesis and proteolysis. Elevated levels of FAs cause insulin resistance in skeletal muscle by directly interfering with the activation of GLUT4 (Fig. 58-13) by insulin. FAs activate a serine/threonine kinase cascade, leading to increased serine phosphorylation of insulin receptor substrate 1 (IRS-1; see Chapter 51), which, in turn, leads to decreased tyrosine phosphorylation of IRS-1 and thus a decrease in the PI3K that is necessary for the insertion of GLUT4 in muscle. This FA-induced decrease in glucose consumption and the parallel increased availability of FAs as a fuel for muscle spare glucose for other tissues under fasting conditions. However, this adaptation may play an important pathologic role in mediating the insulin resistance associated with obesity and type 2 diabetes. In addition to their effects on muscle, FAs enter the liver, where they undergo β oxidation and generate energy. A fall in the insulin-glucagon ratio inhibits ACC (see Fig. 51-12), reduces levels of malonyl CoA, and promotes mitochondrial FA oxidation. Thus, the hormonal changes both increase the supply of FAs and activate the enzymes necessary for FA oxidation. This β oxidation furnishes the energy and reducing power required for gluconeogenesis. If the availability of FAs outstrips the ability of the citric acid cycle to oxidize the resulting acetyl CoA, the result may be the accumulation of ketone bodies, which can serve as a fuel for the CNS as well as for cardiac and skeletal muscle. Prolonged starvation moderates proteolysis but accelerates lipolysis, thereby releasing ketone bodies As the duration of fasting increases, the body shifts from using its limited protein stores for gluconeogenesis to using fat for ketogenesis (Fig. 58-14). Moreover, the brain shifts from oxidizing glucose to oxidizing two ketone bodies, βhydroxybutyrate and acetoacetate, to meet most of its energy requirements. Decreased Proteolysis A fasting human could survive for only ∼10 days if totally dependent on protein utilization to meet whole-body energy requirements. Thus, prolonged survival during starvation requires a major reduction in proteolysis. Indeed, urea excretion decreases from 10 to 15 g/ day during the initial days of a fast to less than 1 g/day after 6 weeks of fasting. Because urea is the major obligatory osmolyte in the urine (see Chapter 38), this reduced urea

Chapter 58 • Metabolism

ECF

Liver

Acetone β-hydroxybutyrate Acetoacetate

Ketone bodies Glucose

RBC Glucose ~3 mM

GLUT2 Acetyl CoA

Lungs

Ketone bodies: Acetone βHB AAc

Insulin independent

G6P

β-oxidation

Brain

Gluconeogenesis Gluconeogenesis

Adipocytes

Acyl CoA

Pyruvate Glycerol

FA

Alanine Lactate

FA

GLUT4 TAG

AQP9

Kidney

Glucose

Muscle Glucose

Glutamine

Gluconeogenic substances: Lactate Alanine Glutamine Fatty acids (FA)

Lactate Alanine Other aa FA

Protein

Ketone bodies

Glycerol

Figure 58-14

Prolonged starvation. AAc, acetoacetate; ECF, extracellular fluid; βHB, β-hydroxybutyrate.

production lessens obligatory water excretion and therefore the daily water requirement. Ammonium excretion also decreases. Decreased Hepatic Gluconeogenesis

The transition from protein to lipid degradation permits humans to extend their survival time during a prolonged fast from weeks to months, as long as fat stores are available and water intake is adequate. During this transition, hepatic gluconeogenesis decreases (Fig. 58-14), mostly because of diminished substrate delivery. During the first few weeks of a fast, muscle releases less alanine, the principal substrate for hepatic gluconeogenesis, thus causing plasma [alanine] to fall markedly, to less than one third of the concentrations seen after the absorption of a meal. Indeed, during a prolonged fast, infusing a small amount of alanine causes plasma [glucose] to rise.

Increased Renal Gluconeogenesis

While hepatic gluconeogenesis falls, renal gluconeogenesis rises (Fig. 58-14), to reach as much as 40% of whole-body glucose production. Renal gluconeogenesis, which consumes H+ (see Fig. 39-5A), most likely is an adaptation to the acidosis that accompanies ketogenesis. Indeed, acidosis stimulates renal ammoniagenesis in parallel with renal gluconeogenesis.

Increased Lipolysis and Ketogenesis During the first 3 to 7 days of fasting, hypoinsulinemia accelerates the mobilization of FAs from adipose tissue and causes plasma levels of FAs to double; FA levels remain stable thereafter. The combination of low insulin and high glucagon levels also increases hepatic oxidation of FAs and leads to a marked increase of hepatic ketogenesis (Fig. 58-14) or ketogenic capacity. The liver achieves peak rates of ketone body production (∼100 g/day) by the third day and maintains them thereafter. Low insulin levels also progressively reduce the extraction of ketone bodies by peripheral tissues. Thus, despite relatively stable rates of ketone body production, circulating levels of ketone bodies continue to rise throughout the next few weeks. As a result, the CNS receives an increasing supply of these water-soluble substrates, which eventually account for more than one half of the brain’s energy requirements. In this way, ketone bodies ultimately supplant the brain’s dependency on glucose. Thus, by limiting the brain’s gluconeogenic demands, the body preserves protein stores. Besides the CNS, other body tissues, especially the heart and skeletal muscle, can use ketone bodies to cover a significant proportion of their energy demands. As the fast progresses, and fat stores are depleted, levels of leptin decrease. This decrease in leptin levels is a protective signal that profoundly affects the hypothalamic-

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pituitary-gonadal axes and reduces the oscillations of luteinizing hormone and follicle-stimulating hormone that cause anovulation. In times of famine, this mechanism protects fertile women from the additional nutritional demands associated with pregnancy. In summary, the body has evolved powerful adaptive mechanisms that ensure adequate substrate supply in the form of glucose and ketone bodies during a prolonged fast to maintain adequate CNS function. Even during a prolonged fast, humans do not lose consciousness because of decreased substrate supply to the brain. Instead, death under these conditions typically occurs when fat stores are depleted and severe protein wasting causes failure of respiratory muscles, which, in turn, leads to atelectasis and terminal pneumonia. REFERENCES Books and Reviews Hillgartner FB, Salati LM, Goodridge AG: Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis. Physiol Rev 1995; 75:47-76.

Jequier E, Tappy J: Regulation of body weight in humans. Physiol Rev 1999; 79:451-480. Koretsky AP: Insights into cellular energy metabolism from transgenic mice. Physiol Rev 1995; 75:667-688. Palmieri F: The mitochondrial transporter family (SLC25): Physiological and pathological implications. Pflugers Arch 2004; 447:689-709. Shulman GI, Landau BR: Pathways of glycogen repletion. Physiol Rev 1992; 72:1019-1035. Stahl A: A current review of fatty acid transport proteins (SLC27). Pflugers Arch 2004; 447:722-727. Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed. Philadelphia: WB Saunders, 1998. Journal Article Abu-Elheiga L, Wonkeun O, Parichher P, Wakil SJ: Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets. Proc Natl Acad Sci U S A 2003; 100:10207-10212.

CHAPTER

59

R E G U L AT I O N O F B O D Y T E M P E R AT U R E John Stitt

HEAT AND TEMPERATURE: THE ADVANTAGES OF HOMEOTHERMY Homeothermy enables an organism to maintain its activity over a wide range of environmental temperatures The ability to regulate internal body temperature has provided higher organisms independence from the environment. Because the rates of most physical and chemical reactions depend on temperature, most physiological functions are sensitive to temperature changes. Thus, the activity levels of poikilotherms (species that do not regulate internal body temperature) generally depend on environmental temperature, whereas those of homeotherms (species that do regulate internal body temperature) are relatively stable over a broad range of ambient conditions. A lizard, for example, is capable of relatively less movement away from its lair on a cold, overcast day than on a hot, sunny day, whereas a prairie dog may be equally mobile on either day. An arctic fox acclimatizes to the extreme cold of winter by maintaining a thick, insulating coat that enables it to resist body cooling and minimizes the necessity to increase metabolic heat generation, which would require increased food intake. The thermoregulatory system of homeotherms creates an internal environment in which reaction rates are relatively high and optimal. At the same time, an effective thermoregulatory system avoids the pathologic consequences of wide deviations in body temperature (Table 59-1). The thermoregulatory system incorporates both anticipatory controls and negative feedback controls. The components of this system are as follows: (1) thermal sensors; (2) afferent pathways; (3) an integration system in the central nervous system (CNS); (4) efferent pathways; and (5) target organs that control heat generation and transfer, such as skeletal muscle (e.g., shivering to generate heat), circulation to the skin (to dissipate heat), and the sweat glands (to dissipate heat). The focus of this chapter is temperature regulation in homeotherms. I examine the physical aspects of heat transfer both within the body and between body and environment, as well as the physiological mechanisms involved in altering

these rates of transfer. Finally, I look at the consequences of extreme challenges to the thermoregulatory mechanism, such as hyperthermia, hypothermia, and dehydration. Body core temperature depends on time of day, physical activity, time in the menstrual cycle, and age Temperature scales are relative scales of heat content. The centigrade scale is divided into 100 equal increments, referenced to the freezing (0ºC) and boiling (100ºC) points of water. The “normal” body temperature of an adult human is approximately 37ºC (i.e., 98.6ºF), but it may be as low as 36ºC or as high as 37.5ºC in active, healthy people. Body temperature usually refers to the temperature of the internal body core, measured under the tongue (sublingually), in the ear canal, or in the rectum. For clinical purposes, the most reliable (although the least practical) among these three is the last, because it is least influenced by ambient (air) temperature. Measurement devices range from traditional mercury-in-glass thermometers to electronic, digital readout thermistors. Nearly all such instruments are accurate to 0.1ºC. The least invasive approach uses an infrared thermometer to measure the radiant temperature over the temporal artery. Body core temperature depends on the time of day, the stage of the menstrual cycle in women, the level of the person’s activity, and the individual’s age. All homeotherms maintain a circadian (24-hour cycle) body temperature rhythm, with variations of ∼1ºC. In humans, body temperature is usually lowest between 3:00 to 6:00 am, and it peaks at 3:00 to 6:00 pm. This circadian rhythmicity is inherent in the autonomic nervous system, independent of the sleepwakefulness cycle, but it is entrained by light-dark cues to a 24-hour cycle. In many women, body temperature increases approximately 0.5ºC during the postovulatory phase of their menstrual cycle (see Chapter 55). An abrupt increase in body temperature of 0.3ºC to 0.5ºC accompanies ovulation and may be useful as a fertility guide. Physical activity generates excess heat as a byproduct of elevated metabolic rate. A portion of this excess heat remains

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Table 59-1 Consequences of Deviations in Body Temperature Temperature (ºC)

Consequence

40-44

Heat stroke with multiple organ failure and brain lesions

38-40

Hyperthermia (as a result of fever or exercise)

36-38

Normal range

34-36

Mild hypothermia

30-34

Impairment of temperature regulation

27-29

Cardiac fibrillation

in the body, causes the core temperature to rise, and triggers appropriately matching heat loss responses. Core temperature remains elevated during activity and for an extended period after exercise ceases. Infants and older people are less able to maintain a normal body temperature than are the rest of the population, particularly in the presence of external challenges. Newborns do not readily shiver or sweat and thus behave more like poikilotherms than like homeotherms. These properties, along with a high surface-to-mass ratio, render infants more susceptible to fluctuations in core temperature when exposed to a hot or cold environment. Older people are also subject to greater fluctuations in core temperature. Aging is associated with a progressive deficit in the ability to sense heat and cold, as well as a reduced ability to generate heat (reduced metabolic rate and metabolic potential because of lower muscle mass) and to dissipate heat (reduced cardiovascular reserve and sweat gland atrophy from disuse).

The body’s rate of heat production can vary from ~70 kcal/hr at rest to 600 kcal/hr during jogging The body’s rate of heat production is closely linked to the . rate of metabolism, the rate of O2 consumption (Vo2). Minor variations occur, depending on the mixture of fuels (foods) being oxidized, a process that determines the respiratory quotient (RQ; see Table 58-6). Because of their inherent inefficiency, metabolic transformations generate heat (see Chapter 58). Ultimately, all the energy contained in fuels appears as heat, mass storage or growth, or physical work done on the environment. The body’s metabolic rate, and thus its rate of heat production, is not constant. The resting metabolic rate (RMR; see Chapter 58) is the rate necessary to maintain the functions of resting cells; these functions include active transport as well as cardiac and respiratory muscle activity. Voluntary or involuntary (e.g., shivering) muscular activity adds to the overall metabolic heat production. Even digesting a meal

increases the metabolic rate (see Chapter 58). An increase in tissue temperature itself raises the metabolic rate, according to the van’t Hoff relation (i.e., a 10ºC increase in tissue temperature more than doubles the metabolic rate). Furthermore, certain hormones, notably thyroxine and epinephrine, increase the cellular metabolic rate. Because the body’s heat production rate is variable, the rate of heat loss must match it closely if the body temperature is to remain constant. At an RQ of 0.8 (see Chapter 58), the average .person under sedentary (i.e., RMR) conditions has a resting Vo2 of 250 mL/ min, which corresponds to a heat production of 72 kcal/hr (∼85 watts). In other words, an adult of average size generates the heat of an 85-watt light bulb (see Chapter 58). During physical exercise, the rate of energy production— and hence, heat generation—increases in proportion to the intensity of exercise. An average adult can comfortably sustain an energy production rate of 400 to 600 kcal/hr for extended periods (e.g., a fast walk or a modest jog). Nearly all this increased heat generation occurs in active skeletal muscle, although a portion arises from increased cardiac and respiratory muscle activity. A thermal load of this magnitude would raise core temperature by 1.0ºC every 8 to 10 minutes if the extra heat could not escape the body. Physical activity would be limited to 25 to 30 minutes, at which time the effects of excessive hyperthermia (>40ºC) would begin to impair body function. This impairment, of course, does not occur, primarily because of the effectiveness of the thermoregulatory system. Within a relatively short period, the increase in body temperature resulting from exercise leads to an increased rate of heat dissipation that matches heat production. Thereafter, the body maintains a new, elevated steady temperature. When exercise ceases, body temperature gradually decreases to its pre-exercise level.

MODES OF HEAT TRANSFER Maintaining a relatively constant body temperature requires a fine balance between heat production and heat losses Temperature homeostasis requires that increases or decreases in heat production balance increases or decreases in heat loss. Physiologists usually express this concept in terms of a whole-body heat balance equation, which, for an adult of constant mass, is as follows: Work Radiative Convective Evaporative Storage done on heat heat heat of loss Metabolism en nvironment loss loss heat (M W) − (R +  C + E) = S −  Heat production (H)

Heat losses

(59-1) All terms in the foregoing equation have the units kcal/hr. Several physiological processes contribute to temperature homeostasis, including modulation of metabolic heat production, physical heat transfer, and elimination of heat.

Chapter 59 • Regulation of Body Temperature

Table 59-2 Contribution of Body Systems to Resting Metabolism

CNS commands

System

RMR

Respiration and circulation

15%

CNS and nerves

20%

Core

Musculature (at rest)

20%

Tcore

Abdominal viscera

45%

RMR

100% (∼70 kcal/hr)

Metabolic control

Vasomotor control

Heat moves from the body core to the skin, primarily by convection Generally, all heat production occurs within the body’s tissues, and all heat elimination occurs at the body surface. Figure 59-1 illustrates a passive system in which heat flows

Skin Pskin (Sweat)

Tcore Heat carried by blood from core to skin Tcore

These processes operate at the level of cells, tissues, and organ systems. Let us discuss in order the terms in Equation 59-1. The rate of metabolism (M) arises from the cellular oxidation of carbohydrates, fats, and proteins. Because of their inherent inefficiency, metabolic transformations generate heat (see Chapter 58). Table 59-2 shows the fractional contributions of different body systems to total heat production under sedentary, resting conditions. Heat production by skeletal muscle can play a vital emergency role in temperature regulation in the cold. Shivering—the rhythmic, clonic activation of the major muscle masses surrounding the head, torso, and upper limbs—can increase total body heat production by as much as 400%. The body can grade the intensity of shivering and heat production to match heat loss. Physical exercise can generate even more heat . than shivering. Under conditions of maximal exercise, Vo2max may correspond to a total energy expenditure (M in Equation 59-1) of 1300 kcal/hr for an endurance athlete. If 75% to 80% of this energy evolves as heat—so that the athlete does ∼300 kcal/hr of useful work on the environment (W)—the rate of heat production (H = M − W) would be 1000 kcal/hr (∼1200 watts) for a brief period of time. This change is equivalent to changing from an 85-watt light bulb to a 1200-watt space heater. Unless the body can dissipate this heat, death from hyperthermia and heat stroke (see box on Heat Stroke) will ensue rapidly. Virtually all heat leaving the body must exit through the skin surface. In the following three sections, the three major routes of heat elimination are discussed: radiation (R), convection (C), and evaporation (E). As the heat balance equation shows, the difference between (M − W) and (R + C + E) is the rate of heat storage (S) within the body. The value of S may be positive or negative, depending on whether (M − W) > (R + C + E) or vice versa. A positive value of S results in a rise of body core temperature, and a negative value results in a fall.

Heat loss from skin

Sweating control

Fixed tissue conductance (minimal)

Ambient environment Pambient Evaporation

Tskin

Tradiant Radiation

Tskin

Tambient Convection

Figure 59-1 Passive or unregulated heat transfer. In the steady state, the rate of heat production by the body core must be matched by the flow of heat from the core to the skin, and from skin to environment. Various homeostatic controls—systems not directly involved in temperature regulation—can affect heat flow. Examples include sweating in response to hypoglycemia, changes in blood flow patterns in response to a fall in blood pressure, and changes in metabolism in response to alterations in thyroid metabolism.

depend on the size, shape, and composition of the body, as well as on the laws of physics. The circulation carries excess heat away from active tissues, such as muscle, to the body core—represented by the heart, lungs, and their central circulating blood volume. How does the body prevents its core from overheating? The answer is that the core transfers this heat to a dissipating heat sink. The organ serving as the body’s greatest potential heat sink is the relatively cool skin, which is the largest organ in the body. Only a minor amount of the body’s generated heat flows directly from the underlying body core to the skin by conduction across the body tissues. Most of the generated body heat flows to the skin by convection in the blood, and blood flow to the skin can increase markedly. There, nearly all the heat transferred to the skin must flow to the environment, facilitated by the skin’s large surface area. The transfer of heat from core to skin occurs by two routes: ⎤ ⎡Heat ⎤ ⎡Heat ⎤ ⎡ Heat ⎢ transfer ⎥ ⎢ passively conductted ⎥ ⎢convected by blood ⎥ ⎥ + ⎢from core to ⎥ ⎢from core ⎥ = ⎢ from core to ⎥ ⎢skin ⎥ ⎢ to skin ⎥ ⎢skin ⎦ ⎣ ⎦ ⎣ ⎦ ⎣ (59-2) Both the conduction and convection terms in the previous equation are proportional to the –temperature gradient from – core to skin (Tcore − Tskin), where Tskin is the average temperature of at least four representative skin sites. The proportionality constant for passive conduction across the subcutaneous fat (the body’s insulation) is relatively fixed. However, the

1239

Section X • Physiology of Everyday Life

Sun Solar radiation

Sweat (evaporation)

Radiation

Heat transfer by radiation occurs between the skin and solid bodies in the environment. The infrared portion of the electromagnetic energy spectrum carries this energy, which is why infrared cameras can detect warm bodies at night. The body gains or loses heat by radiation at a rate that is proportional to the temperature difference between the skin and the radiating body: Rate of radiative heat loss

R 

kcal h

=

Radiative heattransfer coefficient

Mean skin temp

Radiant temp of another object

Body surface area available forr radiative heat exchange

h Tskin − Tradiant )  Aradiat ive radiative    i (    i  2 kcal °C

(59-3)

m

h × °C × m2

R is positive when the body loses heat and negative when it gains heat. One may not be so aware of radiative heat fluxes to and from the body, particularly when the radiating body temperature (Tradiant) differs from the ambient environmental temperature (Tambient) that tends to dominate our attention. Indoors, Tradiant is the same as Tambient because surrounding objects thermally equilibrate with one another. Outdoors, radiating bodies may be at widely different temperatures. The radiant heat load from the sun to the body on a cloud-

Air temperature Air humidity

Skin blood flow (convection)

Respiratory (evaporation)

Body core

Airflow (convection)

Muscle blood flow (convection)

Heat moves from the skin to the environment by radiation, conduction, convection, and evaporation

Radiation from body Ground thermal radiation

Work Contracting muscle

ng

sp

ee

d

Reflected solar radiation

Conduction

ni

Figure 59-2 is a graphic summary of the heat balance equation (Equation 59-1) for an athlete exercising in an outdoor environment. This illustration depicts the movement of heat within the body, its delivery to the skin surface, and its subsequent elimination to the environment by radiation, convection, and evaporation.

Sky thermal radiation

un

proportionality constant for heat convection by blood is a variable term, reflecting the variability of the blood flow to the skin. The ability to alter skin blood flow, under autonomic control, is therefore the primary determinant of heat flow from core to skin. The capacity to limit blood flow to the skin is an essential defense against body cooling in the cold (hypothermia). A side effect, however, is that skin temperature falls. Conversely, the capacity to elevate skin blood flow is an essential defense against hyperthermia. On very hot days when skin temperature may be very high and close to body core temperature, even high skin blood flow may not be adequate to transfer sufficient heat to allow body core temperature to stabilize because the temperature gradient – (Tcore − Tskin) is too small. Although most of the heat leaving the core moves to the skin, a small amount also leaves the body core by the evaporation of water from the respiratory tract. The evaporative rate is primarily a function of the rate of ventilation (see Chapter 31), which, in turn, increases linearly with the metabolic rate over a wide range of exercise intensities.

R

1240

Figure 59-2 Model of energy transfer from the body to the environment.

less summer day may exceed the RMR by a considerable amount. The radiant heat load from a fire or a radiant lamp can provide substantial warming of bodies in the radiant field. Conversely, on a winter evening, the radiant heat loss from the body to a cloudless, dark sky—which has a low radiant temperature—may exceed RMR. Thus, one may feel a sudden chill when walking past an uncurtained window. This chill is caused by the sudden fall in skin temperature owing to increased radiant heat loss. Radiation of heat from the body accounts for ∼60% of heat lost when the body is at rest in a neutral thermal indoor environment. A neutral thermal environment is a set of conditions (air temperature, airflow and humidity, and temperatures of surrounding radiating surfaces) in which the temperature of the body does not change when the subject is at rest (i.e., RMR) and is not shivering. Conduction Heat transfer by conduction occurs when the body touches a solid material of different temperature. For

Chapter 59 • Regulation of Body Temperature

example, lying on the hot sand causes one to gain heat by conduction. Conversely, placing an ice pack on a sore muscle causes heat loss by conduction. However, under most normal circumstances (e.g., when one is standing and wearing shoes or recumbent and wearing clothes), the heat gain or loss by conduction is minimal. Convection

Heat transfer by convection occurs when a fluid such as air or water carries the heat between the body and the environment. The convective heat loss is proportional to the difference between skin and ambient temperature: Rate of Convective Mean Ambient convective heatskin n temp heat transfer temp loss coeffficient

C 

kcal h

Body surface area available for convective heat exchange

(59-4)

= h T − Tambient ) i  A convective convective    i (T   skin  °C

kcal h × °C × m 2

m2

C is positive when the body loses heat and negative when it gains heat. Whereas the radiative heat transfer coefficient (hradiative) is constant, the convective coefficient (hconvective) is variable and can increase up – to 5-fold when air velocity is high. Thus, even when (Tskin − Tambient) is fixed, convective heat loss increases markedly as wind speed increases. In the absence of air movement, the air immediately overlying the skin warms as heat leaves the skin. As this warmer and lighter air rises off the skin, cooler ambient air replaces it and, in turn, is warmed by the skin. This is the process of natural convection. However, with forced air movement, such as by wind or a fan, the cooler “ambient” air replaces the warmer air overlying the skin more rapidly. This change increases the effective convective heat transfer from the skin, even though the temperature of the ambient air is unchanged. This is a process of forced convection, which underlies the wind chill factor. Evaporation

Humans can dissipate nearly all the heat produced during exercise by evaporating sweat from the skin surface. The evaporative rate is independent of the temperature gradient between skin and environment. Instead, it is proportional to the water vapor pressure gradient between skin and environment: Rate of evaporative heat loss

E 

kcal h

Evaporative heattransfer coefficient

= hevaporative  

kcal h × mmHg × m2

H2 O vapor pressure of skin

i

H2 O vapor pressure of environment

(Pskin − Pambient )

mmHg

Body surface area available for evapoorative heat exchange

Aevaporative i  m2

(59-5) E is positive when the body loses heat by evaporation and negative when it gains heat by condensation.

The evaporation of 1 g of water removes ∼0.58 kcal from the body. Because the body’s sweat glands can deliver up to 30 g fluid/min or 1.8 L/hr to the skin surface, evaporation can remove 0.58 × 1800 g or ∼1000 kcal/hr. Thus, under ideal conditions (i.e., when ambient humidity is sufficiently low to allow efficient evaporation), evaporation could theoretically remove nearly all the heat produced during heavy exercise. As with convection, increased air velocity over the skin increases the effective vapor pressure gradient between skin and the overlying air because of the faster movement of water vapor away from the skin. The efficiency of heat transfer from the skin to the environment depends on both physiological and environmental factors. If ambient humidity is high, the gradient of water vapor pressure between skin and air will be low, thus slowing evaporation and increasing the body’s tendency to accumulate excess heat produced during exercise. This phenomenon underlies the temperature humidity index (heat index). Conversely, if ambient humidity is low, as in the desert, net heat loss from the body by evaporation will occur readily, even when ambient temperature exceeds skin temperature and the body is gaining heat by radiation and convection. When the body is immersed in water, nearly all heat exchange occurs by convection, because essentially no exchanges can occur by radiation or evaporation. Because of the high conductivity and thermal capacity of water, the heat transfer coefficient (hconvective) is ∼100 times greater than that of air. Thus, rate of heat exchange underwater is much greater than it is in air. It is therefore not surprising that nearly all the deaths in the Titanic shipwreck disaster resulted from hypothermia in the cold Atlantic waters, rather than from drowning. When heat gain exceeds heat loss, body core temperature rises With a knowledge of the transfer coefficients—hradiative (Equation 59-3), hconvective (Equation 59-4), and hevaporative (Equation 59-5)—and the gradients of temperature and water vapor pressure between the skin and environment, we can calculate the body . heat fluxes (R, C, and E). Knowing M (computed from Vo2 by indirect calorimetry; see Chapter 49) and W (if any), we can use the heat balance equation (Equation 59-1) to calculate the heat storage (S). From this value, we can predict the rate of change in mean body temperature: ΔTbody Rate of heat storagge = Δt 0 .83 ⋅ BW 

Rate of temperature increase

   °C h

Specific heat of body tissues

Body weight



(59-6)

kcal / h [kcal /(kg °C)] × kg

We can verify the accuracy of –this predicted rate of change – in Tbody by comparing it to the Tbody measured by direct thermometry, using a weighted average of the measured Tcore and average Tskin.

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The body has to deal with two types of heat loads that tend to make its temperature rise. In the heat balance equation (Equation 59-1), the term (M – W) constitutes an internal heat load. In contrast, the term (R + C + E)—normally representing a net heat loss—can represent an external heat load if either the radiation or convection terms are heat gains rather than – heat losses. Thus, if we stand in the sun and Tradiant exceeds Tskin (Equation 59-3), we experience a radiant heat – load. If we stand in a hot sauna and Tambient exceeds Tskin (Equation 59-4), we experience a convective heat load. Clearly, both internal and external heat loads can result in net heat storage and thus a rise in body temperature. Changes in environmental temperature (Tradiant and Tambient) exert their influence from the outside, through the body surface. If, starting from relatively low values, Tradiant or Tambient rises, at first the rate at which heat leaves the body decreases, so core temperature tends to rise. Further increases in environmental temperature produce a frank heat load rather than a loss. Conversely, metabolism produces heat inside the body. For the athlete, all the terms of the heat balance equation are important because dissipating the thermal load is essential for prolonging exercise. The clinician must understand these principles to treat thermally related illnesses. For example, excessive heat exposure can lead to heat exhaustion, in which core temperature rises to as high as 39ºC because the body cannot dissipate the heat load. The causes are dehydration (which reduces sweating) and hypovolemia (which reduces blood flow from muscle to core to skin). Heat exhaustion is the most common temperature-related abnormality in athletes. In more severe cases, excessive heat can lead to heat stroke (see the box on Heat Stroke), in which core temperature rises to 41ºC or more, as a result of impaired thermoregulatory mechanisms. Clothing insulates the body from the environment and limits heat transfer from the body to the environment Placing one or more layers of clothing between the skin and the environment insulates the body and retards heat transfer between the core and the environment. In the presence of clothing, heat transfer from a warmer body to a cooler environment occurs by the same means as without clothing (i.e., radiation, conduction, convection, and evaporation), but from the clothing surface rather than from the skin surface. The insulating effect of clothing is described by the clo unit. By definition, one clo is the insulation necessary to maintain a resting person at a thermal steady state in comfort at 21ºC with minimal air movement. Obviously, clo units increase with a greater area of skin coverage by clothing or with thicker clothing.

cipally part of the autonomic nervous system, and thermal effectors that either control heat transfer between the body and environment or regulate the body’s rate of heat production. This active system contrasts with the passive system. Thermal sensors in the skin and in the body core (mainly the hypothalamus) respond to changes in theirlocal temperature The body has specialized sensory neurons that provide the CNS with information about the body’s thermal condition. These thermosensitive elements are free nerve endings that are distributed over the entire skin surface. These elements are also present within the body core, at particularly high densities in the preoptic area and anterior hypothalamus. Skin receptors, although ideal for sensing changes in environmental temperature, do not serve well during exercise because internal temperatures would rise to intolerably high levels before the skin temperature rose to detect this excess heat. Body core thermoreceptors, in contrast, although ideal for detecting changes in core temperature, are inadequate for sensing changes in the environmental temperature. Because of the thermal inertia of the body’s mass, the lag time in using body core sensors to detect externally induced changes in temperature would be too great to achieve effective regulation. Not surprisingly, then, the body is endowed with both peripheral and central thermoreceptors that are integrated within the CNS, to permit a rapid and effective balance of heat loss and heat production while maintaining body core temperature within relatively narrow limits. Skin Thermoreceptors The entire surface of the body has a network of sensory nerve endings that serve as thermoreceptors. Peripheral thermoreceptors fall into two categories—warmth receptors and cold receptors (Fig. 59-3). Each type is anatomically distinct, and each innervates definable warm-sensitive or cold-sensitive spots on the skin surface (see Chapter 15). Thermal discrimination varies over the surface of the body; it is coarsest on the body trunk and

40 30

Warmth receptor fibers

Mean steady firing rate 20 (impulses/ sec) 10

Cold receptor fibers

ACTIVE REGULATION OF HEAT TRANSFER The body actively regulates its temperature by a feedback system that includes temperature sensors, afferent nerve fibers that carry sensory information to the brain, a hypothalamic control center, efferent nerve fibers that are prin-

0 15

20 30 40 Skin temperature (°C)

50

Figure 59-3 Response of warmth and cold receptors to temperature change.

Chapter 59 • Regulation of Body Temperature

Body core thermoreceptors are especially important during exercise, which is one of the few conditions in which the body’s heat production and dissipation rates can differ dramatically and can lead to rapid changes in core temperature.

Tset CNS controller

Tcore inputs

Tskin inputs

Shiver command

The hypothalamic center integrates thermal information and directs changes in efferent activity to modify heat transfer rates

Sweat command

Figure 59-4 regulation.

Heat carried by blood from core to skin

Fixed tissue conductance (minimal)

Thermoreceptors in the skin

Thermoreceptors in the core

Vasomotor command C W Heat loss to the environment

Model of negative feedback in temperature

limbs and finest on the face, lips, and fingers. Increasing local temperature, up to 44ºC to 46ºC, causes warmth receptors to increase their steady firing rate (Fig. 59-3). Cold receptors characteristically increase their steady firing rate as local temperature decreases from ∼40ºC to 24ºC to 28ºC. In either case, a sustained temperature change (see Fig. 15-28) may cause a stable change in the sensor’s firing rate (i.e., tonic or static response) or a temporary change (i.e., phasic or dynamic response). Because of their location, skin thermoreceptors primarily provide the hypothalamic thermoregulatory center with information about ambient temperature (Fig. 59-4). As discussed later, they provide an anticipatory signal in conditions of rapidly changing ambient temperature and allow the autonomic nervous system to exert reflex thermoregulation. Information from skin thermoreceptors also travels through thalamic pathways to the cerebral cortex, thus providing the basis for conscious perception of the thermal environment and appreciation of thermal comfort. We can use this information, for example, to move from the sun to the shade when we sense that we are too hot. Body

Core Thermoreceptors Thermoreceptors are present in the brain, in the spinal cord, and perhaps in the muscles and major blood vessels. However, the hypothalamus clearly plays the major role in detecting changes in deep body temperature (Fig. 59-4). In the preoptic area and anterior hypothalamus (see Chapter 14), ∼10% of neurons will show a positive temperature coefficient when local temperature is cycled over a range of 2ºC to 4ºC about the mean.

Cooling or warming of the skin alters both the tonic and the phasic components of the activity of cold or warmth receptors (Fig. 59-4). The neural activity of these skin thermoreceptors travels through the spinal cord to the hypothalamus, which integrates thermal information from other parts of the body, including the hypothalamus, compares the prevailing thermal status with an idealized set of thermal conditions, and directs efferent commands to alter the rate of heat generation and to modify heat transfer rates within and from the body. The skin receptors provide information mainly about environmental temperature, which affects the body’s heat loss rate and could ultimately cause core temperature to change, if the body does not initiate the appropriate thermoregulatory responses to skin cooling or warming. Thus, reflex responses to changes in the average skin temperature may be thought of as anticipatory rather than negative feedback in nature. Moreover, it is impossible to regulate skin temperature because of the skin’s exposure to the ambient environment. However, these anticipatory reflexes are essential elements for an effective thermoregulatory system because the body’s thermal inertia is too great to rely on central receptors alone. For example, low skin temperature—enhanced by cutaneous vasoconstriction in the cold— ensures a rapid and continuous cold signal that maintains a drive for shivering and thus thermogenesis. Conversely, thermoregulatory responses to changes in core (i.e., hypothalamic) temperature, such as those that occur during exercise, exhibit negative feedback, inasmuch as they modify heat transfer rates that maintain the core temperature at its regulated level. How do skin and core thermoreceptor inputs interact and how does the CNS integrate these inputs to produce appropriate thermoregulatory responses to both external and internal heat loads? The most plausible explanation is that signals from skin thermoreceptors change the sensitivity of the response to signals from core thermoreceptors. For example, in Figure 59-5 a decrease in core temperature produces an effector response (i.e., increased metabolic rate) that depends upon the level of input from skin cold thermoreceptors. Thus, the “gain” of the centrally induced metabolic response increases as skin temperature falls. Thermal effectors include the cutaneous circulation, sweat glands, and skeletal muscles that are responsible for shivering Figure 59-4 summarizes the three effectors of the thermoregulatory system. Adjusting the smooth muscle tone of cutaneous arterioles controls blood flow, and therefore heat flow, from the core to the skin surface, the primary

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Section X • Physiology of Everyday Life

9.0

Tskin = 32.2°C

6.0 Metabolic rate (w/kg)

Tskin = 34.3°C

Exercise

Tskin = 30.5°C Normal hypothalamic temperature Resting metabolic rate

1000

Metabolic heat production

800 Energy flow (kcal/hr)

400

Convective loss

200 0

3.0

Radiative loss

Evaporative loss

600

0

10

20 Time (min)

30

40

Thermal steady state 0

33

36 39 42 Hypothalamic temperature (°C)

45

Figure 59-5 Thermoeffector responses. In these experiments on rabbits, the investigators implanted water-perfused thermodes to control the temperature of the preoptic/anterior hypothalamic area (x-axis) at three different skin temperatures (Tskin).

Body core temperature (⬚C)

37.5 37.0

Mean skin temperature (⬚C)

site of heat dissipation to the environment. Over most of the skin, the autonomic nervous system controls cutaneous blood flow (see Chapter 24). When it is necessary to increase heat dissipation, active vasodilation can increase cutaneous blood flow up to 10-fold above the resting level. Conversely, when it is necessary to conserve heat in a cold environment, cutaneous vasoconstriction—mediated by sympathetic nerves—can elicit a relatively minor reduction in cutaneous blood flow, to half the resting rate. This vasoconstriction occurs at the expense of allowing skin temperature to drop closer to ambient temperature. Even with maximal vasoconstriction in effect, heat losses to a very cold environment do not fall to zero of the minimum tissue conductance. With a moderate heat load, the autonomic response primarily increases the heat transfer rate from core to skin by increasing cutaneous blood flow. However, when the heat load is sufficiently great, the autonomic nervous system also activates the eccrine sweat glands (see Chapter 60), which secrete sweat onto the skin surface, thus elevating the partial pressure of water vapor there and promoting increased evaporation. The innervation of the secretory segment of the sweat gland is sympathetic, but it is unusual in that acetylcholine is the neurotransmitter (see Chapter 14). When a cold stress is sufficiently great, the physiological response includes increasing heat production by involuntary, clonic, rhythmic contractions and relaxations of skeletal muscle. This shivering can double the metabolic rate for extended periods (hours) before fatigue occurs; for brief intervals, shivering can triple or quadruple the metabolic rate. Nonshivering thermogenesis in newborn infants and hibernating animals can also produce substantial amounts of heat, primarily in brown fat cells (see Chapter 57).

38.0

Figure 59-6

0

10

20 Time (min)

30

40

0

10

20 Time (min)

30

40

31.0 30.0 29.0

Whole-body heat balance during exercise.

HYPERTHERMIA, HYPOTHERMIA, AND FEVER Exercise raises heat production, followed by a matching rise in heat loss, but at the cost of a steady-state hyperthermia of exercise At the onset of muscular exercise, the rate of heat production increases in proportion to the exercise intensity and exceeds the current rate of heat dissipation, thus causing heat storage and a rise in core temperature (Fig. 59-6). Hypothalamic thermoreceptors sense this increase in core temperature. The hypothalamic integrator compares this temperature signal with a reference signal, detects an error between the two, and directs neural output that activates heat dissipation (Fig. 59-4). As a result, skin blood flow and sweating increase as core temperature rises. These processes thus promote an increase in the rate of heat transfer from core to environment and slow the rate of temperature rise. At some point, the rising rate of heat dissipation equals the rate of heat production, and the rate of heat storage falls to zero. However, the now elevated steady-state core temperature persists as long as exercise continues. The steady-state core temperature during exercise is not “regulated” at the elevated level; rather, the hyperthermia of exercise is the consequence of the initial imbalance between rates of heat production and dissipation. This imbalance is unavoidable because temperature must increase to provide the error signal that culminates in increased heat dissipation and because the response is not instantaneous. In Figure 59-6, metabolic heat production rises rapidly to its maximal

Chapter 59 • Regulation of Body Temperature

level. However, evaporative heat loss increases only after a delay and then rises slowly to its maximal level, driven by increasing body temperature. The result is net storage during the first 15 minutes. The slight initial drop in core temperature at the onset of exercise is caused by flushing out of blood from the cooler peripheral circulation when the muscle and skin beds vasodilate in response to the onset of exercise. In addition, mean skin temperature decreases during exercise because of the increased evaporative cooling of the skin caused by sweating. Hyperthermia or hypothermia occurs when heat transfer from or to the environment overwhelms the body’s regulatory capacity Although the body’s temperature regulating machinery is impressive, its capabilities are not limitless. Any factor that causes sufficiently large shifts—either positive or negative— in the rate of heat storage (Equation 59-1) could result in progressive hyperthermia or hypothermia (Equation 59-6). Because humans must operate within a fairly narrow core temperature range, such temperature changes could become life-threatening. The most common environmental condition that results in excessive hyperthermia is prolonged exposure to heat and high ambient humidity, particularly when accompanied by physical activity (i.e., elevated heat production rate). The ability to dissipate heat by radiation falls as the radiant temperature of nearby objects increases (Equation 59-3), and the ability to dissipate heat by convection falls as ambient temperature increases (Equation 59-4). When ambient temperature reaches the mid-30s (ºC), evaporation becomes the only effective avenue for heat dissipation. However, high ambient humidity reduces the skin-to-environment gradient for water vapor pressure and thus reduces evaporation (Equation 59-5). The combined reduction of heat loss by these three pathways can markedly increase the rate of heat storage (Equation 59-6) and can cause progressive hyperthermia. It is uncommon for radiative or convective heat gain to cause hyperthermia under conditions of low ambient humidity, because the body has a high capacity for dissipating the absorbed heat by evaporation. Radiative heat gain can be excessively high during full exposure to the desert sun or during exposure to heat sources such as large furnaces. The most obvious protections against radiative hyperthermia are avoiding radiant sources (e.g., sitting in the shade) and covering the skin with loose clothing. Loose clothing screens the radiation while allowing air circulation and thereby maintaining evaporative and convective losses. The most common environmental condition causing excessive hypothermia is prolonged immersion in cold water. Water has a specific heat per unit volume that is approximately 4000 times that of air and a thermal conductivity that is approximately 25 times that of air. Both properties contribute to a convective heat transfer coefficient (hconvective in Equation 59-4) that is approximately 100-fold greater in water than it is in air. The hconvective is ∼200 kcal/(m2/ hr/ºC) at rest in still water but ∼500 kcal/(m2/hr/ºC) while swimming. The body’s physiological defenses against hypothermia include peripheral vasoconstriction (increasing

insulation) and shivering (increasing heat production), but even these measures do not prevent hypothermia during prolonged exposure because of water’s high thermal conductivity. A thick layer of insulating fat retards heat loss to the water and postpones or even prevents hypothermia during prolonged exposures. Endurance swimmers used this knowledge to protect themselves when they applied a thick layer of grease to the skin surface (now, more commonly, they don a wet suit) before an event. Herman Melville noted this principle in 1851, when he referred to the low thermal conductivity of fat: For the whale is indeed wrapt up in his blubber as in a real blanket. . . . It is by reason of this cozy blanketing that the whale is enabled to keep himself comfortable in all seas. . . . this great monster, to whom corporeal warmth is as indispensable as it is to man. . . . — Moby Dick Like blubber, clothing adds insulation between skin and environment and thus reduces heat loss during exposure to the cold. The more skin one covers, the more one reduces the surface area for direct heat loss from skin to environment by convection and radiation. Adding layers of clothing increases the resistance of heat flow by trapping air, which is an excellent insulator. During heat exposure, the major avenue for heat loss is evaporation of sweat. Because evaporation also depends on the surface area available, the amount of clothing should be minimized. Wetting the clothing increases the rate of heat loss from the skin because water is a better conductor than air. Water also can evaporate from the clothing surface, thereby removing heat from the outer layers and increasing the temperature gradient (and rate of heat loss) from skin to clothing.

Heat Stroke

A

s body core temperature rises, excessive cutaneous vasodilation can lead to a fall in arterial pressure (see Chapter 25) and therefore to a decrease in brain perfusion. As core temperature approaches 41ºC, confusion and, ultimately, loss of consciousness occur. Excessive hyperthermia (>41ºC) leads to the clinical condition known as heat stroke. High temperature can cause fibrinolysis and consumption of clotting factors and thus disseminated intravascular coagulation (DIC), which results in uncontrolled vascular thrombosis and hemorrhage. Heat-induced damage to the cell membranes of skeletal and myocardial muscle leads to rhabdomyolysis (in which disrupted muscle cells release their intracellular contents, including myoglobin, into the circulation) and myocardial necrosis. Cell damage may also cause acute hepatic insufficiency and pancreatitis. Renal function, already compromised by low renal blood flow, may be further disrupted by the high plasma levels of myoglobin. Ultimately, the CNS is affected by the combination of high brain temperature, DIC, and metabolic disturbances.

1245

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Section X • Physiology of Everyday Life

A

EXERCISE HYPERTHERMIA Heat storage

Energy flow (kcal/hr)

B

Exercise

Heat production

Heat losses

Heat production

FEVER HYPERTHERMIA

In heat

Heat storage Or

Heat losses

Heat production Heat storage

In cold

Heat losses

Rate of heat storage

Tcore Temperature

Negative error signal is sustained.

Tset Tcore Tset

Positive error signal is corrected.

Figure 59-7 Exercise hyperthermia versus fever. A, The top panel shows how, during exercise, heat production temporarily exceeds heat loss, thus resulting in net heat storage. The middle panel shows that the rate of heat storage is highest initially and falls to zero in the new steady state. Finally, the bottom panel shows that as body core temperature rises away from the set-point, the error signal gradually increases. In the new steady state, the error signal is maximal and sustained. B, The top two panels show how, during fever, net heat storage can occur because of either reduced heat loss or increased heat production. The third panel from the top shows that, as in exercise, the rate of heat storage is highest initially. The bottom panel shows that as body core temperature rises, it approaches the new elevated set-point. Thus, the error signal is initially maximal and gradually decreases to zero in the new steady state.

Fever, unlike other types of hyperthermia, reflects an increase in the set point for temperature regulation Fever is a regulated elevation of core temperature resulting from effects associated with infection or disease. Fever is caused by the action of circulating cytokines called pyrogens, which are low-molecular-weight polypeptides produced by cells of the immune system. As for the hyperthermia of exercise, fever begins when heat production temporarily exceeds heat dissipation. However, fever differs from other hyperthermias in that the hypothalamus actively regulates core temperature to an elevated set point. Figure 59-7 illustrates the basic differences between the events leading to exercise hyperthermia and those leading to fever. During the genesis of exercise hyperthermia (Fig. 59-7A), the rate of heat production increases to more than the rate of heat dissipation for a period and causes net heat storage. Moreover, the temperature set point (Tset) is unchanged, and thus the error signal gradually increases to a new, sustained level. During the genesis of a fever (Fig. 59-7B), Tset suddenly increases to a value higher than the normal temperature, so the integrator interprets the normal temperature as being lower than the new Tset. The fever is an

appropriate response to this condition and develops as the heat loss rate from the body falls or the heat production rate rises until such time as core temperature increases to the new “regulated” level. Thus, the error signal is initially large but becomes smaller as the fever develops. In the new steady state, core temperature remains elevated until the signals responsible for the fever (i.e., pyrogens) subside and Tset returns to normal. The subjective assessments of thermal comfort support this description. During exercise, one perceives the rise in core temperature as body heating and may choose to remove clothing to cool the body. During the onset of a fever, however, the individual feels cold and may choose to put on additional clothing and warm the body. If fever strikes when the patient is in a warm environment in which the cutaneous vessels are dilated (Fig. 59-7B, top panel), the response to the Tset increase will be to vasoconstrict, which decreases heat loss. In contrast, if the patient is in a cold environment in which the cutaneous vessels are already constricted (Fig. 59-7B, second panel), the response will be to shiver. Figure 59-8 summarizes the responses to fever-producing stimuli. Macrophages and, to a lesser extent, lymphocytes release cytokines into the circulation in response to a variety of infectious and inflammatory stimuli. Cytokines, the

Chapter 59 • Regulation of Body Temperature

FEVER-PRODUCING STIMULI Gram-negative bacilli Viruses Fungi Endotoxins Antigen/antibody reactions

BLOODBRAIN BARRIER

CIRCULATION Cytokines Macrophage Immunocompetent and reticuloendothelial cell systems

Liver and metabolic responses

Cytokines breach the blood-brain barrier.

Brain (OVLT: endothelial cells)

T lymphocyte Release of prostaglandin (PGE2) Immune response: activation of lymphocytes, neutrophils, macrophages

Acute-phase response

Hypothalamus

Increases Tset

Fever

Figure 59-8

Host defense response.

messenger molecules of the immune system, are a diverse group of proteins involved in numerous tasks in the host defense response. The first is the immune response to foreign substances including stimulation of T-lymphocyte proliferation, natural killer cells, and antibody production. The second is the acute-phase response to foreign substances, a diffuse collection of nonspecific host reactions to infection or trauma. Finally, cytokines may act as endogenous pyrogens (Table 59-3). However, no one cytokine, administered experimentally, can fully mimic the temperature increase that occurs during fever. Fever production may occur through a cascade that is initiated when interleukin (IL-1β), for example, interacts with the endothelial cells in a leaky portion of the blood-brain barrier (see Chapter 11) located in the capillary bed of the organum vasculosum laminae terminalis (OVLT). The OVLT is highly vascular tissue that lies in the wall of the third ventricle (above the optic chiasm) in the brain. IL-1β triggers endothelial cells within the OVLT to release prostaglandin E2 (see Chapter 3), which then diffuses into the adjacent hypothalamus and—in a manner not yet understood—elevates Tset and initiates the febrile response. The value of fever in fighting infection is still debated. A popular hypothesis is that the elevated temperature enhances the host’s response to infection. This view is supported

Table 59-3

Endogenous Pyrogens

Pyrogen

Symbol

Interleukin 1α

IL-1α

Interleukin 1β

IL-1β

Interleukin 6

IL-6

Interleukin 8

IL-8

Tumor necrosis factor α

TNF-α

Tumor necrosis factor β

TNF-β

Macrophage inflammatory protein 1α

MIP-1α

Macrophage inflammatory protein 1β

MIP-1β

Interferon α

INF-α

Interferon β

INF-β

Interferon γ

INF-γ

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by the in vitro observation that the rate of T-lymphocyte proliferation in response to interleukins is many-fold higher at 39ºC than it is at 37ºC. REFERENCES Books and Reviews Blatteis CM, Sehic E: Fever: How may circulating pyrogens signal the brain? News Physiol Sci 1997; 12:1-9.

Block BA: Thermogenesis in muscle. Annu Rev Physiol 1994; 56:535-577. Horowitz M: Do cellular heat acclimation responses modulate central thermoregulatory activity? News Physiol Sci 1998; 13:218-225. Lee-Chiong TL, Stitt JT Jr: Disorders of temperature regulation: Compr Ther 1995; 21:697-704. Simon HB: Current concepts: Hyperthermia. N Engl J Med 1993; 329:483-487.

CHAPTER

60

E X E R C I S E P H YS I O LO GY A N D S P O RTS S C I E N C E Steven S. Segal

Physical exercise is often the greatest stress that the body encounters in the course of daily life. Skeletal muscle typically accounts for 30% to 50% of the total body mass. Thus, with each bout of muscular activity, the body must make rapid, integrated adjustments at the level of cells and organ systems—and must modulate these adjustments over time. The subdiscipline of exercise physiology and sports science focuses on the integrated responses that enable the conversion of chemical energy into mechanical work. To understand these interdependent processes, one must appreciate where regulation occurs, the factors that limit performance, and the adaptations that occur with repetitive use. The cross-bridge cycle that underlies contraction of skeletal muscle requires energy in the form of ATP (see Chapter 9). To supply this energy, skeletal muscle converts ∼25% of the energy stored in foodstuffs into mechanical work. The rest appears as heat as a result of the inefficiencies of the biochemical reactions (see Chapter 58). Thus, the dissipation of this heat is central to cardiovascular function, fluid balance, and the ability to sustain physical effort—an example of an integrated organ system response. Moreover, because muscle stores of ATP, phosphocreatine (PCr), and glycogen are limited, the ability to sustain physical activity requires another set of integrated cellular and organ system responses to supply O2 and energy sources to active muscles.

MOTOR UNITS AND MUSCLE FUNCTION In Chapter 9, the cellular and molecular physiology of skeletal muscle contraction is discussed. In this major section, we examine the way in which these smaller elements integrate into a contracting whole muscle. The motor unit is the functional element of muscle contraction A typical skeletal muscle receives innervation from ∼100 somatic motor neurons. The motor unit consists of a single motor neuron and all the muscle fibers that it activates.

When the motor neuron generates an action potential, all the fibers in the motor unit fire simultaneously. Thus, the fineness of control for movement varies with the innervation ratio—the number of muscle fibers per motor neuron. As discussed later, the small motor units that are recruited during sustained activity contain a high proportion of type I muscle fibers, which are highly oxidative and resistant to fatigue. In contrast, the large motor units that are recruited for brief periods—for rapid, powerful activity—typically consist of type IIa and IIb (see Chapter 9) muscle fibers, which are glycolytic and are much more susceptible to fatigue. Within a whole muscle, muscle fibers of each motor unit intermingle with those of other motor units so extensively that—in a volume of muscle that contains 100 muscle fibers—nerve endings from perhaps 50 different motor neurons synapse on the 100 end plates. Within some muscles, the fibers of a motor unit are constrained to discrete compartments. This anatomical organization enables different regions of a muscle to exert force in somewhat different directions, thereby enabling more precise control of movement. Muscle force rises with the recruitment of motor units and an increase in their firing frequency During contraction, the force exerted by a muscle depends on (1) how many motor units are recruited and (2) how frequently each of the active motor neurons fire action potentials. Motor units are recruited in a progressive order, from the smallest (i.e., fewest number of muscle fibers) and therefore the weakest motor units to the largest and strongest. This intrinsic behavior of motor unit recruitment is known as the size principle and reflects inherent differences in the biophysical properties of respective motor neurons. For a given amount of excitatory input (i.e., depolarizing synaptic current; Isyn in Fig. 60-1), a neuronal cell body with smaller volume and surface area has a higher membrane input resistance. Therefore, the depolarizing voltage in a neuron with a smaller neuronal cell body rises to threshold more quickly than in a neuron with a larger cell body (Fig. 60-1). Because the neurons with the small cell bodies tend to innervate a small number of slow-twitch (type I) muscle

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Interneuron Isyn Isyn Large-diameter motor neuron

Small-diameter motor neuron

Rm is high... and conduction velocity is low.

Rm is low... and conduction velocity is high.

Slow-twitch (type I) fibers Action potential Vm 0 Rest

EPSP

Fast-twitch (type II) fibers

Vm 0 EPSP Threshold

Threshold Rest

Figure 60-1 The size principle for motor units. Small motor neurons are more excitable, conduct action potentials more slowly, and excite fewer fibers that tend to be slow twitch (type I). Conversely, large motor neurons are less excitable, conduct action potentials more rapidly, and excite many fibers that tend to be fast twitch (type II). EPSP, excitatory postsynaptic potential. Isyn, depolarizing synaptic current; Vm, membrane potential. (Adapted from Kandel ER, Schwartz JH, Jessell TM. Principles of Neural Science, 4th ed. New York: McGraw-Hill, 2000.)

fibers, the motor units with the greatest resistance to fatigue are the first to be recruited. Conversely, the neurons with the larger cell bodies tend to innervate a larger number of fasttwitch (type II) fibers, so the largest and most fatigable motor units are the last to be recruited. Because the relative timing of action potentials in different motor units is asynchronous, the force developed by individual motor units integrates into a smooth contraction. As a muscle relaxes, the firing of respective motor units diminishes in reverse order. At levels of force production lower than the upper limit of recruitment, gradations in force are accomplished through concurrent changes in the number of active motor units and the firing rate of those that have been recruited—rate coding. Once all the motor units in a muscle have been recruited, any further increase in force results from an increase in firing rate. The relative contribution of motor unit recruitment and rate coding varies among muscles. In some cases, recruitment is maximal by the time muscle force reaches ∼50% of maximum, whereas in others, recruitment continues until the muscle reaches nearly 90% of maximal force. In addition to the intrinsic membrane properties of motor neurons (i.e., the size principle), other neurons that originate in the brainstem project to the motor neurons and

release the neuromodulatory neurotransmitters serotonin and norepinephrine (see Fig. 13-6). For example, this neuromodulatory input, acting on the motor neurons of small, slow-twitch motor units, can promote self-sustained levels of firing of the motor neurons during the maintenance of posture. In contrast, the withdrawal of this excitatory neuromodulatory input during sleep promotes muscle relaxation. Thus, the brain can control the overall gain of a pool of motor neurons. Compared with type I motor units, type IIb units are faster and stronger but more fatigable Within a given motor unit, each muscle fiber is of the same functional type. As summarized in Tables 9-1 and 9-2, the three muscle fiber types—type I, type IIa, and type IIb— differ in contractile and regulatory proteins, the content of myoglobin (and thus color) and mitochondria and glycogen, and the metabolic pathways used to generate ATP (i.e., oxidative versus glycolytic metabolism). These biochemical properties determine a range of functional parameters, including (1) speeds of contraction and relaxation, (2) maximal force, and (3) susceptibility to fatigue (Fig. 60-2). In response to an action potential evoked through the motor axon, slow-twitch (type I) motor units (top row of Fig. 60-2A) require relatively long times to develop tension and return to rest. In contrast, fast-twitch (types IIa and IIb) motor units exhibit relatively short contraction and relaxation times (top row of Fig. 60-2B, C). Accordingly, during repetitive stimulation (middle row of Fig. 60-2), slow-twitch motor units summate to a fused tetanus at lower stimulation frequencies than do fast-twitch motor units. Indeed, the α motor neurons in the spinal cord that drive slow motor units fire at frequencies of 10 to 50 Hz, whereas those that drive fast motor units fire at frequencies ranging from 30 Hz to more than 100 Hz. The maximal force that can develop per cross-sectional area of muscle tissue is constant across fiber types (∼25 N/ cm2). Therefore, the ability of different motor units to develop active force is directly proportional to the number and diameter of fibers each motor unit contains. In accord with the innervation ratios of motor units, peak force production (middle row of Fig. 60-2) increases from type I motor units (used for fine control of movement) to type II motor units (recruited during more intense activities). The susceptibility to fatigue of a motor unit depends on the metabolic profile of its muscle fibers. The red type I muscle fibers have greater mitochondria content and can rely largely on the aerobic metabolism of sugars and lipid for energy because they are well supplied with capillaries for delivery of O2 and nutrients. Type I motor units, although smaller in size (and innervation ratio), are recruited during sustained activity of moderate intensity and are highly resistant to fatigue (bottom row of Fig. 60-2A). In contrast, the larger type II motor units are recruited less often—during brief periods of intense activity—and rely to a greater extent on short-term energy stores (e.g., glycogen stored within the muscle fiber). Among type II motor units, type IIa motor units have a greater mitochondrial content, a larger capacity for aerobic energy metabolism, a greater O2 supply, and a

Chapter 60 • Exercise Physiology and Sports Science

A

TYPE I (SLOW)

B

TYPE IIa (FAST, FATIGUE-RESISTANT)

C

TYPE IIb (FAST, FATIGABLE)

Twitch 50 g

2g

10 g 50 msec

50 msec

50 msec

Unfused tetanic force

13 Hz

2g 500 msec Fatigability

250 msec

30 g

2 0

25 Hz

250 msec

4 g

50 g

20 Hz

10 g

50

20

g

10 0

2

4

6

60

0

0

2

min

4 min

6

50

0

0

2

4

6

15

min

Figure 60-2 A to C, Properties of fiber types (i.e., motor units in gastrocnemius muscle). The top row shows the tension developed during single twitches for each of the muscle types; the arrows indicate the time of the electrical stimulus. The middle row shows the tension developed during an unfused tetanus at the indicated stimulus frequency (pps, pulses per second). The bottom row shows the degree to which each of the fiber types can sustain force during continuous stimulation. The time scales become progressively larger from the top to bottom rows, with a break in the bottom row. In addition, the tension scales become progressively larger from left (fewer fibers per motor unit) to right (more fibers per motor unit). (Data from Burke RE, Levine DN, Tsairis P, et al. J Physiol 1977; 234:723–748.)

higher endurance capacity and hence are classified as fast fatigue-resistant units (bottom row of Fig. 60-2B). In contrast, type IIb motor units have greater capacity for rapid energy production through nonoxidative (i.e., anaerobic) glycolysis, so they can produce rapid and powerful contractions. However, type IIb units tire more rapidly and are therefore classified as fast fatigable units (bottom row of Fig. 60-2C). As external forces stretch muscle, series elastic elements contribute a larger fraction of total tension As sarcomeres contract, some of their force acts laterally— through membrane-associated and transmembrane proteins—on the extracellular matrix and connective tissue that surrounds each muscle fiber. Ultimately, the force is transmitted to bone, typically (but not always) through a tendinous insertion. The structural elements that transmit force from the cross-bridges to the skeleton comprise the series elastic elements of the muscle and behave as a spring with a characteristic stiffness. Stretching resting muscle causes passive tension to increase exponentially with length (see Fig. 9-9C). Thus, muscle stiffness increases with length. During an isometric contraction (see Chapter 9), when the external length of a muscle (or muscle fiber) is held constant, the sarcomeres shorten at the expense of stretching the series

elastic elements. An isometric contraction can occur at modest levels of force development, such as holding a cup of coffee, as well as during maximal force development, such as when opposing wrestlers push and pull against each other, with neither gaining ground. Physical activity typically involves contractions in which muscles are shortening and lengthening, as well as periods during which muscle fibers are contracting isometrically. During cyclic activity such as running, muscles undergo a stretch-shorten cycle that may increase total tension while decreasing active tension. For example, as the calf muscles relax as the foot lands and decelerates, the series elastic elements of the calf muscles (e.g., the Achilles tendon, connective tissue within muscles) are stretched and develop increased passive tension (see Fig. 9-9C). Thus, when the calf muscles contract to begin the next cycle, they start from a higher passive tension and thus use a smaller increment in active tension to reach a higher total tension. This increased force helps to propel the runner forward. In a concentric contraction (e.g., climbing stairs), the force developed by the cross-bridges exceeds the external load, and the sarcomeres shorten. During a concentric contraction, a muscle performs positive work (force × distance) and produces power (work/time; see Chapter 9). As shown in Figure 9-9E, the muscle achieves peak power at relatively moderate loads (30% to 40% of isometric tension) and velocities (30% to 40% of maximum shortening velocity).

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The capacity of a muscle to perform positive work determines physical performance. For example, a stronger muscle can shorten more rapidly against a given load, and a muscle that is metabolically adapted to a particular activity can sustain performance for a longer period of time before it succumbs to fatigue. In an eccentric contraction (e.g., descending stairs), the force developed by the cross-bridges is less than the imposed load, and the sarcomeres lengthen. During an eccentric contraction, the muscle performs negative work, thus providing a brake to decelerate the applied force being applied, and absorbs power. Eccentric contractions can occur with light loads, such as lowering a cup of coffee to the table, as well as with much heavier loads, such as decelerating after jumping off a bench onto the floor. At the same absolute level of total force production, eccentric contractions—with increasingly stretched sarcomeres—develop less active tension than do concentric or isometric contractions. Conversely, the passive tension developed by the series elastic elements make a greater contribution to total tension. As a result, the tension generated eccentrically is greater than that generated isometrically. When the external force stretches the muscle sufficiently, all the tension is passive, and the limit is the breaking point (see Fig. 19-9B) of the series elastic elements. Thus, eccentric contractions are much more likely than isometric or isotonic contractions to damage muscle fibers and connective tissue.

A

AXIS OF MUSCLE FIBERS A muscle with a few long fibers in parallel with the axis of shortening... produces rapid shortening, but less tension.

Tendon

A muscle with many short fibers at an angle to the axis of shortening... produces slow shortening, but more tension. B

ORIGIN AND INSERTION OF A MUSCLE Humerus

Brachialis muscle Ulna Center of rotation

The action of a muscle depends on the axis of its fibers and its origin and insertion on the skeleton In addition to the contractile and metabolic properties of muscle fibers discussed earlier, two anatomical features determine the characteristics of the force produced by a muscle. The first anatomical determinant of muscle function is the arrangement of fibers with respect to the axis of force production (i.e., the angle of pennation). With other determinants of performance (e.g., fiber type and muscle mass) being equal, muscles that have a relatively small number of long fibers oriented parallel to the axis of shortening (e.g. the sartorius muscle of the thigh, Fig. 60-3A, top) shorten more rapidly. Indeed, the more sarcomeres are arranged in series, the more rapidly the two ends of the muscle will approach each other. In contrast, muscles that have many short fibers at an angle to the axis (e.g. the soleus muscle of the calf, Fig. 60-3A, bottom) develop more force. Indeed, the greater the number of fibers (and sarcomeres) in parallel with each other, the greater is the total cross-sectional area for developing force. The second anatomical determinant of limb movement consists of the locations of the origin and insertion of the muscle to the skeleton. Consider, for example, the action of the brachialis muscle on the elbow joint. The distance between the insertion of the muscle on the ulna and the joint’s center of rotation is D, which may be 5 cm. The torque that the muscle produces on the joint is the component of total muscle force that is perpendicular to the ulna, multiplied by D (Fig. 60-3B). An equivalent definition is that torque is the product of the total muscle force multiplied by the moment arm, which is the length of the line segment

60° of flexion

Torque = (F cos a)  D Perpendicular component of force

F

a

F cos a

D

Torque = F  (D sin q ) Moment arm F

Moment arm = D sin q

q D

Figure 60-3 A and B, Determinants of the mechanical action of a muscle.

that runs perpendicular to the muscle and through the center of rotation (Fig. 60-3B). As we flex our elbow, the moment arm is constantly changing, and muscle force changes as well. For this joint, we achieve maximum torque at 60 degrees of flexion.

Chapter 60 • Exercise Physiology and Sports Science

To perform a desired movement—whether playing the piano or serving a tennis ball—the nervous system must activate a combination of muscles with the appropriate contractile properties, recruit motor units in defined patterns, and thereby create suitable mechanical interactions among body segments. When we perform movements with uncertainty—as in learning a new skill—actions tend to be stiff because of concurrent recruitment of motor units in antagonistic muscles that produce force in opposite directions. Such superfluous muscle fiber activity also increases the energy requirements for the activity. Even in someone who is skilled, the fatigue of small motor units leads to the recruitment of larger motor units in the attempt to maintain activity, but with loss of fine control and greater energy expenditure. With learning, recruitment patterns become refined and coordinated, and muscle fibers adapt to the task. Thus, movements become fluid and more energetically efficient, as exemplified by highly trained musicians and athletes who can make difficult maneuvers appear almost effortless. Strength versus endurance training differentially alters the properties of motor units The firing pattern of the α motor neuron—over time—ultimately determines the contractile and metabolic properties of the muscle fibers in the corresponding motor unit. This principle was demonstrated elegantly by classic experiments in which the motor nerve to a muscle consisting primarily of fast motor units was cut and switched with that of a muscle consisting primarily of slow motor units. As the axons regenerated and the muscles recovered contractile function over several weeks, the fast muscle became progressively slower and more fatigue resistant, whereas the slow muscle became faster and more susceptible to fatigue. Varying the pattern of efferent nerve impulses through chronic stimulation of implanted electrodes elicits similar changes in muscle properties. A corollary of this principle is that physical activity leads to adaptation only in those motor units that are actually recruited during the activity. The effects of physical activity on motor unit physiology depend on the intensity and duration of the exercise. In general, sustained periods of low to moderate intensity performed several times per week—endurance training—result in a greater oxidative capacity of muscle fibers and are manifested by increases in O2 delivery, capillary supply, and mitochondrial content. These adaptations reduce the susceptibility of the affected muscle fibers to fatigue. The lean and slender build of long-distance runners reflects highly oxidative muscle fibers of relatively small diameter that promote O2 and CO2 diffusion between capillaries and mitochondria for high levels of aerobic energy production. Further, the high ratio of surface area to volume of the slender body also facilitates cooling of the body during prolonged activity and in hot environments. In contrast, brief sets of high-intensity contractions performed several times per week—strength training—result in motor units that can produce more force and can shorten

against a given load at greater velocity by increasing the amount of contractile protein. The hypertrophied muscles of sprinters and weight lifters exemplify this type of adaptation, which relies more on rapid, anaerobic sources of energy production.

CONVERSION OF CHEMICAL ENERGY TO MECHANICAL WORK At rest, skeletal muscle has a low metabolic rate. In response to contractile activity, the energy consumption of skeletal muscle can easily rise more than 100-fold. The body meets this increased energy demand by mobilizing energy stores both locally from muscle glycogen and triacylglycerols and systemically from liver glycogen and adipose tissue triacylglycerols. The integrated physiological response to exercise involves the delivery of sufficient O2 and fuel to ensure that the rate of ATP synthesis rises in parallel with the rate of ATP breakdown. Indeed, skeletal muscle precisely regulates the ratio of ATP to ADP even with these large increases in ATP turnover. Physical performance can be defined in terms of power (work/time), speed, or endurance. Skeletal muscle has three energy systems, each designed to support a particular type of performance (Fig. 60-4). For power events, which typically last a few seconds or less (e.g., hitting a ball with a bat), the immediate energy sources include ATP and PCr. For spurts of activity that last several seconds to a minute (e.g., sprinting 100 m), muscles rely primarily on the rapid nonoxidative breakdown of carbohydrate stored as muscle glycogen to form ATP. For activities that last 2 minutes or longer but have low power requirements (e.g., jogging several kilometers), the generation of ATP through the oxidation of fat and glucose derived from the circulation becomes increasingly important. Next I consider the key metabolic pathways for producing the energy that enables skeletal muscle to have such a tremendous dynamic range of activity.

Relative energy potential of each system (%)

Fluid and energetically efficient movements require learning

100 Oxidative Nonoxidative Immediate

75

50

25

0

1

2

3

4

5

Time (min)

Figure 60-4 Energy sources for muscle contraction. (Modified from Edington DW, Edgerton VR: The Biology of Physical Activity. Boston: Houghton Mifflin, 1976.)

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ATP and phosphocreatine provide immediate but limited energy At the onset of exercise, or during the transition to a higher intensity of contractile activity, the immediate energy sources are ATP and PCr. As for any other cell, muscle cells break down ATP to ADP and inorganic phosphate (Pi) and release ∼11.5 kcal/mol of free energy (ΔG) under physiological conditions: ATP → ADP + Pi + ΔG

(60-1)

Muscle cells rapidly regenerate ATP from PCr in a reaction that is catalyzed by creatine kinase: creatine kinase

ADP + PCr ⎯⎯⎯⎯⎯→ ATP + Creatine

(60-2)

Resting skeletal muscle cells contain 5 to 6 mmol/kg of ATP but 25 to 30 mmol/kg of PCr—representing nearly 5-fold more energy. These two energy stores are sufficient to support intense contractile activity only for a few seconds. When rates of ATP breakdown (Equation 60-1) are high, ADP levels (normally very low) increase and can actually interfere with muscle contraction. Under such conditions, adenylate kinase (also known as myokinase) transfers the second phosphate group from one ADP to another and thereby regenerates ATP: adenylate kinase → ATP + AMP (60-3) ADP + ADP ⎯⎯⎯⎯⎯⎯

The foregoing reaction is limited by the initial pool of ADP, which is small. In contrast, creatine kinase (Equation 60-2) so effectively buffers ATP that [ATP]i changes very little. Although changes in [ATP]i cannot provide an effective signal to stimulate metabolic pathways of energy production, the products of ATP hydrolysis—Pi, ADP, and AMP— are powerful signals. The high-energy phosphates ATP + PCr are historically referred to as phosphagens and are recognized as the immediate energy supply because they are readily available, albeit for only several seconds (Fig. 60-4). This role is of particular importance at the onset of exercise and during transitions to more intense activity, before other metabolic pathways have time to respond. Anaerobic glycolysis provides a rapid but self-limited source of ATP When high-intensity exercise continues for more than several seconds, the breakdown of ATP and PCr is followed almost instantly by the accelerated breakdown of intramuscular glycogen to glucose and then to lactate. This anaerobic metabolism of glucose has the major advantage of providing energy quickly to meet the increased metabolic demands of an intense workload, even before O2, glucose, or fatty acid delivery from blood increases. However, because of the low ATP yield of this pathway, muscle rapidly depletes its glycogen stores and thereby limits intense activity to durations of ∼1 minute (Fig. 60-4). Muscle fibers store 300 to 400 g of carbohydrate in the form of glycogen and, particularly in the case of type II

fibers, are rich in the enzymes required for glycogenolysis and glycolysis. In glycogenolysis, phosphorylase breaks down glycogen to glucose-1-phosphate. Activation of the sympathetic nervous system during exercise elevates levels of epinephrine and promotes the breakdown of muscle glycogen. Subsequently, phosphoglucomutase converts glucose1-phosphate to glucose-6-phosphate (G6P). Muscle fibers can also take up blood-borne glucose using the GLUT4 transporter (see Chapter 58) and use hexokinase to phosphorylate it to G6P. Intracellular glycogen is more important than blood-borne glucose in rapidly contributing G6P for entry into glycolysis—breakdown of glucose to pyruvate (see Fig. 59-6A). In the absence of O2, or when glycolysis generates pyruvate more rapidly than the mitochondria can oxidize it (see later), muscle cells can divert pyruvate to lactic acid, which readily dissociates into H+ and lactate. The overall process generates two ATP molecules/glucose: C 6 H12O6 + 2 ADP + 2 Pi → 2 C 3 H5O3− Glucose

+ 2 ATP + 2 H+ + heat

Lactate

(60-4)

This anaerobic regeneration of ATP through breakdown of intramuscular glycogen, although faster than oxidative metabolism, captures only a fraction of the energy stored in glucose. Moreover, the process is self-limiting because the H+ generated from the dissociation of lactic acid can lower pHi from 7.1 to nearly 6.2, a process that inhibits glycolysis, impairs the contractile process, and thereby contributes to muscle fatigue.

Oxidation of glucose, lactate, and fatty acids provides a slower but long-term source of ATP The body stores only a small amount of O2 in the blood, and the cardiovascular and respiratory systems require 1 to 2 minutes to increase O2 delivery to muscle to support oxidative metabolism. Endurance training speeds these adjustments. Nevertheless, before the increase in O2 delivery is complete, muscle must rely on the immediate release of energy from ATP and PCr, as well as anaerobic glycolysis, as just discussed. To sustain light and intermediate physical activity for more than ∼1 minute, muscle regenerates ATP through oxidative metabolism in the mitochondria of type I and type IIa muscle fibers (Fig. 60-4). Muscle also uses oxidative metabolism to recover from intense activities of short duration that relied on the immediate and anaerobic systems of energy supply. The anaerobic metabolism of glucose provides nearly 100-fold more energy than is available through the immediate breakdown of ATP and PCr. Oxidative metabolism of glucose, lactate, and fat provides far more than even the anaerobic metabolism of glucose. Oxidation of Non-Muscle Glucose

The aerobic metabolism of glucose, although slower than anaerobic glycolysis (Equation 60-4), provides nearly 15-fold more ATP molecules per glucose (see Table 58-4):

Chapter 60 • Exercise Physiology and Sports Science

Epinephrine stimulates energy mobilization at several sites. Gluconeogenesis in liver

Epinephrine Glucagon

Glycolysis in muscle

Blood

Triacylglycerol breakdown in fat cells

Epinephrine

Blood

Epinephrine

Epinephrine receptor

4 4

Type IIb

cAMP

cAMP

+2

Glycogen

Glycogen

Glucose

Glucose

+6

2

Pi

+2

Triacylglycerol Hormonesensitive lipase Fatty acids

Pi

2

+2

2 Pyruvate

2 Pyruvate

2 Lactate

2 Lactate

= Cori cycle Type I

cAMP

Glycogen Glucose

2 2

2 Pyruvate

Pi

+2

Fatty acids Aerobic β oxidation

Acetyl CoA Citric acid cycle 2 Lactate

Figure 60-5

ATP

Steady-state energy supply to muscle from energy stores in muscle, liver, and adipose tissue.

Glucose    C 6 H12 O6 + 6 O2 + 30 ADP + 30 Pi → 6 CO2 + 6 H2 O + 30 ATP + heat

(60-5)

The glucose that muscle oxidizes comes from the breakdown of hepatic glycogen stores of 75 to 100 g. Glucose uptake by exercising muscle can increase 7- to 40-fold above rest. However, increased glucose release from liver (through glycogenolysis) balances the glucose uptake from the blood by active muscle, thereby stabilizing blood [glucose]. An increase

in portal vein levels of glucagon in particular (see Fig. 51-12) and a decrease in insulin—together with an increase in epinephrine—are the main signals for this elevated hepatic glucose output during exercise. However, hepatic denervation in dogs does not prevent accelerated rates of glucose production during exercise, a finding showing that sympathetic innervation of the liver is not essential. Contracting skeletal muscle is an important sink for blood-borne glucose (Fig. 60-5). Moreover, contractile activ-

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ity triggers the translocation of additional GLUT4 transporters (see Chapter 51) from the cytosol to the plasma membrane. This process, which is insulin independent and is likely mediated by activation of AMP kinase, supports increased glucose uptake. Because exercise-induced translocation of GLUT4 is insulin independent, endurance exercise is an important adjunct in controlling elevated levels of blood [glucose] in patients with diabetes.

muscles undergoing prolonged exercise. During recovery, muscle glycogenolysis and lactate release from previously resting muscle continue, and lactate enters the liver for conversion to glucose and release. The subsequent glucose uptake by previously exercising muscles thereby replenishes their glycogen stores. In this way, the body ensures an adequate supply of fuel for the next fight or flight response.

Oxidation of Lactate

During the first minutes of exercise, active muscle fibers use glycogenolysis to liberate glucose and then use glycolysis to form either pyruvate or lactate, depending on the relative activities of glycolysis and mitochondrial respiration. Indeed, lactate production occurs even in fully aerobic contracting muscles with high oxidative capacity. As blood flow and O2 delivery increase during the initial minutes of the cardiovascular and respiratory adjustments to exercise, muscle fibers convert accumulated lactate back to pyruvate for uptake and subsequent oxidation by the mitochondria. In addition, glycolytic (type IIb) muscle fibers release lactate that can diffuse to nearby oxidative muscle fibers (type I and IIa), which can oxidize it (Fig. 60-5). The lactate that escapes into the bloodstream can enter the heart for oxidation, the distant skeletal muscles for oxidation (Fig. 60-5), or the liver for gluconeogenesis (discussed later). This shuttling of lactate provides a link between anaerobic and oxidative cells. After the initial few minutes of moderateintensity exercise—and after the cardiovascular and respiratory adjustments—exercising muscle takes up and oxidizes blood-borne glucose and simultaneously diminishes its release of lactic acid.

Oxidation of Non-Muscle Lipid Most stored energy is in the form of triglycerides. In the prototypic 70-kg person, adipocytes store ∼132,000 kcal of potential energy. The mobilization of lipid from adipocytes during exercise is largely under the control of the sympathetic nervous system, complemented by the release of growth hormone during exercise lasting longer than 30 to 40 minutes. The result of this mobilization is an increase in circulating levels of fatty acids, which can enter skeletal muscle—especially type I fibers (Fig. 60-5). In addition to fatty acids that enter muscle from adipocytes, skeletal muscle itself stores ∼8000 kcal of potential energy as intracellular triacylglycerols, which contribute to fatty acid oxidation, particularly during recovery following prolonged exertion. In the presence of adequate O2, fatty acids provide up to 60% of the oxidized fuel supply of muscle during prolonged exercise. The oxidation of fatty acids, using palmitate as an example, has a very high yield of ATP:

Gluconeogenesis

Lipids are an important source of energy when O2 is available, that is, during prolonged low- to moderate-intensity activity and during recovery following exercise.

Hepatic gluconeogenesis becomes increasingly important as exercise is prolonged beyond an hour and hepatic glycogen stores become depleted. The most important substrates for hepatic gluconeogenesis are lactate and alanine. During prolonged exercise, the key substrate is lactate released into the circulation by contracting skeletal muscle (see later) for uptake by the liver, which resynthesizes glucose for uptake by the muscle—the Cori cycle (see Fig. 58-13 and 60-5). At workloads exceeding 65% of maximal O2 uptake by . the lungs (VO2max), lactate production rises faster than removal and causes an exponential increase in blood [lactate]. Endurance training increases the rate of lactate clearance from the blood at any given [lactate]. Oxidation is responsible for ∼75% of lactate removal, and hepatic gluconeogenesis is responsible for the remainder. Also during prolonged exercise, the oxidation of branched-chain amino acids by skeletal muscle leads to the release of alanine into the circulation for uptake by the liver, followed by hepatic gluconeogenesis and the release of glucose into the blood for uptake by muscle— the glucose-alanine cycle (see Fig. 58-13). The Cori and glucose-alanine cycles play an important role in redistributing glycogen from resting muscle to exercising muscle during prolonged exercise and during recovery from exercise. For example, after prolonged arm exercise, lactate release from leg muscle is 6- to 7-fold greater than in the pre-exercise basal state. Conversely, after leg exercise, lactate release from forearm muscle increases. Thus, the Cori cycle redistributes glycogen from resting muscle to fuel

Palmitic acid   C15 H31COOH + 23 O2 + 106 ADP + 106 Pi → 16 CO2 + 16 H2 O + 106 ATP + heat

(60-6)

Choice of Fuel Sources For sustained activity of moderate intensity, fat is the preferred substrate, given ample O2 . availability. For example, at 50% of VO2max, fatty acid oxidation accounts for more than half of muscle energy production, and glucose accounts for the remainder. As the duration of exercise further lengthens, fatty acid oxidation progressively increases, and it becomes the dominant oxidative fuel as glucose utilization by the muscle declines. However, as exercise intensity increases, active muscle relies increasingly on glucose derived from intramuscular glycogen as well as on blood-borne glucose. This crossover from lipid to carbohydrate metabolism has the advantage that, per liter of O2 consumed, carbohydrate provides slightly more energy than lipid. Conversely, as muscle depletes its glycogen stores, it loses its ability to consume O2 at high rates. At a given metabolic demand, the increased availability and utilization of fatty acids translate to lower rates of glucose oxidation and muscle glycogenolysis, thereby prolonging the ability to sustain activity. Endurance training promotes these adaptations of skeletal muscle. Under conditions of carbohydrate deprivation (e.g., starvation), extremely prolonged exercise (e.g., ultramarathon), and impaired glucose utilization (e.g., diabetes), muscle can also oxidize ketone bodies as their plasma levels rise.

Chapter 60 • Exercise Physiology and Sports Science

MUSCLE FATIGUE

Impaired excitability and impaired Ca2+ release can produce peripheral fatigue

Fatigued muscle produces less force and has a reduced velocity of shortening

Transmission block at the neuromuscular junction does not cause muscle fatigue, even though the release of neurotransmitter can decline. Peripheral fatigue reflects a spectrum of events at the level of the muscle fiber, including impairments in the initiation and propagation of muscle action potentials, the release and handling of intracellular Ca2+ for cross-bridge activation, depletion of substrates for energy metabolism, and the accumulation of metabolic byproducts. The nature of fatigue and the time required for recovery vary with the recruitment pattern of motor units and the metabolic properties of their constitutive muscle fibers (Fig. 60-2).

Muscle fatigue is defined as the inability to maintain a desired power output—resulting from muscle contraction against a load—with a decline in both force and velocity of shortening. A decline in maximal force production with fatigue results from a reduction in the number of active cross-bridges as well as the force produced per cross-bridge. As fatigue develops, the production of force usually declines earlier and to a greater extent than shortening velocity. Other characteristics of fatigued skeletal muscle are lower rates of both force production and relaxation, owing to impaired release and reuptake of Ca2+ from the sarcoplasmic reticulum (SR). As a result, fast movements become difficult or impossible, and athletic performance suffers accordingly. Nevertheless, fatigue may serve an important protective role in allowing contractions at reduced rates and lower forces while preventing extreme changes in cell composition that could cause damage. Muscle fatigue is reversible with rest, which contrasts with muscle damage or weakness, in which even muscles that are well rested are compromised in their ability to develop force. For example, muscle damage induced by eccentric contractions can easily be mistaken for fatigue, except the recovery period can last for days. Factors contributing to fatigue include motivation, physical fitness, nutritional status, and the types of motor units (i.e., fibers) recruited with respect to the intensity and duration of activity. As discussed in this major section, fatigue during prolonged activity of moderate intensity involving relatively low frequencies of motor unit activation is caused by different factors than fatigue during bursts of high intensity involving high frequencies of motor unit activation. Moreover, fatigue can result from events occurring in the central nervous system (CNS; central fatigue) as well as from changes within the muscle (peripheral fatigue). Changes in the CNS produce central fatigue Central fatigue reflects changes in the CNS and may involve altered input from muscle sensory nerve fibers, reduced excitatory input to motor control centers of the brain and spinal cord, and altered excitability of α and γ motor neurons (see Fig. 15-30). The contributions of these factors vary with the individual and with the nature of activity. For example, central fatigue is likely to play only a minor role in limiting performance of highly trained athletes who have learned to pace themselves according to the task and are mentally conditioned to discomfort and stress. In contrast, central fatigue is likely of greater importance in novice athletes and during repetitive (i.e., boring) tasks. The identification of specific sites involved in central fatigue is difficult because of the complexity of the CNS. Nevertheless, external sensory input, such as shouting and cheering, can often increase muscle force production and physical performance, a finding indicating that pathways proximal to corticospinal outputs can oppose central fatigue.

High-Frequency Fatigue With continuous firing of action potentials during intense exercise, Na+ entry and K+ exit exceed the ability of the Na-K pump to restore and maintain normal resting ion concentration gradients. As a result, [K+]o and [Na+]i increase, thus making the resting membrane potential of muscle fibers more positive by 10 to 20 mV. This depolarization inactivates voltage-gated Na+ channels and makes it more difficult to initiate and propagate action potentials. Within the T tubule, such depolarization impairs the ability of L-type Ca2+ channels to activate Ca2+ release channels in the SR (see Fig. 9-3). Fatigue resulting from impaired membrane excitability is particularly apparent at high frequencies of stimulation during recruitment of type II motor units—high-frequency fatigue. On cessation of contractile activity, ionic and ATP homeostasis recovers within 30 minutes; thus, the recovery from high-frequency fatigue occurs relatively quickly. Low-Frequency Fatigue In prolonged, moderate-intensity exercise, the release of Ca2+ from the SR falls—perhaps reflecting change in either the Ca2+ release channel or its associated proteins—thus leading to a depression in the amplitude of the [Ca2+]i transient that accompanies the muscle twitch. A diminution of Ca2+ release is apparent at all stimulation frequencies. However, the effect on force development is most apparent at low stimulation frequencies, for the following reason. During unfused tetanus (see Fig. 9-11), [Ca2+]i does not continuously remain at high enough levels to saturate troponin C (see Chapter 9). As a result, cross-bridge formation is highly sensitive to the amount of Ca2+ released from the SR with each stimulus. In contrast, with high frequencies of stimulation that produce fused tetanus, [Ca2+]i is at such high levels that Ca2+ continuously saturates troponin C and thereby maximizes crossbridge interactions and masks the effects of impaired Ca2+ release with each stimulus. Fatigue resulting from impaired Ca2+ release is thus particularly apparent at low frequencies of stimulation during recruitment of type I motor units— low-frequency fatigue. Recovery requires several hours.

Fatigue can result from ATP depletion, lactic acid accumulation, and glycogen depletion ATP Depletion

As outlined in Chapter 9, muscle fibers require ATP for contraction, relaxation, and the activity of

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Section X • Physiology of Everyday Life

Intense activity also activates glycolysis—again, particularly in type IIb fibers—resulting in a high rate of lactic acid production and thus reducing pHi to as low as 6.2 (Equation 60-4). This fall in pHi inhibits myosin ATPase activity and thereby reduces the velocity of shortening. The fall in pHi also inhibits cross-bridge interaction and the binding of Ca2+ to troponin, the Na-K pump, as well as to phosphofructokinase (see Chapter 51), the ratelimiting step of muscle glycolysis. The combined effects of low pHi and high Pi interact to impair the peak force production of muscle fibers more than either agent alone. The mechanisms are reductions in the number of cross-bridges and in the force per cross-bridge by impairing the transition from weak to strong binding states between actin and myosin. In addition, both H+ and Pi reduce Ca2+ sensitivity of contractile proteins, such that higher free [Ca2+]i is required for a given level of force production. Glycogen Depletion

During prolonged exercise of moderate intensity (∼50% of maximal aerobic power), and with well maintained O2 delivery, the eventual decrease in glycogen stores in oxidative (type I and IIa) muscle fibers decreases power output. Long-distance runners describe this phenomenon as “hitting the wall.” Muscle glycogen stores are critical because the combination of blood-borne delivery of substrates and the availability of intramuscular fatty acids is inadequate to accommodate the energy requirements. In long-distance running, endurance depends on the absolute amount of glycogen stored in the leg muscles before exercise. To postpone hitting the wall, the athlete must either begin the event with an elevated level of muscle glycogen or race more slowly. Because glycogen storage is primarily a function of diet, carbohydrate loading can increase resting muscle glycogen stores and can postpone the onset of fatigue. Lowcarbohydrate diets have the opposite effect. Although physical training has little effect on the capacity for glycolysis, it can promote glycogen storage, particularly if it is combined with a carbohydrate-rich diet. Aerobic training can spare muscle glycogen by adaptations such as mitochondrial proliferation that shift the mix of oxidized fuels toward fatty acids. Indeed, well-trained athletes can maintain moderateintensity exercise for hours. During exercise at relatively high intensities (>65% of maximal aerobic power), fatigue develops on the order of

DETERMINANTS OF MAXIMAL O2 UPTAKE AND CONSUMPTION The O2 required for oxidative metabolism by exercising muscle travels from the atmosphere to the muscle mitochondria in three discrete steps: 1. The uptake of O2 by the lungs depends on pulmonary ventilation. 2. O2 delivery to muscle depends on blood flow and O2 content. 3. The extraction of O2 from blood by muscle depends on O2 delivery and the PO2 gradient between blood and mitochondria. Maximal O2 uptake by the lungs can exceed resting O2 uptake by more than 20-fold The respiratory and cardiovascular systems can readily deliver O2 to active skeletal muscle at mild and moderate exercise intensities. As power output increases, the body eventually reaches a point at which the capacity of O2 transport systems can no longer keep. pace with demand, so the rate of. O2 uptake by the lungs (VO2) plateaus (Fig. 60-6). At rest, VO2 is typically 250 mL/min for a 70-kg person (see Chapter 29), a value that corresponds to 3.6 mL of O2 consumed. per minute for each kg of body mass [mL O2/(min × kg)]. VO2max is an objective index of the functional capacity of the body’s ability to generate aerobic power. In people who have a deficiency in any part of the O2 transport system (e.g., chronic obstructive pulmonary disease or advanced heart . disease), VO2max can be as low as 10 to 20 mL O2/(min × kg). The range for mildly active middle-aged adults is 30 to

· VO2 max

Trained Untrained

50 40

· VO2 max

2

Lactic Acid Accumulation

tens of minutes. One explanation for this decrement in performance is that type IIb muscle fibers fatigue when their glycogen supplies become exhausted, and the result is a decline in whole-muscle power output.

O ( min  kgmLbody weight )

the membrane pumps that maintain ionic homeostasis. Therefore, the cells must maintain [ATP]i to avoid fatigue (see Chapter 9). Intense stimulation of muscle fibers (particularly type IIb) requires high rates of ATP utilization, with PCr initially buffering [ATP]i. As fatigue develops, [PCr]i diminishes and [ATP]i can fall from 5 mM to less than 2 mM, particularly at sites of cross-bridge interaction and in the vicinity of membrane pumps, thereby impairing respective ATPase activities. Simultaneously, Pi, ADP, Mg2+, lactate, and H+ accumulate in the sarcoplasm. Impairment of the Ca2+ pump at the SR prolongs the Ca2+ transient while reducing the electrochemical driving force for Ca2+ release from the SR. Independently, the fall in [ATP]i and the increase in [Mg2+] can also inhibit Ca2+ release through the ryanodine receptor (see Chapter 9).

· Oxygen consumption (VO2)

1258

30 20

Maximal trained work capacity

Maximal untrained work capacity

10 0 0

3.5 External power output watts kg body weight

(

)

. Figure 60-6 Dependence of VO2 on mechanical power output. . Training increases VO2max.

Chapter 60 • Exercise Physiology and Sports Science

40 mL O2/(min × kg); for people in this category, . a 3-month program of physical conditioning can increase VO2max by 20%. . In elite endurance athletes, VO2max may be as high as 80 to 90 mL O2/(min × kg), more than a 20-fold elevation above . . the resting VO2. Hemorrhage or high altitude decreases VO2max, whereas blood transfusion or training increases it. . The typical method for determining VO2max is an incremental exercise test on a stationary cycle ergometer or treadmill. Such tests assess training status, predict performance in athletes, and provide an index of functional impairment in patients. During the test, the technician monitors the PO2 and PCO2 of the subject’s expired. air, as well as total ventilation. The criteria for achieving VO2max include (1) an inability to continue the pace. at the prescribed power requirement, (2) a leveling off of VO2 with an increasing power requirement . . (Fig. 60-6), and (3) a respiratory exchange ratio (VCO2/VO2) . . greater than 1.15. This VCO2/VO2 is a transient/non–steadystate occurrence (i.e., not a real respiratory quotient, see Chapter 54) and indicates that a significant hyperventilation, triggered by low blood pH (see Chapter 32), is reducing the body’s CO2 stores. O2 uptake by muscle is the product of muscle blood flow and O2 extraction The body’s total store of O2 is ∼1 L (mainly in the form of O2 bound to hemoglobin), a volume that (if used completely) could support moderate exercise for 30 seconds at best, heavy exercise for not more than 15 seconds, and maximal exercise for less than 10 seconds. If activity is to persist, the body must continually transport O2 from the ambient air to the muscle mitochondria at a rate that is equivalent to the O2 utilization by the muscle. This increased O2 transport is accomplished by increasing alveolar ventilation to maintain alveolar PO2 levels that are sufficient to saturate arterial blood fully (see Chapter 31) and by increasing cardiac output to ensure a sufficiently high flow of oxygenated blood to the muscles (see Chapter 20). The integrated organ system response to the new, elevated metabolic load involves the close coupling of the pulmonary and the cardiovascular O2 delivery systems to the O2 acceptor mechanisms in the muscle; the response includes sophisticated reflexes to ensure matching of the two processes. The convective O2 delivery rate is the product of cardiac output (i.e., heart rate × stroke volume) and arterial O2 content: Arterial O2 delivery rate to whole body

 O Va 2  mL O2 min

Heart rate

=

HR 

beats min

Cardiac stroke volume

⋅

SV 

mL blood beat

Arterial O2 content

⋅

Ca O2 (60-7) 

mL O2 mL blood

. The rate of O2 uptake by skeletal muscle (VO2) depends on both the O2 delivery to skeletal muscle and the extraction of O2 by.the muscle. According to the Fick equation (see Chapter 17), VO2 is the product of blood flow to muscle (F) and the arteriovenous (a-v) difference for O2:

a−v difference of O2 content

Rate of O2 Blood flow uptake by to skeletal muscle skeletal muscle

V O2 

mL O2 min

=

F

mL blood min

⋅

(Ca O2 − Cv O2 )  

(60-8)

mL O2 mL blood

. The VO2, established by the rate of oxidative phosphorylation in muscle mitochondria, requires an adequate rate of O2 delivery to the active muscle. Exercise triggers a complicated series of changes in the cardiovascular system that has the net effect of increasing F and redistributing cardiac output away from the splanchnic and renal vascular beds, as well as from inactive to active muscle (see Chapter 25). Increased O2 extraction from the blood by active skeletal muscle occurs at the onset of exercise in response to elevated mitochondrial respiration and the attendant fall in intracellular PO2, which increases the gradient for O2 diffusion from blood to mitochondria. At the onset of exercise, the content of O2 in the arterial blood (CaO2) actually increases somewhat (e.g., from 20.0 to 20.4 mL O2/mL blood; see Table 29-3) secondary to the increase in alveolar ventilation triggered by the CNS (see Chapter 14). Also as a consequence of the anticipatory hyperventilation, PCO2 actually falls with the onset of exercise. Possible mechanisms of this ventilatory increase include a response to mechanoreceptors in joints and muscles, descending cortical input, or resetting of peripheral chemoreceptors by a reduction in their blood supply (see Chapter 32). The increase in ventilation in anticipation of future needs is enhanced in well-trained athletes.

O2 delivery by the cardiovascular system is the limiting step for maximal O2 utilization For years, exercise and. sports scientists have debated over the factors that limit VO2max and thus contribute to performance limitations. As noted earlier, the transport of O2 from atmosphere to muscle mitochondria occurs in three steps: uptake, delivery, and extraction. A limitation in any step could be rate limiting for maximal O2 utilization by muscle. Limited . O2 Uptake by Lungs

One view is that the lungs limit VO2max. An inability of alveolar O2 diffusion to saturate arterial blood fully occurs in a subset of elite athletes (including race horses). Thus, a decrease in CaO2 occurs at maximal effort on an incremental test. The inability to saturate arterial blood in athletes could be the consequence of a ventilation-perfusion mismatch at very high cardiac outputs (see Chapter 31). Limited O2 Delivery by Cardiovascular System

According to the prevalent view, a limitation in O transport by the 2 . cardiovascular system determines VO2max. That is, according to the convective flow model, maximal cardiac output, and hence O2 delivery, is the limiting step. Support for this view comes from the observation that training can considerably augment maximal cardiac output and muscle blood flow

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6 Elite athletes 5 Endurance athletes Maximal 4 oxygen uptake, . VO2 max 3 (liters/min)

Physically active

Sedentary 2

Bed rest

1 1

5 2 3 .4 Maximal oxygen delivery, VaO2 max (liters/min)

6

Figure 60-7 Dependence of maximal O2 utilization on O2 delivery. The graph illustrates the relationship between maximal O2 delivery . . to the peripheral tissues (VaO2max) and VO2max for five individuals with different lifestyles. Training increases both O2 delivery and O2 uptake. (Data from Saltin B, Strange S: Med Sci Sports Exerc 1992; 24:30-37.)

. (see the following section). Moreover, VO2max largely increases in parallel with these adaptations (Fig. 60-7). Limited O2 Extraction by Muscle A third point of view is that, with increasing demand, extraction by muscle of O2 from blood becomes inadequate despite adequate O2 delivery. According to this diffusive flow model, a major limitation in O2 transport is the kinetics of O2 diffusion from hemoglobin in the red blood cell to the muscle mitochondrial matrix. Thus, anything that lowers either the muscle’s diffusing capacity for O2 or the PO2. gradient between hemoglobin and mitochondria reduces VO2max.

Effective circulating volume takes priority over cutaneous blood flow for thermoregulation When we exercise in the heat, our circulatory systems must simultaneously support a large blood flow to both the skin (see Chapter 59) and the contracting muscles, an effort that taxes the cardiac output and effective circulating volume (see Chapter 40). During exercise, the ability to maintain both arterial blood pressure and body core temperature (Tcore) within acceptable physiological limits depends on maintaining an adequate effective circulating volume. Effective circulating volume depends on total blood volume, which, in turn, relies on extracellular fluid volume and on overall vasomotor (primarily venomotor) tone, which is important for distributing blood between central and peripheral pools. Effective circulating volume tends to fall during prolonged exercise, especially exercise in the heat, for three reasons (Fig. 60-8). First, exercise causes a shift in plasma water from the intravascular to the interstitial space. This transcapillary

movement of fluid during exercise primarily reflects increased capillary hydrostatic pressure (see Chapter 20). In addition, increased osmolality within muscle cells removes water from the extracellular space. When exercise intensity . exceeds 40% of VO2max, this loss of plasma water is proportional to the exercise intensity. In extreme conditions, the loss of plasma water can amount to more than 500 mL, or approximately one sixth of the total plasma volume. Second, exercise causes a loss of total body water through sweating (discussed in the next major section). If exercise is prolonged, without concomitant water intake, sweat loss will cost the body an important fraction of its total water. A loss of body water in excess of 3% of body weight is associated with early signs of heat-related illness, including lightheadedness and disorientation, and it constitutes clinical dehydration. Third, exercise causes a redistribution of blood volume to the skin because of the increase in cutaneous blood flow in response to body heating (Fig. 60-8). Venous volume in the skin increases as a result of the increased pressure in the compliant vessels as blood flow to the skin rises. No compensatory venoconstriction occurs in the skin because of the overriding action of the temperature control system. In response to this decrease in effective circulating volume that occurs during exercise, the cardiopulmonary, low-pressure baroreceptors (see Chapter 19) initiate compensatory responses to increase total vascular resistance (Fig. 60-8). This increase in resistance is accomplished through the sympathetic nervous system by (1) increasing the splanchnic vascular resistance, (2) offsetting some of the vasodilatory drive to the skin initiated by the temperature control system, and (3) offsetting some of the vasodilatory drive to the active skeletal muscles. In conditions of heavy thermal demand, the restriction of peripheral blood flow has the benefit of helping to maintain arterial blood pressure and effective circulating volume, but it carries two liabilities. First, it reduces convective heat transfer from the core to the skin because of the reduced skin blood flow and consequently contributes to excessive heat storage and, in the extreme, heat stroke (see Chapter 59 for the box on this topic). Second, the limitation of blood flow to active muscle may compromise O2 delivery and hence aerobic performance. In conditions of low thermal and metabolic demand, no serious conflict arises among the systems that regulate effective circulating volume, arterial blood pressure, and body temperature. The cutaneous circulation is capable of handling the heat transfer requirements of the temperature regulatory mechanism without impairing muscle blood flow or cardiac filling pressure.

SWEATING Eccrine, but not apocrine, sweat glands contribute to temperature regulation Sweat glands are exocrine glands of the skin, formed by specialized infoldings of the epidermis into the underlying dermis. Sweat glands are of two types: apocrine and eccrine (Fig. 60-9A). The apocrine sweat glands, located in the

Chapter 60 • Exercise Physiology and Sports Science

Venous reservoir

3 Effective circulating volume falls.

Left ventricle Right ventricle

Pulmonary blood volume

Splanchnic circulation

2a Fluid leaves capillaries, entering interstitial space.

Muscle

4 In response to signals from low-pressure baroreceptors, splanchic valve closes and valves to muscle and skin become less open.

1 Exercise causes these valves to open.

Skin

2c Increased blood flow raises venous volume in skin.

2b Fluid is lost from all compartments due to sweating.

Figure 60-8

Effect of exercise on central blood volume. “Valves” refer to the resistance vasculature of respective organs.

axillary and anogenital regions of the body, are relatively few in number (∼100,000) and large in diameter (2 to 3 mm). Their ducts empty into hair follicles. These glands, which often become active during puberty, produce a turbid and viscous secretion that is rich in lipids and carbohydrates and carries a characteristic body odor that has spawned an entire industry to conceal. Apocrine sweat glands have no role in temperature regulation in humans, although they may act as a source of pheromones. Eccrine sweat glands are distributed over the majority of the body surface, are numerous (several million), and are small in diameter (50 to 100 μm). The palms of the hands and soles of the feet tend to have both larger and more densely distributed eccrine glands than elsewhere. The full complement of eccrine sweat glands is present at birth and becomes functional within a few months, and the density of these glands decreases as the skin enlarges during normal growth. The essential role of eccrine sweating is temperature regulation, although stimuli such as food, emotion, and pain

can evoke secretory activity. Regionally, the trunk, head, and neck show more profuse sweating than the extremities. Sweat production is quantitatively less in women than in men, a finding reflecting less output per gland rather than fewer eccrine sweat glands.

Eccrine sweat glands are tubules comprising a secretory coiled gland and a reabsorptive duct An eccrine sweat gland is a simple tubular epithelium composed of a coiled gland and a duct (Fig. 60-9B). A rich microvascular network surrounds the entire sweat gland. The coiled gland, located deep in the dermis (see Fig. 15-26), begins at a single blind acinus innervated by postganglionic sympathetic fibers that are cholinergic. The release of acetylcholine stimulates muscarinic receptors on the acinar cells and causes them to secrete into the lumen a clear, odorless solution, similar in composition to protein-free plasma. This

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A

APOCRINE AND ECCRINE SWEAT GLANDS

Sebaceous gland Apocrine sweat gland

COMPONENTS OF AN ECCRINE SWEAT GLAND Pore Epidermis

Lumen Na+

Interstitial space ENaC

Duct cell 3 Na+

–

Cl

–

2 K+

CFTR K Cl

Absorption of Na+ and Cl–

+

–

Dermis

B

Eccrine sweat gland

3 Na+

Coil cell

Primary secretion of a proteinfree filtrate

2 K+ 6

Cl– 5 K+ 3 K+

Sympathetic nerve

3 Na+ 6 Na

–

+

6 Cl–

+ This cotransporter cycles three times.

Figure 60-9 A and B, Sweat glands. The sebaceous gland—the duct of which empties into the hair follicle independently of the duct apocrine sweat gland—secretes sebum, a mixture of fat and the remnants of the cells that secrete the fat.

Chapter 60 • Exercise Physiology and Sports Science

primary secretion flows through a long, wavy duct that passes outward through the dermis and epidermis. Along the way, duct cells reabsorb salt and water until the fluid reaches the skin surface through an opening, the sweat pore. Although these pores are too small to be seen with the naked eye, the location of sweat pores is readily identified as sweat droplets form on the skin surface. Both the secretory cells in the coil and the reabsorptive cells in the duct are rich in mitochondria, which are essential for providing sustained energy for the high rates of ion transport necessary for prolonged periods of intense sweating, for example, during exercise in hot environments. Surrounding the secretory cells in the coil is a layer of myoepithelial cells that resemble smooth muscle and may contract, thereby expressing sweat to the skin surface in a pulsatile fashion. However, this action is not essential because the hydrostatic pressures generated within the gland can exceed 500 mm Hg. Secretion by Coil Cells

The release of acetylcholine onto the secretory coil cells activates muscarinic G protein– coupled receptors (see Chapter 3) and thus leads to the activation of phospholipase C, which, in turn, stimulates protein kinase C and raises [Ca2+]i. These signals somehow trigger the primary secretion, which follows the general mechanism for Cl− secretion (see Chapter 5). A Na/K/Cl cotransporter (see Chapter 5) mediates the uptake of Cl− across the basolateral membrane, and the Cl− exits across the apical membrane through a Cl− channel (Fig. 60-9B, lower inset). As Cl− diffuses into the lumen, the resulting lumennegative voltage drives Na+ secretion through the paracellular pathway. The secretion of NaCl, as well as of urea and lactate, into the lumen sets up an osmotic gradient that drives the secretion of water, so the secreted fluid is nearly isotonic with plasma. This secretion of fluid into the lumen increases hydrostatic pressure at the base of the gland and thereby provides the driving force for moving the fluid along the duct to reach the skin surface.

Reabsorption by Duct Cells As the secreted solution flows along the sweat gland duct, the duct cells reabsorb Na+ and Cl− (Fig. 60-9B, upper inset). Na+ enters the duct cells across the apical membrane through epithelial Na+ channels (ENaCs), and Cl− enters through the cystic fibrosis transmembrane regulator (CFTR). The Na-K pump is responsible for the extrusion of Na+ across the basolateral membrane, and Cl− exits through a pathway such as a Cl− channel. Because the water permeability of the epithelium lining the sweat duct is low, water reabsorption is limited, resulting in a final secretory fluid that is always hypotonic to plasma. Because sweat is hypotonic, sweating leads to the loss of solute-free water, that is, the loss of more water than salt. As a result, the extracellular fluid contracts and becomes hyperosmolar, thereby causing water to exit from cells. Thus, intracellular fluid volume decreases, and intracellular osmolality increases (see Chapter 5). This water movement helps to correct the fall in extracellular fluid volume. The solutefree water lost in perspiration therefore is ultimately derived from all body fluid compartments.

The NaCl content of sweat increases with the rate of secretion but decreases with acclimatization to heat Flow Dependence With mild stimulation of acinar cells, the small volume of primary secretion travels slowly along the duct, and the ducts reabsorb nearly all the Na+ and Cl−, which can fall to final concentrations as low as ∼5 mM (Fig. 60-10). In contrast, with strong cholinergic stimulation, a large volume of primary secretion travels rapidly along the duct, so the load exceeds the capacity of the ductal epithelium to reabsorb Na+ and Cl−. Thus, a greater fraction of the secreted Na+ and Cl− remains within the lumen, with resulting levels of 50 to 60 mM. In contrast, [K+] in the sweat remains nearly independent of flow at 5 to 10 mM. Cystic Fibrosis In patients with cystic fibrosis (see Chapter 43 for the box on this topic), abnormal sweat gland function is attributable to a defect in the CFTR, a cAMP-regulated Cl− channel that is normally present in the apical membrane of sweat gland duct cells. These individuals secrete normal volumes of sweat into the acinus but have a defect in Cl− (and, therefore, Na+) absorption as the fluid travels along the duct. As a result, the sweat is relatively rich in NaCl (Fig. 60-10). Replenishment During a thermoregulatory response in a healthy individual, the rate of sweat production can commonly reach 1 to 2 L/hour, which, after a sufficient time, can represent a substantial fraction of total body water. Such a loss of water and salt requires adequate repletion to pre-

100

Na+ and Cl– in C.F. sweat

80 Na+ Cl– Concentration in mM

60

40

20 Lactate– K+ 0

0

10 20 Sweat flow (mL/[min · m2])

30

Figure 60-10 Flow dependence of sweat composition. Defective Cl− (and therefore Na+) reabsorption in cystic fibrosis (CF) patients leads to greater salt loss in sweat.

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serve fluid and electrolyte balance. Restoring body fluid volume following dehydration is often delayed in humans despite the consumption of fluids. The reason for this delay is that dehydrated persons drink free water, which reduces the osmolality of the extracellular fluid and thus reduces the osmotic drive for drinking (see Chapter 40). This consumed free water distributes into the cells as well as the extracellular space and dilutes the solutes. In addition, the reduced plasma osmolality leads to decreased secretion of arginine vasopressin (i.e., antidiuretic hormone), thus increasing free water excretion by the kidney (see Fig. 40-7). A more effective means of restoring body fluid volume is to ingest NaCl with water. When Na+ is taken with water (as in many exercise drinks), plasma [Na+] remains elevated throughout a greater duration of the rehydration period and is significantly higher than with the ingestion of water alone. In such conditions, the salt-dependent thirst drive is maintained, and the stimulation of urine production is delayed, thereby leading to more complete restoration of body water content. Acclimatization

With ample, continuing hydration, a heat-acclimatized individual can sweat up to 4 L/hour during maximal sweating. Over several weeks, as the body acclimates to high rates of eccrine sweat production, the ability to reabsorb NaCl increases, and the result is more hypotonic sweat. This adaptation is mediated by aldosterone (see Chapter 35) in response to the net loss of Na+ from the body during the early stages of acclimatization. For example, an individual who is not acclimatized and who sweats profusely can lose more than 30 g of salt per day for the first few days. In contrast, after several weeks of acclimatization, salt loss falls to several grams per day. Thus, an important benefit of physical training and heat acclimatization is the development of more dilute perspiration, which conserves NaCl content and thus effective circulating volume (see Chapter 5) during dehydration. The hyperthermia of exercise stimulates eccrine sweat glands As discussed in Chapter 59, the rate of perspiration increases with body Tcore, which, in turn, increases during exercise. The major drive for increased perspiration is the sensing by the hypothalamic centers of increased body Tcore. Physical training increases the sensitivity of the hypothalamic drive to higher Tcore. Indeed, the hyperthermia of exercise causes sweating to begin at a lower skin temperature than does sweating elicited by external heating. The efferent limb of the sweating reflex is mediated by postganglionic sympathetic cholinergic fibers. Sweating is especially important for thermoregulation under hot ambient conditions and with exercise-induced increases in body temperature. Indeed, as ambient temperature rises to more than 30ºC, heat loss through radiation, convection, and conduction (see Chapter 59) becomes progressively ineffective, and evaporative cooling becomes by far the most important mechanism of regulating body temperature. Conversely, evaporative cooling becomes progressively less effective as ambient humidity rises (see Equation 59-5).

AEROBIC TRAINING Aerobic training requires regular periods of stress and recovery The body improves its capacity to perform work through physical exertion. However, one must meet four conditions to achieve a training effect, or adaptation to exercise. First, the intensity of the activity must be higher than a critical threshold. For aerobic training (e.g., running, cycling, and swimming), the level of stress increases with the speed of the activity. Second, each period of activity must be of sufficient duration. Third, one must repeat the activity over time on a regular basis (e.g., several times per week). Finally, sufficient rest must occur between each training session because adaptations occur during the recovery period. A great deal of research has focused on optimizing the foregoing four factors, as well as task specificity for individual athletes competing in specific events. Increasing levels of exertion progressively recruit and thereby adapt type I muscle fibers, followed by type IIa and then type IIb fibers. However, regardless of how long or intensely an individual trains, inactivity reverses these adaptations with an associated decrement in performance. Aerobic conditioning . increases Vo2max as well as the body’s ability to eliminate excess heat that is produced during exercise (see Fig. 59-5). Aerobic training increases maximal O2 delivery by increasing plasma volume and maximal cardiac output . VO2max could increase as the result of either optimizing O2 delivery to active muscle or optimizing O2 extraction by active muscle, as demonstrated in the following modification of Equation 60-7: Maximal rate of O2 uptake

Optimal heart rate

 O Va = max   2

HR opt ⋅ SVopt ⋅ (Ca O2 − Cv O2 )max   

 

mL O2 min

Optimal stroke volume

Maximal mL blood cardiac output min

Maximal a − v difference of O2 content

mL O2 mL blood

(60-9) In fact, aerobic training improves both O2 delivery and O2 extraction; the problem for physiologists has been to determine to what extent each system contributes to the wholebody response. For example, an increase in the circulatory system’s capacity to deliver O2 could reflect an increase in either the maximal arterial O2 content or the maximal cardiac output, or both. Maximizing Arterial O2 Content

Several factors could theoretically contribute to maximizing CaO2: 1. Increasing the maximal alveolar ventilation enhances the driving force for O2 uptake by the lungs (see Fig. 31-4). 2. Increasing the capacity for gases to diffuse across the alveolar-capillary barrier in the lungs could enhance O2

Chapter 60 • Exercise Physiology and Sports Science

uptake at very high cardiac output, particularly at high altitude (see Fig. 27-7). 3. Improving the matching of pulmonary ventilation to perfusion should increase arterial PO2 and the saturation of hemoglobin (see Chapter 31). 4. Increasing the concentration of hemoglobin enables a given volume of arterial blood to carry a greater amount of O2 (see Chapter 29). In nearly all conditions of exercise, the pulmonary system maintains alveolar PO2 at levels that are sufficiently high to ensure nearly complete (i.e., ∼97%) saturation of hemoglobin with O2, even at maximal power output. Thus, it is unlikely that an increase in the maximal alveolar ventilation or pulmonary . diffusing capacity could explain the large increase in VO2max that occurs with training. CaO2 would be increased by elevating the blood’s hemoglobin concentration. However, no evidence indicates that physical training induces such an increase. On the contrary, [hemoglobin] tends to be slightly lower in endurance athletes, a phenomenon called sports anemia, which reflects an expansion of the plasma compartment (discussed later). Whereas increasing [hemoglobin] provides a greater O2-carrying capacity in blood, maximal O2 transport does not necessarily increase accordingly because blood viscosity and therefore total vascular resistance also increase. The heart would be required to develop higher arterial pressure to generate an equivalent cardiac output. The resultant increased cardiac work would thus be counterproductive to the overall adaptive response. Blood doping—transfusion of blood before competition—is thus not only illegal, but also hazardous to athletes, particularly when water loss through sweating leads to further hemoconcentration. Maximizing Cardiac Output Factors that contribute to increasing maximal cardiac output include optimizing the increases in heart rate and cardiac stroke volume so their product (i.e., cardiac output) is maximal (Equation 60-9; see Chapter 25). Because training does not increase maximal heart rate and has a relatively small effect on O2 extraction, . nearly all the increase in VO2max that occurs with training must be the result of an increase in maximal cardiac output, the product of optimal heart rate and optimal stroke volume (Equation 60-9). The athlete achieves this increased cardiac output by increasing maximal cardiac stroke volume. Maximal cardiac output can increase by ∼40% during physical conditioning that also increases maximal aerobic power by 50%. The difference between 40% and 50% is accounted for by increased extraction: (CaO2 − CvO2)max. This increased extraction is the consequence of capillary proliferation and of increasing the content of mitochondria in muscle fibers that have adapted to endurance training, thereby creating a greater O2 sink under maximal aerobic conditions. Maximal cardiac stroke volume increases during aerobic training because expansion of the plasma compartment increases the heart’s preload (see Chapter 22), with concomitant hypertrophy of the heart. An increase in preload increases ventricular filling and proportionally increases stroke volume (Starling’s law of the heart), thereby elevating maximal cardiac output accordingly. An additional benefit is that a trained athlete achieves a given cardiac output at a

lower heart rate, both at rest and during moderate exercise. Because it is more efficient to increase stroke volume than heart rate, increasing stroke volume reduces the myocardial metabolic load for any particular level of activity. The expansion of plasma volume probably reflects an increase in albumin content (1 g albumin is dissolved in 18 g of plasma H2O). This increase appears to be caused both by translocation from the interstitial compartment and by increased synthesis by the liver. The result of more colloid in the capillaries is a shift of fluid from the interstitium to the blood. Although the total volume of red blood cells also increases with aerobic training, the plasma volume expansion is greater than the red blood cell expansion, thus reducing the hemoglobin concentration. This sports anemia occurs in highly trained endurance athletes, particularly those acclimatized to hot environments. The increased blood volume has another beneficial effect. It enhances the ability to maintain high skin blood flow in potentially compromising conditions (e.g., heavy exercise in the heat), thus providing greater heat transport from core to skin and relatively lower storage of heat (see Chapter 59). Aerobic training enhances O2 diffusion into muscle Whereas an increase in maximal cardiac output accounts for most of the increased O2 delivery to muscle with training, a lesser fraction reflects increased O2 extraction from blood. Fick’s law describes the diffusion of O2 between the alveolar air and pulmonary capillary blood (see Equation 30-7). A similar relationship describes the diffusion of O2 from the systemic capillary blood to the mitochondria. The factors that contribute to O2 diffusion are analogous to those that affect the diffusing capacity in the lung. Trained muscle can accommodate a greater maximal blood flow because of the growth of new microvessels, particularly capillaries. Indeed, well-conditioned individuals have a 60% greater number of capillaries per cross-sectional area of muscle than do sedentary people. This increased capillary density increases O2 delivery and thus provides a greater surface area for diffusion. Increase in capillary density also reduces the diffusion distance for O2 between the capillary and muscle fibers (see Fig. 20-4). In addition, training increases total capillary length and volume, prolongs the transit time of red blood cells along capillaries, and thereby promotes the extraction of O2 and nutrients from the blood as well as the removal of metabolic byproducts. Finally, training increases cardiac output and muscle blood flow and preserves a relatively high capillary PO2 throughout the muscle that maintains the driving force for O2 diffusion from capillaries to mitochondria. Aerobic training increases mitochondrial content In untrained (but otherwise healthy) individuals, the maximum ability of mitochondria to consume O2 is considerably greater than that of the cardiovascular system to supply O2. Thus, mitochondrial content does not limit . VO2max. We have already seen that endurance training markedly increases O2 delivery. In parallel, endurance training

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Section X • Physiology of Everyday Life

Training

No training

220

200

Citric acid cycle enzymes

180 Conversion of glycogen phosphorylase b to a

Control (%) 160

Capillaries 140

Maximal oxygen uptake

120 Size of muscle fibers 100

0

1

2

6

12 Time (wk)

18

AMPK-dependent pathways

24

30

Figure 60-11 Enzyme adaptation during training. Training causes a slow increase in the level of several enzymes, as. well as in the number of capillaries, VO2max, and size of muscle fibers. These changes reverse rapidly on the cessation of training. (Data from Saltin B, Henriksson J, Nygaard E, Andersen P: Ann N Y Acad Sci 1977; 301:3-29.)

AMPK AMP ADP+Pi

Ca2+ signaling

mRNA

ATP

Nucleus Ca2+

Translation TFAM Import machinery

Sarcoplasmic reticulum

mtDNA Electron-transport chain I

II III

IV

V

Exercise Muscle cell

can also increase the mitochondrial content of skeletal muscle fibers nearly 2-fold by stimulating the synthesis of mitochondrial enzymes and other proteins (Fig. 60-11). The stimulus for mitochondrial biogenesis is the repeated activity of the muscle fiber during training, leading to increases in the time-averaged [Ca2+]i, which may act in two ways (Fig. 60-12). One is by directly modulating the transcription of nuclear genes. The other is by increasing cross-bridge cycling and raising [AMP]i, thereby stimulating the fuel sensor AMP

Figure 60-12 Exercise-induced mitochondrial biogenesis. (Data from Chabi B, Adhihetty PJ, Ljubicic V, Hood DA: Med Sci Sports Exerc 2005; 37:2102-2110.)

kinase—AMPK—which, in turn, can modulate transcription. Some of the newly synthesized proteins are themselves transcription factors that modulate the transcription of nuclear genes. At least one protein (Tfam) enters the mitochondrion and stimulates the transcription and translation of mitochondrial genes for key elements of the electron transport chain. Finally, some newly synthesized proteins encoded by genomic DNA, guided by cytoplasmic chaperones, target the mitochondrial import machinery and

Chapter 60 • Exercise Physiology and Sports Science

become part of multiple subunit complexes—together with proteins of mitochondrial origin. Because mitochondria create the sink for O2 consumption during the oxidative phosphorylation of ADP to ATP, increased mitochondrial content promotes O2 extraction from the blood. However, the primary benefit from mitochondrial adaptation in aerobic conditioning is the capacity to oxidize substrates, particularly fat, an ability that enhances endurance of muscle. Recall that mitochondria are responsible not only for the citric acid cycle and oxidative phosphorylation but also for β oxidation of fatty acids. In athletes trained for . endurance, the greater reliance on fat at a given level of VO2 is the metabolic basis of glycogen sparing and thus reduced production of lactate and H+. REFERENCES Books and Reviews Brooks GA, Fahey TD, Baldwin KM: Exercise Physiology: Human Bioenergetics and Its Applications, 4th ed. Boston: McGrawHill, 2004. Freinkel RK, Woodley DT (eds): The Biology of the Skin. New York: Parthenon, 2001, pp 47-76. Hurley HJ: The eccrine sweat gland: Structure and function. In Freinkel RK, Woodley DT (eds): The Biology of the Skin. New York: Parthenon, 2001, pp 47-76. Rowell LR, Shepherd JT (eds): American Physiological Society’s Handbook of Physiology, sect 12: Exercise: Regulation and Inte-

gration of Multiple Systems. New York: Oxford University Press, 1995. Tipton CM (ed): American College of Sports Medicine’s Advanced Exercise Physiology. Baltimore: Lippincott Williams & Wilkins, 2005. Journal Articles Burke RE, Levine DN, Tsairis P, Zajac FE 3rd: Physiological types and histochemical profiles in motor units of the cat gastrocnemius. J Physiol 1973; 234:723-748. Chabi B, Adhihetty PJ, Ljubicic V, Hood DA: How is mitochondrial biogenesis affected in mitochondrial disease? Med Sci Sports Exerc 2005; 37:2102-2110. Enoka RM: Morphological features and activation patterns of motor units. J Clin Neurophysiol 1995; 12:538-559. Holloszy JO: Biochemical adaptations in muscle: Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J Biol Chem 1967; 242:2278-2282. Salmons S, Sreter FA: Significance of impulse activity in the transformation of skeletal muscle type. Nature 1976; 263:30-34. Saltin B, Henriksson J, Nygaard E, Andersen P: Fiber types and metabolic potentials of skeletal muscles in sedentary man and endurance runners. Ann N Y Acad Sci 1977; 301:3-29. Saltin B, Strange S: Maximal oxygen uptake: Old and new arguments for a cardiovascular limitation. Med Sci Sports Exerc 1992; 24:30-37. Sato K, Kang WH, Saga K, Sato KT: Biology of sweat glands and their disorders. I. Normal sweat gland function. J Am Acad Dermatol 1989; 20:537-563. Thomas GD, Segal SS: Neural control of muscle blood flow during exercise. J Appl Physiol 2004; 97:731-738.

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CHAPTER

61 E N V I R O N M E N TA L P H Y S I O L O G Y Arthur DuBois

The earth and its atmosphere provide environments that are compatible with an extraordinary number of diverse life forms, each adapted to its particular ecologic niche. However, not all the earth’s surface is equally friendly for human survival, let alone comfort and function. Mountain climbers and deep sea divers know the profound effects of barometric pressure (PB) on human physiology, and astronauts quickly learn how the physically equivalent forces of gravity and acceleration affect the body. Humans can adapt to changes in PB and gravity up to a point, but survival under extreme conditions requires special equipment; otherwise, our physiological limitations would restrict our occupancy of this planet to its lowland surfaces. Much can be learned from exposure to extreme environmental conditions. Although most people do not seek out these extreme environments, the same physiological responses that occur under extreme environmental conditions may also occur, to a lesser extent, in everyday life. In this chapter, I first discuss general principles of environmental physiology and then focus on extreme environments encountered in three activities: deep sea diving, mountain climbing, and space flight.

THE ENVIRONMENT Voluntary feedback-control mechanisms can modulate the many layers of our external environment Chapter 1 describes Claude Bernard’s concept of the milieu intérieur (basically, the extracellular fluid in which cells of the organism live) and his notion that “fixité du milieu intérieur” (the constancy of this extracellular fluid) is the condition of “free, independent life.” Chapters 2 through 57 focus mainly on the interaction between cells and their extracellular fluid. In this chapter, I consider how the milieu extérieur, which physically surrounds the whole organism, affects our body functions and how we, in turn, modify our surroundings when it is necessary to improve our comfort or to extend the range of habitable environments. The milieu extérieur, in fact, has several layers: the skin surface, the air that surrounds the skin, clothing that may

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surround that air, additional air that may surround the clothing, a structure (e.g., a house) that may surround that air, and finally a natural environment that surrounds that structure. As we interact with our multilayered environment, sensors monitor multiple aspects of the milieu intérieur, and involuntary physiological feedback-control mechanisms— operating at a subconscious level—make appropriate adjustments to systems that control a panoply of parameters, including blood pressure, ventilation, effective circulating volume, gastric secretions, blood glucose levels, and temperature. The sensory input can also rise to a conscious level and, if perceived as discomfort, can motivate us to take voluntary actions that make the surroundings more comfortable. For example, if we sense that we are uncomfortably hot, we may move out of the sun or, if indoors, turn on the air conditioning. If we then sense that we are too cool, we may move into the sun or turn off the air conditioning. Such conscious actions are part of the effector limb in a complex negative feedback system that includes sensors, afferent pathways, integration and conscious decision making in the brain, efferent pathways to our muscles, and perhaps inanimate objects such as air conditioners. For a voluntary feedback system to operate properly, the person must be aware of a signal from the surroundings and must be able to determine the error by which this signal deviates from a desirable set-point condition. Moreover, the person must respond to this error signal by taking actions that reduce the error signal and thereby restore the milieu intérieur to within a normal range. Humans respond to discomfort by a wide variety of activities that may involve any layer of the environment. Thus, we may adjust our clothing, build housing, and eventually even make equipment that allows us to explore the ocean depths, mountain heights, and outer space. Physiological control mechanisms—involuntary or voluntary—do not always work well. Physicians are acutely aware that factors such as medication, disease, or the extremes of age can interfere with involuntary feedback systems. These same factors can also interfere with voluntary feedback systems. For example, turning on the air conditioning is a difficult or even impossible task for an unconscious person, a bedridden patient, or a perfectly healthy baby. In these

Chapter 61 • Environmental Physiology

situations, a caregiver substitutes for the voluntary physiological control mechanisms. However, to perform this role effectively, the caregiver must understand how the environment would normally affect the care recipient and must anticipate how the involuntary and voluntary physiological control mechanisms would respond. Environmental temperature provides conscious clues for triggering voluntary feedback mechanisms Involuntary control mechanisms—discussed in Chapter 60—can only go so far in stabilizing body core temperature in the presence of extreme environmental temperatures. Thus, voluntary control mechanisms can become extremely important. As summarized in Table 60-1, the usual range of body core temperature is 36ºC to 38ºC. At an environmental temperature of 26ºC to 27ºC and a relative humidity of 50%, a naked person is in a neutral thermal environment (see Chapter 60), feeling comfortable and being within the zone of vasomotor regulation of body temperature. At 28ºC to 29ºC, the person feels warm, and ∼25% of the skin surface becomes wet with perspiration. At 30ºC to 32ºC, the person becomes slightly uncomfortable. At 35ºC to 37ºC, one becomes hot and uncomfortable, ∼50% of the skin area is wet, and heat stroke (see the box on heat stroke in Chapter 60) may be possible. The environmental temperature range of 39ºC to 43ºC is very hot and uncomfortable, and the body may fail to regulate core temperature. At 46ºC, the heat is unbearable, and heat stroke is imminent—the body heats rapidly, and the loss of extracellular fluid to sweat may lead to circulatory collapse and death (see Chapter 60). At the other extreme, we regard environmental temperatures of 24ºC to 25ºC as cool and 21ºC to 22ºC as slightly uncomfortable. At temperatures of 19ºC to 20ºC, we feel cold, vasoconstriction occurs in the hands and feet, and muscles may be painful. Room ventilation should maintain PO2, PCO2, and toxic substances within acceptable limits . Ventilation of a room (VRoom) must be sufficient to supply enough O2 and to remove enough CO2 to keep the partial pressures of these gases within acceptable limits. In addition, . it may be necessary to increase VRoom even more, to lower relative humidity and to reduce odors. As outlined in Table 26-1, dry air in the natural environment at sea level has a PO2 of ∼159 mm Hg (20.95%) and a PCO2 of ∼0.2 mm Hg (0.03%). Acceptable limits for PO2 and PCO2 The acceptable lower limit for PO2 for work environments is 148 mm Hg in dry air, which is 19.5% of dry air at sea level. The environmental atmosphere of a submarine may be kept at this slightly low PO2 to minimize the chance of fires, yet retain the mental capacity of the occupants. An acceptable upper limit for PCO2 in working environments is 3.8 mm Hg, or 0.5% of dry air at sea level. This level of CO2 would increase total ventilation by ∼7%, a hardly

noticeable rise. Exposures to 3% CO2 in the ambient air— which initially would cause more substantial respiratory acidosis—could be tolerated for at least 15 minutes, by the end of which it would nearly double total ventilation. With longer exposures to 3% CO2, the metabolic compensation to respiratory acidosis (see Chapters 28 and 39) would have already begun to increase plasma [HCO3−] noticeably. Measuring Room Ventilation .

Two approaches are available for determining VRoom. The first is a steady-state method . that requires knowing (1) the rate of CO2 production (VCO2) by the occupants of the room and (2) the fraction of the room air that is CO2. The equation is analogous to the one introduced for determining alveolar ventilation, beginning with Equation 31-9: V Room =

V CO2 ⎛ Fraction of ⎞ ⎜ room air ⎟ ⎝ that is CO2 ⎠

(61-1)

We could use a similar equation based on PO2 and the rate of O2 extraction by the occupants. In the exponential decay method, the second approach . for determining VRoom, one monitors the washout of a gas from the room. The approach is to add a test gas (e.g., CO2) to the room and then measure. the concentrations of the gas at time zero (Cinitial) and—as VRoom washes out the gas over some time interval (Δt)—at some later time (Cfinal). The equation for exponential decay is as follows:  Room = VRoom ln ⎛ Cinitial ⎞ V ⎜⎝ C ⎟ Δt final ⎠

(61-2)

For example, imagine that we wish to measure the ventilation of a room that is 3 × 3 × 3 m—a volume of 27 m3 or 27,000 L. Into this room, we place a tank of 100% CO2 and a fan to mix the air. We then open the valve on the tank until an infrared CO2 meter reads 3% CO2 (Cinitial = 3%), at which point we shut off the valve on the tank. Ten minutes later (Δt = 10 minutes), the meter reads 1.5% (Cfinal = 1.5%). Substituting these measured values into Equation 61-2 leads to the following:

( )

27, 000 liters 3.0% ln = 1871 liters /min V Room = 10 min 1.5%

(61-3)

This approach requires that the incoming air contain virtually no CO2 and that the room contain no CO2 sources (e.g., people). Carbon Monoxide

More insidious than hypoxia, and less noticeable, is the symptomless encroachment of carbon monoxide (CO) gas on the oxyhemoglobin dissociation curve (see Chapter 29). CO—which can come from incomplete combustion of fuel in furnaces, charcoal burners, or during house fires—suffocates people without their being aware of its presence. Detectors for this gas are thus essential for providing an early warning. CO can be lethal when it occupies approximately half of the binding sites on hemoglobin (Hb), which occurs at a PCO of ∼0.13 mm Hg or

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0.13/760 ≅ 170 parts per million (ppm). However, the halftime for washing CO into or out of the body is ∼4 hours. Thus, if the ambient CO level were high enough to achieve a 50% saturation of Hb at equilibrium, then after a 2-hour exposure (i.e., one half of the half-time), the CO saturation would be 1/2 × 1/2 × 50% or 12.5%. The symptoms at this point would be mild and nonspecific and would include headache, nausea, vomiting, drowsiness, and interference with night vision. Victims with limited coronary blood flow could experience angina. After a 4-hour exposure (i.e., one half-time), the CO saturation would be 1/2 × 50%, or 25%. The symptoms would be more severe and would include impaired mental function and perhaps unconsciousness. Threshold Limit Values and Biological Exposure Indices Threshold limit values (TLVs) are reasonable environ-

mental levels of toxic substances or physical agents (e.g., heat or noise) to which industrial workers can be exposed without causing predictable harm. Rather than depending on concentrations measured in air or food, we can use biological exposure indices (BEIs) to limit exposure to toxic substances by measuring the effects of these substances on animals and humans. The changes detected in the body are called biomarkers of exposure and correlate with the intensity and duration of exposure to toxic substances. Tissues must resist the G force produced by gravity and other mechanisms of acceleration Standing motionless on the earth’s surface at sea level, we experience a gravitational force—our weight—that is the product of our mass and the acceleration resulting from gravity (g = 9.8 ms−2): FN newtons

= m N × Ng kg

ms−2

(61-4)

In a particular condition, we may experience a different acceleration (a) from that caused by gravity. The G force is a dimensionless number that describes force (m · a) that we experience under a particular condition, relative to the gravitational force (m · g): G=

m⋅a a = m⋅ g g

(61-5)

Thus, we normally experience a force of +1G that would cause us to fall with an acceleration of 9.8 ms−2 if we were not supported in some way. Accelerations besides that caused by gravity also affect physiology. An accelerometer, placed on a belt, would show that we can jump upward with an acceleration of ∼3G. It would also show that, on landing, we would strike the ground with a force of 3G—a force that our bones and other tissues must be able to tolerate. Later, we discuss G forces from the perspective of air and space flight. At +1G, each cm2 of the cross section of a vertebral body, for example, can withstand the compressive force generated by a mass of ∼20 kg before the trabeculae begin to be crushed. Thus, at +1G, a vertebral body with a surface area of 10 cm2 could support the compressive force generated by a mass of

∼200 kg, far more than enough to support 35 kg, the mass of the upper half of the body of a 70-kg person. In fact, this strength would be adequate to withstand a G force of a (200 kg)/(35 kg) = +5.7G—provided the backbone is straight. However, if the backbone is not straight, the tolerance could be +3G, or approximately the acceleration achieved by jumping upward and landing on the feet with the back curved. When a pilot ejects from an aircraft, the thrust of the explosive cartridges accelerates the seat upward, and this can crush a vertebral body unless the pilot keeps the back straight. With increasing age, our bones tend to demineralize (see Chapter 60), a process that weakens them and also causes us to grow shorter because of the demineralization of the vertebrae. Stepping off a curb, an elderly person with demineralized bones may fracture the neck of the femur or crush a vertebra. Demineralization also occurs with immobilization and space flight. In one study, a 6- to 7-week period of immobilization from bed rest led to losses of 14 g of calcium from bones, 1.7 kg of muscle cytoplasm, 21% in the strength of the gastrocnemius muscle, and 6% in average blood volume. The subjects became faint when they were suddenly tilted on a board, head above feet. Although the changes were reversible after these subjects resumed ambulation, it took 4 weeks for muscle strength to return to normal during remobilization. The partial pressures of gases—other than water—inside the body depend on barometric pressure As discussed in the next two major sections of this chapter, extremely high or extremely low values of PB create special challenges for the physiology of the body, particularly the physiology of gases. Dalton’s law (see Chapter 26 for the box on this topic) states that PB is the sum of the partial pressures of the individual gases in the air mixture. Thus, in the case of ordinary dry air (see Table 26-1), most of the sea level PB of 760 mm Hg is the result of N2 (∼593 mm Hg) and O2 (∼159 mm Hg), with smaller contributions from trace gases such as argon (∼7 mm Hg) and CO2 (∼0.2 mm Hg). As PB increases during diving beneath the water, or as PB decreases during ascent to high altitude, the partial pressure of each constituent gas in dry ambient air changes in proportion to the change in PB. At high values of PB, this relationship is especially important for ambient PN2 and PO2, which can rise to toxic levels. At low values of PB, this relationship is important for ambient PO2, which can fall to levels low enough to compromise the O2 saturation of Hb (see Chapter 31) and thus the delivery of O2 to the tissues. The proportionality between PB and the partial pressure of constituent gases breaks down in the presence of liquid water. When a gas is in equilibrium with liquid water—as it is for inspired air by the time it reaches the trachea (see Chapter 26)—the partial pressure of water vapor (PH2O) depends not on PB but on temperature. Thus, at the very high pressures associated with deep sea diving, PH2O becomes a negligible fraction of PB, whereas PH2O becomes an increasingly dominant factor as we ascend to altitude.

Chapter 61 • Environmental Physiology

DIVING PHYSIOLOGY For every 10 m of depth of immersion, barometric pressure increases by 1 atm, thereby compressing gases in the lungs The average PB at sea level is 760 mm Hg. In other words, if you stand at sea level, the column of air extending from your feet upward for several tens of kilometers through the atmosphere exerts a pressure of 1 atmosphere (atm). In a deep mine shaft, over which the column of air is even taller, PB is higher still. However, it is only when diving under water that humans can experience extreme increases in PB. A column of fresh water extending from the earth’s surface upward 10.3 m exerts an additional pressure of 760 mm Hg—as much as a column of air extending from sea level to tens of kilometers skyward. The same is true for a column of water extending from the surface of a lake to a depth of 10.3 m. For seawater, which has a density ∼2.5% greater than that of fresh water, the column must be only 10 m to exert 1 atm of pressure. Because liquid water is virtually incompressible, PB increases linearly with the height (weight) of the column of water (Fig. 61-1). Ten meters below the surface of the sea, PB is 2 atm, 1 atm for the atmospheric pressure plus 1 atm for the column of water. As the depth increases to 20 m and then to 30 m, PB increases to 3 atm, then 4 atm, and so on. Increased external water pressure does not noticeably compress the body’s fluid and solid components until a depth of ∼1.5 km. However, external pressure compresses each of the body’s air compartments to an extent that depends on the compliance of the compartment. In compliant cavities such as the intestines, external pressure readily compresses internal gases. In relatively stiff cavities, or those that cannot equilibrate readily with external pressure, increases of external pressure can distort the cavity wall, with resulting pain

20 Breathing a mixture of O2 and helium to “saturation” at this depth requires nearly 4 days of decompression.

18 16

Pressure (atm)

14 Human record for breath-hold dive.

12 10 8 6

Breathing air for 2–4 hr (“saturation”) at this depth requires 40 h of decompression.

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Figure 61-1 Pressures at increasing depth of immersion. The pressure at the surface of the ocean is 1 atm and increases by 1 atm for each 10 m of immersion in sea water.

or damage. For example, when the eustachian tube is blocked, the middle ear pressure cannot equilibrate with external pressure, and blood fills the space in the middle ear or the tympanic membrane ruptures. According to Boyle’s law, pressure and volume vary inversely with each other. Thus, if the chest wall were perfectly compliant, a breath-holding dive to 10 m below the surface would double the pressure and compress the air in the lungs to half its original volume. Aquatic mammals can dive to extreme depths because rib flexibility allows the lungs to empty. Whales, for example, can extend a breath-hold dive for up to 2 hours and can descend to depths as great as 900 m (91 atm) without suffering any ill effects. The human chest wall does not allow complete emptying of the lungs, and, indeed, the human record for a breath-hold dive is 160 m below the surface. In a breath-hold dive that is deep enough to double PB, alveolar PCO2 could also double to 80 mm Hg. Because this value is substantially higher than the PCO2 of mixed venous blood at sea level (46 mm Hg), the direction of CO2 diffusion across the blood-gas barrier reverses, and alveolar CO2 enters pulmonary capillary blood and increases arterial PCO2. In time, metabolically generated CO2 accumulates in the blood and eventually raises mixed venous PCO2 to values higher than alveolar PCO2 so CO2 diffusion again reverses direction, and CO2 accumulates in the alveoli. The increase in arterial PCO2 can reduce the duration of the dive by increasing ventilatory drive (see Chapter 32). During the ascent phase of the dive, the fall in PB leads to a fall in alveolar PCO2 and PO2, promoting the exit of both gases from the blood, and thus a fall in arterial PCO2 and PO2. The fall in arterial PO2 can lead to shallow water blackout. Divers breathe compressed air to keep their lungs normally expanded Technical advances have made it possible for divers to remain beneath the water surface for periods longer than permitted by a single breath-hold. One of the earliest devices was a diving bell that surrounded the diver on all sides except the bottom. Such a bell was reportedly used by Alexander the Great in 330 bc and then improved by Sir Edmund Halley in 1716 (Fig. 61-2). By the early 19th century, pumping compressed air from above the water surface through a hose to the space underneath the bell kept water out of the bell. In all these cases, the diver breathed air at the same pressure as the surrounding water. Although the pressures both surrounding the diver’s chest and inside the airways were far higher than at sea level, the pressure gradient across the chest wall was normal. Thus, the lungs were normally expanded. The conditions are essentially the same in a modern-day caisson, a massive, hollow, pressurized structure that functions like a large diving bell. Once again, the pressure inside the caisson (3 to 4 atm) has to be high enough to prevent water at the bottom of the caisson from entering. Several workers (“sand hogs”) at the bottom of the caisson may excavate material from the bottom of a river for constructing tunnels or foundations of bridges. Technical advances also extended to individual divers, who first wore diving suits with spherical helmets over their heads (Fig. 61-3A). The air inside these helmets was pressur-

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2000 liters Sea Level

1000 liters

ized to match exactly the pressure of the water in which they were diving. In 1943, Jacques Cousteau perfected the selfcontained underwater breathing apparatus, or SCUBA, that replaced cumbersome gear and increased the mobility and convenience of an underwater dive (Fig. 61-3B). Although the foregoing techniques permit deep dives for extended periods of time, they require training and carry the risk of drowning secondary to muscle fatigue and hypothermia. Air floatation and thermal insulation of the diving suit lessen these hazards. For reasons that will become apparent, use of any of these techniques while breathing room air carries additional hazards, including nitrogen narcosis, O2 toxicity, and problems with decompression. Increasing alveolar P N2 can cause narcosis

10 m

500 liters

30 m

Descending beneath the water causes the inspired PN2— nearly 600 mm Hg at sea level (see Table 26-1)—to increase as PB increases. According to Henry’s law (see Chapter 26 for the box on this topic), the increased PN2 will cause more N2 to dissolve in pulmonary capillary blood and, eventually, the body’s tissues. The dissolved [N2] in various compartments begins to increase immediately but may take many hours to reach the values predicted by Henry’s law, as discussed later. Because of its high lipid solubility, N2 dissolves readily in adipocytes and in membrane lipids. A high PN2 reduces the ion conductance of membranes, and therefore neuronal excitability, by mechanisms that are similar to those of gas anesthetics. Diving to increased depths (e.g., 4 to 5 atm) while breathing compressed air causes nitrogen narcosis. Mild nitrogen narcosis resembles alcohol intoxication (e.g., loss of psychosocial inhibitions). According to “Martini’s law,” each 15 m of depth has the effects of drinking an additional martini. Progressive narcosis occurs with increasing depth or time of the dive and is accompanied by lethargy and drowsiness, rapid onset of fatigue, and, eventually, loss of consciousness. Because it develops insidiously, nitrogen narcosis poses a potentially fatal threat to divers who are not aware of the risks. Increasing alveolar PO2 can lead to O2 toxicity At sea level, dry inspired air has a PO2 of 159 mm Hg. However, the alveolar PO2 of a healthy person at sea level air

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Figure 61-2 Diving bell. Between 1716 and 1721, Halley, the astronomer who gave his name to the comet, designed and built a wooden diving bell with an open bottom. Because the bell was at a relatively shallow depth (∼12 m), the water level rose only partly into the bell. In Halley’s system, the air was replenished from a barrel that was open at the bottom and weighted with lead to sink beneath the diving bell. Thus, the air pressure in the barrel was higher than in the bell. The diver used a valve to regulate airflow into the bell. This design was in use for a century, until a practical pump was available for pumping air directly from the surface. The lower part of the figure illustrates what would have happened if Halley’s bell had been lowered to much greater depths. The greater the depth, the greater the water pressure. Because the air pressure inside the bell must be the same as the water pressure, the air volume progressively decreases at greater depths, and the water level rises inside the bell.

Chapter 61 • Environmental Physiology

A

DIVING HELMET

Air from surface

B

SCUBA SYSTEM Hose

Air hose Diving helmet

Reducing valve Air cylinder

Demand and exhaust valve

Figure 61-3 Devices for breathing under water. A, Compressed air, pumped from the surface to the diver, keeps the pressure inside the helmet slightly higher than that of the surrounding water. B, SCUBA is an acronym for self-contained underwater breathing apparatus.

is ∼101 mm Hg, reduced from 159 mm Hg by humidification in the airways and removal of O2 by gas exchange with the blood (see Chapter 31). Arterial PO2 at sea level is very close to alveolar PO2 (within ∼10 mm Hg) and nearly saturates Hb, to yield an arterial O2 content of ∼20 mL/dL blood (Fig. 61-4, red curve). As PB—and therefore arterial PO2— increases at greater depths, the O2 bound to Hb increases very little. However, according to Henry’s law (see Chapter 26 for the box on this topic), the O2 that is physically dissolved in the water of blood increases linearly (Fig. 61-4, black line). Thus, the increment of total O2 content at depth reflects dissolved O2 (Fig. 61-4, blue curve). During a breath-hold dive to 5 atm, or in a hyperbaric chamber pressurized to 5 atm, arterial PO2 increases to ∼700 mm Hg, slightly higher than breathing 100% O2 at sea level. Exposure to such a high PO2 has no ill effects for up to several hours. However, prolonged exposure damages the airway epithelium and smooth muscle and causes bronchiolar and alveolar membrane inflammation and, ultimately, pulmonary edema, atelectasis, fibrin formation, and lung consolidation. These effects are the result of inactivating several structural repair enzymes and oxidizing certain cellular constituents. A prolonged, elevated PO2 also has detrimental effects on nonpulmonary tissues, including the central nervous system (CNS). Exposure to an ambient PO2 of ∼1500 mm Hg (e.g., breathing room air at ∼10 atm) for as little as 30 to 45 minutes can cause seizures and coma. Preliminary symptoms of O2 toxicity include muscle twitching, nausea, disorientation, and irritability. The toxic effects of O2 occur because O2 free radicals (e.g., superoxide and peroxide free radicals) oxidize the polyunsaturated fatty acid component of cell membranes as well as enzymes that are involved in energy metabolism. At the more modest PO2 levels that normally

prevail at sea level, scavenger enzymes (see Chapter 62) eliminate the relatively few radicals formed. Using helium to replace inspired N2 and O2 avoids nitrogen narcosis and O2 toxicity Several occupations—including deep mining caisson work and deep diving—require people to spend extended periods at a PB greater than that at sea level. During an extended dive or other exposure to high pressure (one exceeding several hours), the body’s tissues gradually equilibrate with the highpressure gases that one has been breathing. This equilibrated state is referred to by the misnomer saturation. At sea level, the human body normally contains ∼1 L of dissolved N2, equally distributed between the body’s water and fat compartments. As PN2 rises, the N2 equilibrates only slowly with the body’s lipid stores because adipose tissue is relatively underperfused. Although a deep dive of several minutes does not provide sufficient time to equilibrate the fat with N2, one of several hours’ duration does. At equilibrium—as required by Henry’s law—the volume of N2 dissolved in the tissues is proportional to alveolar PN2. Thus, if the body normally dissolves 1 L of N2 at a PB of 1 atm, it will ultimately dissolve 4 L of N2 at a PB of 4 atm. These same principles apply to O2, although the degree to which O2 dissolves in various tissues, and the speed at which equilibration takes place, is different. The adverse effects of N2 and O2 depend on the amount of gas that is dissolved in tissues. The amount, in turn, increases with the dive’s depth (i.e., partial pressure of the gas) and duration (i.e., how close the gas is to achieving equilibrium with various tissues). Thus, the length of time that a diver can spend safely underwater is inversely proportional to the depth of the dive.

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Oxygen toxicity

Following an extended dive, a diver must decompress slowly to avoid decompression sickness

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Figure 61-4 O2 content of blood at high pressures. The red curve is the same Hb-O2 dissociation curve as that in Figure 29-3, except the range is extended to very high values of PO2.

To prevent nitrogen narcosis in saturation diving conditions, divers must partly or completely replace N2 with another inert gas. Helium is the replacement gas of choice for four reasons: 1. Helium has only a fraction of the narcotizing effect of N2. 2. Helium dissolves in the tissues to a lesser extent than N2. 3. Helium has a lower density than N2, and this lowers effective airway resistance. However, the low density of helium facilitates convective “air” cooling around the body, thereby increasing heat loss. Thus, ambient temperature must be higher in a high-helium compression chamber. 4. During the decompression phase of a dive, helium diffuses out of the tissues more rapidly than does N2 and thereby alleviates most of the problems associated with decompression. To prevent O2 toxicity in saturation diving conditions, divers must reduce the fraction of inspired air that is O2 in the compressed gas mixture. Thus, at a PB of 10 atm, a mixture of 2% O2 in helium will provide the same inspired PO2 as room air does at sea level (i.e., ∼20% O2 at a PB of 1 atm).

Although I have focused on problems divers face while at great depths, serious difficulties also arise if—after a deep saturation dive—the diver returns to the surface too quickly. At the end of a saturation dive, PN2 is at the same high value in the alveoli and most tissues. As PB falls during ascent, alveolar PN2 will fall as well, thus creating a PN2 gradient from the mixed venous blood to the alveolar air. Washout of N2 from the blood creates a PN2 gradient from tissues to blood. To allow enough time for the dissolved N2 to move from tissues to blood to alveoli, a diver must rise to the surface slowly (no faster than ∼3 m/hr). Because N2 exits from water much faster than it does from fat, the total elimination of N2 has two components: some compartments empty quickly (e.g., blood), and some empty slowly (e.g., joints, fat, eyeballs). Too rapid an ascent causes the N2 in the tissues—previously dissolved under high pressure—to leave solution and to form bubbles as PB falls. This process is identical to the formation of gas bubbles when one opens a bottle of a carbonated beverage that had been capped under high pressure. Similar problems can occur in pilots who bail out from a pressurized aircraft at high altitude or in divers who ascend to altitude or become aircraft passengers (i.e., exposed to a lower-than-normal PB) too soon after completing a dive that, by itself, would not cause difficulties. During a too-rapid decompression, bubble formation can occur in any tissue in which N2 has previously dissolved. Decompression sickness (DCS) is the general term for the clinical disorder. The pathologic process has three general causes: (1) local formation of bubbles in tissue; (2) bubbles that form emboli in blood; the blood can carry them along until they become wedged in and obstruct a vessel, and a patent foramen ovale can allow bubbles to enter the arterial circulation; and (3) arterial gas embolization; if air is trapped behind an obstructed bronchus, expansion can cause it to tear the lung tissue, enter a pulmonary vein and then a systemic artery, and lodge in the brain or other organ. Clinicians recognize three categories of DCS. Mild or type I DCS can include short-lived mild pains (“niggles”), pruritus, a skin rash, and deep throbbing pain (“bends”) resulting from bubbles that form in muscles and joints. Serious or type II DCS can include symptoms in the CNS, lungs, and circulatory system. The CNS disorder—most commonly involving the spinal cord—reflects bubble formation in the myelin sheath of axons that compromises nerve conduction. Symptoms may range from dizziness (“staggers”) to paralysis. Pulmonary symptoms (“chokes”)—resulting from gas emboli in the pulmonary circulation—include burning pain on inspiration, cough, and respiratory distress. In the circulatory system, bubbles can not only obstruct blood flow but also trigger the coagulation cascade and lead to the release of vasoactive substances. Hypovolemic shock is also a part of this syndrome. The third category is arterial gas embolization, in which large gas emboli can have catastrophic consequences unless the victim receives immediate recompression treatment. Figure 61-5 shows how long a diver can spend at various depths—breathing room air—without having to undergo a

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0

800 No decompression required 700

Denver Mexico City

15 600 PB (mm Hg)

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45 0

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Figure 61-5 The need for decompression as a function of depth and duration of dive. If the dive is sufficiently brief or sufficiently shallow, no decompression is required (teal area). For deeper depths or longer durations, a decompression protocol is required (salmon area). (Data from Duffner GJ: Ciba Clin Symp 1958; 10:99-117.)

500 400 300 200

HIGH-ALTITUDE PHYSIOLOGY Barometric pressure and ambient PO2 on top of Mount Everest are approximately one third of their sea level values Unlike a column of water, which is relatively noncompressible and has a uniform density, the column of air in the atmosphere is compressible and has a density that decreases exponentially ascending from sea level. Half of the mass of the earth’s atmosphere is contained in the lowest 5500 m. Another fourth is contained in the next 5500 m (i.e., 5500 to 11,000 m of altitude). In other words, at higher and higher altitudes, the number of gas molecules pressing down on a mountain climber also falls exponentially; PB falls by half for each ∼5500 m of ascent (Fig. 61-6). Everest Base Camp At an altitude of 5500 m—which also happens to be the altitude of the first base camp used in most ascents of Mount Everest—PB is half the value at sea level (PB ≅ 380 mm Hg), as is the ambient PO2 (PO2 ≅ 80 mm Hg). At this altitude, arterial O2 delivery (arterial blood O2 content

84

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Altitude (m)

Figure 61-6

decompression protocol during the ascent. For example, a dive to 8 m can last indefinitely without any ill effects during the ascent. A dive of 25 minutes’ duration will not provide sufficient time to saturate the tissues unless the dive exceeds 40 m. However, a longer dive at 40 m will require a decompression program. For instance, a 20-minute dive to a depth of 90 m requires nearly 3 hours of decompression time. Thus, the rate at which a diver should ascend to avoid DCS depends on both the depth and the duration of the dive. Divers use detailed tables to plan their rate of ascent from a deep dive. The best treatment for DCS is to recompress the diver in a hyperbaric chamber. Recompression places the gases back under high pressure and forces them to dissolve again in the tissues, a process that instantly relieves many symptoms. Once the diver is placed under high pressure, decompression can be carried out at a deliberate and supervised pace.

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Lhasa, Tibet, and LaPaz, Bolivia Every 5500 m, Summit of Mt. Blanc the barometric pressure falls Summit of by half. Base Mt. McKinley camp at Summit of Mt. Everest Mt. Everest (8848 m)

PO2 (mm Hg)

Depth of submersion (m)

168

Hong Kong (sea level)

Altitude dependence of PB and PO2 in dry air.

× cardiac output) can still meet O2 demands in most healthy, active persons, even during mild physical activity. However, the body’s compensatory responses to reduced ambient PO2 at high altitude vary among different people. Thus, exposure to an altitude of 5500 m is problematic for a significant portion of the population. Peak of Mount Everest

The peak of Mount Everest— 8848 m above sea level—is the highest point on earth. PB at the peak is ∼255 mm Hg, approximately one third that at sea level, and the ambient PO2 is only ∼53 mm Hg. For a climber at the peak of Mount Everest, the PO2 of the humidified inspired air entering the alveoli is even lower because of the effects of water vapor (PH2O = 47 mm Hg at 37ºC). Therefore, the inspired PO2 = 21% × (255 − 47) = 44 mm Hg, compared with 149 mm Hg at sea level (see Table 26-1). Hypoxia is thus a major problem at the summit of Mount Everest. Air Travel Pressurized cabins in passenger planes maintain an ambient pressure equivalent to ∼1800 m of altitude (∼79% of sea level pressure) in cross-continental flights, or ∼2400 m of altitude (∼74% of sea level pressure) in transoceanic flights. Considering that most people do not need supplemental O2 in the inspired air at Denver (∼1500 m) or at some ski resorts (∼3000 m), most airline passengers are not bothered by the slight reduction in arterial O2 saturation (89% saturation at 3000 m) associated with these airline cabin pressures. However, passengers with chronic obstructive pulmonary disease may need to carry supplemental O2 onto the plane even if they do not require it at sea level.

Up to an altitude of ~3000 m, arterial O2 content falls proportionally less than P B because of the shape of the hemoglobin-O2 dissociation curve Although PB and ambient PO2 decrease by the same fraction with increasing altitude, the O2 saturation of Hb in arterial blood decreases relatively little at altitudes up to ∼3000 m.

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The reason is that, at this altitude, arterial PO2 is 60 to 70 mm Hg, which corresponds to the relatively flat portion of the O2-Hb dissociation curve (see Fig. 29-3), where almost all the O2 in blood is bound to Hb. Decreasing arterial PO2 has relatively little effect on arterial O2 content until arterial PO2 falls to less than this flat portion of the curve. Thus, the characteristics of Hb protect the arterial O2 content, despite modest reductions of PO2. At higher altitudes, aviators are advised to breathe supplemental O2. Although the amount of O2 in the blood leaving the lung is important, even more important is the amount of O2 taken up by systemic tissues. This uptake is the product of cardiac output and the arteriovenous (a-v) difference in O2 content (see Chapter 29). At sea level, arterial PO2 is ∼100 mm Hg, corresponding to an Hb saturation of ∼97.5%, whereas the mixed venous PO2 is ∼40 mm Hg, corresponding to an Hb saturation of ∼75%. The difference between the arterial and the venous O2 contents is ∼22.5% of Hb’s maximal carrying capacity for O2. However, at an altitude of 3000 m, arterial PO2 is only ∼60 mm Hg, which may correspond to an Hb saturation of only 88%. This reduction in blood O2 content is called hypoxemia. Assuming that everything else remains the same (e.g., O2 utilization by the tissues, hematocrit, 2,3diphosphoglycerate levels, pH, cardiac output), then the mixed a-v difference in Hb saturation must still be 22.5%. Thus, the mixed venous blood at 3000 m must have an Hb saturation of 88% − 22.5% = 65.5%, which corresponds to a PO2 of ∼33 mm Hg. As a result, the a-v difference of PO2 is much larger at sea level (100 − 40 = 60 mm Hg) than at 3000 m (60 − 33 = 27 mm Hg), even though the a-v difference in O2 content is the same. The reason for the discrepancy is that the O2-Hb dissociation curve is steeper in the region covered by the PO2 values at high altitude. At very high altitudes, still another factor comes into play. The uptake of O2 by pulmonary capillary blood slows at high altitudes and thereby reflects the smaller O2 gradient from alveolus to blood (see Fig. 30-10D). As a result, at sufficiently high altitudes, particularly during exercise, O2 may no longer reach diffusion equilibrium between alveolar air and pulmonary capillary blood by the time the blood reaches the end of the capillary. Thus, at increasing altitude, not only does alveolar PO2—and hence the maximal attainable arterial PO2—fall in a predictable way, but also the actual arterial PO2 may fall to even a greater extent because of a failure of pulmonary capillary blood to equilibrate with alveolar air. During the first few days at altitude, compensatory adjustments to hypoxemia include tachycardia and hyperventilation A reduction in arterial PO2 stimulates the peripheral chemoreceptors and causes an immediate increase in ventilation. Increased ventilation has two effects. First, it brings alveolar PO2 (and thus arterial PO2) closer to the ambient PO2. Second, hyperventilation blows off CO2, the effect of which is respiratory alkalosis that inhibits the peripheral but especially the central chemoreceptors and decreases ventilatory drive (see Chapters 31 and 32). Thus, total ventilation during an acute exposure to 4500 m is only about twice that at sea level, whereas the hypoxia by itself would have produced a much larger stimulation. Accompanying the increased ventilatory

drive during acute altitude exposure is an increase in heart rate, probably owing to the heightened sympathetic drive that accompanies acute hypoxemia (see Chapter 23). The resultant increase in cardiac output enhances O2 delivery. During the next few days to weeks at an elevation of 4500 m, acclimatization causes ventilation to increase progressively by about the same amount as the acute response. As a result, PO2 continues to improve, and PCO2 falls. Two mechanisms appear to cause this slower phase of increased ventilation. First, the pH of the cerebrospinal fluid (CSF) decreases, an effect that counteracts the respiratory alkalosis induced by the increase in ventilation and thus offsets the inhibition of central chemoreceptors. However, the time course of the pH increase in CSF does not correlate tightly with the time course of the increase in ventilation. The pH at the actual site of the central chemoreceptors may fall with the appropriate time course. Long-term hypoxia appears to increase the sensitivity of the peripheral chemoreceptors to hypoxia, and this effect may better account for acclimatization. In the second mechanism for acclimatization, the kidneys respond over a period of several days to the respiratory alkalosis by decreasing their rate of acid secretion (see Chapter 39) so blood pH decreases toward normal (i.e., metabolic compensation for respiratory alkalosis). Another result of this compensation is spillage of HCO3− into the urine that leads to osmotic diuresis and production of alkaline urine. The consequence of reducing both CSF and plasma pH is to remove part of the inhibition caused by alkaline pH and thus allow hypoxia to drive ventilation to higher values. An extreme case of adaptation to high altitude occurs in people climbing very high mountains. In 1981, a team of physiologists ascended to the peak of Mount Everest. Although on their way up to the summit the climbers breathed supplemental O2, at the summit they obtained alveolar gas samples while breathing ambient air—trapping exhaled air in an evacuated metal container. The alveolar PCO2 at the summit was a minuscule 7 to 8 mm Hg, or ∼20% of the value of 40 mm Hg at sea level. Thus, assuming a normal rate of CO2 production, the climber’s alveolar ventilation must have been 5-fold higher than normal (see Chapter 31). Because the work of heavy breathing and increased cardiac output at the summit (driven by hypoxia) would increase CO2 production substantially, the increase in alveolar ventilation must have been much greater than 5-fold. The climbers’ alveolar PO2 at the peak of Mount Everest was ∼28 mm Hg, which is marginally adequate to provide a sufficient arterial O2 content to sustain “resting” metabolic requirement at the summit. However, the term resting is somewhat of a misnomer, because the work of breathing and the cardiac output are markedly elevated. Long-term adaptations to altitude include increases in hematocrit, pulmonary diffusing capacity, capillarity, and oxidative enzymes Although the increases in ventilation and cardiac output help to maintain O2 delivery during acute hypoxia, they are costly from an energy standpoint and cannot be sustained for extended periods. During prolonged residence at a high altitude, the reduced arterial PO2 triggers profound

Chapter 61 • Environmental Physiology

adaptations that enhance O2 delivery to tissues at a cost that is lower than that exacted by short-term compensatory strategies. Many of these adaptations are mediated by an increase in hypoxia-inducible factor 1 (HIF-1), a transcription factor that activates genes involved in erythropoiesis, angiogenesis, and other processes. Hematocrit

Red blood cell (RBC) mass slowly increases with prolonged hypoxemia. The Hb concentration of blood increases from a sea level value of 14 to 15 g/dL to more than 18 g/dL, and hematocrit increases from 40% to 45% to more than 55%. Normally, the body regulates RBC mass within fairly tight limits. However, renal hypoxia and norepinephrine stimulate the production and release of erythropoietin (EPO) from fibroblast-like cells in the kidney (see p. 453 for the box on EPO). EPO is a growth factor that stimulates production of proerythroblasts in bone marrow and also promotes accelerated development of RBCs from their progenitor cells.

Pulmonary Diffusing Capacity Acclimatization to high altitude also causes a 2- to 3-fold increase in pulmonary diffusing capacity. Much of this increase appears to result from a rise in the blood volume of pulmonary capillaries and from the associated increase in capillary surface area available for diffusion (see Chapter 30). This surface area expands even further because hypoxia stimulates an increase in the depth of inspiration. Finally, right ventricular hypertrophy raises pulmonary arterial pressure and increases perfusion to the upper, well-ventilated regions of the lungs (see Chapter 31). Capillary Density

Hypoxia causes a dramatic increase in tissue vascularity. Tissue angiogenesis (see Chapter 20) occurs within days of exposure to hypoxia, triggered by growth factors released by hypoxic tissues. Among these angiogenic factors are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and angiogenin.

Oxidative Enzymes Hypoxia promotes expression of oxidative enzymes in the mitochondria and thereby enhances the tissues’ ability to extract O2 from the blood (see Chapter 60). Thus, acclimatization to high altitude increases not only O2 delivery to the periphery, but also O2 uptake by the tissues.

Altitude causes mild symptoms in most people and acute or chronic mountain sickness in susceptible individuals Symptoms of Hypoxia

The first documented evidence of the ill effects of high altitude was in 35 bc, when Chinese travelers called the Himalayas the “Headache Mountains.” Recreational mountain climbing became popular in the mid-19th century, and with modern transportation, many people can now travel rapidly to mountain resorts. In fact, it is possible to ascend passively from sea level to high altitude in a matter of minutes (e.g., in a balloon) to hours. A rapid ascent may precipitate a constellation of relatively mild symptoms: drowsiness, fatigue, headache, nausea, and a gradual decline in cognition. These uncomfortable effects of

acute hypoxia are progressive with increasing altitude. They occur in some people at altitudes as low as 2100 m and occur in most people at altitudes higher than 3500 m. Initially, these symptoms reflect an inadequate compensatory response to hypoxemia that results in reduced O2 delivery to the brain. In the longer term, symptoms may stem from mild cerebral edema, which probably results from dilation of the cerebral arterioles, thus leading to increased capillary filtration pressure and enhanced transudation (see Chapter 20). Acute Mountain Sickness

Some people who ascend rapidly to altitudes as seemingly moderate as 3000 to 3500 m develop acute mountain sickness (AMS). The constellation of symptoms is more severe than those described in the previous paragraph and includes headache, fatigue, dizziness, dyspnea, sleep disturbance, peripheral edema, nausea, and vomiting. The symptoms usually develop within the first day and last for 3 to 5 days. The primary problem in AMS is hypoxia, and the symptoms probably have two causes. The first is thought to be a progressive, more severe case of cerebral edema. The second cause of the symptoms is pulmonary edema, which occurs as hypoxia leads to hypoxic pulmonary vasoconstriction (see Chapter 31), which, in turn, increases total pulmonary vascular resistance, pulmonary capillary pressure, and transudation. Certain people have an exaggerated pulmonary vascular response to hypoxia, and they are especially susceptible to AMS. Cerebral or pulmonary edema can be fatal if the exposure to hypoxia is not rapidly reversed, first by providing supplemental O2 to breathe and then by removing the individual from the high altitude. Although being physically fit provides some protection against AMS, the most important factor is an undefined constitutional difference. Persons who are least likely to develop symptoms ventilate more in response to the hypoxia and therefore tend to have a higher PO2 and a lower PCO2. The higher PO2 and lower PCO2 lead to less cerebral vasodilation, and the higher PO2 minimizes pulmonary vasoconstriction. Chronic Mountain Sickness

After prolonged residence at high altitude, chronic mountain sickness may develop. The cause of this disorder is an overproduction of RBCs—an exaggerated response to hypoxia. In such conditions, the hematocrit can exceed 60% (polycythemia), thereby dramatically increasing blood viscosity and vascular resistance and increasing the risk of intravascular thrombosis. The combination of pulmonary hypoxic vasoconstriction and increased blood viscosity is especially onerous for the right side of the heart, which experiences a greatly increased load. These conditions eventually lead to congestive heart failure of the right ventricle.

FLIGHT AND SPACE PHYSIOLOGY Acceleration in one direction shifts the blood volume in the opposite direction To accelerate a rocket from rest, we must apply enough force to overcome its inertial force (i.e., its weight, the product of

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4 3 Acceleration 2 (G) 1 0

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Figure 61-7 G forces during ascent into space on the space shuttle. Before liftoff, astronauts experience +1G, the acceleration that results from the earth’s gravity. After liftoff, the solid rockets burn for ∼2 minutes, during which time the G force increases to slightly more than +3G. After the solid-rocket burn, the G force falls back to +1G. Thereafter, the main engine gradually builds up the G force to ∼+4G before engine cut-off. These G-force data were generated in a human centrifuge to simulate the profile of a shuttle launch. (Data from Buckey JC, Goble RL, Blomqvist CG: Med Instrum 1987; 87:238-243.)

its mass, and the acceleration caused by gravity), as well as the frictional forces of the environment. This requirement is merely a restatement of Newton’s second law of motion. With the rocket accelerating vertically, astronauts inside experience an inertial G force, as required by Newton’s third law, a force that presses the astronauts into their seats in the direction opposite that of the rocket’s acceleration. Before liftoff, an astronaut experiences only the force of gravity, +1G. As a rocket blasts off from earth, the astronaut experiences higher G forces. In early rockets, astronauts sometimes experienced G forces as high as +10G. Maximal G forces in the space shuttle are only ∼+4G (Fig. 61-7). Similarly, pilots of high-performance aircraft experience positive G forces as they pull out of a dive, and we all experience negative G forces when an aircraft hits turbulence, suddenly loses altitude, and lifts us out of our seats. Although G forces can frequently have potentially large effects on aircraft pilots, they affect astronauts only during the liftoff and re-entry phases of space flight. To ensure that acceleration effects have a minimal influence on body function, astronauts sit with their backs perpendicular to the direction of the accelerating force, so the G force acts across the chest from front to back. G forces propel the body’s tissues in the direction opposite that of acceleration; these forces compress soft tissues against underlying structural elements (e.g., bone) or pull these tissues away from overlying structural elements. In addition, G forces tend to shift the blood volume away from the direction of acceleration, thereby adding to the other component forces that determine blood pressure (see Chapter 17). In high-performance aircraft, the rapid motions associated with changes in flight direction or altitude produce G forces that can be considerable for several minutes, exceeding 8Gs. Even in relatively primitive aircraft, aerobatic maneuvers can shift blood volume away from the head and can result in transient reductions in cerebral blood flow and O2

delivery. If these reductions are sufficiently large, they can result in loss of consciousness. The early warnings of such an event are narrowing of the visual field (i.e., loss of peripheral vision) and loss of color perception as the retina is deprived of O2, a phenomenon called gray-out. The term blackout describes a total loss of consciousness that occurs during acceleration that lasts for tens of seconds or minutes. Pilots experiencing gray-out or blackout are at extreme risk. As early as World War II, fighter pilots used G-suits that provided counterpressure to the lower extremities during repeated tight maneuvers during dogfights. The counterpressure opposed the pooling of blood in the extremities and maintained sufficient cardiac filling, cardiac output, and blood flow to the brain, thereby eliminating the tendency toward gray-out. “Weightlessness” causes a cephalad shift of the blood volume An astronaut in an orbiting spacecraft experiences “weightlessness,” a state of near-zero G force, also called a microgravity environment. Although an astronaut at an altitude of 200 km still experiences ∼94% of the force of the earth’s gravity at sea level (i.e., the astronaut truly has weight), the centrifugal force of the spacecraft’s orbital trajectory balances the earth’s gravitational force, and the astronaut experiences no net acceleration forces and thus has the sensation of weightlessness. This weightlessness, however, differs from the true near-zero-gravity environment in “outer space.” We are adapted to life at +1G, and arteriolar tone in the lower extremities prevents pooling of blood in the capacitance vessels (see Chapter 25), thereby ensuring adequate venous return to the right heart. The acute effects of microgravity on the circulatory system are exactly what you would expect for a system designed to oppose the effect of gravity in a standing person: blood volume redistributes toward the head. This cephalad shift of blood volume—away from the capacitance vessels of the legs—expands the central blood volume, increases the cardiac preload, and increases the filtration of plasma water into the interstitium of the facial region. The resulting edema explains the dramatically bloated facial appearance of astronauts in microgravity within 24 hours of the launch. From this discussion, you would think that the central venous pressure (CVP) is higher in space. However, such an increase in CVP has been difficult to confirm. In laboratory studies involving prolonged head-down tilt (i.e., a model intended to simulate microgravity exposure), the cephalad shift of blood volume produces the expected increase in CVP and rapid reflex responses to the apparent volume overload. First, the increased stretch on the right atrium causes release of atrial natriuretic peptide (ANP). Second, stimulation of the low-pressure baroreceptors inhibits secretion of arginine vasopressin or antidiuretic hormone from the posterior pituitary (see Chapter 23). These two events increase excretion of salt and water by the kidneys (see Chapter 40). They also correct the perceived volume overload and explain the tendency for astronauts to remain relatively underhydrated during space flight. In orbiting spacecraft, the cephalad shift of blood volume, even without an increase in CVP, causes a small increase in

Chapter 61 • Environmental Physiology

cerebral arterial pressure and thus in blood flow to the brain. Such regional alterations in blood volume and flow do not substantially affect total peripheral resistance in space. Thus, mean arterial pressure and cardiac output are not significantly different from their values on the earth’s surface. Space flight leads to motion sickness and to decreases in muscle and bone mass Despite training (e.g., in three-dimensional motion simulators), more than half of all astronauts experience motion sickness during the initial days of microgravity. Motion sickness (i.e., nausea and vomiting) results from conflicting sensory input to the brain regarding the position of the body. In space flight, motion sickness is the consequence of altered inertial stimulation of the vestibular system in the absence of normal gravitational forces. Nearly all cases of motion sickness resolve within the first 96 hours of microgravity exposure as the vestibular system or the CNS accommodates to the novel input. The increased cerebral blood flow and blood volume in microgravity, accompanied by increased capillary filtration of fluid from the intravascular space, contribute to the increased incidence of headache, nausea, and motion sickness, at least during the transition period to microgravity. These symptoms reduce performance. Astronauts attempt to minimize these effects by restricting water intake before launch. Numerous other changes occur during prolonged residence in microgravity, many of which are related to the markedly diminished aerobic power output in space, where the force of gravity does not oppose muscle contraction. The major physiological alterations include reductions in body water content, plasma and RBC volume, total body N2 stores, muscle mass, and total body Ca2+ and phosphate (associated with a loss in bone mass). The bone loss appears to be continuous with time in a weightless environment, whereas the other changes occur only during the first weeks in space. The reductions in plasma and RBC volumes result in a marked decrease in maximum cardiac output, a determinant of maximal aerobic power. The reduction of muscle mass decreases the maximal force developed by muscle. The reduction in bone mass similarly decreases bone strength. Although these changes are appropriate adaptations to a microgravity environment, in which great strength and high aerobic capacity have little inherent value, they are decidedly disadvantageous on return to the earth’s surface.

ral pressure differences—across the blood vessels in the feet—that are similar to pressure differences when standing upright on the earth’s surface. However, this arrangement greatly exaggerates transmural pressure differences near the waist. For this reason, these astronauts also wear positivepressure pants that compress the tissues by 70 mm Hg at the level of the waist and—decrementally—by 0 mm Hg at the feet. The net effect of the negative-pressure chamber and the graded positive-pressure pants is to create a physiological toe-to-waist gradient of transmural pressures across the blood vessels of the lower body. The aerobic activity, the impact of the feet on the treadmill, and the generation of physiological transmural pressure gradients appear to be sufficient to simulate exercise at +1G. This regimen can reduce or even eliminate the deconditioning effects of space flight. Return to earth requires special measures to maintain arterial blood pressure The problems associated with re-entry reflect a return to full gravity on earth’s surface. The most dramatic effects result from reduced blood volume and decreased tone of the leg vessels. Both factors contribute to reductions in cardiac preload, orthostatic tolerance, and exercise capacity. It has been common practice to shield astronauts from public view immediately after return to the earth’s surface, until they have regained a good orthostatic response. In recent years, astronauts have employed various strategies just before re-entry to counter the adaptations to microgravity. The countermeasure to orthostatic intolerance is restoration of blood volume before re-entry. One means of attenuating the reduction of blood volume in space flight is an exercise program. Even a brief period (e.g., 30 minutes) of intense exercise expands plasma albumin content and increases plasma oncotic pressure and plasma volume by 10% within 24 hours. The problems with exercise programs are difficulties in logistics and the astronauts’ lack of motivation. A second means of minimizing the reduced blood volume is increasing salt and fluid intake. However, this practice has proven difficult to implement because of the consequent increase in urine flow. Currently, astronauts are educated about the effects of prolonged space flight and are then maintained under continuous scrutiny after re-entry until they have regained a normal orthostatic response. This usually occurs within hours, and certainly within 1 day, of re-entry.

Exercise partially overcomes the deconditioning of muscles during space flight

REFERENCES

The intermittent loading of muscles, bone, and the cardiovascular system prevents—to some extent—the deconditioning effects of space flight on muscle mass and performance. Astronauts have used bungee (i.e., elastic) cords and ergometric (i.e., work-measuring) stationary bicycles to provide resistance against which to exert force. The most effective exercise regimen appears to be walking on a motor-driven treadmill with the lower body encased in a negativepressure chamber. Reducing the chamber pressure to 100 mm Hg lower than ambient pressure creates transmu-

Books and Reviews Bunn HF, Poyton RO: Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 1996; 76:839-885. Crystal RG, West JB: The Lung. New York: Raven Press, 1991. Duffner GJ: Medical problems involved in underwater compression and decompression. Ciba Clin Symp 1958; 10:99-117. Krakauer J: Into Thin Air. New York: Anchor Books–Doubleday, 1997. Monge C: Chronic mountain sickness. Physiol Rev 1943; 23:166-184. West JB: Man in space. News Physiol Sci 1986; 1:189-192.

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Journal Articles Buckey JC, Goble RL, Blomqvist CG: A new device for continuous ambulatory central venous pressure measurement. Med Instrum 1987; 87:238-243. Cain SM, Dunn JE II: Low doses of acetazolamide to aid the accommodation of men to altitude. J Appl Physiol 1966; 21:1195-1200.

Schoene RB, Lahiri S, Hackett PH, et al: Relationship of hypoxic ventilatory response to exercise performance on Mount Everest. J Appl Physiol 1984; 56:1478-1483. West JB: Human physiology at extreme altitudes on Mount Everest. Science 1984; 223:784-788.

CHAPTER

62

T H E P H YS I O LO GY O F A G I N G Edward J. Masoro

Biomedical science paid surprisingly little attention to a remarkable change in human biology during the 20th century—the marked increase in human life expectancy in developed nations. For example, in the United States, life expectancy for men progressively increased from 47.9 years in 1900 to 74.5 years in 2002, and for women, it increased from 50.7 years in 1900 to 79.9 years in 2002. Not until 1974 did the United States establish the National Institute on Aging (NIA) in the National Institutes of Health. The NIA has had a major impact in the United States and throughout the world in the promotion of research on aging and on the development of geriatric medicine.

CONCEPTS IN AGING During the 20th century, the age structure of populations in developed nations shifted toward older individuals The fraction of the U.S. population 65 years of age or older was only 4% in 1900 but 13% in 1990. This trend in age structure is projected to continue (Fig. 62-1). Moreover, because women have a greater life expectancy, they comprised 70.5% of the population that was more than 80 years old in 1990 in developed nations. The shift in age structure of the U.S. population during the 20th century depended only modestly on an increase in life expectancy from birth. More important was the progressive decrease in birth rates, which led the elderly to become an ever-increasing fraction of the population, particularly in developed nations. Indeed, the effect of the post-World War II “Baby Boom Generation” on population age structure is clearly apparent in Figure 62-1. However, because birth rates are unlikely to fall much further, future changes in the age structure of the U.S. population will depend mainly on further projected increases in life expectancy. The definition, occurrence, and measurement of aging are fundamental but controversial issues The age of an organism usually refers to the length of time the individual has existed. Biogerontologists and members

of the general public alike usually use aging to mean the process of senescence. For example, we may say that a person is young for her age, an expression meaning that the processes of senescence appear to be occurring slowly in that person. Aging—the synonym for senescence that we use throughout this chapter—is the progressive deteriorative changes, during the adult period of life, which underlie an increasing vulnerability to challenges and thereby decrease the ability of the organism to survive. Biogerontologists distinguish biological age from chronologic age. Although we easily recognize the biological aging of family members, friends, and pets, it would be helpful to have a quantitative measure of the rate of aging of an individual. Biomarkers of aging—morphologic and functional changes that occur with time in the adult organism—could in principle serve as a measure of senescent deterioration. Alas, a generally agreed on panel of biomarkers of aging has yet to emerge, so it is currently impossible to quantitate the aging of individuals. In contrast to the aging of individuals, it has long been possible to measure the rate of aging of populations. In 1825, Benjamin Gompertz, a British actuary, published a report on human age-specific death rate—the fraction of the population entering an age interval (e.g., 60 to 61 years of age) that dies during the age interval. Gompertz found that, after early adulthood, the age-specific death rate increases exponentially with increasing adult age. The same is true for other human populations (Fig. 62-2) and for many animal populations. Based on the assumption that the death rate reflects the vulnerability caused by senescence, it has generally been accepted that the slope in Figure 62-2 reflects the rate of population aging. Although gompertzian and related analyses had long been viewed as the “gold standard” for measuring population aging, some biogerontologists have challenged this approach. Aging is an evolved trait Most evolutionary biologists no longer accept the once popular belief that aging is an evolutionary adaptation with a genetic program similar to that for development. The current view is that aging evolved by default and reflected the absence of forces of natural selection that would

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otherwise eliminate mutations that promote senescence. For example, consider a cohort of a species that reaches reproductive maturity at age X. At that age, all members of the cohort will be involved in generating progeny. Furthermore, assume that this species is evolving in a hostile environment—the case for most species. As the age of this cohort increases past X, fewer and fewer members survive, so that all members of the cohort die before exhibiting senescence. In this cohort, genes with detrimental actions—expressed only at advanced ages—would not be subjected to natural selection. If we now move the progeny of our cohort to a highly protective environment, many may well live to ages at which the deleterious genes can express their effects, thereby giving rise to the aging phenotype. This general concept led to three genetic mechanisms, discussed in the following paragraphs. These mechanisms are not mutually exclusive, and each has experimental support. In 1952, Peter Medawar proposed a variant of the foregoing model, now referred to as the mutation-accumulation mechanism. He proposed that most deleterious mutations

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Figure 62-1 The age structure of the 1955 U.S. population and the projected age structure of the 2010 U.S. population. (From Tauber C: Sixty-Five Plus in America. Washington, DC: U. S. Bureau of the Census, 1992, rev. 1993.)

in gametes will result in progeny that are defective during most of life, and natural selection removes such genes from the population. However, a very few mutated genes will not have deleterious effects until advanced ages, and natural selection would fail to eliminate such genes. George Williams proposed another variant in 1957. He postulated that the genes with deleterious actions in late life actually increase evolutionary fitness in early adulthood. Natural selection will strongly favor such alleles because they promote the ability of the young adult to generate progeny and because they have a negative impact only after reproduction—antagonistic pleiotropy. In this situation, aging is a byproduct of natural selection. In 1977, Tom Kirkwood proposed the Disposable Soma Theory, according to which the fundamental life role of organisms is to generate progeny. Natural selection would apportion the use of available energy between reproduction and body (i.e., somatic) maintenance, to maximize the individual’s lifetime yield of progeny. As a consequence, less energy is available for somatic maintenance than is needed

Chapter 62 • The Physiology of Aging

is, different cohorts have had different environmental experiences. For example, in studies of the effects of aging on cognition, a confounding factor could be that younger cohorts have had the benefit of a relatively higher level of education. If aware of a potential confounder, the investigator may be able to modify the study’s design to avoid the confounder. The second potential confounder is selective mortality—individuals with risk factors for diseases that cause death at a relatively young age are underrepresented in older age groups. For example, in a study on the effect of age on plasma lipoproteins, mortality at a young age from cardiovascular disease would preferentially eliminate individuals with the highest low-density lipoprotein levels.

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Longitudinal Design To circumvent the confounders encountered in cross-sectional designs, investigators can repeatedly study a subject over a significant portion of his or her lifetime. However, this longitudinal design has other problems. Long-term longitudinal studies require a special organizational structure that can outlive an individual investigator and complete the study. Even shorter longitudinal studies are very costly. Some problems are inherent in the course of longitudinal studies, including the effect of repeated measurements on the function assessed, changes in subjects’ lifestyle (e.g., diet), dropout of subjects from the study, and changes in professional personnel and technology.

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for indefinite survival. This theory further proposes that a hostile environment increases the fraction of energy expended in reproduction and leaves a smaller fraction for somatic maintenance. Measurements of human aging can be either cross sectional or longitudinal Measuring the effects of aging on the human physiology presents investigators with a difficulty—the subjects’ life span is greater than the investigator’s scientific life span. Cross-Sectional Design

The usual approach to the foregoing difficulty is a cross-sectional design in which investigators study cohorts with several different age ranges (e.g., 20 to 29 year olds, 30 to 39 year olds) over a brief period (e.g., a calendar year). However, this design suffers from two serious potential confounders. One is the cohort effect; that

Age-associated diseases are those that do not cause morbidity or mortality until advanced ages. Examples are coronary artery disease, stroke, many cancers, type 2 diabetes, osteoarthritis, osteoporosis, cataracts, Alzheimer disease, and Parkinson disease. These are either chronic diseases or acute diseases that result from long-term processes (e.g., atherogenesis). Most gerontologists have held the view that age-associated diseases are not an integral part of aging. These gerontologists developed the concept of primary and secondary aging to explain why age-associated diseases occur in almost all elderly people. Primary aging refers to intrinsic changes occurring with age, unrelated to disease or environmental influences. Secondary aging refers to changes caused by the interaction of primary aging with environmental influences or disease processes. In contrast, some gerontologists adhere to the following view, expressed by Robin Holliday: “The distinction between age-related changes that are not pathological and those that are pathological is not at all fundamental.” Moreover, the genetic mechanisms proposed for the evolution of aging may apply equally to the processes underlying both primary and secondary aging.

CELLULAR AND MOLECULAR MECHANISMS OF AGING In this major section, I consider three major classes of cellular and molecular processes that may be proximate causes of organismic aging: (1) damage caused by oxidative stress

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and other factors, (2) inadequate repair of damage, and (3) dysregulation of cell number. No single one of these processes is the underlying mechanism of aging. The basic mechanism of aging is likely to be the long-term imbalance between damage and repair. During growth and development, the genetic program not only creates a complex structure, but also repairs damaged molecules that arise in the process. Following development is a brief adult period when damage and repair are in balance, and then begins long-term imbalance in favor of damage. The factors underlying the imbalance vary among species and among individuals within species, as a result of both genetic and environmental variability. For example, oxidative stress is one of many damaging processes that underlie aging, and the genome of the animal as well as the environment will determine the extent to which it is an important causal factor.

Oxidative stress and related processes that damage macromolecules may have a causal role in aging One gram of tissue from a small mammal has a higher resting metabolic rate (RMR; see Chapter 58) than the same mass of tissue from a larger mammal (e.g., a human). Because smaller mammals have a shorter life span than humans, Max Rubner reported in 1908 that a gram of tissue from diverse domestic animals and humans has similar lifetime energy expenditure. Based on these findings, Raymond Pearl in 1928 proposed that organisms have a finite amount of a “vital principle” that they deplete at a rate proportional to the rate of energy expenditure. However, later experimental evidence did not support this Rate of Living Theory of Aging. Reactive O2 Species As illustrated in Figure 62-3A, reactive oxygen species (ROS) include molecules such as hydrogen peroxide (H2O2), neutral free radicals such as the hydroxyl radical (•OH), and anionic radicals such as the superoxide anion radical (O.-2 ). Free radicals have an unpaired electron in the outer orbital, shown in red in Figure 62-3A. These free radicals are extremely unstable because they react with a target molecule to capture an electron and thus become a stable molecule with only paired electrons in the outer shell. However, the target molecule left behind becomes a free radical, initiating a chain reaction that continues until two free radicals meet to create a product with a covalent bond. ROS—particularly •OH, which is the most reactive of them all—have the potential to damage important biological molecules, such as proteins, lipids and DNA. However, ROS also play important physiological roles in the oxidation of iodide anions by thyroid peroxidase in the formation of thyroid hormone (see Chapter 49), as well as in the destruction of certain bacteria by NADPH oxidase and myeloperoxidase in phagocytic cells. Finally, the highly reactive signaling molecule nitric oxide (see Chapter 3) is a free radical (Fig. 62-3A). ROS can also form as the result of ionizing radiation. Quantitatively, the most important source of ROS is the mitochondrial electron transport chain (see Chapter 5).

A

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MITOCHONDRIAL GENERATION OF ROS Electron transport chain Complex I and Complex III

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Figure 62-3

H2O2 Hydrogen peroxide

Fenton reaction Fe3+

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ROS. A, Structures. B, Mitochondrial generation.

Complex I and complex III of the electron transport chain generate O2.− as byproducts (Fig. 62-3B). The enzyme superoxide dismutase (SOD) converts O2.− to H2O2, which, in turn, can yield the highly reactive •OH. Only a small fraction of the oxygen (