Berne & Levy - Physiology [8 ed.] 9780323847902, 9780323847919

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Berne & Levy Physiology

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EIGHTH EDITION

Editors

Bruce M. Koeppen, MD, PhD Dean Emeritus Frank H. Netter, MD School of Medicine, Quinnipiac University Hamden, Connecticut United States

Bruce A. Stanton, PhD Andrew C. Vail Professor Microbiology and Immunology Geisel School of Medicine at Dartmouth, Hanover, New Hampshire United States Associate Editors

Julianne M. Hall, PhD Professor of Medical Sciences Frank H. Netter, MD School of Medicine, Quinnipiac University Hamden, Connecticut United States

Agnieszka Swiatecka-Urban, MD Professor of Pediatrics

Division Head of Pediatric Nephrology University of Virginia Charlottesvile, Virginia United States

Copyright ELSEVIER 1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899 BERNE AND LEVY PHYSIOLOGY, EIGHTH EDITION ISBN: 978-0-323-84790-2 INTERNATIONAL EDITION ISBN: 978-0-323-84791-9 Copyright © 2024 by 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. Details on how to seek permission, further information about the Publisher's permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notice Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Previous editions copyrighted 2018, 2010, 2008, 2004, 1998, 1993, 1988, and 1983. Executive Content Strategist: Elyse O'Grady Content Development Specialist: Grace Onderlinde Publishing Services Manager: Deepthi Unni Project Manager: Nayagi Anandan Book Designer: Patrick Ferguson

Printed in the United States of America Last digit is the print number: 9 8 7 6 5 4 3 2 1

Dedication This eighth edition of Physiology is dedicated to the memory of Dr. Gerhard Giebisch (1927–2020). Gerhard was an internationally recognized physiologist and expert on the functioning of the kidneys. He was a great friend and important mentor to us both. Bruce M. Koeppen, MD, PhD Bruce A. Stanton, PhD

Section Authors Alix Ashare, MD, PhD Associate Professor Medicine, Microbiology and Immunology Geisel School of Medicine at Dartmouth Hanover, New Hampshire, United States   Section 5: Respiratory Physiology Kim E. Barrett, PhD Vice Dean for Research and Distinguished Professor Physiology and Membrane Biology UC Davis School of Medicine Sacramento, California, United States   Section 6: Gastrointestinal Physiology James L. Carroll Jr., MD Associate Clinical Professor Medicine Geisel School of Medicine at Dartmouth Hanover, New Hampshire, United States Section of Pulmonary and Critical Care Dartmouth-Hitchcock Medical Center Lebanon, New Hampshire, United States   Section 5: Respiratory Physiology Julianne M. Hall, PhD Associate Professor Medical Sciences Frank H. Netter, MD School of Medicine Quinnipiac University Hamden, Connecticut, United States   Section 7: Renal Physiology   Section 8: Endocrine Physiology John R. Harrison, PhD Associate Professor Orthodontics University of Connecticut School of Dental Medicine Farmington, Connecticut, United States   Section 8: Endocrine Physiology

Robert D. Harvey, PhD Professor Pharmacology University of Nevada-Reno Reno, Nevada, United States   Section 4: Cardiovascular Physiology Bruce M. Koeppen, MD, PhD Dean Emeritus Frank H. Netter, MD School of Medicine Quinnipiac University Hamden, Connecticut, United States   Section 1: Cellular Physiology   Section 7: Renal Physiology Helen E. Raybould, PhD Professor Anatomy, Physiology and Cell Biology UC Davis School of Veterinary Medicine Davis, California, United States   Section 6: Gastrointestinal Physiology Bruce A. Stanton, PhD Andrew C. Vail Professor Microbiology and Immunology Geisel School of Medicine at Dartmouth Hanover, New Hampshire, United States   Section 1: Cellular Physiology   Section 7: Renal Physiology Agnieszka Swiatecka-Urban, MD Professor of Pediatrics Division Head of Pediatric Nephrology University of Virginia Charlottesville, Virginia, United States   Section 7: Renal Physiology James M. Watras, PhD Associate Professor Cell Biology University of Connecticut School of Medicine Farmington, Connecticut, United States   Section 3: Muscle Physiology Bruce White, PhD Professor Emeritus Cell Biology University of Connecticut School of Medicine

Farmington, Connecticut, United States   Section 8: Endocrine Physiology Withrow Gil Wier, PhD Emeritus Professor Physiology University of Maryland School of Medicine Baltimore, Maryland, United States   Section 4: Cardiovascular Physiology Mark Yeckel, PhD Professor Medical Sciences Frank H. Netter, MD School of Medicine Quinnipiac University Hamden, Connecticut, United States   Section 2: Neurophysiology

Preface We are pleased that the following section authors have continued as members of the eighth edition team: Dr. James Watras (muscle), Dr. Withrow Gil Wier (cardiovascular system), Drs. Kim Barrett and Helen Raybould (gastrointestinal system), and Drs. Bruce White and John Harrison (endocrine and reproductive systems). We would like to thank the following individuals for their contributions to previous editions: Drs. Kalman Rubinson and Eric Lang (nervous system), Dr. Achilles Pappano (cardiovascular system), and Drs. Michelle Cloutier and Roger Thrall (respiratory system). We also welcome the following authors: Dr. Mark Yeckel (nervous system), Dr. Robert Harvey (cardiovascular system), and Drs. Alix Ashare and James Carroll (respiratory system). Importantly, Drs. Julianne Hall and Agnes SwiateckaUrban have joined us as authors and associate editors. As in the previous editions of this textbook, we have attempted to emphasize broad concepts and to minimize the compilation of isolated facts. Each chapter has been written to make the text as lucid, accurate, and current as possible. We have included both clinical and molecular information in each section, as feedback on these features has indicated that this information serves to provide clinical context and new insights into physiologic phenomena at the cellular and molecular levels. The human body consists of billions of cells that are organized into tissues (e.g., muscle, epithelia, and nervous tissue) and organ systems (e.g., nervous, cardiovascular, respiratory, renal, gastrointestinal, endocrine, and reproductive). For these tissues and organ systems to function properly, and thus allow humans to live and carry out daily activities, several general conditions must be met. First and foremost, the cells within the body must survive. Survival requires adequate cellular energy supplies, maintenance of an appropriate intracellular milieu, and defense against a hostile external environment. Once cell survival is ensured, the cell can then perform its designated or specialized function (e.g., contraction by skeletal muscle cells). Ultimately, the function of cells, tissues, and organs must be coordinated and regulated. All of these functions are the essence of the discipline of physiology and are presented throughout this book. What follows is a brief introduction to these general concepts. Cells need a constant supply of energy. This energy is derived from the hydrolysis of adenosine triphosphate (ATP). If not replenished, the cellular ATP supply would be depleted in most cells in less than 1 minute. Thus, ATP must be continuously synthesized. This in turn requires a steady supply of cellular fuels. However, the cellular fuels (e.g., glucose, fatty acids, and ketoacids) are present in the blood at levels that can support cellular metabolism only for a few minutes. The blood levels of these cellular fuels are maintained through the ingestion of precursors (i.e., carbohydrates, proteins, and fats). In addition, these fuels can be stored and then mobilized when ingestion of the precursors is not possible. The storage forms of these fuels are triglycerides (stored in adipose tissue), glycogen (stored in the liver and skeletal muscle), and protein. The maintenance of adequate levels of cellular fuels in the blood is a complex process involving the following tissues, organs, and organ systems: • Liver: Converts precursors into fuel storage forms (e.g., glucose → glycogen) when food is ingested, and converts storage forms to cellular fuels during fasting (e.g., glycogen → glucose

and amino acids → glucose). • Skeletal muscle: Like the liver, stores fuel (glycogen and protein) and converts glycogen and protein to fuels (e.g., glucose) or fuel intermediates (e.g., protein → amino acids) during fasting. • Gastrointestinal tract: Digests and absorbs fuel precursors. • Adipose tissue: Stores fuel during feeding (e.g., fatty acids → triglycerides) and releases the fuels during fasting. • Cardiovascular system: Delivers the fuels to the cells and to and from their storage sites. • Endocrine system: Maintains the blood levels of the cellular fuels by controlling and regulating their storage and their release from storage (e.g., insulin and glucagon). • Nervous system: Monitors oxygen levels and nutrient content in the blood and, in response, modulates the cardiovascular, pulmonary, and endocrine systems and induces feeding and drinking behaviors. In addition to energy metabolism, the cells of the body must maintain a relatively constant intracellular environment to survive. This includes the uptake of fuels needed to produce ATP, the export from the cell of cellular wastes, the maintenance of an appropriate intracellular ionic environment, the establishment of a resting membrane potential, and the maintenance of a constant cellular volume. All of these functions are carried out by specific membrane transport proteins. The composition of the extracellular fluid (ECF) that bathes the cells must also be maintained relatively constant. In addition, the volume and temperature of the ECF must be regulated. Epithelial cells in the lungs, gastrointestinal tract, and kidneys are responsible for maintaining the volume and composition of the ECF, while the skin plays a major role in temperature regulation. On a daily basis, H2O and food are ingested, and essential components are absorbed across the epithelial cells of the gastrointestinal tract. This daily intake of solutes and water must be matched by excretion from the body, thus maintaining steady-state balance. The kidneys are critically involved in the maintenance of steadystate balance for water and many components of the ECF (e.g., Na+, K+, HCO3−, pH, Ca++, organic solutes). The lungs ensure an adequate supply of O2 to “burn” the cellular fuels for the production of ATP and excrete the major waste product of this process (i.e., CO2). Because CO2 can affect the pH of the ECF, the lungs work with the kidneys to maintain ECF pH. Because humans inhabit many different environments and often move between environments, the body must be able to rapidly adapt to the challenges imposed by changes in ambient temperature and availability of food and water. Such adaptation requires coordination of the function of cells in different tissues and organs as well as their regulation. The nervous and endocrine systems coordinate and regulate cell, tissue, and organ function. The regulation of function can occur rapidly (seconds to minutes), as is the case for levels of cellular fuels in the blood, or over much longer periods of time (days to weeks), as is the case for acclimatization when an individual moves from a cool to a hot environment or changes from a high-salt to a low-salt diet. The function of the human body represents complex processes at multiple levels. This book explains what is currently known about these processes. Although the emphasis is on the normal function of the human body, discussion of disease and abnormal function is also appropriate, as these often illustrate physiologic processes and principles at the extremes. The authors for each section have presented what they believe to be the most likely mechanisms responsible for the phenomena under consideration. We have adopted this compromise to achieve brevity, clarity, and simplicity.

Bruce M. Koeppen, MD, PhD Bruce A. Stanton, PhD

Note to Instructors Contact your Elsevier Sales Representative for teaching resources, including slides and image banks, for Berne and Levy Physiology, 8e or request these supporting materials at: http://evolve.elsevier.com/BerneLevy/physiology/

Table of Contents Cover Image Title Page Copyright Dedication Section Authors Preface Note to Instructors Table of Contents

Section 1 Cellular Physiology Chapter 1 Principles of Cell and Membrane Function Learning Objectives Overview of Eukaryotic Cells Vesicular Transport Electrochemical Gradient Osmosis and Osmotic Pressure Key Points

Chapter 2 Homeostasis: Volume and Composition of Body Fluid Compartments Learning Objectives Concept of Steady-State Balance Volumes and Composition of Body Fluid Compartments Maintenance of Cellular Homeostasis Principles of Epithelial Transport Key Concepts Chapter 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression Learning Objectives Cell-to-Cell Communication Receptors Receptors and Signal Transduction Pathways Regulation of Gene Expression by Signal Transduction Pathways Key Points

Section 2 Neurophysiology Chapter 4 The Nervous System: Introduction to Cells and Systems Learning Objectives Cellular Components of the Nervous System The Peripheral Nervous System The Central Nervous System Nervous Tissue Reactions to Injury Key Points

Chapter 5 Generation and Conduction of Action Potentials Learning Objectives Membrane Potentials Suprathreshold Response: The Action Potential Conduction of Action Potentials Key Points Chapter 6 Synaptic Transmission Learning Objectives Electrical Synapses Chemical Synapses Synaptic Integration Modulation of Synaptic Activity Neurotransmitters Neurotransmitter Receptors Key Points Chapter 7 The Somatosensory System Learning Objectives Subdivisions of the Somatosensory System Discriminatory Touch and Proprioception Thalamic and Cortical Somatosensory Areas Pain and Temperature Sensation Transduction in the Somatosensory System Centrifugal Control of Somatosensation Key Points

Chapter 8 The Special Senses Learning Objectives The Visual System The Auditory and Vestibular Systems The Chemical Senses Key Points Chapter 9 Organization of Motor Function Learning Objectives Principles of Spinal Cord Organization Descending Motor Pathways Brainstem Control of Posture and Movement Motor Control by the Cerebral Cortex Motor Control by the Cerebellum Motor Control by the Basal Ganglia Eye Movement Key Points Chapter 10 Integrative Functions of the Nervous System Learning Objectives The Cerebral Cortex Key Points Chapter 11 The Autonomic Nervous System and Its Central Control Learning Objectives Organization of the Autonomic Nervous System

Autonomic Ganglia Neurotransmitters Central Control of Autonomic Function Key Points

Section 3 Muscle Physiology Chapter 12 Skeletal Muscle Physiology Learning Objectives Skeletal Muscle Physiology Organization of Skeletal Muscle Control of Skeletal Muscle Activity Skeletal Muscle Types Modulation of the Force of Contraction Modulation of Force by Reflex Arcs Skeletal Muscle Tone Energy Sources During Contraction Oxygen Debt Fatigue Growth and Development Denervation, Reinnervation, and Cross-Innervation Response to Exercise Delayed-Onset Muscle Soreness Biophysical Properties of Skeletal Muscle Key Points

Chapter 13 Cardiac Muscle Learning Objectives Basic Organization of Cardiac Muscle Cells Control of Cardiac Muscle Activity Regulation of the Force of Contraction Cardiac Muscle Metabolism Cardiac Muscle Hypertrophy Key Concepts Chapter 14 Smooth Muscle Learning Objectives Overview of Smooth Muscle Structure of Smooth Muscle Cells Control of Smooth Muscle Activity Innervation of Smooth Muscle Regulation of Contraction Regulation of Myoplasmic Calcium Concentration Development and Hypertrophy Synthetic and Secretory Functions Biophysical Properties of Smooth Muscle Key Points

Section 4 Cardiovascular Physiology Chapter 15 Overview of Circulation Learning Objectives

The Heart The Cardiovascular Circuit Blood Vessels Key Points Chapter 16 Elements of Cardiac Function Learning Objectives Overview of Cardiac Function Electrical Properties of the Heart Normal Sinus Rhythm Arrhythmogenic Mechanisms Electrocardiography Arrhythmias The Cardiac Pump Key Points Chapter 17 Properties of the Vasculature Learning Objectives Hemodynamics The Arterial System The Venous System Microcirculation and Lymphatic System Coronary Circulation Cutaneous Circulation Skeletal Muscle Circulation Cerebral Circulation

Intestinal Circulation Hepatic Circulation Fetal Circulation Key Points Chapter 18 Regulation of the Heart and Vasculature Learning Objectives Regulation of Heart Rate and Myocardial Performance Nervous Control of the Heart Rate Regulation of Myocardial Performance Regulation of the Peripheral Circulation Key Points Chapter 19 Integrated Control of the Cardiovascular System Learning Objectives Regulation of Cardiac Output and Blood Pressure Vascular Function Curve Relating the Cardiac Function Curve to the Vascular Function Curve A More Complete Theoretical Model: The Two-Pump System Role of the Heart Rate in Control of Cardiac Output Ancillary Factors That Affect the Venous System and Cardiac Output Interplay of Central and Peripheral Factors in Control of the Circulation Key Points

Section 5 Respiratory Physiology Chapter 20 Introduction to the Respiratory System

Learning Objectives Lung Anatomical Structure/Function Relationships Circulatory Systems in the Lung Innervation Lung Embryology, Development, Aging, and Repair Key Points Chapter 21 Static Lung and Chest Wall Mechanics Learning Objectives Pressures in the Respiratory System How a Pressure Gradient Is Created Lung Volumes and Their Measurement Lung Volumes and Capacities Determinants of Lung Volume Pressure-Volume Relationships Lung Compliance Surface Tension and Surfactant Key Points Chapter 22 Dynamic Lung and Chest Wall Mechanics Learning Objectives Dynamic Lung Mechanics Work of Breathing Key Concepts Chapter 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

Learning Objectives Ventilation Dead Space Ventilation: Anatomical and Physiological Alveolar Ventilation Pulmonary Vascular Resistance Distribution of Pulmonary Blood Flow Active Regulation of Blood Flow Ventilation/Perfusion Relationships Arterial Blood Hypoxemia, Hypoxia, and Hypercarbia Low Ventilation/Perfusion Alveolar Hypoventilation Diffusion Abnormalities Mechanisms of Hypercapnia Effect of 100% Oxygen on Arterial Blood Gas Abnormalities Regional Differences Key Points Chapter 24 Oxygen and Carbon Dioxide Transport Learning Objectives Gas Diffusion Oxygen Transport Carbon Dioxide Transport Regulation of Hydrogen Ion Concentration and Acid-Base Balance Carbon Dioxide Dissociation Curve Key Points

Chapter 25 Control of Respiration Learning Objectives Ventilatory Control: An Overview Response to Carbon Dioxide Control of Ventilation: The Details Exercise Abnormalities in the Control of Breathing Key Points Chapter 26 Host Defense and Metabolism in the Lung Learning Objectives Host Defense Epithelial Cells and Commensal Microbiota Protect the Lumen of the Airways Clinical Manifestations Associated With Abnormalities in Mucosal Innate and Adaptive Immunity Metabolic Functions of the Lung Key Points

Section 6 Gastrointestinal Physiology Chapter 27 Functional Anatomy and General Principles of Regulation in the Gastrointestinal Tract Learning Objectives Functional Anatomy Regulatory Mechanisms in the Gastrointestinal Tract Response of the GI Tract to a Meal Key Concepts

Chapter 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal Learning Objectives Cephalic and Oral Phases Esophageal Phase Key Concepts Chapter 29 The Gastric Phase of the Integrated Response to a Meal Learning Objectives Functional Anatomy of the Stomach Gastric Secretion Digestion in the Stomach Gastrointestinal Motility Gastric Motility Key Concepts Chapter 30 The Small Intestinal Phase of the Integrated Response to a Meal Learning Objectives Gastric Emptying in the Small Intestinal Phase Carbohydrate Assimilation Protein Assimilation Uptake of Peptides and Amino Acids Lipid Assimilation Water and Electrolyte Secretion and Absorption Absorption of Minerals and Water-Soluble Vitamins Motor Patterns of the Small Intestine Key Concepts

Chapter 31 The Colonic Phase of the Integrated Response to a Meal Learning Objectives Overview of the Large Intestine Key Concepts Chapter 32 Transport and Metabolic Functions of the Liver Learning Objectives Overview of the Liver and its Functions Structural Features of the Liver and Biliary System Bile Formation and Secretion Ammonia Handling by the Liver Clinical Assessment of Liver Function Key Concepts

Section 7 Renal Physiology Chapter 33 Elements of Renal Function Learning Objectives Overview of Renal Function Functional Anatomy of the Kidneys Assessment of Renal Function Glomerular Filtration Renal Blood Flow Regulation of Renal Blood Flow and Glomerular Filtration Rate Key Points Chapter 34 Solute and Water Transport Along the Nephron: Tubular Function

Learning Objectives Solute and Water Reabsorption Along the Nephron Regulation of NaCl and Water Reabsorption Key Points Chapter 35 Control of Body Fluid Osmolality and Volume Learning Objectives Control of Body Fluid Osmolality: Urine Concentration and Dilution Control of Extracellular Fluid Volume and Regulation of Renal NaCl Excretion Key Concepts Chapter 36 Potassium, Calcium, and Phosphate Homeostasis Learning Objectives K+ Homeostasis Regulation of Plasma [K+] Alterations in Plasma [K+] K+ Excretion by the Kidneys Cellular Mechanism of K+ Transport by Principal and Intercalated Cells in the DT and CCD K+ Excretion by the DT and CCD Overview of Calcium and Inorganic Phosphate Homeostasis Integrative Review of Parathyroid Hormone and Calcitriol on Ca++ and Pi Homeostasis Key Concepts Chapter 37 Role of the Kidneys in the Regulation of Acid-Base Balance Learning Objectives The HCO3− Buffer System

Overview of Acid-Base Balance Net Acid Excretion by the Kidneys Response to Acid-Base Disorders Simple Acid-Base Disorders Key Concepts

Section 8 Endocrine Physiology Chapter 38 Introduction to the Endocrine System Learning Objectives Configuration of Feedback Loops Within the Endocrine System Chemical Nature of Hormones Transport of Hormones in the Circulation Cellular Responses to Hormones Key Concepts Chapter 39 Hormonal Regulation of Energy Metabolism Learning Objectives Continual Energy Supply and Demand: The Challenge Integrated Overview of Energy Metabolism Pancreatic Hormones Involved in Metabolic Homeostasis During Different Metabolic Phases Hormonal Regulation of Specific Metabolic Reactions and Pathways Leptin and Energy Balance Key Concepts Chapter 40 Hormonal Regulation of Calcium and Phosphate Metabolism

Learning Objectives Crucial Roles of Calcium and Phosphate in Cellular Physiology Hormonal Regulation of Calcium and Phosphate: PTH, Vitamin D, and FGF23 Hormonal Effects on Target Organs Regulation of Bone Formation Integrated Physiological Regulation of Ca++/Pi Metabolism Key Concepts Chapter 41 The Hypothalamus and Pituitary Gland Learning Objectives Anatomy The Neurohypophysis The Adenohypophysis Key Concepts Chapter 42 The Thyroid Gland Learning Objectives Anatomy and Histology of the Thyroid Gland Thyroid Hormones Transport of Thyroid Hormones Cellular Entry and Peripheral Conversion of Thyroid Hormones Regulation of Thyroid Function Physiological Effects of Thyroid Hormone Key Concepts Chapter 43 The Adrenal Gland

Learning Objectives Anatomy Adrenal Medulla Adrenal Cortex Key Concepts Chapter 44 The Male and Female Reproductive Systems Learning Objectives The Male Reproductive System The Female Reproductive System Mammogenesis and Lactation Menopause Key Concepts Acknowledgment Index

S E CT I ON 1

Cellular Physiology Bruce M. Koeppen and Bruce A. Stanton

Chapter 1 Principles of Cell and Membrane Function Chapter 2 Homeostasis: Volume and Composition of Body Fluid Compartments Chapter 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression

C H AP T E R 1

Principles of Cell and Membrane Function LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. What organelles are found in a typical eukaryotic cell, and what is their function? 2. What is the composition of the plasma membrane? 3. What are the major classes of membrane transport proteins, and how do they transport biologically important molecules and ions across the plasma membrane? 4. What is the electrochemical gradient, and how is it used to determine whether the transport of a molecule or an ion across the plasma membrane is active or passive? 5. What are the driving forces for movement of water across cell membrane and the capillary wall? In addition, the student should be able to define and understand the following properties of physiologically important solutions and fluids: • Molarity and equivalence • Osmotic pressure • Osmolarity and osmolality • Oncotic pressure • Tonicity

The human body is composed of billions of cells. Although cells can perform different functions, they share certain common elements. This chapter provides an overview of some of these common elements with a focus on the transport of molecules and water into and out of the cell across its plasma membrane.

Overview of Eukaryotic Cells Eukaryotic cells are distinguished from prokaryotic cells by the presence of a membrane-delimited nucleus. With a few exceptions (e.g., mature human red blood cells and cells within the lens of the eye), all cells within the human body contain a nucleus. The cell is therefore effectively divided into two compartments: the nucleus and the cytoplasm. The cytoplasm is an aqueous solution containing numerous organic molecules, ions, cytoskeletal elements, and a number of organelles. Many of the organelles are ​membrane-enclosed compartments that carry out specific cellular function. An idealized eukaryotic cell is

depicted in Fig. 1.1, and the primary functions of some components and compartments of the cell are summarized in Table 1.1. Readers who desire a more in-depth presentation of this material are encouraged to consult one of the many textbooks on cell and molecular biology that are currently available.

FIG. 1.1 Schematic drawing of a eukaryotic cell. The top portion of the cell is omitted to illustrate the nucleus and various intracellular organelles. See text for details.

Table 1.1 Primary Functions of Some Eukaryotic Cellular Components and Compartments Component

Primary Function

Cytosol

Metabolism, protein synthesis (free ribosomes)

Cytoskeleton

Cell shape and movement, intracellular transport

Nucleus

Genome (22 autosomes and 2 sex chromosomes—in humans), DNA and RNA synthesis

Mitochondria

ATP synthesis by oxidative phosphorylation, Ca2+ storage

Smooth Synthesis of lipids, Ca2+ storage endoplasmic reticulum Free ribosomes

Translation of mRNA into cytosolic proteins

Rough Translation of mRNA into membrane-associated proteins or for secretion out of the cell endoplasmic reticulum Lysosome

Intracellular degradation

Endosome

Cellular uptake of cholesterol, removal of receptors from the plasma membrane, uptake of small molecules and water into the cell, internalization of large particles (e.g., bacteria, cell debris)

Golgi apparatus

Modification, sorting, and packaging of proteins and lipids for delivery to other organelles within the cell or for secretion out of the cell

Proteosome

Degradation of intracellular proteins

Peroxisome

Detoxification of substances

ATP, Adenosine triphosphate; mRNA, messenger RNA.

The Plasma Membrane The cells within the body are surrounded by a plasma membrane that separates the intracellular contents from the extracellular environment. Because of the properties of this membrane and, in particular, the presence of specific membrane proteins, the plasma membrane is involved in a number of important cellular functions, including the following: • Selective transport of molecules into and out of the cell. A function carried out by membrane transport proteins. • Cell recognition through the use of cell surface antigens. • Cell communication through neurotransmitter and hormone receptors and through signal transduction pathways. • Tissue organization, such as temporary and permanent cell junctions, and interaction with the extracellular matrix, with the use of a variety of cell adhesion molecules. • Membrane-dependent enzymatic activity. • Determination of cell shape by linkage of the cytoskeleton to the plasma membrane.

In this chapter, the structure and function of the plasma membrane of eukaryotic cells are considered. More specifically, the chapter focuses on the transport of molecules and water across the plasma membrane. Only the principles of membrane transport are presented here. Additional details that relate to specific cells are presented in the various sections and chapters of this book. Structure and Composition The plasma membrane of eukaryotic cells consists of a 5-nm-thick lipid bilayer with associated proteins (Fig. 1.2). Some of the membrane-associated proteins are integrated into the lipid bilayer; others are more loosely attached to the inner or outer surfaces of the membrane, often by binding to the integral membrane proteins.

FIG. 1.2 Schematic diagram of the cell plasma membrane. Not shown are lipid rafts. See text for details. GPI, ​Glycosylphosphatidylinositol. (Modified from Cooper GM. The Cell—A Molecular Approach. 2nd ed. Washington, DC: Sinauer; 2000, Fig. 12.3.)

Membrane Lipids The major lipids of the plasma membrane are phospholipids and phosphoglycerides. Phospholipids are amphipathic molecules that contain a charged (or polar) hydrophilic head and two (nonpolar) hydrophobic fatty acyl chains (Fig. 1.3). The amphipathic nature of the phospholipid molecule is critical for the formation of the bilayer: The hydrophobic fatty acyl chains form the core of the bilayer, and the polar head groups are exposed on the surface.

FIG. 1.3 Models of the major classes of plasma membrane lipids, depicting the hydrophilic and hydrophobic regions of the molecules. The molecules are arranged as they exist in one leaflet of the bilayer. The opposing leaflet is not shown. One of the fatty acyl chains in the phospholipid molecule is unsaturated. The presence of this double bond produces a “kink” in the fatty acyl chain, which prevents tight packing of membrane lipids and increases membrane fluidity. (Modified from Hansen JT, Koeppen BM. Netter’s Atlas of Human Physiology. Teterboro, NJ: Icon Learning Systems; 2002.)

The majority of membrane phospholipids have a glycerol “backbone” to which are attached the fatty acyl chains, and an alcohol is linked to glycerol via a phosphate group. The common alcohols are choline, ethanolamine, serine, inositol, and glycerol. Another important phospholipid, sphingomyelin, has the amino alcohol sphingosine as its “backbone” instead of glycerol. Table 1.2 lists these common phospholipids. The fatty acyl chains are usually 14 to 20 carbons in length and may be saturated or unsaturated (i.e., contain one or more double bonds). Table 1.2 Plasma Membrane Lipids Phospholipid

Primary Location in Membrane

Phosphatidylcholine

Outer leaflet

Sphingomyelin

Outer leaflet

Phosphatidylethanolamine

Inner leaflet

Phosphatidylserine

Inner leaflet

Phosphatidylinositol*

Inner leaflet

*Involved in signal transduction.

The phospholipid composition of the membrane varies among different cell types and even between the bilayer leaflets. For example, in the erythrocyte plasma membrane, phosphatidylcholine and sphingomyelin are found predominantly in the outer leaflet of the membrane, whereas phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are found in the inner leaflet. As described in detail in Chapter 3, phosphatidylinositol plays an important role in signal transduction, and its location in the inner leaflet of the membrane facilitates this signaling role. The sterol molecule cholesterol is also a critical component of the bilayer (see Fig. 1.3). It is found in both leaflets and serves to stabilize the membrane at normal body temperature (37°C). As much as 50% of the lipids found in the membrane can be cholesterol. A minor lipid component of the plasma membrane is glycolipids. These lipids, as their name indicates, consist of two fatty acyl chains linked to polar head groups that consist of carbohydrates (see Fig. 1.3). As discussed in the section on Membrane Proteins, one glycolipid, glycosylphosphatidylinositol (GPI), plays an important role in anchoring proteins to the outer leaflet of the membrane. Both cholesterol and glycolipids, like the phospholipids, are amphipathic, and they are oriented with their polar groups on the outer surface of the leaflet in which they are located. Their hydrophobic portion is thus located within the interior of the bilayer. The lipid bilayer is not a static structure. The lipids and associated proteins can diffuse within the plane of the membrane. The fluidity of the membrane is determined by temperature and by its lipid composition. As temperature increases, the fluidity of the membrane increases. The presence of unsaturated fatty acyl chains in the phospholipids and glycolipids also increases membrane fluidity. If a fatty acyl chain is unsaturated, the presence of a double bond introduces a “kink” in the molecule (see Fig. 1.3). This kink prevents the molecule from associating closely with surrounding lipids, and, as a result, membrane fluidity is increased. Although the lipid bilayer is “fluid,” movement of proteins in the membrane can be constrained or limited. For example, membrane proteins can be anchored to components of the intracellular cytoskeleton, which limits their movement. Membrane domains can also be isolated from one another. An important example of this can be found in epithelial tissues. Junctional complexes (e.g., tight junctions) separate the plasma membrane of epithelial cells into two domains: apical and basolateral (see Chapter 2). The targeted localization of membrane proteins into one or other of these domains allows epithelial cells to carry out vectorial transport of substances from one side of the epithelium to the opposite side. The ability to carry out vectorial transport is crucial for the functioning of several organ systems (e.g., the gastrointestinal tract and kidneys). In addition, some regions of the membrane contain lipids (e.g., sphingomyelin and cholesterol) that aggregate into what are called lipid rafts. These lipid rafts often have an association with specific proteins, which diffuse in the plane of the membrane as a discrete unit. Lipid rafts appear to serve a number of functions. One important function of these rafts is to segregate signaling molecules. Membrane Proteins As much as 50% of the plasma membrane is composed of proteins. These membrane proteins are classified as integral, lipid-anchored, or peripheral. Integral membrane proteins are imbedded in the lipid bilayer, where hydrophobic amino acid residues are associated with the hydrophobic fatty acyl chains of the membrane lipids. Many integral membrane proteins span the bilayer; such proteins are termed transmembrane proteins. Transmembrane proteins have both hydrophobic and hydrophilic regions. The hydrophobic region, often in the form of an α helix, spans the membrane. Hydrophilic amino acid residues are then exposed to the aqueous environment on either side of the membrane. Transmembrane proteins may pass through the membrane multiple times.

AT THE C ELLU LAR LEVEL There is a superfamily of membrane proteins that serve as receptors for many hormones, neurotransmitters, and numerous drugs. These receptors are coupled to heterotrimeric G proteins and are termed G protein–coupled receptors (see Chapter 3). These proteins span the membrane with seven α-helical domains. The binding site of each ligand is either on the extracellular portion of the protein (large ligands) or in the membrane-spanning portion (small ligands), whereas the cytoplasmic portion binds to the G protein. This superfamily of membrane proteins makes up the third largest family of genes in humans. Nearly half of all nonantibiotic prescription drugs are targeted toward G protein– coupled receptors. A protein can also be attached to the membrane via lipid anchors. The protein is covalently attached to a lipid molecule, which is then embedded in one leaflet of the bilayer. GPI anchors proteins to the outer leaflet of the membrane. Proteins can be attached to the inner leaflet via their amino-terminus by fatty acids (e.g., myristate or palmitate) or via their carboxyl-terminus by prenyl anchors (e.g., farnesyl or geranylgeranyl). Peripheral proteins may be associated with the polar head groups of the membrane lipids, but they more commonly bind to integral or lipid-anchored proteins. In many cells, some of the outer leaflet lipids, as well as many of the proteins exposed on the outer surface of the membrane, are glycosylated (i.e., have short chains of sugars, called oligosaccharides, attached to them). These glycolipids and glycoproteins are components of the “cell coat” called the glycocalyx. The glycocalyx establishes an extracellular microenvironment at the surface of the cell membrane. Depending on the cell, the glycocalyx may be involved in metabolism (e.g., in the gastrointestinal tract), cell recognition (e.g., cell surface antigens), and formation of cell-cell interactions (e.g., attachment of neutrophils to vascular endothelial cells). Membrane Transport Although plasma membrane proteins perform many important cellular functions, as noted previously, the remainder of this chapter focuses on one group of plasma membrane proteins: the membrane transport proteins, or transporters. It has been estimated that approximately 10% of human genes (≈2000) code for transporters. They are also targets for numerous drugs. The normal function of cells requires the continuous movement of water and solutes into and out of the cell. The intracellular and extracellular fluids are composed primarily of H2O, in which solutes (e.g., ions, glucose, amino acids) are dissolved. The plasma membrane, with its hydrophobic core, is an effective barrier to the movement of virtually all of these biologically important solutes. It also restricts the movement of water across the membrane. The presence of specific membrane transporters in the membrane is responsible for the movement of these solutes and water across the membrane. Membrane Transport Proteins Membrane transporters have been classified in several different ways. In this chapter, the transporters are divided into four general groups: water channels, ion channels, solute carriers, and adenosine triphosphate (ATP)–dependent transporters. Table 1.3 lists these groups of membrane transporters, their modes of transport, and estimates of the rates at which they transport molecules or ions across the membrane.

Table 1.3 Major Classes of Plasma Membrane Transporters Class

Transport Mode

Transport Rate

Porea

Open (not gated)

Up to 109 molecules/sec

Channel

Gated

106–108 molecules/sec

Solute carrier

Cycle

102–104 molecules/sec

ATP-dependent

Cycle

102–104 molecules/sec

a

Examples include porins that are found in the outer membrane of mitochondria, and water channels (i.e., aquaporins) that function as a pore. ATP, Adenosine triphosphate.

Water Channels Water channels, or aquaporins (AQPs), are the main routes for water movement into and out of the cell. Although water can cross the plasma membrane through other membrane transporters (e.g., glucose transporter, urea transporter), these routes for water movement across the plasma membrane are secondary to the AQPs. The AQPs are widely distributed throughout the body (e.g., the brain, lungs, kidneys, salivary glands, gastrointestinal tract, and liver). Cells express different AQP isoforms, and some cells even express multiple isoforms. For example, cells in the collecting ducts of the kidneys express AQP3 and AQP4 in their basolateral membrane and AQP2 in their apical membrane. Moreover, the abundance of AQP2 in the apical membrane is regulated by antidiuretic hormone (also called arginine vasopressin), which is crucial for the ability of the kidneys to concentrate the urine (see Chapter 35). Although all AQP isoforms allow the passive movement of H2O across the membrane, some isoforms also provide a pathway for other molecules (e.g., glycerol, urea, mannitol, purines, pyrimidines, CO2, and NH3) to cross the membrane. Because glycerol was one of the first molecules identified as crossing the membrane via some AQPs, this group of AQPs is collectively called aquaglyceroporins (see also Chapter 34). Regulation of the amount of H2O that can enter or leave the cell via AQPs occurs primarily by altering the number of AQPs in the membrane.

AT THE C ELLU LAR LEVEL Each AQP molecule consists of six membrane-spanning domains and a central water-transporting pore. Four AQP monomers assemble to form a homotetramer in the plasma membrane, with each monomer functioning as a water channel. Ion Channels Ion channels are found in all cells and are especially important for the function of excitable cells (e.g., neurons and muscle cells). Ion channels are classified by their selectivity, conductance, and mechanism of channel gating (i.e., opening and closing). Selectivity is defined as the nature of the ions that pass through the channel. At one extreme, ion channels can be highly selective, in that they allow only a specific ion through. At the other extreme, they may be nonselective, allowing all or a group of cations or anions through. Channel conductance refers to the number of ions that pass through the channel and is typically

expressed in picosiemens (pS). The range of conductance is considerable: Some channels have a conductance of only 1 to 2 pS, whereas others have a conductance of more than 100 pS. For some channels, the conductance varies, depending on the direction in which the ion is moving. For example, if the channel has a larger conductance when ions are moving into the cell than when they are moving out of the cell, the channel is said to be an inward rectifier. Moreover, ion channels fluctuate between an open state and a closed state, a process called gating (Fig. 1.4). Factors that can control gating include membrane voltage, extracellular agonists or antagonists (e.g., acetylcholine is an extracellular agonist that controls the gating of a cation-selective channel in the motor end plate of skeletal muscle cells; see Chapter 6), intracellular messengers (e.g., Ca++, ATP, cyclic guanosine monophosphate), and mechanical stretch of the plasma membrane. Ion channels can be regulated by a change in the number of channels in the membrane or by gating of the channels.

FIG. 1.4 Recording of current flow through a single ion channel. The channel spontaneously fluctuates between an open state and a closed state. The amplitude of the current is approximately 2 pA (2 × 10−12 amps); that is, 12.5 million ions/second cross the membrane.

Solute Carriers Solute carriers (denoted SLCs by the HUGO Gene Nomenclature Committee) represent a large group of membrane transporters categorized into more than 50 families; almost 400 specific transporters have been identified to date. These carriers can be divided into three groups according to their mode of transport. One group, uniporters (or facilitated transporters), transports a single molecule across the membrane. The transporter that brings glucose into the cell (glucose transporter 1 [GLUT-1], or SLC2A1) is an

important member of this group. The second group, symporters (or cotransporters), couples the movement of two or more molecules/ions across the membrane. As the name implies, the molecules/ions are transported in the same direction. The Na+,K+,2Cl− symporter found in the kidney (NKCC2, or SLC12A1), which is crucial for diluting and concentrating the urine (see Chapter 34), is a member of this group. The third group, antiporters (or exchange transporters), also couples the movement of two or more molecules/ions across the membrane; in this case, however, the molecules/ions are transported in opposite directions. The Na+-H+ antiporter is a member of this group of solute carriers. One isoform of this antiporter (NHE-1, or SLC9A1) is found in all cells and plays an important role in regulating intracellular pH. Adenosine Triphosphate–Dependent Transporters The ATP-dependent transporters, as their name implies, use the energy in ATP to drive the movement of molecules/ions across the membrane. There are two groups of ATP-dependent transporters: the ATPase ion transporters and the ATP-binding cassette (ABC) transporters. The ATPase ion transporters are subdivided into P-type ATPases and V-type ATPases.a The P-type ATPases are phosphorylated during the transport cycle. Na+,K+-ATPase is an important example of a P-type ATPase. With the hydrolysis of each ATP molecule, it transports three Na+ ions out of the cell and two K+ ions into the cell. Na+,K+-ATPase is present in all cells and plays a critical role in establishing cellular ion and electrical gradients, as well as maintaining cell volume (see Chapter 2).

AT THE C ELLU LAR LEVEL Na+,K+-ATPase (also called the Na+,K+-pump or just the Na+-pump) is found in all cells and is responsible for establishing the gradients of Na+ and K+ across the plasma membrane. These gradients in turn provide energy for several essential cell functions (see Chapter 2). Na+,K+-ATPase is composed of three subunits (α, β, and γ), and the protein exists in the membrane with a stoichiometric composition of 1α, 1β, 1γ. The α subunit contains binding sites for Na+,K+ and ATP. It is also the subunit that binds cardiac glycosides (e.g., ouabain), which specifically inhibit the enzyme. It has a transmembrane domain and three intracellular domains: phosphorylation (P-domain), nucleotide binding (N-domain), and actuator (A-domain). Although the α subunit is the functional subunit of the enzyme (i.e., it hydrolyzes ATP, binds Na+ and K+, and translocates them across the membrane), it cannot function without the β subunit. The β subunit is responsible for targeting the α subunit to the membrane and also appears to modulate the kinetic properties of the Na+,K+-ATPase. The α and β subunits can carry out Na+ and K+ transport in the absence of the γ subunit. However, like the β subunit, the γ subunit appears to play a regulatory role by modulating Na+ affinity and the kinetics of the enzyme. V-type H+-ATPases are found in the membranes of several intracellular organelles (e.g., endosomes, lysosomes); as a result, they are also referred to as vacuolar H+-ATPases. The H+-ATPase in the plasma membrane plays an important role in urinary acidification (see Chapter 37). ABC transporters represent a large group of membrane transporters. They are found in both prokaryotic and eukaryotic cells, and they have amino acid domains that bind ATP (i.e., ABC domains). Seven subgroups of ABC transporters are found in humans and more than 40 specific transporters have been identified to date. They transport a diverse group of molecules/ions, including Cl−, cholesterol, bile

acids, drugs, iron, and organic anions.

IN THE C LIN IC Cystic fibrosis is an autosomal recessive disease characterized by chronic lung infections, pancreatic insufficiency, and infertility in boys and men. Death usually occurs because of respiratory failure. It is most prevalent in white people and is the most common lethal genetic disease in this population, occurring in 1 per 3000 live births. It is a result of mutations in a gene on chromosome 7 that codes for an ABC transporter. To date, more than 2000 mutations in the gene have been identified. The most common mutation is a deletion of a phenylalanine at position 508 (Phe508del). Because of this deletion, degradation of the protein by the endoplasmic reticulum in enhanced, and, as a result, the transporter does not reach the plasma membrane. This transporter, called cystic fibrosis transmembrane conductance regulator (CFTR), normally functions as a Cl− and HCO3− channel and also regulates other membrane transporters (e.g., the epithelial Na+ channel [ENaC]). Thus in individuals with cystic fibrosis, epithelial transport is defective, which is responsible for the pathophysiologic process. For example, in patients not affected by cystic fibrosis, the epithelial cells that line the airway of the lung are covered with a layer of mucus that entraps inhaled particulates and bacteria. Cilia on the epithelial cells then transport the entrapped material out of the lung, a process termed mucociliary transport (see Chapter 26 for more details). In patients with cystic fibrosis, the inability to secrete Cl−, Na+, HCO3−, and H2O results in an increase in the viscosity of the airway surface mucus; thus the cilia cannot transport the entrapped bacteria and other pathogens out of the lung. This in turn leads to recurrent and chronic lung infections. The inflammatory process that accompanies these infections ultimately destroys the lung tissue, causing respiratory failure and death. In 2019, the U.S. Food and Drug Administration approved elexacaftor/ivacaftor/tezacaftor (Trikafta), a drug that increases the amount of Phe508del CFTR in the plasma membrane. Trikafta, approved for patients with at least one Phe508del mutation (∼90% of the cystic fibrosis population), reduces clinical exacerbations and substantially improves lung function.

AT THE C ELLU LAR LEVEL Proteins within the plasma membrane of cells are constantly being removed and replaced with newly synthesized proteins. One mechanism by which membrane proteins are “tagged” for replacement is by the attachment of ubiquitin to the cytoplasmic portion of the protein. Ubiquitin is a 76–amino acid protein that is covalently attached to the membrane protein (usually to lysine) by a class of enzymes called ubiquitin protein ligases. One important group of these ligases is the developmentally downregulated protein 4 (Nedd4)/Nedd4-like family. Once a membrane protein is ubiquitinated, it undergoes endocytosis (see below) and is degraded either by lysosomes or by the proteosome. Cells also contain deubiquitinating enzymes (DUBs). Thus the amount of time a protein stays in the plasma membrane depends on the rate that ubiquitin groups are added by the ligases versus the rate that they are removed by the DUBs. For example, Na+ reabsorption by the collecting ducts of the kidneys is stimulated by the adrenal hormone aldosterone (see Chapters 34 and 35). One of the actions of aldosterone is to inhibit Nedd4-2. This prevents ubiquitination of ENaC in the apical membrane of epithelial cells. Thus the channels are retained for a longer period of time in the membrane, and as a result, more Na+ enters the cell and is thereby reabsorbed.

Because biologically important molecules enter and leave cells through membrane transporters, membrane transport is specific and regulated. Although some membrane transporters are ubiquitously expressed in all cells (e.g., Na+,K+-ATPase), the expression of many other transporters is limited to specific cell types. This specificity of expression tailors the function of the cell to the organ system in which it is located (e.g., the sodium-glucose–linked transporters SGLT-1 and SGLT-2 in the epithelial cells of the intestines and renal proximal tubules). In addition, the amount of a molecule being transported across the membrane can be regulated. Such regulation can take place through altering the number of transporters in the membrane or altering the rate or kinetics of individual transporters (e.g., the time an ion channel stays in the open versus closed state), or both.

Vesicular Transport Solute and water can be brought into the cell through a process of endocytosis and released from the cell through the process of exocytosis. Endocytosis is the process whereby a piece of the plasma membrane pinches off and is internalized into the cell interior, and exocytosis is the process whereby vesicles inside the cell fuse with the plasma membrane. In both of these processes, the integrity of the plasma membrane is maintained, and the vesicles allow for the transfer of the contents among cellular compartments. In some cells (e.g., the epithelial cells lining the gastrointestinal tract), endocytosis across one membrane of the cell is followed by exocytosis across the opposite membrane. This allows the transport of substances inside the vesicles across the epithelium, a process termed transcytosis. Endocytosis occurs by three mechanisms. The first is pinocytosis, which consists of the nonspecific uptake of small molecules and water into the cell. Pinocytosis is a prominent feature of the endothelial cells that line capillaries and is responsible for a portion of the fluid exchange that occurs across these vessels. The second form of endocytosis, phagocytosis, allows for the cellular internalization of large particles (e.g., bacteria, cell debris). This process is an important characteristic of cells in the immune system (e.g., neutrophils and macrophages). Often, but not always, phagocytosis is a receptor-mediated process. For example, macrophages have receptors on their surface that bind the Fc portion of immunoglobulins. When bacteria invade the body, they are often coated with antibody, a process called opsonization. These bacteria then attach to the membrane of macrophages via the fragment crystallizable (Fc) portion of the immunoglobulin, undergo phagocytosis, and are destroyed inside the cell. The third mechanism of endocytosis is receptor-mediated endocytosis, which allows the uptake of specific molecules into the cell. In this form of endocytosis, molecules bind to receptors on the surface of the cell. Endocytosis involves a number of accessory proteins including adaptin, clathrin, and the GTPase dynamin (Fig. 1.5).

FIG. 1.5 Receptor-mediated endocytosis. Receptors on the surface of the cell bind the ligand. A clathrincoated pit is formed with adaptin linking the receptor molecules to clathrin. Dynamin, a guanosine triphosphatase (GTPase), assists in separation of the endocytic vesicle from the membrane. Once inside the cell, the clathrin and adaptin molecules dissociate and are recycled. The uncoated vesicle is then ready to fuse with other organelles in the cell (e.g., lysosomes). (Adapted from Ross MH, Pawlina W. Histology. 5th ed. Baltimore: Lippincott Williams & Wilkins; 2006.)

IN THE C LIN IC Cholesterol is an important component of cells (e.g., it is a key component of membranes). However, most cells are unable to synthesize cholesterol and therefore must obtain it from the blood. Normally, cholesterol is ingested in the diet, and it is transported through the blood in association with lipoproteins. Low-density lipoproteins (LDLs) in the blood carry cholesterol to cells, where they bind to LDL receptors in the plasma membrane. After the receptors bind LDL, they collect into “coated pits” and undergo endocytosis as clathrin-coated vesicles. Once inside the cell, the endosomes release LDL and then recycle the LDL receptors back to the cell surface. Inside the cell, LDL is then degraded in lysosomes, and the cholesterol is made available to the cell. Defects in the LDL receptor prevent cellular uptake of LDL. Individuals with this defect have elevated levels of blood LDL, often called “bad cholesterol,” because it is associated with the development of cholesterol-containing plaques in the smooth muscle layer of arteries. This process, atherosclerosis, is associated with an increased risk for heart attacks as a result of occlusion of the coronary arteries. Exocytosis can be either constitutive or regulated. Constitutive exocytosis occurs, for example, in plasma cells that are secreting immunoglobulin or in fibroblasts secreting collagen. Regulated secretion occurs in endocrine cells, neurons, and exocrine glandular cells (e.g., pancreatic acinar cells). In these cells, the secretory product (e.g., hormone, neurotransmitter, or digestive enzyme), after synthesis and processing in the rough endoplasmic reticulum and Golgi apparatus, is stored in the cytoplasm in

secretory granules until an appropriate signal for secretion is received. These signals may be hormonal or neural. Once the cell receives the appropriate stimulus, the secretory vesicle fuses with the plasma membrane and releases its contents into the extracellular fluid. Fusion of the vesicle with the membrane is mediated by a number of accessory proteins. One important group is the SNARE (soluble Nethylmaleimide sensitive fusion protein [NSF] attachment protein receptors) proteins. These membrane proteins help target the secretory vesicle to the plasma membrane. The process of secretion is usually triggered by an increase in the concentration of intracellular Ca++ ([Ca++]). However, two notable exceptions to this general rule exist: (1) Renin secretion by the juxtaglomerular cells of the kidney occurs with a decrease in intracellular Ca++ (see Chapters 34 and 35), as does (2) the secretion of parathyroid hormone by the parathyroid gland (see Chapter 40).

Basic Principles of Solute and Water Transport As already noted, the plasma membrane, with its hydrophobic core, is an effective barrier to the movement of virtually all biologically important molecules into or out of the cell. Thus membrane transport proteins provide the pathway that allows transport to occur into and out of cells. However, the presence of a pathway is not sufficient for transport to occur; an appropriate driving force is also required. In this section, the basic principles of diffusion, active and passive transport, and osmosis are presented. These topics are discussed in greater depth, as appropriate, in the other sections of the book.

Diffusion Diffusion is the process by which molecules move spontaneously from an area of high concentration to one of low concentration. Thus wherever a concentration gradient exists, diffusion of molecules from the region of high concentration to the region of low concentration dissipates the gradient (as discussed later, the establishment of concentration gradients for molecules requires the expenditure of energy). Diffusion is a random process driven by the thermal motion of the molecules. Fick’s first law of diffusion quantifies the rate at which a molecule diffuses from point A to point B:

(Equation 1.1) where J = the flux or rate of diffusion per unit time D = the diffusion coefficient A = the area across which the diffusion is occurring ΔC = the concentration difference between points A and B ΔX = the distance along which diffusion is occurring The diffusion coefficient takes into account the thermal energy of the molecule, its size, and the viscosity of the medium through which diffusion is taking place. For spherical molecules, D is

approximated by the Stokes-Einstein equation:

(Equation 1.2)

where k = Boltzmann’s constant T = temperature in degrees Kelvin r = radius of the molecule η = viscosity of the medium According to Eqs. 1.1 and 1.2, the rate of diffusion will be faster for small molecules than for large molecules. In addition, diffusion rates are high at elevated temperatures, in the presence of large concentration gradients, and when diffusion occurs in a low-viscosity medium. With all other variables held constant, the rate of diffusion is linearly related to the concentration gradient. Fick’s equation can also be applied to the diffusion of molecules across a barrier, such as a lipid bilayer. When applied to the diffusion of a molecule across a bilayer, the diffusion coefficient (D) incorporates the properties of the bilayer and especially the ability of the molecule to diffuse through the bilayer. To quantify the interaction of the molecule with the bilayer, the term partition coefficient (β) is used. For a molecule that “dissolves” equally in the fluid bathing the lipid bilayer (e.g., water) and in the lipid bilayer, β = 1. If the molecule dissolves more easily in the lipid bilayer, β > 1; and if it dissolves less easily in the lipid bilayer, β < 1. For a simple lipid bilayer, the more lipid soluble the molecule is, the larger the partition coefficient is, and thus the diffusion coefficient—therefore the rate of diffusion of the molecule across the bilayer—is greater. In this situation, ΔC represents the concentration difference across the membrane, A is the membrane area, and ΔX is the thickness of the membrane. Another useful equation for quantitating the diffusion of molecules across the plasma membrane (or any membrane) is as follows:

(Equation 1.3) where J = the flux or rate of diffusion across the membrane P = the permeability coefficient Ci = the concentration of the molecule inside the cell Co = the concentration of the molecule outside the cell This equation is derived from Fick’s equation (Eq. 1.1). P incorporates D, ΔX, A, and the partition

coefficient (β). P is expressed in units of velocity (e.g., centimeters per second), and C the units of moles/cm3. Thus the units of flux are moles per square centimeter per second (mol/cm2/sec). Values for P can be obtained experimentally for any molecule and bilayer. As noted, the phospholipid portion of the plasma membrane represents an effective barrier to many biologically important molecules. Consequently, diffusion through the lipid phase of the plasma membrane is not an efficient process for movement of these molecules across the membrane. It has been estimated that for a cell 20 µm in diameter, with a plasma membrane composed only of phospholipids, dissipation of a urea gradient imposed across the membrane would take approximately 8 minutes. Similar gradients for glucose and amino acids would take approximately 14 hours to dissipate, whereas ion gradients would take years to dissipate. As noted previously, the vast majority of biologically important molecules cross cell membranes via specific membrane transporters, rather than by diffusing through the lipid portion of the membrane. Nevertheless, Eq. 1.3 can be and has been used to quantitate the diffusion of molecules across many biological membranes. When this is done, the value of the permeability coefficient (P) reflects the properties of the pathway (e.g., membrane transporter or, in some cases, multiple transporters) that the molecule uses to cross the membrane. Despite the limitations of using diffusion to describe and understand the transport of molecules across cell membranes, it is also important for understanding gas exchange in the lungs (see Chapter 24), the movement of molecules through the cytoplasm of the cell, and the movement of molecules between cells in the extracellular fluid. For example, one of the physiological responses of skeletal muscle to exercise is the recruitment or opening of capillaries that are not perfused at rest. This opening of previously closed capillaries increases capillary density and thereby reduces the diffusion distance between the capillary and the muscle fiber so that oxygen and cellular fuels (e.g., fatty acids and glucose) can be delivered more quickly to the contracting muscle fiber. In resting muscle, the average distance of a muscle fiber from a capillary is estimated to be 40 µm. However, with exercise, this distance decreases to 20 µm or less.

Electrochemical Gradient The electrochemical gradient (also called the electrochemical potential difference) is used to quantitate the driving force acting on a molecule to cause it to move across a membrane. The electrochemical gradient for any molecule (Δµx) is calculated as follows:

(Equation 1.4)

where R = the gas constant T = temperature in degrees Kelvin ln = natural logarithm [X]i = the concentration of X inside the cell

[X]o = the concentration of X outside the cell zx = the valence of charged molecules F = the Faraday constant Vm = the membrane potential (Vm = Vi − Vo)b The electrochemical gradient is a measure of the free energy available to carry out the useful work of transporting the molecule across the membrane. It has two components: The first component represents the energy in the concentration gradient for X across the membrane (chemical potential difference). The second component (electrical potential difference) represents the energy associated with moving charged molecules (e.g., ions) across the membrane when a membrane potential exits (i.e., Vm ≠ 0 mV). Thus for the movement of glucose across a membrane, only the concentrations of glucose inside and outside of the cell need to be considered (Fig. 1.6A). However, the movement of K+ across the membrane, for example, would be determined both from the K+ concentrations inside and outside of the cell and from the membrane voltage (see Fig. 1.6B).

FIG. 1.6 Electrochemical gradients and cellular transport of molecules. A, Because glucose is uncharged, the electrochemical gradient is determined solely by the concentration gradient for glucose across the cell membrane. As shown, the glucose concentration gradient would be expected to drive glucose into the cell. B, Because K+ is charged, the electrochemical gradient is determined by both the concentration gradient and the membrane voltage (Vm ). The Nernst equilibrium potential for K+ (

),

calculated with Eq. 1.5a, is −90.8 mV ( at equilibrium). The energy in the concentration gradient, + which drives K out of the cell, is thus proportional to +90.8 mV. The membrane voltage of −60 mV drives K+ into the cell. Thus the electrochemical gradient, or net driving force, is 2.97 kJ/mol (equivalent to 30.8 mV), which drives K+ out of the cell.

Eq. 1.4 can be used to derive the Nernst equation for the situation in which a molecule is at equilibrium across the membrane (i.e., Δµ = 0):

(Equation 1.5a)

Alternatively,

(Equation 1.5b)

The value of Vm calculated with the Nernst equation represents the equilibrium condition and is referred to as the Nernst equilibrium potential (Ex, the Vm at which there is no net transport of the molecule across the membrane). It should be apparent that the Nernst equilibrium potential quantitates the energy in a concentration gradient and expresses that energy in millivolts. For example, for the cell depicted in Fig. 1.6B, the energy in the K+ gradient (derived from the Nernst equilibrium potential for K+ [ ]) is proportional to 90.8 mV (causing K+ to move out of the cell). This is opposite to, and of greater magnitude than, the energy in the membrane voltage (Vm = −60 mV), which causes K+ to enter the cell. As a result, the electrochemical gradient is such that the net movement of K+ across the membrane will be out of the cell. Another way to state this is that the net driving force for K+ (Vm −  ) is 30.8 mV (driving K+ out of the cell). This is described in more detail in Chapter 2. The Nernst equation, at 37° C, can be written as follows by replacing the natural logarithm function with the base 10 logarithm function:

(Equation 1.6a)

or

(Equation 1.6b)

These are the most common forms of the Nernst equation in use. In these equations, it is apparent that for a univalent ion (e.g., Na+, K+, Cl−), a 10-fold concentration gradient across the membrane is equivalent in energy to an electrical potential difference of 61.5 mV (at 37° C), and a 100-fold gradient is equivalent to an electrical potential difference of 123 mV. Similarly, for a divalent ion (e.g., Ca++), a 10fold concentration gradient is equivalent to a 30.7-mV electrical potential difference, because z in Eqs. 1.6a and 1.6b is equal to 2.

Active and Passive Transport When the net movement of a molecule across a membrane occurs in the direction predicted by the electrochemical gradient, that movement is termed passive transport. Thus for the examples given in Fig. 1.6, the movement of glucose into the cell and the movement of K+ out of the cell would be considered passive transport. Transport that is passive is sometimes referred to as either “downhill transport” or “transport with the electrochemical gradient.” In contrast, if the net movement of a molecule across the membrane is opposite to that predicted by the electrochemical gradient, that movement is termed active transport, a process that requires the input of energy (e.g., ATP). Active transport is sometimes referred to as either “uphill transport” or “transport against the electrochemical gradient.” In the various classes of plasma membrane transport proteins, the movement of H2O through water channels is a passive process (see later discussion), as is the movement of ions through ion channels and the transport of molecules via uniporters (e.g., transport of glucose via GLUT-1). The ATPase-dependent transporters can use the energy in ATP to drive active transport of molecules (e.g., Na+,K+-ATPase, H+ATPase, or ABC transporters). Because the transport is directly coupled to the hydrolysis of ATP, it is referred to as primary active transport. Solute carriers that couple movement of two or more molecules (e.g., 3Na+,Ca++ antiporter) often transport one or more molecules (one Ca++ molecule in this example) against their respective electrochemical gradient through the use of the energy in the electrochemical gradient of the other molecule or molecules (three Na+ in this example). When this occurs, the molecule or molecules transported against their electrochemical gradient are said to be transported by secondary active transport mechanisms (Fig. 1.7).

FIG. 1.7 Examples of several membrane transporters, illustrating primary active, passive, and secondary active transport. See text for details. ATP, Adenosine triphosphate.

IN THE C LIN IC Glucose is transported by the epithelial cells that line the gastrointestinal tract (small intestine), and by cells that form the proximal tubules of the kidneys. In the gastrointestinal tract, the glucose is absorbed from ingested food. In the kidney, the proximal tubule reabsorbs the glucose that was filtered across the glomerular capillaries and thereby prevents it from being lost in the urine. The uptake of glucose into the epithelial cell from the lumen of the small intestine and from the lumen of the proximal tubule is a secondary active process involving the sodium-glucose–linked transporters SGLT-1 and SGLT-2. SGLT-2 transports one glucose molecule with one Na+ ion, and the energy in the electrochemical gradient for Na+ (into the cell) drives the secondary active uptake of glucose. According to the following equation, for calculating the electrochemical gradient, and if the membrane potential (Vm) is −60 mV and there is a 10-fold [Na+] gradient across the membrane, an approximate 100-fold glucose gradient could be generated by SGLT-2:

Thus, if the intracellular glucose concentration was 2 mmol/L, the cell could lower the extracellular glucose concentration to approximately 0.02 mmol/L. However, by increasing the number of Na+ ions transported with glucose from one to two, SGLT-1 can generate a nearly 10,000-fold glucose gradient:

Again, if the intracellular glucose concentration is 2 mmol/L, SGLT-1 could remove virtually all glucose from either the lumen of the small intestine or the lumen of the proximal tubule (i.e., the luminal glucose concentration 0.0002 mmol/L).

Osmosis and Osmotic Pressure The movement of water across cell membranes occurs by the process of osmosis. The movement of water is passive, with the driving force for this movement being the osmotic pressure difference across the cell membrane. Fig. 1.8 illustrates the concept of osmosis and the measurement of the osmotic pressure of a solution.

FIG. 1.8 Schematic representation of osmotic water movement and the generation of an osmotic pressure. Compartment A and compartment B are separated by a semipermeable membrane (i.e., the membrane is highly permeable by water but impermeable by solute). Compartment A contains a solute, whereas compartment B contains only distilled water. Over time, water moves by osmosis from compartment B to compartment A. (Note: This water movement is driven by the concentration gradient for water. Because of the presence of solute particles in compartment A, the concentration of water in compartment A is less than that in compartment B. Consequently, water moves across the semipermeable membrane from compartment B to compartment A down its concentration gradient.) This causes the level of fluid to be raised in compartment A and lowered in compartment B. At equilibrium, the hydrostatic pressure exerted by the column of water (h) stops the net movement of water from compartment B to A. Thus at equilibrium, the hydrostatic pressure is equal and opposite to the osmotic pressure exerted by the solute particles in compartment A. (Redrawn from Koeppen BM, Stanton BA. Renal Physiology. 4th ed. St. Louis: Mosby; 2006.)

Osmotic pressure is determined by the number of solute molecules dissolved in the solution. It is not dependent on factors such as the size of the molecules, their mass, or their chemical nature (e.g., valence). Osmotic pressure (π), measured in atmospheres (atm), is calculated by van’t Hoff’s law as follows:

(Equation 1.7) where n = number of dissociable particles per molecule C = total solute concentration R = gas constant T = temperature in degrees Kelvin For a molecule that does not dissociate in water, such as glucose or urea, a solution containing 1 mmol/L of these molecules at 37°C can exert an osmotic pressure of 2.54 × 10−2 atm, as calculated with Eq. 1.7 and the following values: n = 1 C = 0.001 mol/L R = 0.082 atm L/mol K T = 310 °K

Because 1 atm equals 760 mm Hg at sea level, π for this solution can also be expressed as 19.3 mm Hg. Alternatively, osmotic pressure is expressed in terms of osmolarity (see the following section). Regardless of the molecule, a solution containing 1 mmol/L of the molecule therefore exerts an osmotic pressure proportional to 1 mOsm/L. For molecules that dissociate in a solution, n of Eq. 1.7 will have a value other than 1. For example, a 150-mmol/L solution of NaCl has an osmolarity of approximately 300 mOsm/L because each molecule of NaCl dissociates into a Na+ and a Cl− ion (i.e., n = 2).c If dissociation of a molecule into its component ions is not complete, n will not be an integer. Accordingly, osmolarity for any solution can be calculated as follows:

(Equation 1.8)

Osmolarity Versus Osmolality The terms osmolarity and osmolality are frequently confused and incorrectly interchanged. Osmolarity refers to the osmotic pressure generated by the dissolved solute molecules in 1 L of solvent, whereas osmolality is the number of molecules dissolved in 1 kg of solvent. For dilute solutions, as encountered in most physiologic settings, the difference between osmolarity and osmolality is insignificant, as is the contribution of the solute particles to volume and mass of the solvent. Importantly measurements of osmolarity are temperature dependent because the volume of the solvent varies with temperature (i.e., the volume is larger at higher temperatures). In contrast, osmolality, which is based on the mass of the solvent, is temperature independent. For this reason, osmolality is the preferred term for biologic systems and is used throughout this book. Because the solvent in biological solutions and bodily fluids is water, and because of the dilute nature of biological solutions and bodily solutions, osmolalities are expressed as milliosmoles per kilogram of water (mOsm/kg H2O).

Tonicity The tonicity of a solution is related to the effect of the solution on the volume of a cell. Solutions that do not change the volume of a cell are said to be isotonic. A hypotonic solution causes a cell to swell, whereas a hypertonic solution causes a cell to shrink. Although related to osmolality, tonicity also accounts for the ability of the molecules in solution to cross the cell membrane. Consider two solutions: a 300-mmol/L solution of sucrose and a 300-mmol/L solution of urea. Both solutions have an osmolality of 300 mOsm/kg H2O and therefore are said to be isosmotic (i.e., they have the same osmolality). When red blood cells—which for the purpose of this illustration also have an intracellular fluid osmolality of 300 mOsm/kg H2O—are placed in the two solutions, those in the sucrose solution maintain their normal volume, whereas those placed in urea swell and eventually burst. Thus the sucrose solution is isotonic and the urea solution is hypotonic. The differential effect of these solutions on red blood cell volume is related to the permeability of the red blood cell plasma membrane to sucrose

and urea. The red blood cell membrane contains uniporters for urea. Thus urea easily crosses the cell membrane (i.e., the cell is permeable by urea), driven by the concentration gradient (i.e., extracellular urea concentration > intracellular urea concentration). In contrast, the red blood cell membrane does not contain sucrose transporters, and sucrose cannot enter the cell (i.e., the cell is impermeable by sucrose). To exert an osmotic pressure across a membrane, a molecule must not cross the membrane. Because the red blood cell membrane is impermeable by sucrose, it exerts an osmotic pressure equal and opposite to the osmotic pressure generated by the contents within the red blood cell (in this case, 300 mOsm/kg H2O). In contrast, urea is readily able to cross the red blood cell membrane, and it cannot exert an osmotic pressure to balance that generated by the intracellular solutes of the red blood cell. Consequently, sucrose is termed an effective osmole, whereas urea is an ineffective osmole. To take into account the effect of a molecule’s ability to permeate the membrane on osmotic pressure, it is necessary to rewrite Eq. 1.7 as follows:

(Equation 1.9) where σ is the reflection coefficient (or osmotic coefficient) and is a measure of the relative ability of the molecule to cross the cell membrane, and π e is the “effective osmotic pressure.” For a molecule that can freely cross the cell membrane, such as urea in the preceding example, σ = 0, and no effective osmotic pressure is exerted (e.g., urea is an ineffective osmole for red blood cells). In contrast, σ = 1 for a solute that cannot cross the cell membrane (in the preceding example, sucrose). Such a substance is said to be an effective osmole. Many molecules are neither completely able nor completely unable to cross cell membranes (i.e., 0 < σ < 1) and generate an osmotic pressure that is only a fraction of what is expected from the molecules’ concentration in solution.

Oncotic Pressure Oncotic pressure is the osmotic pressure generated by large molecules (especially proteins) in solution. As illustrated in Fig. 1.9, the magnitude of the osmotic pressure generated by a solution of protein does not conform to van’t Hoff’s law. The cause of this anomalous relationship between protein concentration and osmotic pressure is not completely understood, but it appears to be related to the size and shape of the protein molecule. For example, the correlation with van’t Hoff’s law is more precise with small, globular proteins than with larger protein molecules.

FIG. 1.9 Relationship between the concentration of plasma proteins in solution and the osmotic pressure (oncotic pressure) they generate. Protein concentration is expressed in grams per deciliter. Normal plasma protein concentration is indicated. Note how the actual pressure generated exceeds that predicted by van’t Hoff’s law.

The oncotic pressure exerted by proteins in human plasma has a normal value of approximately 26 to 28 mm Hg. Although this pressure appears to be small in relation to osmotic pressure (28 mm Hg 1.4 mOsm/kg H2O), it is an important force involved in fluid movement across capillaries (see Chapter 17).

Specific Gravity The total concentration of all molecules in a solution can also be measured as specific gravity. Specific gravity is defined as the weight of a volume of solution divided by the weight of an equal volume of distilled water. Thus the specific gravity of distilled water is 1. Because biological fluids contain a number of different molecules, their specific gravities are greater than 1. For example, normal human plasma has a specific gravity in the range of 1.008 to 1.010.

IN THE C LIN IC The specific gravity of urine is sometimes measured in clinical settings and used to assess the urineconcentrating ability of the kidneys. The specific gravity of urine varies in proportion to its osmolality. However, because specific gravity depends both on the number of molecules and on their weight, the relationship between specific gravity and osmolality is not always predictable. For example, in patients who have received an injection of radiocontrast dye (molecular weight > 500 g/mole) for xray studies, values of urine specific gravity can be high (1.040 to 1.050), even though the urine osmolality is similar to that of plasma (e.g., 300 mOsm/kg H2O).

Key Points • The plasma membrane is a lipid bilayer composed of phospholipids and cholesterol, into which are embedded a wide range of proteins. One class of these membrane proteins (membrane transport proteins or transporters) is involved in the selective and regulated transport of molecules into and out of the cell. These transporters include water channels (aquaporins), ion channels, solute carriers, and ATP-dependent transporters. • The movement of molecules across the plasma membrane through ion channels and solute carriers is driven by chemical concentration gradients and electrical potential differences (charged molecules only). The electrochemical gradient is used to quantitate this driving force. ATPdependent transporters use the energy in ATP to transport molecules across the membrane and often establish the chemical and electrical gradients that then drive the transport of other molecules through channels and by the solute carriers. Water movement through aquaporins is driven by an osmotic pressure difference across the membrane. • Transport across the membrane is classified as passive or active. Passive transport is the movement of molecules as expected from the electrochemical gradient for that molecule. Active transport represents transport against the electrochemical gradient. Active transport is further divided into primary active and secondary active transport. Primary active transport is directly coupled to the hydrolysis of ATP (e.g., ATP-dependent transporters). Secondary active transport occurs with coupled solute carriers, for which passive movement of one or more molecules drives the active transport of other molecules (e.g., Na+-glucose symporter, Na+-H+ antiporter). a

Another type of ATPases, F-type ATPases, is found in the mitochondria, and they are responsible for ATP synthesis. They are not considered in this chapter. b

By convention, membrane voltages are determined and reported with regard to the exterior of the cell. In a typical cell, the resting membrane potential (Vm) is negative. Positive Vm values can be observed in some excitable cells at the peak of an action potential. c

NaCl does not completely dissociate in water. The value for n is 1.88 rather than 2. However, for simplicity, the value of 2 is most often used.

C H AP T E R 2

Homeostasis: Volume and Composition of Body Fluid Compartments LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. What is steady-state balance, and, with water balance as an example, what are the elements needed to achieve steady-state balance? 2. What are the volumes of the body fluid compartments, and how do they change under various conditions? 3. How do the body fluid compartments differ with regard to their composition? 4. What determines the resting membrane potential of cells? 5. How do cells regulate their volume in isotonic, hypotonic, and hypertonic solutions? 6. What are the structural features of epithelial cells, how do they carry out vectorial transport, and what are the general mechanisms by which transport is regulated?

Normal cellular function requires that the intracellular composition—with regard to ions, small molecules, water, pH, and a host of other substances—be maintained within a narrow range. This is accomplished by the transport of many substances and water into and out of the cell via membrane transport proteins, as described in Chapter 1. In addition, each day, food and water are ingested, and waste products are excreted from the body. In a healthy individual, these processes occur without significant changes in either the volume of the body fluid compartments or their composition. The maintenance of constant volume and composition of the body fluid compartments (and their temperature in warm-blooded animals and humans) is termed homeostasis. The human body has multiple systems designed to achieve homeostasis, the details of which are explained in the various chapters of this book. In this chapter, the basic principles that underlie the maintenance of homeostasis are outlined. In addition, the volume and composition of the various body fluid compartments are defined.

Concept of Steady-State Balance The human body is an “open system,” which means that substances are added to the body each day and, similarly, substances are lost from the body each day. The amounts added to or lost from the body can vary widely, depending on the environment, access to food and water, disease processes, and even cultural norms. In such an open system, homeostasis occurs through the process of steady-state balance.

To illustrate the concept of steady-state balance, consider a river on which a dam is built to create a man-made lake. Each day, water enters the lake from the various streams and rivers that feed it. In addition, water is added by underground springs, rain, and snow. At the same time, water is lost through the spillways of the dam and by the process of evaporation. For the level of the lake to remain constant (i.e., steady-state balance), the rate at which water is added, regardless of source, must be exactly matched by the amount of water lost, again regardless of route. Because the addition of water is not easily controlled and the loss by evaporation cannot be controlled, the only way to maintain a constant level of the lake is to regulate the amount that is lost through the spillways. To understand steady-state balance as it applies to the human body, the following key concepts are important. 1. There must be a “set point” so that deviations from this baseline can be monitored (e.g., the level of the lake in the preceding example, or setting the temperature in a room by adjusting the thermostat). 2. The sensor or sensors that monitor deviations from the set point must generate “effector signals” that can lead to changes in either input or output, or both, to maintain the desired set point (e.g., electrical signals to adjust the spillway in the dam analogy, or electrical signals sent to either the furnace or air conditioner to maintain the proper room temperature). 3. “Effector organs” must respond in an appropriate way to the effector signals generated by the set point monitor (i.e., the spillway gates must operate, and the furnace or air conditioner must turn on). 4. The sensitivity of the system (i.e., how much of a deviation from the set point is tolerated) depends on several factors, including the nature of the sensor (i.e., how much of a deviation from the set point is needed for the sensor to detect the deviation), the time necessary for generation of the effector signals, and how rapidly the effector organs respond to the effector signals. It is important to recognize that deviations from steady-state balance do occur. When input is greater than output, a state of positive balance exists. When input is less than output, a state of negative balance exists. Although transient periods of imbalance can be tolerated, prolonged states of positive or negative balance are generally incompatible with life. Fig. 2.1 illustrates several important concepts for the maintenance of steady-state water balance (details related to the maintenance of steady-state water balance are presented in Chapter 35). As depicted in Fig. 2.1, there are multiple inputs and outputs of water, many of which can vary but nevertheless cannot be regulated. For example, the amount of water lost through the lungs depends on the humidity of the air and the rate of respiration (e.g., low humidity and rapid breathing increase water loss from the lungs). Similarly, the amount of water lost as sweat varies according to ambient temperature and physical activity. Finally, water loss via the gastrointestinal tract can increase from a normal level of 100 to 200 mL/day to many liters with acute diarrhea. Of these inputs and outputs, the only two that can be regulated are increased ingestion of water in response to thirst and alterations in urine output by the kidneys (see Chapter 35).

FIG. 2.1 Whole-body steady-state water balance. See text for details. ADH, Antidiuretic ​hormone (also called arginine vasopressin); CNS, central nervous system; GI, gastrointestinal.

Water balance determines the osmolality of the body fluids. Cells within the hypothalamus of the brain monitor body fluid osmolality for deviations from the set point (normal range: 280–295 mOsm/kg H2O). When deviations are sensed, two effector signals are generated. One is neural and relates to the individual’s sensation of thirst. The other is hormonal (antidiuretic hormone, also called arginine vasopressin), which regulates the amount of water excreted by the kidneys. With appropriate responses to these two signals, water input, water output, or both are adjusted to maintain balance and thereby keep body fluid osmolality at the set point.

Volumes and Composition of Body Fluid Compartments Unicellular organisms maintain their volume and composition through exchanges with the environment they inhabit (e.g., sea water). The billions of cells that constitute the human body must maintain their volume and composition as well, but their task is much more difficult. This challenge, as well as its solution, was first articulated by the French physiologist Claude Bernard (1813–1878). He recognized that although cells within the body cannot maintain their volume and composition through exchanges with the environment, they can do so through exchanges with the fluid environment that surrounds them (i.e., the extracellular fluid). Bernard referred to the extracellular fluid as the milieu intérieur (“the environment within”). He also recognized that the organ systems of the body are designed and function to maintain a constant milieu interieur or a “constant internal environment.” This in turn allows all cells to maintain their volume and composition through exchanges with the extracellular fluid as a result of membrane transport (see Chapter 1). Transport by the epithelial cells of the gastrointestinal tract, kidneys, and lungs is the body’s interface with the external environment and control both the intake and excretion of numerous substances, as well

as water. The cardiovascular system delivers nutrients to and removes waste products from the cells and tissues and keeps the extracellular fluid well mixed. Finally, the nervous and endocrine systems provide regulation and integration of these important functions. To provide background for the study of all organ systems, this chapter presents an overview of the normal volume and composition of the body fluid compartments and describes how cells maintain their intracellular composition and volume. Included is a presentation on how cells generate and maintain a membrane potential, which is fundamental for understanding the function of excitable cells (e.g., neurons and muscle cells). Finally, because epithelial cells are so central to the process of regulating the volume and composition of the body fluids, the principles of solute and water transport by epithelial cells are also reviewed.

Definition and Volumes of Body Fluid Compartments Water makes up approximately 60% of the body’s weight; variability among individuals is a function of the amount of adipose tissue. Because the water content of adipose tissue is lower than that of other tissue, increased amounts of adipose tissue reduce the fraction of water in the total body as a percentage of weight. The percentage of body weight attributed to water also varies with age. In newborns, it is approximately 75%. This decreases to the adult value of 60% by the age of 1 year. As illustrated in Fig. 2.2, total body water is distributed between two major compartments, which are divided by the cell membrane.a The intracellular fluid (ICF) compartment is the larger compartment and contains approximately two-thirds of the total body water. The remaining third is contained in the extracellular fluid (ECF) compartment. The volumes of total body water, ICF, and ECF in liters are calculated as follows:

FIG. 2.2 Relationship between the volumes of the various body fluid compartments. The actual values shown are for an individual weighing 70 kg. (Modified from Levy MN, Koeppen BM, Stanton BA. Berne & Levy’s Principles of Physiology. 4th ed. St. Louis: Mosby; 2006.)

The ECF compartment is further subdivided into interstitial fluid and plasma. The ECF also includes fluid contained within bone and dense connective tissue, as well as the cerebrospinal fluid. The interstitial fluid surrounds the cells in the various tissues of the body and makes up three-fourths of the ECF volume. Plasma is contained within the vascular compartment and represents the remaining fourth of the ECF. In some pathological conditions, additional fluid may accumulate in what is referred to as a third space. Third-space collections of fluid are part of the ECF; an example is the accumulation of fluid in the peritoneal cavity (ascites) of individuals with liver disease.

Movement of Water Between Body Fluid Compartments As depicted in Fig. 2.2, water moves between the ICF and ECF compartments across the plasma membranes of cells, and it moves between the vascular (plasma) and interstitial compartments across capillary walls. The pathways and driving forces for this water movement are different across cell membranes, in comparison to the capillary walls. Movement of water between the ICF and ECF compartments, across cell membranes, occurs through aquaporins expressed in the plasma membrane (see Chapter 1). The driving force for this water

movement is an osmotic pressure difference. The osmotic pressure of both the ICF and ECF is determined by the molecules/ions present in these fluids. For simplicity, these can be divided into (1) molecules of low molecular weight (e.g., glucose) and ions (e.g., Na+) and (2) macromolecules (e.g., proteins). The osmotic pressures of both the ICF and ECF are in the range of 280 to 295 mOsm/kg H2O. For the ECF, the low-molecular-weight molecules and ions account for nearly all of this pressure because the osmotic pressure contributed by proteins is only 1 to 2 mOsm/kg H2O. The molecules/ions contributing to the osmotic pressure within the cell are less well understood, but they also include low-molecular-weight molecules (e.g., glucose), ions (e.g., Na+), and macromolecules (e.g., proteins). The fact that cell volume remains constant when ECF osmolality is constant means that the osmotic pressure inside the cells is equal to that of the ECF. If an osmotic pressure difference did exist, the cells would either swell or shrink, as described in the section “Nonisotonic Cell Volume Regulation.” Movement of water between the vascular (plasma) compartment and the interstitial fluid compartment occurs across the capillary wall. The amount of water that moves across the capillary wall and the mechanism of the water movement vary depending on the capillary. For example, in the capillary sinusoids of the liver, endothelial cells are often separated by large gaps (discontinuous capillary). As a result, water and all components of the plasma (and some cellular elements) can pass easily across the wall. Other capillaries are lined by endothelial cells that contain fene​strations that are up to 80 to 100 nm in diameter (e.g., in the kidneys). These fenestrations allow all components of the plasma (only cellular elements of blood cannot pass through the fenestrations) to move across the capillary wall. Some capillaries (e.g., in the brain) form a relatively tight barrier to water and small molecules and ions, and water movement occurs through small pores on the endothelial cell surface or through clefts between adjacent endothelial cells. These pores and clefts allow water and molecules smaller than 4 nm to pass. In addition, a small amount of water traverses the capillary wall via pinocytosis by endothelial cells. The driving forces for fluid (water) movement across the capillary wall are hydrostatic pressure and oncotic pressure (i.e., osmotic pressure generated by proteins). Collectively, these are called the Starling forces. Capillary fluid movement is discussed in detail in Chapter 17; in brief, hydrostatic pressure within the capillary (as a result of the pumping of the heart and the effect of gravity on the column of blood in the vessels feeding a capillary) is a force that causes fluid to move out of the capillary. Hydrostatic pressure in the surrounding interstitial tissue opposes the effect of the capillary hydrostatic pressure. The oncotic pressure of the plasma in the capillary tends to draw fluid from the interstitium into the capillary. The oncotic pressure of the interstitial fluid opposes this. Depending on the capillary bed, proteins can cross the capillary wall to varying degrees. For example, very little protein crosses the wall of skeletal muscle capillaries and the capillaries in the glomerulus of the kidneys. In contrast, proteins readily cross the wall of the liver capillaries (i.e., sinusoids). The degree to which proteins cross the capillary wall is quantitated by a reflection coefficient (σ). If no protein crosses the capillary wall, σ = 1, and if proteins freely cross the capillary wall, σ = 0. Thus the amount of fluid moving across the wall of the capillary is determined as follows:

(Equation 2.1) where

Qf = fluid movement Kf = filtration constant (measure of surface area + intrinsic permeability) Pc = capillary hydrostatic pressure Pi = interstitial fluid hydrostatic pressure πc = capillary (plasma) oncotic pressure πi = interstitial fluid oncotic pressure σ = reflection coefficient for protein across the capillary wall. Depending on the magnitude of these forces, fluid may move out of the capillary or into the capillary. The compositions of the various body fluid compartments differ; however, as described later, the osmolalities of the fluid within these compartments are essentially identical.b Thus the compartments are in “osmotic equilibrium.” In addition, any change in the osmolality of one compartment quickly causes water to redistribute across all compartments, which brings them back into osmotic equilibrium. Because of this rapid redistribution of water, measuring the osmolality of plasma or serum,c which is easy to do, reveals the osmolality of the other body fluid compartments (i.e., interstitial fluid and intracellular fluid). As described later, Na+ is a major constituent of the ECF. Because of its high concentration in comparison with other molecules and ions, Na+ (and its attendant anions, primarily Cl− and HCO3−) is the major determinant of the osmolality of this compartment. Accordingly, it is possible to obtain an approximate estimate of the ECF osmolality by simply doubling the sodium concentration [Na+]. For example, if a blood sample is obtained from an individual, and the [Na+] of the serum is 145 mEq/L, its osmolality can be estimated as follows:

(Equation 2.2)

IN THE C LIN IC In some clinical situations, it is possible to obtain a more accurate estimate of the serum osmolality, and thus the osmolalities of the ECF and ICF, by also considering the osmoles contributed by glucose and urea, as these are the next most abundant solutes in the ECF (the other components of the ECF contribute only a few additional milliosmoles). Accordingly, serum osmolality can be estimated as follows:

The glucose and urea concentrations are expressed in units of milligrams per deciliter (dividing by 18 for glucose and 2.8 for urea* allows conversion from the units of milligrams per deciliter to millimoles

per liter and thus to milliosmoles per kilogram of H2O). This estimation of serum osmolality is especially useful in treating patients who have an elevated serum glucose concentration secondary to diabetes mellitus, and in patients with chronic renal failure, whose serum urea concentration is elevated because of reduced renal excretion. As discussed in Chapter 1, the ability of a substance to cause water to move across the plasma membrane of a cell depends on whether the substance itself crosses the membrane. Recall Eq. 1.9:

where Πε = the effective osmotic pressure and σ = the reflection coefficient for the substance. For many cells, glucose and urea cross the cell membrane. Although they contribute to serum osmolality, as measured by a laboratory osmometer where all molecules are “effective osmoles,” they are ineffective osmoles for water movement across many, but not all, cell membranes. In contrast, Na+ is an “effective osmole” for water movement across the plasma membrane of virtually all cells. Eq. 2.2 gives the best estimate of the effective osmolality of the serum. *The urea concentration in plasma is measured as the nitrogen in the urea molecule, or blood urea nitrogen (BUN).

In contrast to water, the movement of ions across cell membranes is more variable from cell to cell and depends on the presence of specific membrane transport proteins (see the section “Composition of BoDy Fluid Compartments”). Consequently, in trying to understand the physiology of fluid shifts between body fluid compartments, it can be assumed that while water moves freely between the compartments, there is little net movement of solutes. For most situations, this is a reasonable assumption. To illustrate the physiologic characteristics of fluid shifts, consider what happens when solutions containing various amounts of NaCl are added to the ECF.d Example 1: Addition of Isotonic Sodium Chloride to the Extracellular Fluid Addition of an isotonic NaCl solution (e.g., intravenous infusion of 0.9% NaCl: osmolality ≈ 290 mOsm/kg H2O)e to the ECF increases the volume of this compartment by the volume of fluid administered. Because this fluid has the same osmolality as does the ECF, and therefore the ICF, there is no driving force for fluid movement between these compartments, and the volume of the ICF remains unchanged. Although Na+ can cross cell membranes, it is effectively restricted to the ECF by the activity of the Na+,K+-ATPase, which is present in the plasma membrane of all cells (see the section “Ionic Composition of Cells”). Therefore, there is no net movement of the infused isotonic NaCl solution into cells.

IN THE C LIN IC Neurosurgical procedures and cerebrovascular accidents (strokes) often result in the accumulation of interstitial fluid in the brain (i.e., edema) and swelling of the neurons. Because the brain is enclosed

within the skull, edema can raise intracranial pressure and thereby disrupt neuronal function, which leads to coma and death. The blood-brain barrier, which separates the cerebrospinal fluid and brain interstitial fluid from blood, can be permeated freely by water but not by most other substances. As a result, excess fluid in brain tissue can be removed by imposing an osmotic gradient across the bloodbrain barrier. Mannitol can be used for this purpose. Mannitol is a sugar (molecular weight, 182 g/mol) that does not readily cross the blood-brain barrier and membranes of cells (neurons and other cells in the body). Therefore, mannitol is an effective osmole, and intravenous infusion results in the movement of interstitial fluid out of the brain by osmosis. Example 2: Addition of Hypotonic Sodium Chloride to the Extracellular Fluid Addition of a hypotonic NaCl solution to the ECF (e.g., intravenous infusion of 0.45% NaCl; osmolality 145 mOsm/kg H2O) decreases the osmolality of this fluid compartment, which results in the movement of water into the ICF. After osmotic equilibration, the osmolalities of the ICF and ECF are again equal but lower than before the infusion, and the volume of each compartment is increased. The increase in ECF volume is greater than the increase in ICF volume. Example 3: Addition of Hypertonic Sodium Chloride to the Extracellular Fluid Addition of a hypertonic NaCl solution to the ECF (e.g., intravenous infusion of 3% NaCl: osmolality 1000 mOsm/kg H2O) increases the osmolality of this compartment, which results in the movement of water out of cells. After osmotic equilibration, the osmolalities of the ECF and ICF are again equal but higher than before the infusion. The volume of the ECF is increased, whereas that of the ICF is decreased.

IN THE C LIN IC Fluid and electrolyte disorders are observed commonly in clinical practice (e.g., in patients with vomiting or diarrhea, or both). In most instances, these disorders are self-limited, and correction of the disorder occurs without need for intervention. However, more severe or prolonged disorders may necessitate fluid replacement therapy. Such therapy may be administered orally, with special electrolyte solutions, or intravenously. Intravenous solutions are available in many formulations. The type of fluid administered to a particular patient is dictated by the patient’s need. For example, if an increase in the patient’s vascular volume is necessary, a solution containing substances that do not readily cross the capillary wall is infused (e.g., 5% protein or dextran solutions). The oncotic pressure generated by the albumin molecules causes fluid to be retained in the vascular compartment, which expands its volume. Expansion of the ECF is accomplished most often with isotonic saline solutions (e.g., 0.9% NaCl or lactated Ringer solution). As already noted, administration of an isotonic NaCl solution does not result in the development of an osmotic pressure gradient across the plasma membrane of cells. Therefore, the entire volume of the infused solution remains in the ECF. Patients whose body fluids are hyperosmotic need hypotonic solutions. These solutions may be hypotonic NaCl (e.g., 0.45% NaCl) or 5% dextrose in water (D5W). Administration of the D5W solution is equivalent to the infusion of distilled water because the dextrose is metabolized to CO2 and water. Administration of these fluids increases the volumes of both the ICF and ECF. In addition, patients whose body fluids are hypotonic need hypertonic solutions. These are typically NaClcontaining solutions (e.g., 3% or 5% NaCl). These solutions expand the volume of the ECF but

decrease the volume of the ICF. Other constituents, such as electrolytes (e.g., K+) or drugs, can be added to intravenous solutions to tailor the therapy to the patient’s fluid, electrolyte, and metabolic needs.

Composition of Body Fluid Compartments The compositions of the ECF and ICF differ considerably. The ICF has significantly more proteins and macromolecules than the ECF. There are also differences in the concentrations of many ions. The composition of the ICF is maintained by the action of a number of specific cell membrane transport proteins. Principal among these transporters is the Na+,K+-adenosine triphosphatase (Na+,K+-ATPase), which converts the energy in ATP into ion and electrical gradients, which can in turn be used to drive the transport of other ions and molecules by means of ion channels and solute carriers (e.g., symporters and antiporters). The compositions of the plasma and interstitial fluid compartments of the ECF are similar because those compartments are separated only by the capillary endothelium, a barrier that ions and small molecules can permeate. The major difference between the interstitial fluid and plasma is that the latter contains significantly more protein. Although this differential concentration of protein can affect the distribution of cations and anions between these two compartments by the Gibbs-Donnan effect (see the section “Isotonic Cell Volume Regulation” for details), this effect is small, and the ionic compositions of the interstitial fluid and plasma can be considered to be identical.

Maintenance of Cellular Homeostasis Normal cellular function requires that the ionic composition of the ICF be tightly controlled. For example, the activity of some enzymes is pH dependent; therefore, intracellular pH must be regulated. In addition, the intracellular composition of other electrolytes is similarly held within a narrow range. This is necessary for the establishment of the membrane potential, a cell property especially important for the normal function of excitable cells (e.g., neurons and muscle cells) and for intracellular signaling (e.g., intracellular [Ca++]; see Chapter 3 for details). Finally, the volume of cells must be maintained because shrinking or swelling of cells can lead to cell damage or death. The regulation of intracellular composition and cell volume is accomplished through the activity of specific transporters in the plasma membrane of the cells. This section is a review of the mechanisms by which cells maintain their intracellular ionic environment and their membrane potential and by which they control their volume.

Ionic Composition of Cells The intracellular ionic composition of cells varies from tissue to tissue. For example, the intracellular composition of neurons is different from that of muscle cells, both of which differ from that of blood cells. Nevertheless, there are similar patterns, and these are presented in Table 2.1. In comparison with the ECF, the ICF is characterized by a low [Na+] and a high [K+]. This is the result of the activity of the Na+,K+-ATPase, which transports three Na+ ions out of the cell and two K+ ions into the cell for each ATP molecule hydrolyzed. As discussed later in this chapter, the activity of the Na+,K+-ATPase not only is important for establishing the cellular Na+ and K+ gradients but also is involved in determining, indirectly, the cellular gradients for many other ions and molecules. Of importance is that the cellular K+

gradient generated by the activity of the Na+,K+-ATPase is a major determinant of the membrane voltage because of the leak of K+ out of the cell through K+-selective channels (see the section “Membrane Potential”). Thus the Na+,K+-ATPase converts the energy in ATP into ion gradients (i.e., Na+ and K+) and a voltage gradient (i.e., membrane voltage). Table 2.1 Ionic Composition of a Typical Cell Ion

Extracellular Fluid

Intracellular Fluid

Na+

135–147 mEq/L

10–15 mEq/L

K+

3.5–5.0 mEq/L

120–150 mEq/L

Cl−

95–105 mEq/L

20–30 mEq/L

HCO3−

22–28 mEq/L

12–16 mEq/L

*Ca++

2.1–2.8 (total) mmol/L





1.1–1.4 (ionized) mmol/L

≈10−7 M (ionized) mmol/L

*Pi

1.0–1.4 (total) mmol/L





0.5–0.7 (ionized) mmol/L

0.5–0.7 (ionized) mmol/L

*Ca++ and Pi (H2PO4−/HPO4−2) are bound to proteins and other organic molecules. In addition, large amounts of Ca++ can be sequestered within cells. Large amounts of Pi are present in cells as part of organic molecules, such as adenosine triphosphate (ATP).

The Na+,K+-ATPase–generated ion and electrical gradients are used to drive the transport of other ions and molecules into or out of the cell (Fig. 2.3). For example, as described in Chapter 1, a number of solute carriers couple the transport of Na+ to that of other ions or molecules. The Na+-glucose and Na+– amino acid symporters use the energy in the Na+ electrochemical gradient, directed to bring Na+ into the cell, to drive the secondary active cellular uptake of glucose and amino acids. Similarly, the inwardly directed Na+ gradient drives the secondary active extrusion of H+ from the cell and thus contributes to the maintenance of intracellular pH. The 3Na+-Ca++ antiporter, along with the plasma membrane Ca++ATPase, extrudes Ca++ from the cell and thus contributes to the maintenance of a low intracellular [Ca++].f In addition, the membrane voltage drives Cl− out of the cell through Cl−-selective channels, thus lowering the intracellular concentration below that of the ECF.

FIG. 2.3 Cell model depicting how cellular gradients and the membrane potential (Vm ) are established. (1) The Na+,K+-ATPase decreases the intracellular [Na+] and increases the intracellular [K+]. Some K+ exits the cell via K+-selective channels and generates the Vm (cell’s interior is electrically negative). (2) The energy in the Na+ electrochemical gradient drives the transport of other ions and molecules through the use of various solute carriers. (3) The Vm drives Cl− out of the cell via Cl−-selective channels. (4) The Ca++-ATPase and the 3Na+-Ca++ antiporters maintain the low intracellular [Ca++]. ATP, Adenosine triphosphate.

Membrane Potential As described previously, the Na+,K+-ATPase and K+-selective channels in the plasma membrane are important determinants of the membrane potential (Vm) of the cell. For all cells within the body, the resting Vm is oriented with the interior of the cell electrically negative in relation to the ECF. However, the magnitude of the Vm can vary widely. To understand what determines the magnitude of the Vm, it is important to recognize that any transporter that transfers charge across the membrane has the potential to influence the Vm. Such transporters are said to be electrogenic. As might be expected, the contribution of various electrogenic transporters to the Vm is highly variable from cell to cell. For example, the Na+,K+-ATPase transports three Na+ and two K+ ions and thus transfers one net positive charge across the membrane. However, the direct contribution of the Na+,K+-ATPase to the Vm of most cells is only a few millivolts at the most. Similarly, the contribution of other electrogenic transporters, such as the 3Na+-Ca++ antiporter and the Na+-glucose symporter, is minimal. The major determinants of the Vm are ion channels. The type (e.g., selectivity), number, and activity (e.g., gating) of these channels determine the magnitude of the Vm. As described in Chapter 5, rapid changes in ion channel activity underly the action potential in neurons and other excitable cells, such as those of skeletal and cardiac muscle (see Chapters 12 and 13). As ions move across the membrane through a channel, they generate a current. As described in Chapter 1, this current can be measured, even at the level of a single channel. By convention, the current generated by the movement of cations into the cell, or the movement of anions out of the cell, is defined as negative

current. Conversely, the movement of cations out of the cell, or the movement of anions into the cell, is defined as positive current. Also by convention, the magnitude of the Vm is expressed in relation to the outside of the cell; thus for a cell with a Vm of −80 mV, the interior of the cell is electrically negative in relation to the outside of the cell. The current carried by ions moving through a channel depends on the driving force for that ion and on the conductance of the channel. As described in Chapter 1, the driving force is determined by the energy in the concentration gradient for the ion across the membrane (Ei), as calculated by the Nernst equation (Eq. 1.5a) and the Vm:

(Equation 2.3) Thus as defined by Ohm’s law, the ion current through the channel (Ii) is determined as follows:

(Equation 2.4) where gi is the conductance of the channel. For a cell, the conductance of the membrane to a particular ion (Gi) is determined by the number of ion channels in the membrane and by the amount of time each channel is in the open state. As illustrated in Fig. 2.4, the Vm is the voltage at which there is no net ion flow into or out of the cell. Thus for a cell that has ion channels selective for Na+, K+, and Cl−,

(Equation 2.5) or

(Equation 2.6)

FIG. 2.4 Current-voltage relationship of a hypothetical cell containing Na+-, K+-, and Cl−-selective channels. Membrane currents are plotted over a range of membrane voltages (i.e., current-voltage relationships). Each ion current is calculated with the use of Ohm’s law, the Nernst equilibrium potential for the ion (ECl, EK, and ENa), and the membrane conductance for the ion. The current-voltage relationship for the whole cell is also shown. Total cell current (Icell) was calculated with the chord conductance equation (see Eq. 2.7). Because 80% of cell conductance is due to K+, the resting membrane voltage (Vm ) of −64.4 mV is near to that of the Nernst equilibrium potential for K+.

Solving for Vm yields

(Equation 2.7)

where . Inspection of Eq. 2.7, which is often called the chord conductance equation, reveals that the Vm will be near to the Nernst equilibrium potential of the ion to which the membrane has the highest conductance. In Fig. 2.4, 80% of the membrane conductance is attributable to K+; as a result, Vm is near to the Nernst equilibrium potential for K+ ( and thus the Vm approximates

). For most cells at rest, the membrane has a high conductance to K+, . Moreover, the Vm is greatly influenced by the magnitude of

,

which in turn is greatly influenced by changes in the [K+] of the ECF. For example, if the intracellular [K+] is 120 mEq/L and the extracellular [K+] is 4 mEq/L, extracellular [K+] is increased to 7 mEq/L,

has a value of −90.8 mV. If the

would be −79.9 mV. This change in

depolarizes

the Vm (i.e., Vm is less negative). Conversely, if the extracellular [K+] is decreased to 2 mEq/L, becomes −109.4 mV, and the Vm hyperpolarizes (i.e., Vm is more negative).

IN THE C LIN IC Changes in the extracellular [K+] can have important effects on excitable cells, especially those of the heart. A decrease in extracellular [K+] (hypokalemia) hyperpolarizes the Vm of cardiac myocytes and, in so doing, makes initiating an action potential more difficult, because a larger depolarizing current is needed to reach the threshold potential (see Chapter 16). If severe, hypokalemia can lead to cardiac arrhythmias, and eventually the heart can stop contracting (asystole). An increase in the extracellular [K+] (hyperkalemia) can be equally deleterious to cardiac function. With hyperkalemia, the Vm is depolarized, and it is easier to initiate an action potential. However, once the action potential fires the channels become inactivated, and are unable to initiate another action potential, until they are reactivated by normal repolarization of the Vm. Because the Vm is depolarized in hyperkalemia, the channels stay in an inactivated state. Thus depolarization of the Vm with hyperkalemia can lead to cardiac arrhythmias and loss of cardiac muscle contraction. Eq. 2.7 also defines the limits for the membrane potential. In the example depicted in Fig. 2.4, it is apparent that the Vm cannot be more negative than

(−90.8 mV), as would be the case if the membrane

were only conductive to K+. Conversely, the Vm could not be more positive than (66.6 mV); such a condition would be met if the membrane were conductive only to Na+. The dependence of the Vm on the conductance of the membrane to specific ions is the basis by which action potentials in excitable cells are generated (Fig. 2.5). As noted previously, in all excitable cells, the membrane at rest is conductive predominantly to K+, and thus Vm is near

. When an action potential is initiated, Na+-channels open

and the membrane is now conductive predominantly to Na+. As a result, Vm now approaches generation of action potentials is discussed in more detail in Chapter 5.

The

FIG. 2.5 Nerve action potential showing the changes in Na+ and K+ conductances ( and , + Respectively) and the membrane potential (Vm ). At rest, the membrane has a high K conductance, and Vm is near the Nernst equilibrium potential for K+ (

). With the initiation of the action potential, there is a

large increase in the Na+ conductance of the membrane, and the Vm approaches the Nernst equilibrium potential for Na+ (

). The increase in Na+ conductance is transient, and the K+ conductance then

increases above its value before the action potential. This hyperpolarizes the cell as Vm approaches

.

+

As the K conductance returns to its baseline value, Vm returns to its resting value of −70 mV. (Modified from Levy MN, Koeppen BM, Stanton BA. Berne & Levy’s Principles of Physiology. 4th ed. St. Louis: Mosby; 2006.)

Regulation of Cell Volume As already noted, changes in cell volume can lead to cell damage and death. Cells have developed mechanisms to regulate their volume. Most cells are highly permeable to water because of the presence of aquaporins in their plasma membranes. As discussed in Chapter 1, osmotic pressure gradients across the cell membrane that are generated by effective osmoles cause water to move either into or out of the cell, which result in changes in cell volume. Thus cells swell when placed in hypotonic solutions and shrink when placed in hypertonic solutions (see the section “Nonisotonic Cell Volume Regulation”). However, even when a cell is placed in an isotonic solution, the maintenance of cell volume is an active process requiring the expenditure of ATP and specifically the activity of the Na+,K+-ATPase. Isotonic Cell Volume Regulation The importance of the Na+,K+-ATPase in isotonic cell volume regulation can be appreciated by the observation that red blood cells swell when chilled (i.e., reduced ATP synthesis) or when the Na+,K+ATPase is inhibited by cardiac glycosides (e.g., ouabain, digoxin [Lanoxin]). The necessity for energy expenditure to maintain cell volume in an isotonic solution is the result of the effect of intracellular proteins on the distribution of ions across the plasma membrane: the so-called Gibbs-Donnan effect (Fig. 2.6).

FIG. 2.6 The Gibbs-Donnan effect. Top, Two solutions are separated by a membrane that is permeable by Na+, Cl−, and H2O but not permeable by protein (P−). The osmolality of solution A is identical to that of solution B. Bottom, Cl− diffuses from compartment B to compartment A down its concentration gradient. This causes compartment A to become electrically negative with regard to compartment B. The membrane voltage then drives the diffusion of Na+ from compartment B to compartment A. The accumulation of additional Na+ and Cl− in compartment A increases its osmolality and causes water to flow from compartment B to compartment A (Note: the increase volume of compartment A results in a lower [P−]). If the container containing the two solutions were sealed at the top so that water could not move from compartment B to compartment A, the pressure in compartment A would increase as the number of osmotically active particles increases in that compartment.

The Gibbs-Donnan effect occurs when a membrane separating two solutions can be permeated by some but not all of the molecules in solution. As noted previously, this effect accounts for the small differences in the ionic compositions of the plasma and the interstitial fluid. In this case, the capillary endothelium represents the membrane, and the plasma proteins are the molecules whose ability to permeate across the capillary is restricted. For cells, the membrane is the plasma membrane, and the impermeant molecules are the intracellular proteins and organic molecules. As depicted in Fig. 2.6, the presence of impermeant molecules (e.g., protein) in one compartment results over time in the accumulation of permeant molecules/ions in the same compartment. This increases the number of osmotically active particles in the compartment containing the impermeant anions, which in turn increases the osmotic pressure, and water thereby enters that compartment. For cells, the GibbsDonnan effect would increase the number of osmotically active particles in the cell and result in cell swelling. However, the activity of the Na+,K+-ATPase counteracts the Gibbs-Donnan effect by actively extruding cations (three Na+ ions are extruded, whereas two K+ ions are brought into the cell). In addition, the K+ gradient established by the Na+,K+-ATPase allows for the development of the Vm (in which the cell’s interior is electrically negative), that in turn drives Cl− and other anions out of the cell. Thus through the activity of the Na+,K+-ATPase, the number of intracellular osmotically active particles is reduced from what would be caused by the Gibbs-Donnan effect, and cell volume is maintained in isotonic solutions.

Nonisotonic Cell Volume Regulation Most cells throughout the body are bathed with isotonic ECF, the composition of which is tightly regulated (see Chapter 35). However, certain regions within the body are not isotonic (e.g., the medulla of the kidney), and with disorders of water balance, the ECF can become either hypotonic or hypertonic. When this occurs, cells either swell or shrink. Cell swelling or shrinkage can result in cell damage or death, but many cells have mechanisms that limit the degree to which the cell volume changes. These mechanisms are particularly important for neurons, in which swelling within the confined space of the skull can lead to serious neurological damage. In general, when a cell is exposed to nonisotonic ECF, volume-regulatory responses are activated within seconds to minutes to restore cell volume (Fig. 2.7). With cell swelling, a regulatory volume decrease response transports osmotically active particles (osmolytes) out of the cell, reducing the intracellular osmotic pressure and thereby restoring cell volume to normal. Conversely with cell shrinking a regulatory volume increase response transports osmolytes into the cell, raising the intracellular osmotic pressure and thereby restoring cell volume to normal. These osmolytes include ions and organic molecules such as polyols (sorbitol and myo-inositol), methylamines (glycerophosphorylcholine and betaine), and some amino acids (taurine, glutamate, and β-alanine). If the cell is exposed to the nonisotonic ECF for an extended period of time, the cell alters the intracellular levels of the organic osmolytes through metabolic processes.

FIG. 2.7 Volume regulation of cells in hypotonic and hypertonic media. Top, When cells are exposed to a hypotonic medium, they swell and then undergo a volume-regulatory decrease (RVD). The RVD involves loss of KCl and organic osmolytes from the cell. The decrease in cellular KCl and organic osmolytes causes intracellular osmotic pressure to decrease, water leaves the cell, and the cell returns to nearly its original volume. Bottom, When cells are exposed to a hypertonic medium, they shrink and then undergo a volume-regulatory increase (RVI). During the RVI, NaCl and organic osmolytes enter the cell. The increase in the activity of Na+,K+-ATPase (not depicted) enhances the exchange of Na+ for K+ so that the K+ (and Cl−) content of the cell is increased. The increase in cellular KCl, along with a rise in intracellular organic osmolytes, increases intracellular osmotic pressure, which brings water back into the cell, and the cell volume returns to nearly its original volume. π, The oncotic pressure inside the cell.

IN THE C LIN IC The ECF of individuals with disorders in water balance may be either hypotonic (positive water balance) or hypertonic (negative water balance). With a decrease in ECF osmolality, neurons and glial cells swell as water enters the cell. To minimize this swelling, the neurons and glial cells reduce intracellular osmolytes. If the ECF osmolality is corrected (i.e., increased) too quickly, the neurons and glial cells then shrink because of the reduced number of intracellular osmolytes. This response to a rapid correction of ECF osmolality can lead to cell damage. Damage to the glial cells that synthesize myelin within the brain can result in demyelinization. This demyelinization response, termed osmotic demyelinization syndrome, can affect any of the white matter of the brain, but especially regions of the pons. These effects are often irreversible. Therefore, correction of disorders of water balance is usually accomplished slowly to avoid this serious neurological complication. The regulatory volume increase response results in the rapid uptake of NaCl and a number of organic osmolytes. To increase cell volume there is an activation of the Na+-H+ antiporter (NHE-1), the 1Na+,1K+,2Cl− symporter (NKCC-1), and a number of cation-selective channels, which together bring NaCl into the cell. The Na+,K+-ATPase then extrudes the Na+ in exchange for K+, so that ultimately the KCl content of the cell is increased. Several organic osmolyte transporters are also activated to increase cell volume. These include a 3Na+,1Cl−-taurine symporter, a 3Na+,2Cl−-betaine symporter, a 2Na+–myoinositol symporter, and a Na+–amino acid symporter. These transporters use the energy in the Na+ and Cl− gradients to drive the secondary active uptake of these organic osmolytes into cells. The regulatory volume decrease response results in the loss of KCl and organic osmolytes from the cell. The loss of KCl occurs through the activation of a wide range of K+-selective, Cl−-selective, and anion-selective channels (the specific channels involved vary depending on the cell), as well as through activation of K+-Cl− symporters. Some of the organic osmolytes appear to leave the cell via anion channels (e.g., volume-sensitive organic osmolyte-anion channels). Several mechanisms are involved in activation of these various transporters during the volumeregulatory responses. Changes in cell volume appear to monitored by the cytoskeleton, by changes in macromolecular crowding and ionic strength of the cytoplasm, and by channels whose gating is influenced, either directly or indirectly, by stretch of the plasma membrane (e.g., stretch-activated cation channels). A number of second messenger systems may also be involved in these responses (e.g., intracellular [Ca++], calmodulin, protein kinase A, and protein kinase C), but the precise mechanisms have not been defined completely.

Principles of Epithelial Transport Epithelial cells are arranged in sheets and provide the interface between the external world and the internal environment (i.e., ECF) of the body. Depending on their location, epithelial cells serve many important functions, such as establishing a barrier to microorganisms (lungs, gastrointestinal tract, and skin), prevention of the loss of water from the body (skin), and maintenance of a constant internal environment (lungs, gastrointestinal tract, and kidneys). The latter function is a result of the ability of epithelial cells to carry out regulated vectorial transport (i.e., transport from one side of the epithelial cell sheet to the opposite side). In this section, the principles of epithelial transport are reviewed. The transport functions of specific epithelial cells are discussed in the appropriate chapters throughout this book.

Epithelial Structure Fig. 2.8 shows a schematic representation of an epithelial cell. The free surface of the epithelial layer is referred to as the apical membrane. It is in contact with the external environment (e.g., air within the alveoli and larger airways of the lungs and the contents of the gastrointestinal tract) or with extracellular fluids (e.g., glomerular filtrate in the nephrons of the kidneys and the secretions of the ducts of the pancreas or sweat glands). The basal side of the epithelium rests on a basal lamina, which is secreted by the epithelial cells, and this in turn is attached to the underlying connective tissue.

FIG. 2.8 Schematic of an epithelial cell, illustrating the various adhering junctions. The tight junction separates the apical membrane from basolateral membrane (see text for details).

Epithelial cells are connected to one another and to the underlying connective tissue by a number of specialized junctions (see Fig. 2.8). The adhering junction, desmosomes, and hemidesmosomes provide mechanical adhesion by linking together the cytoskeleton of adjacent cells (adhering junction and desmosome) or to the underlying connective tissue (hemidesmosome). The gap junction and tight junction play important physiological roles. Gap junctions provide low-resistance connections between cells.g The functional unit of the gap junction is the connexon. The connexon is composed of six integral membrane protein subunits called connexins. A connexon in one cell is aligned with the connexon in the adjacent cell, forming a channel. The channel may be gated, and when it is open, it allows the movement of ions and small molecules between cells. Because of their low electrical resistance, they effectively couple electrically one cell to the adjacent cell. The tight junction serves two main functions. It divides the cell into two membrane domains (apical and basolateral) and, in so doing, restricts the movement of membrane lipids and proteins between these two

domains. This so-called fence function allows epithelial cells to carry out vectorial transport from one surface of the cell to the opposite surface by segregating membrane transporters to one or other of the membrane domains. They also serve as a pathway for the movement of water, ions, and small molecules across the epithelium. This pathway between the cells is referred to as the paracellular pathway, as opposed to the transcellular pathway through the cells.

AT THE C ELLU LAR LEVEL Epithelial cell tight junctions (also called zonula occludens) are composed of several integral membrane proteins, including occludins, claudins, and several members of the immunoglobulin superfamily (e.g., the junctional adhesion molecule [JAM]). Occludins and claudins are transmembrane proteins that span the membrane of one cell and link to the extracellular portion of the same molecule in the adjacent cell. Cytoplasmic linker proteins (e.g., tight junction protein [ZO-1, ZO2, and ZO-3]) then link the membrane spanning proteins to the cytoskeleton of the cell. Of these junctional proteins, claudins appear to be important in determining the permeability characteristics of the tight junction, especially with regard to cations and anions. Certain claudins serve as barrier proteins that restrict the movement of ions through the tight junction, whereas others form a “pore” that facilitates the movement of ions through the junction. Thus the permeability characteristics of the tight junction of an epithelium are determined by the complement of claudins expressed by the cell. For example, the proximal tubule of the kidney is termed a “leaky” epithelium, in which water and solutes (e.g., Na+) move through the junction. Claudin 4 and claudin 10 are expressed in the tight junction of proximal tubule cells. In contrast, the collecting duct of the kidney is considered a “tight” epithelium, with restricted movement of ions through the tight junction. Collecting duct cells express claudins 3, 4, 7, 8, 10, and 18. The function of claudins can be regulated at several levels, including gene expression, posttranslational modification, interactions with cytoplasmic scaffolding proteins, and interactions with other claudins in the same membrane (cis-interaction), as well as with claudins of adjacent cells (transinteraction). The mineralocorticoid hormone aldosterone stimulates Na+ reabsorption by distal segments of the renal nephron (see Chapters 34 and 35). In addition to the hormone’s effect on Na+ transporters in the cell, aldosterone also upregulates expression of claudin 8 in the tight junction. The increased expression of claudin 8 reduces the ability of Na+ to permeate the tight junction, which then reduces the backward leak of Na+ from the interstitium into the tubule lumen, thereby allowing more efficient Na+ reabsorption by the epithelium.

IN THE C LIN IC Mutations in the gene that codes for claudin 16 result in the autosomal recessive condition known as familial hypomagnesemia, hypercalciuria, and nephrocalcinosis (FHHNC). Claudin 16 is found in the tight junction of the thick ascending portion of Henle’s loop in the kidneys and serves as a route for the paracellular reabsorption of Ca++ and Mg++ from the tubular fluid. Individuals with FHHNC lack functional copies of claudin 16, and reabsorption of these divalent ions is thus reduced, which leads to hypomagnesemia, hypercalciuria, and nephrocalcinosis. The apical surface of epithelial cells may have specific structural features. One such feature is microvilli (Fig. 2.9A). Microvilli are small (typically 1–3 µm in length), nonmotile projections of the

apical plasma membrane that serve to increase surface area. They are commonly located on cells that must transport large quantities of ions, water, and molecules (e.g., epithelial cells lining the small intestine and cells of the renal proximal tubule). The core of the microvilli is composed of actin filaments and a number of accessory proteins. This actin core is connected to the cytoskeleton of the cell via the terminal web (a network of actin fibers at the base of the microvilli) and provides structural support for the microvilli. Another surface feature is stereocilia (see Fig. 2.9B). Stereocilia are long (up to 120 µm), nonmotile membrane projections that, like microvilli, increase the surface area of the apical membrane. They are found in the epididymis of the testis and in the “hair cells” of the inner ear. Their core also contains actin filaments and accessory proteins.

FIG. 2.9 Illustration of apical membrane specializations of epithelial cells (Not drawn to scale). A, Microvilli 1 to 3 µm in length serve to increase the surface area of the apical membrane (e.g., those of the epithelial cells of the small intestine). B, Stereocilia can be up to 120 µm in length (e.g., those of the epididymis of the male reproductive tract). Both microvilli and stereocilia have a core structure composed primarily of actin, with a number of associated proteins. Both are nonmotile. (Redrawn from Pawlina W. Histology: A Text and Atlas, with Correlated Cell and Molecular Biology. 7th ed. Philadelphia: Wolters Kluwer Health; 2016.)

A third apical membrane feature is cilia (Fig. 2.10). Cilia may be either motile (called secondary cilia) or nonmotile (called primary cilia). The motile cilia contain a microtubule core arranged in a characteristic “9+2” pattern (nine pairs of microtubules around the circumference of the cilium, and one pair of microtubules in the center). Dynein is the molecular motor that drives the movement of the cilium. Motile cilia are characteristic features of the epithelial cells that line the respiratory tract. They pulsate in a synchronized manner and serve to transport mucus and inhaled particulates out of the lung, a process termed mucociliary transport (see Chapter 26). Nonmotile cilia serve as mechanoreceptors and are involved in determining left-right asymmetry of organs during embryological development, as well as sensing the flow rate of fluid in the nephron of the kidneys (see Chapter 33). Only a single nonmotile

cilium is found in the apical membrane of cells. Nonmotile cilia have a microtubule core (“9+0” arrangement) and lack a motor protein.

FIG. 2.10 Cilia are apical membrane specializations of some epithelial cells. Cilia are 5 to 10 µm in length and contain arrays of microtubules, as depicted in these cross-sectional diagrams. Left, The primary cilium has nine peripheral microtubule arrays. It is nonmotile and serves as a mechanoreceptor (e.g., cells of the renal collecting duct). Cells that have a primary cilium have only a single cilium. Right, The secondary cilium has a central pair of microtubules in addition to the nine peripheral microtubule arrays. Also in the secondary cilium, the motor protein dynein is associated with the microtubule arrays and therefore is motile. A single cell can have thousands of secondary cilia on its apical surface (e.g., epithelial cells of the respiratory tract). (Redrawn from Rodat-Despoix L, Delmas P. Ciliary functions in the nephron. Pflugers Archiv. 2009;458:179.)

As noted previously, the tight junction effectively divides the plasma membrane of an epithelial cell into two domains: an apical surface and a basolateral surface. The basolateral membrane of many epithelial cells is folded or invaginated. This is especially so for epithelial cells that have high transport rates. These invaginations serve to increase the membrane surface area to accommodate the large number of membrane transporters (e.g., Na+,K+-ATPase) needed in the membrane.

Vectorial Transport Because the tight junction divides the plasma membrane into two domains (i.e., apical and basolateral), epithelial cells are capable of vectorial transport, whereby an ion or molecule can be transported from one side of the epithelial sheet to the opposite side (Fig. 2.11). The accomplishment of vectorial transport requires that specific membrane transport proteins be targeted to and remain in one or the other of the membrane domains. In the example shown in Fig. 2.11, the Na+ channel is present only in the apical membrane, whereas the Na+,K+-ATPase and the K+ channels are confined to the basolateral membrane. The operation of the Na+,K+-ATPase and the leakage of K+ out of the cell across the basolateral

membrane set up a large electrochemical gradient for Na+ to enter the cell across the apical membrane through the Na+ channel (intracellular [Na+] < extracellular [Na+], and Vm which is oriented with the cell’s interior electrically negative with respect to the cell’s exterior). The Na+ is then pumped out of the cell by the Na+,K+-ATPase, and vectorial transport from the apical side of the epithelium to the basolateral side of the epithelium occurs. Transport from the apical side to the basolateral side of an epithelium is termed either absorption or reabsorption: For example, the uptake of nutrients from the lumen of the gastrointestinal tract is termed absorption, whereas the transport of NaCl and water from the lumen of the renal nephrons is termed reabsorption. Transport from the basolateral side of the epithelium to the apical side is termed secretion.

FIG. 2.11 In symmetrical cells (A; e.g., red blood cells), membrane transport proteins are distributed over the entire surface of the cell. Epithelial cells (B), in contrast, are asymmetrical and target various membrane transport proteins to either the apical or the basolateral membrane. When the transporters are confined to a membrane domain, vectorial transport can occur. In the cell depicted, Na+ is transported from the apical surface to the basolateral surface. ATP, Adenosine triphosphate.

As noted previously, the Na+,K+-ATPase and K+-selective channels play an important role in

establishing cellular ion gradients for Na+ and K+ and in generating the Vm. In all epithelial cells except the choroid plexus and retinal pigment epithelium,h the Na+,K+-ATPase channel is located in the basolateral membrane of the cell. Numerous K+-selective channels are in epithelial cells and may be located in either membrane domain. Through the establishment of these chemical and voltage gradients, the transport of other ions and solutes can be driven (e.g., Na+-glucose symporter, Na+-H+ antiporter, 1Na+,1K+,2Cl− symporter, 1Na+-3HCO3− symporter). The direction of transepithelial transport (reabsorption or secretion) depends simply on which membrane domain the transporters are located. Because of the dependence on the Na+,K+-ATPase, epithelial transport requires the expenditure of energy. Other ATP-dependent transporters, such as the H+-ATPase, H+,K+-ATPase, and a host of ABC transporters—such as P-glycoprotein (PGP) and multidrug resistance-associated protein 2 (MRP2), which transport xenobiotics (drugs), and cystic fibrosis transmembrane conductance regulator (CFTR), which transports Cl−—are involved in epithelial transport. Solutes and water can be transported across an epithelium by traversing both the apical and basolateral membranes (transcellular transport) or by moving between the cells across the tight junction (paracellular transport). Solute transport via the transcellular route is a two-step process, in which the solute molecule is transported across both the apical and basolateral membrane. Uptake into the cell, or transport out of the cell, may be either a passive or an active process. Typically, one of the steps is passive, and the other is active. For the example shown in Fig. 2.11B, the uptake of Na+ into the cell across the apical membrane through the Na+-selective channel is passive and driven by the electrochemical gradient for Na+. The exit of Na+ from the cell across the basolateral membrane is primary active transport via the Na+,K+-ATPase. Because a transepithelial gradient for Na+ can be generated by this process (i.e., the [Na+] in the apical compartment can be reduced below that of the basolateral compartment), the overall process of transepithelial Na+ transport is said to be active. Any solute that is actively transported across an epithelium must be transported via the transcellular pathway. Depending on the epithelium, the paracellular pathway is an important route for transepithelial transport of solute and water. As noted, the permeability characteristics of the paracellular pathway are determined, in large part, by the specific claudins that are expressed by the cell. Thus the tight junction can have low permeability for solutes, water, or both, or it can have a high permeability. For epithelia in which there are high rates of transepithelial transport, the tight junctions typically have a high permeability (i.e., are leaky). Examples of such epithelia include the proximal tubule of the renal nephron and the early segments of the small intestine (e.g., duodenum and jejunum). If the epithelium must establish large transepithelial gradients for solutes, water, or both, the tight junctions typically have low permeability (i.e., are tight). Examples of this type of epithelium include the collecting duct of the renal nephron, the urinary bladder, and the terminal portion of the colon. In addition, the tight junction may be selective for certain solutes (e.g., cation versus anion selective). All solute transport that occurs through the paracellular pathway is passive in nature. The two driving forces for this transport are the transepithelial concentration gradient for the solute and, if the solute is charged, the transepithelial voltage (Fig. 2.12). The transepithelial voltage may be oriented with the apical surface electrically negative in relation to the basolateral surface as shown in Fig. 2.12, or it may be oriented with the apical surface electrically positive in relation to the basolateral surface. The polarity and magnitude of the transepithelial voltage are determined by the specific membrane transporters in the apical and basolateral membranes, as well as by the permeability characteristics of the tight junction.

FIG. 2.12 The electrical profile across an epithelial cell. The magnitude of the membrane voltages, and the transepithelial voltage are determined by the various membrane transport proteins in the apical and basolateral membranes. The transepithelial voltage is equal to the sum of the apical and basolateral membrane voltages (see text for details).

It is important to recognize that transcellular transport processes set up the transepithelial chemical and voltage gradients, which in turn can drive paracellular transport. This is illustrated in Fig. 2.13 for an epithelium that reabsorbs NaCl and for an epithelium that secretes NaCl. In both epithelia, the transepithelial voltage is oriented with the apical surface electrically negative in relation to the basolateral surface. For the NaCl-reabsorbing epithelium, the transepithelial voltage is generated by the active, transcellular reabsorption of Na+. This voltage in turn drives Cl− reabsorption through the paracellular pathway. In contrast, for the NaCl-secreting epithelium, the transepithelial voltage is generated by the active transcellular secretion of Cl−. Na+ is then secreted passively via the paracellular pathway, driven by the negative transepithelial voltage.

FIG. 2.13 The role of the paracellular pathway in epithelial transport. A, Na+ transport through the cell generates a transepithelial voltage that then drives the passive movement of Cl− through the tight junction. NaCl reabsorption results. B, Cl− transport through the cell generates a transepithelial voltage that then drives the passive transport of Na+ through the tight junction. NaCl secretion results.

Transepithelial Water Movement Water movement across epithelia is passive and driven by transepithelial osmotic pressure gradients. Water movement can occur by a transcellular route involving aquaporins in both the apical and basolateral membranes.i In addition, water may also move through the paracellular pathway. In the NaClreabsorbing epithelium depicted in Fig. 2.13A, the reabsorption of NaCl from the apical compartment lowers the osmotic pressure in that compartment, whereas the addition of NaCl to the basolateral compartment raises the osmotic pressure in that compartment. As a result, a transepithelial osmotic pressure gradient is established that drives the movement of water from the apical to the basolateral compartment (i.e., reabsorption). The opposite occurs with NaCl-secreting epithelia (see Fig. 2.13B), in which the transepithelial secretion of NaCl establishes a transepithelial osmotic pressure gradient that drives water secretion. In some epithelia (e.g., proximal tubule of the renal nephron), the movement of water across the epithelium via the paracellular pathway can drive the movement of additional solute. This process is termed solvent drag and reflects the fact that solutes dissolved in the water will traverse the tight junction with the water. As is the case with the establishment of transepithelial concentration and voltage gradients, the establishment of transepithelial osmotic pressure gradients requires transcellular transport of solutes by the epithelial cells.

Regulation of Epithelial Transport Epithelial transport must be regulated to meet the homeostatic needs of the individual. Depending on the epithelium, this regulation involves neural or hormonal mechanisms, or both. For example, the enteric nervous system of the gastrointestinal tract regulates solute and water transport by the epithelial cells that line the intestine and colon. Similarly, the sympathetic nervous system regulates transport by the epithelial cells of the renal nephron. Aldosterone, a steroid hormone produced by the adrenal cortex (see Chapter 43), is an example of a hormone that stimulates NaCl transport by the epithelial cells of the colon, renal nephron, and sweat ducts. Epithelial cell transport can also be regulated by locally produced and locally acting substances, a process termed paracrine regulation. The stimulation of HCl secretion in the stomach by histamine is an example of this process. Cells that are located near the epithelial cells of the stomach release histamine, which acts on the HCl-secreting cells of the stomach (parietal cells) and stimulates them to secrete HCl. When acted upon by a regulatory signal, the epithelial cell may respond in several different ways, including: • Retrieval of transporters from the membrane, by endocytosis, or insertion of transporters into the membrane from an intracellular vesicular pool, by a process called exocytosis • Change in activity of membrane transporters (e.g., ion channel gating)

• Synthesis of specific transporters, and their insertion into the membrane The first two mechanisms can occur quite rapidly (seconds to minutes), but the synthesis of transporters takes additional time (minutes to days).

Key Concepts • The body maintains steady-state balance for water and a number of important solutes. This occurs when input into the body equals output from the body. For each solute and water, there is a normal set point. Deviations from this set point are monitored (i.e., when input ≠ output), and effector mechanisms are activated that restore balance. This balance is achieved by adjustment of either intake or excretion of water and solutes. Thereafter, input and output are again equal to maintain balance. • The Na+,K+-ATPase and K+-selective channels are critically important in establishing and maintaining the intracellular composition, the membrane potential (Vm), and cell volume. Na+,K+-ATPase converts the energy in ATP into potential energy of ion gradients and the membrane potential. The ion and electrical gradients created by this process are then used to drive the transport of other ions and other molecules, especially by solute carriers (i.e., symporters and antiporters). • Epithelial cells constitute the interface between the external world and the internal environment of the body. Vectorial transport of solutes and water across epithelia helps maintain steady-state balance for water and a number of important solutes. Because the external environment constantly changes, and because dietary intake of food and water is highly variable, transport by epithelia is regulated to meet the homeostatic needs of the individual. a

In these and all subsequent calculations, it is assumed that 1 L of fluid (e.g., ICF and ECF) has a mass of 1 kg. Although 1 L of the ICF and ECF has a mass of slightly more than 1 kg, this simplification allows conversion from measurements of body weight to volume of body fluids. b

Some exceptions do exist. The cerebrospinal fluid is part of the ECF, but its osmolality is slightly higher than that of the ECF elsewhere in the body. Also, regions within the kidney can have osmolalities that are either less than or greater than that of the ECF. However, these volumes are small (≈150 mL) in comparison with the total volume of the ECF (≥12 L). c

Serum is derived from clotted blood. Thus serum differs from plasma by the absence of clotting factors. With regard to osmolality and the concentrations of other molecules and ions, the osmolality and concentrations in plasma and serum are virtually identical. d

Fluids are usually administered intravenously. When electrolyte solutions are infused by this route, equilibration between plasma and interstitial fluid is rapid (i.e., minutes) because of the high permeability of many capillary walls for water and electrolytes. Thus these fluids are essentially added to the entire ECF. e

A 0.9% NaCl solution (0.9 g NaCl/100 mL) contains 154 mmol/L of NaCl. Because NaCl does not dissociate completely in solution (i.e., 1.88 Osm/mol), the osmolality of this solution is 290 mOsm/kg H2O, which is very similar to that of normal ECF.

f

In muscle cells, in which contraction is regulated by the intracellular [Ca++], the maintenance of a low intracellular [Ca++] during the relaxed state involves not only the activity of the plasma membrane 3Na+Ca++ antiporter and the Ca++-ATPase but also a Ca++-ATPase molecule located in the smooth endoplasmic reticulum (see Chapters 12 to 14). g

Gap junctions are not limited to epithelial cells. A number of other cells also have gap junctions (e.g., cardiac myocytes and smooth muscle cells). h

The choroid plexus is located in the ventricles of the brain and secretes the cerebrospinal fluid. The Na+,K+-ATPase is located in the apical membrane of these cells. i

Different aquaporin isoforms are often expressed in the apical and basolateral membrane. In addition, multiple isoforms may be expressed in one or more of the membrane domains.

C H AP T E R 3

Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. How do cells communicate with each other? 2. What are the four classes of receptors, and what signal transduction pathways are associated with each class of receptors? 3. How do steroid and thyroid hormones, cyclic adenosine monophosphate, and receptor tyrosine kinases regulate gene expression?

Cell-to-Cell Communication An overview of how cells communicate with each other is presented in Fig. 3.1. Cells communicate by releasing extracellular signaling molecules (e.g., hormones and neurotransmitters) that bind to receptor proteins located in the plasma membrane, cytoplasm, or nucleus. This signal is transduced into the activation, or inactivation, of one or more intracellular messengers by interacting with receptors. Receptors interact with a variety of intracellular signaling proteins, including kinases, phosphatases, and guanosine triphosphate (GTP)–binding proteins (G proteins). These signaling proteins interact with and regulate the activity of target proteins and thereby modulate cellular function. Target proteins include, but are not limited to, ion channels and other transport proteins, metabolic enzymes, cytoskeletal proteins, gene regulatory proteins, and cell cycle proteins that regulate cell growth and division. Signaling pathways are characterized by (1) multiple, hierarchical steps; (2) amplification of the signal-receptor binding event, which magnifies the response; (3) activation of multiple pathways and regulation of multiple cellular functions; and (4) antagonism by constitutive and regulated feedback mechanisms, which minimize the response and provide tight regulatory control over these signaling pathways. A brief description of how cells communicate follows. Readers who desire a more in-depth presentation of this material are encouraged to consult one of the many cellular and molecular biology textbooks currently available.

FIG. 3.1 An overview of how cells communicate. A signaling molecule (i.e., hormone or neurotransmitter) binds to a receptor, which may be in the plasma membrane, cytosol, or nucleus. Binding of ligand to a receptor activates intracellular signaling proteins, which interact with and regulate the activity of one or more target proteins to change cellular function. Signaling molecules regulate cell growth, division, and differentiation and influence cellular metabolism. In addition, they modulate the intracellular ionic composition by regulating the activity of ion channels and transport proteins. Signaling molecules also control cytoskeleton-associated events, including cell shape, division, and migration and cell-to-cell and cell-to-matrix adhesion. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

IN THE C LIN IC The significance of signaling pathways in medicine is illustrated by the following short list of popular drugs that act by regulating signaling pathways. Details on these pathways are presented later in this and other chapters. • Aspirin, the first pharmaceutical (1899), inhibits cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) and therefore is antithrombotic (i.e., reduces the formation of blood clots). • β-Adrenergic receptor agonists and antagonists are used to treat a variety of medical conditions. β1-Agonists increase cardiac contractility and heart rate in patients with low blood pressure. β2-Agonists (albuterol [ProAir HFA], levalbuterol [Xopenex HFA], metaproterenol [Alupent],

and terbutaline [Bricanyl]) dilate bronchi and are used to treat asthma and chronic obstructive lung disease. In contrast, β-adrenergic antagonists (bisoprolol [Zebeta], carvedilol [Coreg], and metoprolol [Toprol]) are used to treat hypertension, angina, cardiac arrhythmias, and congestive heart failure (see Chapter 18). • Fluoxetine (Prozac) is an antidepressant medication that inhibits reuptake of the neurotransmitter serotonin into the presynaptic cell, which results in enhanced activation of serotonin receptors (see Chapter 6). • Several monoclonal antibodies are used to treat cancer caused by the activation of growth factor receptors in cancer cells. For example, trastuzumab (Herceptin) is a monoclonal antibody used to treat metastatic breast cancer in women who overexpress HER2/neu, a member of the family of epidermal growth factor (EGF) receptors, which stimulate cell growth and differentiation. Cetuximab (Erbitux) and bevacizumab (Avastin) are monoclonal antibodies that are used to treat metastatic colorectal cancer and cancers of the head and neck. These antibodies bind to and inhibit the EGF receptor and thereby inhibit EGF-induced cell growth in cancer cells. • Drugs that inhibit cyclic guanosine monophosphate (cGMP)–specific phosphodiesterase type 5, such as sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra), prolong the vasodilatory effects of nitric oxide and are used to treat erectile dysfunction and pulmonary arterial hypertension (see Chapter 17).

Cells in higher animals release into the extracellular space hundreds of chemicals, including (1) peptides and proteins (e.g., insulin); (2) amines (e.g., epinephrine and norepinephrine); (3) steroid hormones (e.g., aldosterone, estrogen); and (4) small molecules, including amino acids, nucleotides, ions (e.g., Ca++), and gases, such as nitric oxide and carbon dioxide. Secretion of signaling molecules is celltype specific. For example, beta cells in the pancreas release insulin, which stimulates glucose uptake into cells. The ability of a cell to respond to a specific signaling molecule depends on the expression of receptors that bind the signaling molecule with high affinity and specificity. Receptors are located in the plasma membrane, the cytosol, and the nucleus (Fig. 3.2).

FIG. 3.2 Signaling molecules, especially ones that are hydrophilic and cannot cross the plasma membrane, bind directly to their cognate receptors in the plasma membrane (A). Other signaling molecules—including steroid hormones, triiodothyronines, retinoic acids, and vitamin D—bind to carrier proteins in blood and readily diffuse across the plasma membrane, where they bind to cognate nuclear receptors in the cytosol or nucleus (B). Still other signaling molecules, including nitric oxide, can diffuse without carrier proteins and cross the membrane to act on intracellular protein targets (B). Both classes of receptors, when ligand bound, regulate gene transcription. mRNA, Messenger RNA. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

Signaling molecules can act over long or short distances and can require cell-to-cell contact or very close cellular proximity (Fig. 3.3). Contact-dependent signaling, in which a membrane-bound signaling molecule of one cell binds directly to a plasma membrane receptor of another cell, is important during development, in immune responses, and in cancer (see Fig. 3.3A). Molecules that are released and act locally are called paracrine (see Fig. 3.3B ) or autocrine (see Fig. 3.3C ) hormones. Paracrine signals are released by one type of cell and act on another type; they are usually taken up by target cells or rapidly degraded (within minutes) by enzymes. For example, enterochromaffin-like cells in the stomach secrete histamine, which stimulates the production of acid by neighboring parietal cells (see Chapter 27 for details). Autocrine signaling involves the release of a molecule that affects the same cell or other cells of the same type (e.g., cancer cells). In synaptic signaling (see Fig. 3.3D), neurons transmit electrical signals along their axons and release neurotransmitters at synapses that affect the function of other neurons or cells that are distant from the neuron cell body. The close physical relationship between the nerve terminal and the target cell ensures that the neurotransmitter is delivered to a specific cell. Details on synaptic signaling are discussed in Chapter 6. Endocrine signals are hormones that are secreted into the

blood and are widely dispersed in the body (see Fig. 3.3E). Details on endocrine signaling are discussed in Chapter 38.

FIG. 3.3 Cell-to-cell communication is mediated by five basic mechanisms: contact-dependent (A), paracrine (B), autocrine (C), synaptic (D), and endocrine signaling (E). These mechanisms are described in detail in the text. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

In addition to paracrine, autocrine, endocrine, and synaptic signaling, cell-to-cell communication also

occurs via gap junctions that form between adjacent cells (see Chapter 2). Gap junctions are specialized junctions that allow intracellular signaling molecules, generally less than 1200 daltons (Da) in size, to diffuse from the cytoplasm of one cell to an adjacent cell. The permeability of gap junctions is regulated by cytosolic [Ca++], [H+], and cyclic adenosine monophosphate (cAMP) and by the membrane potential. Gap junctions also allow cells to be electrically coupled, which is vitally important for the coordinated activity of cardiac and smooth muscle cells (see Chapters 13 and 14). The speed of a response to an extracellular signal depends on the mechanism of delivery. Endocrine signals are relatively slow (seconds to minutes) because time is required for diffusion and blood flow to the target cell, whereas synaptic signaling is extremely fast (milliseconds). If the response involves changes in the activity of proteins in the cell, the response may occur in milliseconds to seconds. However, if the response involves changes in gene expression and the de novo synthesis of proteins, the response may take hours to occur, and a maximal response may take days. For example, the stimulatory effect of aldosterone on sodium transport by the kidneys requires days to develop fully (see Chapter 35). The response to a particular signaling molecule also depends on the ability of the molecule to reach a particular cell, on expression of the cognate receptor (i.e., receptors that recognize a particular signaling molecule or ligand with a high degree of specificity), and on the cytoplasmic signaling molecules that interact with the receptor. Thus signaling molecules frequently have many different effects that are dependent on the cell type. For example, the neurotransmitter acetylcholine stimulates contraction of skeletal muscle but decreases the force of contraction in heart muscle. This is because skeletal muscle and heart cells express different acetylcholine receptors.a

Receptors All signaling molecules bind to specific receptors that act as signal transducers, thereby converting a ligand-receptor binding event into intracellular signals that affect cellular function. Receptors can be divided into four basic classes on the basis of their structure and mechanism of action: (1) ligand-gated ion channels, (2) G protein–coupled receptors (GPCRs), (3) enzyme-linked receptors, and (4) nuclear receptors (Table 3.1; Figs. 3.4 and 3.5). Table 3.1 Classes of Membrane Receptors Receptor Class

Ligand

Signal Transduction Pathway/Target

LigandExtracellular ligand: gated ion channels

Membrane currents:



GABA

Cl−



ACh (muscle)

Na+, K+, Ca++



ATP

Ca++, Na+, K+



Glutamate: NMDA

Na+, K+, Ca++



Intracellular ligand:





cAMP (olfaction)

K+



cGMP (vision)

Na+, K+



InsP3

Ca++

G protein– coupled receptors

Neurotransmitters (ACh) Peptides (PTH, oxytocin) Odorants Cytokines, lipids

βγ Subunits activate ion channels α Subunit activates enzymes: Cyclases that generate cAMP, cGMP, phospholipases that generate InsP3 and diacylglycerol, and phospholipases that generate arachidonic acid and its metabolites. Monomeric G proteins

Enzymelinked receptors

ANP

Receptor guanylyl cyclase



TGF-β

Receptor serine/threonine kinase



Insulin, EGF

Receptor tyrosine kinase



Interleukin-6, erythropoietin

Tyrosine kinase–associated receptor

Nuclear receptors

Steroid hormones: Mineralocorticoids Glucocorticoids Androgens Estrogens Progestins

Bind to regulatory sequences in DNA and increase or decrease gene transcription



Miscellaneous hormones: Thyroid Vitamin D Retinoic acid Prostaglandins

Bind to regulatory sequences in DNA and increase or decrease gene transcription

ACh, Acetylcholine; ANP, atrial natriuretic peptide; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; EGF, epidermal growth factor; GABA, gamma-aminobutyric acid; InsP3, inositol 1,4,5triphosphate; NMDA, N-methyl-D-aspartate; PTH, parathyroid hormone; TGF, transforming growth factor.

FIG. 3.4 Three of the four classes of plasma membrane receptors. See text for details. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

FIG. 3.5 Steroid hormones stimulate the transcription of early-response genes and late-response genes. See text for details. (Redrawn from Alberts B, et al. Molecular Biology of Cell. 6th ed. New York: Garland Science; 2015.)

Ligand-gated ion channels mediate direct and rapid synaptic signaling between electrically excitable cells (see Fig. 3.4A). Neurotransmitters bind to receptors and either open or close ion channels, thereby changing the ionic permeability of the plasma membrane and altering the membrane potential. For examples and more details, see Chapter 6. GPCRs regulate the activity of other proteins, such as enzymes and ion channels (see Fig. 3.4B ). In the example in Fig. 3.4B, the interaction between the receptor and the target protein is mediated by heterotrimeric G proteins, which are composed of α, β, and γ subunits. Stimulation of G proteins by ligand-bound receptors activates or inhibits downstream target proteins that regulate signaling pathways if the target protein is an enzyme or changes membrane ion permeability if the target protein is an ion channel. Enzyme-linked receptors either function as enzymes or are associated with and regulate enzymes (see Fig. 3.4C ). Most enzyme-linked receptors are protein kinases or are associated with protein kinases, and ligand binding causes the kinases to phosphorylate a specific subset of proteins on specific amino acids, which in turn activates or inhibits protein activity. Nuclear receptors are small hydrophobic molecules, including steroid hormones, thyroid hormones, retinoids, and vitamin D, that have a long biological half-life (hours to days), diffuse across the plasma

membrane, and bind to nuclear receptors or to cytoplasmic receptors that, once bound to their ligand, translocate to the nucleus (see Fig. 3.5). Some nuclear receptors, such as those that bind cortisol and aldosterone, are located in the cytosol and enter the nucleus after binding to hormone, whereas other receptors, including the thyroid hormone receptor, are located in the nucleus. In both cases, inactive receptors are bound to inhibitory proteins, and binding of hormone results in dissociation of the inhibitory complex. Hormone binding causes the receptor to bind coactivator proteins that activate gene transcription. Once activated, the hormone-receptor complex regulates the transcription of specific genes. Activation of specific genes usually occurs in two steps: an early primary response (≈30 minutes), which activates genes that stimulate other genes to produce a delayed (hours to days) secondary response (see Fig. 3.5). Each hormone elicits a specific response that is based on cellular expression of the cognate receptor, as well as on cell type–specific expression of gene regulatory proteins that interact with the activated receptor to regulate the transcription of a specific set of genes (see Chapter 38 for more details). In addition to steroid receptors that regulate gene expression, evidence also suggests the existence of membrane and juxtamembrane steroid receptors that mediate the rapid, nongenomic effects of steroid hormones. Some membrane proteins do not fit the classic definition of receptors, but they subserve a receptor-like function in that they recognize extracellular signals and transduce the signals into an intracellular second messenger that has a biological effect. For example, on activation by a ligand, some membrane proteins undergo regulated intramembrane proteolysis (RIP), which elaborates a cytosolic peptide fragment that enters the nucleus and regulates gene expression (Fig. 3.6). In this signaling pathway, binding of ligand to a plasma membrane receptor leads to ectodomain shedding, facilitated by members of the metalloproteinase-disintegrin family, and produces a carboxy-terminal fragment that is the substrate for γsecretase. γ-Secretase induces RIP, thereby causing the release of an intracellular domain of the protein that enters the nucleus and regulates transcription (see Fig. 3.6). The best characterized example of RIP is the sterol regulatory element–binding protein (SREBP), a transmembrane protein expressed in the membrane of the endoplasmic reticulum. When cellular cholesterol levels are low, SREBP undergoes RIP, and the proteolytically cleaved fragment is translocated into the nucleus, where it transcriptionally activates genes that promote cholesterol biosynthesis.

FIG. 3.6 Regulated intramembrane proteolysis. See text for details. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

IN THE C LIN IC Alzheimer’s disease, a progressive neurodegenerative brain disease characterized by the formation of amyloid plaques, affects approximately 44 million people worldwide. In Alzheimer’s disease, regulated intramembrane proteolysis of amyloid β-protein precursor (APP) causes the accumulation of amyloid β-protein (Aβ), which forms amyloid plaques that are thought to contribute to the pathogenesis of Alzheimer’s disease. APP is a type I transmembrane protein (i.e., its spans the membrane only once). After ectodomain shedding, its sequential proteolysis by β-secretase and γ-secretase produces the Aβ40 and Aβ42 peptides that are normally produced throughout life but accumulate in individuals with Alzheimer’s disease. Missense mutations in presenilins, proteins that regulate γ-secretase protease activity, enhance the production of Aβ42, which is more hydrophobic and prone to aggregation into amyloid fibrils than is the more abundant Aβ40 protein.

Receptors and Signal Transduction Pathways When hormones bind to plasma membrane receptors, signals are relayed to effector proteins via intracellular signaling pathways. When hormones bind to nuclear or cytosolic receptors, they relay signals primarily through regulation of gene expression. Signaling pathways can amplify and integrate signals but can also downregulate and desensitize signals, reducing or terminating the response, even in the continued presence of hormone. Intracellular signaling molecules—so-called second messengers (the first messenger of the signal is the ligand that binds to the receptor)—include small molecules such as cAMP, cGMP, Ca++, and diacylglycerol. Signaling pathways often include dozens of small molecules that form complicated networks within the cell (Fig. 3.7). Some proteins in the intracellular signaling pathways relay the signal by passing the message directly to another protein (e.g., by phosphorylating a target, or by binding and causing an allosteric change). Such intracellular signaling proteins act as reversible molecular switches:

When a signal is received, they switch from an inactive to an active form or vice versa, until another signaling molecule reverses the process. This principle of reversibility is central to many signaling pathways. In many cases, activation is achieved by reversing inhibition: For example, the thyroid hormone receptor is bound to an inhibitory protein in the absence of signal.

FIG. 3.7 Illustration of how intracellular signals are amplified and integrated. Signaling pathways often include dozens of proteins and small molecules that form complicated networks within the cell. Some signaling proteins relay the signal by passing the message to another protein. Many proteins amplify the

signal either by producing large amounts of additional signaling molecules or by activating a large number of downstream signaling proteins. Other proteins carry the signal from one region of the cell to another. See text for more details. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

Signaling complexes, composed of multiple proteins that interact physically, enhance the speed, efficiency, and specificity of signaling. Many proteins, usually enzymes or ion channels, transduce the signal into a different chemical form and simultaneously amplify the signal either by producing large amounts of additional signaling molecules or by activating a large number of downstream signaling proteins. For example, adenylyl cyclase, the enzyme that makes cAMP, transduces a signal (receptor activation of G proteins) and amplifies the signal by generating large amounts of cAMP. Other types of signaling proteins include those that integrate multiple signals. Other proteins carry the signal from one region of the cell to another: for example, by translocating from the cytosol to the nucleus. Cells can respond quickly and in a graded manner to increasing concentrations of hormone, and the effect of a signaling molecule can be either long- or short-lived. Cells can also adjust their sensitivity to a signal by desensitization, whereby prolonged exposure to a hormone decreases the cell’s response over time. Desensitization is a reversible process that can involve a reduction in the number of receptors expressed in the plasma membrane, inactivation of receptors, or changes in signaling proteins that mediate the downstream effect of the receptors. Homologous desensitization involves a reduction in the response only to the signaling molecule that caused the response (e.g., opioid dependence and tolerance), whereas heterologous desensitization is when one ligand desensitizes the response to another ligand. Table 3.1 summarizes the four general classes of receptors and provides a few examples of the signal transduction pathways associated with each class of receptors.

Ligand-Gated Ion Channel Signal Transduction Pathways This class of receptors transduces a chemical signal into an electrical signal, which elicits a response. For example, the ryanodine receptor, located in the membrane of the sarcoplasmic reticulum of skeletal muscle, is activated by Ca++, caffeine, adenosine triphosphate (ATP), or metabolites of arachidonic acid to release Ca++ into the cytosol, which facilitates muscle contraction (see Chapter 12 for details). In glutamatergic synapses in which high levels of prior synaptic activity have led to partial membrane depolarization, activation of the N-methyl-D-aspartate receptor by glutamate stimulates Ca++ influx important for synaptic plasticity.

G Protein–Coupled Signal Transduction Pathways There are two classes of GTP-binding proteins (i.e., GTPases, which are named for their ability to hydrolyze GTP to guanosine diphosphate [GDP] and an inorganic phosphate): low-molecular-weight, monomeric G proteins and heterotrimeric G proteins composed of α, β, and γ subunits. GTP binding activates, whereas hydrolysis of GTP to GDP inactivates, GTP-binding proteins (Fig. 3.8A). All GTPases are controlled by regulatory proteins, including GTPase-activating proteins, which induce the hydrolysis of GTP to GDP and thus inactivate the GTPase, and guanine nucleotide exchange factors (GEFs) that cause the GTPase to release GDP, which is rapidly replaced by GTP, thereby activating the GTPase (see Fig. 3.8B).

FIG. 3.8 GTP-binding proteins. GTP binding activates whereas hydrolysis of GTP to GDP inactivates GTP-binding proteins (A). All GTPases are controlled by regulatory proteins, including GTPase-activating proteins (GAP), which induce the hydrolysis of GTP to GDP, thus inactivating the GTPase, and guanine nucleotide exchange factors (GEF), which cause the GTPase to release GDP, which is rapidly replaced by GTP, thereby activating the GTPase (B). (Redrawn from Kantrowitx ER, Lipscomb WN. Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition. Trends Biochem Sci. 1990;15:53-59.)

Monomeric G proteins are composed of a single 20- to 40-kDa protein and can be membrane bound because of the addition of lipids post-translationally. Monomeric G proteins have been classified into five families (Ras, Rho, Rab, Ran, and Arf ), play a central role in many enzyme-linked receptor pathways, and regulate gene expression and cell proliferation, differentiation, and survival. Rho GTPases regulate actin cytoskeletal organization, cell cycle progression, and gene expression. The Rab GTPases regulate intravesicular transport and trafficking of proteins between organelles in the secretory and endocytic pathways. Ran GTPases regulate nucleocytoplasmic transport of RNA and proteins. Ras GTPases are involved in many signaling pathways that control cell division, proliferation, and death. Arf GTPases, like Rab GTPases, regulate vesicular transport. Heterotrimeric G proteins couple to more than 1000 different receptors and thereby mediate the cellular response to an incredibly diverse set of signaling molecules, including hormones, neurotransmitters, peptides, and odorants. Like monomeric G proteins, they can be membrane bound because of the addition of lipids post-translationally. Heterotrimeric complexes are composed of three subunits: α, β, and γ. There exist 16 α subunits, 5 β subunits, and 12 γ subunits, which can assemble into

hundreds of different combinations and thereby interact with a diverse number of receptors and effectors. The assembly of subunits and the association with receptors and effectors depend on the cell type. An overview of heterotrimeric G protein activation is illustrated in Fig. 3.9. In the absence of ligand, these G proteins are inactive and form a heterotrimeric complex in which GDP binds to the α subunit. Binding of a signal molecule to an inactive GPCR induces a conformational change in the G protein that promotes the release of GDP and the subsequent binding of GTP to the α subunit. Binding of GTP to the α subunit stimulates dissociation of the α subunit from the heterotrimeric complex and results in release of the α subunit from the βγ dimer, each of which can interact with and regulate downstream effectors such as adenylyl cyclase and phospholipases (see Fig. 3.9). Activation of downstream effectors by the α subunit and βγ dimer is terminated when the α subunit hydrolyzes the bound GTP to GDP and inorganic phosphate (Pi). The α subunit bound to GDP associates with the βγ dimer and terminates the activation of effectors.

FIG. 3.9 Activation of a G protein–coupled receptor (GPCR) and effector activation. In the absence of ligand, heterotrimeric G proteins are in an inactive state because GDP binds to the α subunit. Binding of a signal molecule to an inactive GPCR induces a conformational change in the G protein that promotes the release of GDP and the subsequent binding of GTP to the α subunit. Binding of GTP to the α subunit stimulates dissociation of the α subunit from the heterotrimeric complex and results in release of the α subunit from the βγ dimer, each of which can interact with and regulate downstream effectors. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

Another way to attenuate or terminate signaling through a GPCR involves desensitization and endocytic removal of receptors from the plasma membrane. Binding of hormone to a GPCR increases the ability of GPCR kinases to phosphorylate the intracellular domain of GPCRs, which recruits proteins called βarrestins to bind to the GPCR. β-Arrestins inactivate the receptor and promote endocytic removal of the GPCR from the plasma membrane. GPCR kinase/β-arrestin inactivation with endocytosis of GPCRs is an important mechanism whereby cells downregulate (desensitize) a response during prolonged exposure to elevated hormone levels. One of the major benefits of β blockers, given for congestive heart failure, is that they reverse chronic desensitization and the associated recovery of adrenergic responsiveness. Activated G protein α subunits couple to a variety of effector proteins, including adenylyl cyclase,

phosphodiesterases, and phospholipases (A2, C, and D). A very common downstream effector of heterotrimeric G proteins is adenylyl cyclase, which facilitates the conversion of ATP to cAMP (Fig. 3.10). When a signal molecule binds to a GPCR that interacts with a G protein composed of an α subunit of the αs class, adenylyl cyclase is activated, which causes an increase in cAMP levels and, as a result, activation of protein kinase A (PKA). By phosphorylating specific serine and threonine residues on downstream effector proteins, PKA regulates effector protein activity. In contrast, when a ligand binds to a receptor that interacts with a G protein composed of an α subunit of the αi class, adenylyl cyclase is inhibited, which causes reductions in cAMP levels and, consequently, in PKA activity.

FIG. 3.10 GPCR stimulation of adenylyl cyclase, cAMP, and protein kinase A (PKA). Binding of a signal molecule to a GPCR mediates Gs stimulation of adenylyl cyclase, which increases cytosolic cAMP, which in turn activates PKA. Activated PKA phosphorylates a number of target proteins to elicit many effects. PKA also enters the nucleus where it phosphorylates CREB (cyclic adenosine monophosphate [cyclic AMP] response element–binding protein). Phosphorylated CREB recruits coactivator CBP, which stimulates gene transcription. GPCR, G Protein–Coupled Receptor. (Redrawn from Alberts B, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

Some effector proteins, such as ion-gated channels, are also regulated directly by cAMP. cAMP is degraded to AMP by cAMP phosphodiesterases, which are inhibited by caffeine and other methylxanthines. Thus by interfering with a constitutive “off” signal, caffeine can prolong a cellular response mediated by cAMP and PKA. Because these effects target existing proteins, they can be

extremely rapid (e.g., adrenaline response). In addition to cytoplasmic signaling, the catalytic subunit of PKA can enter the nucleus of cells and phosphorylate and activate the transcription factor cAMP response element–binding (CREB) protein (see Fig. 3.10). Phospho-CREB protein increases the transcription of many genes, which can in turn produce a distinct set of responses with much slower kinetics. Hence, cAMP has many cellular effects, including direct and indirect effects mediated by PKA.

IN THE C LIN IC Cholera toxin, secreted by Vibrio cholera, catalyzes the ADP ribosylation of the G-protein αs subunit, which inhibits the GTPase activity of αs. Thus αs remains in its activated, GTP-bound state, which in turn causes activation of adenylyl cyclase and an increase in cAMP/PKA levels. In the intestines, elevated levels of PKA increase cystic fibrosis transmembrane conductance regulator (CFTR)– mediated chloride secretion, which leads to secretory diarrhea and extensive loss of fluids, characteristic of cholera. Bordetella pertussis, the bacterium that causes whooping cough, secretes pertussis toxin, which ADP ribosylates the αi subunit. In this case, the ribosylation inactivates αi, reducing the inhibition of adenylyl cyclase and thus also leading to increased levels of cAMP/PKA. Heterotrimeric G proteins also regulate phototransduction. In rod cells in the eye, absorption of light by rhodopsin activates the G protein transducin, which via the αt subunit activates cGMP phosphodiesterase. Activation of this phosphodiesterase lowers the concentration of cGMP and thereby closes a cGMPactivated cation channel. The ensuing change in cation channel activity alters the membrane voltage. The exquisite sensitivity of rods to light—rods can detect a single photon of light—is due to the abundance of rhodopsin in rods and amplification of the signal (a photon of light) by the G protein–cGMP phosphodiesterase–cGMP channel signaling pathway (see Chapter 8 for more details). Heterotrimeric G proteins also regulate phospholipases, a family of enzymes that modulate a variety of signaling pathways. Ligands that activate receptors that are coupled to the αq subunit stimulate phospholipase C, an enzyme that converts phosphatidylinositol 4,5-biphosphate to inositol 1,4,5triphosphate (InsP3) and diacylglycerol. InsP3 is a second messenger that diffuses to the endoplasmic reticulum, where it activates a ligand-activated Ca++ channel to release Ca++ into the cytosol, whereas diacylglycerol activates protein kinase C, which phosphorylates effector proteins. As noted earlier, both Ca++ and protein kinase C influence effector proteins, as well as other signaling pathways, to elicit responses. Ligand binding to GPCRs can also activate phospholipase A2, an enzyme that releases arachidonic acid from membrane phospholipids. Arachidonic acid, which can also be released from diacylglycerol via an indirect pathway, can be released from cells and thereby regulate neighboring cells or stimulate inflammation. It can also be retained within cells, where it is incorporated into the plasma membrane or is metabolized in the cytosol to form intracellular second messengers that affect the activity of enzymes and ion channels. In one pathway, cytosolic cyclooxygenases facilitate the metabolism of arachidonic acid to prostaglandins, thromboxanes, and prostacyclins. Prostaglandins mediate aggregation of platelets, cause constriction of the airways, and induce inflammation. Thromboxanes also induce platelet aggregation and constrict blood vessels, whereas prostacyclin inhibits platelet aggregation and causes dilation of blood vessels. In a second pathway of arachidonic acid metabolism, the enzyme 5-lipoxygenase initiates the conversion of arachidonic acid to leukotrienes, which participate in allergic and inflammatory responses, including those causing asthma, rheumatoid arthritis, and inflammatory bowel disease. The

third pathway of arachidonic acid metabolism is initiated by epoxygenase, an enzyme that facilitates the generation of hydroxyeicosatetraenoic acid (HETE) and cis-epoxyeicosatrienoic acid (cis-EET). HETE and cis-EET and their metabolites increase release of Ca++ from the endoplasmic reticulum, stimulate cell proliferation, and regulate inflammatory responses. Ca++ is also an intracellular messenger that elicits cellular effects via Ca++-binding proteins, most notably calmodulin (CaM). When Ca++ binds to CaM, its conformation is altered, and the structural change in CaM allows it to bind to and regulate other signaling proteins, including cAMP phosphodiesterase, an enzyme that degrades cAMP to AMP, which is inactive and unable to activate PKA. By binding to CaM-dependent kinases, CaM also phosphorylates specific serine and threonine residues in many proteins, including myosin light-chain kinase, which facilitates smooth muscle contraction (see Chapter 14).

Protein Phosphatases and Phosphodiesterases Counteract the Activation of Cyclic Nucleotide Kinases There are two ways to terminate a signal initiated by cAMP and cGMP: enhancing degradation of these cyclic nucleotides by phosphodiesterases and dephosphorylation of effectors by protein phosphatases. Phosphodiesterases facilitate the breakdown of cAMP and cGMP to AMP and GMP, respectively, and are activated by ligand activation of GPCRs. Phosphatases dephosphorylate effector proteins that were phosphorylated by kinases such as PKA. The balance between kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation allows rapid and exquisite regulation of the phosphorylated state and thus the activity of signaling proteins.

Enzyme Receptor–Linked Signal Transduction Pathways There are several classes of receptors that have enzymatic activity or are intimately associated with proteins that have enzymatic activity. Four of these classes are discussed next, including receptors that mediate the cellular responses to atrial natriuretic peptide (ANP) and nitric oxide (guanylyl cyclase receptors); transforming growth factor-β (TGF-β; threonine/serine kinase receptors); EGF, plateletderived growth factor (PDGF), and insulin (tyrosine kinase receptors); and interleukins (tyrosine kinase–associated receptors).

IN THE C LIN IC There are two isoforms of cyclooxygenase: COX1 and COX2. When activated in endothelial cells, COX1 facilitates the production of prostacyclins, which inhibit blood clots. In vascular smooth muscle cells and platelets, COX1 facilitates the production of thromboxane A2, which is prothrombotic (i.e., promotes blood clots). Thus cardiovascular health depends in part on the balance between prostacyclins and thromboxane A2, generated by distinct cell types. Low doses of aspirin, a nonsteroidal anti-inflammatory drug (NSAID), reduce thromboxane A2 production by platelets with little effect on endothelial prostacyclin production. Thus low-dose aspirin is antithrombotic (i.e., reduces blood clots). COX2 is activated by inflammatory stimuli. Thus the ability of NSAIDs (e.g., aspirin, ibuprofen, naproxen, acetaminophen, indomethacin) to suppress the inflammatory response is due to inhibition of COX2. Both COX1 and COX2 facilitate the production of prostanoids that protect the stomach. Several lines of evidence suggest that both COX1 and COX2 must be inhibited to elicit

damage to the gastrointestinal tract. Consequently, the negative effects of NSAIDs on the gastric mucosa (e.g., increased incidence of gastrointestinal bleeding) are most likely due to inhibition of COX1 and COX2 by these nonselective COX inhibitors. Selective COX2 inhibitors (e.g., Celecoxib, Rofecoxib) are very effective in selectively inhibiting COX2 and are used extensively to reduce the inflammatory response. Because COX2 inhibitors are thought to lack the negative effects elicited by NSAIDs on the gastrointestinal tract, their use has increased dramatically. However, in 2005, the U.S. Food and Drug Administration (FDA) announced that selective COX2 inhibitors, as well as nonselective NSAIDs, increase the risk for heart attacks and strokes and required that COX2-selective and nonselective NSAIDs carry a warning label on product packaging that highlighted the potential for the increased risk for adverse cardiovascular events and stroke. In addition, although much evidence suggests that COX2-selective inhibitors do not cause gastrointestinal bleeding, in 2005 the FDA also required the pharmaceutical industry to add to the warning label on COX2-selective drugs a caution about the potential for increased risk for gastrointestinal bleeding. In 2015, the FDA strengthened warnings that both COX2-selective and COX2-nonselective NSAIDs increase the risk of heart attacks and strokes.b

AT THE C ELLU LAR LEVEL Ras GTPases, monomeric G proteins, are involved in many signaling pathways that control cell division, proliferation, and death. Many mutations of proteins in the Ras signaling pathway are oncogenic (cancer causing) or inactivate tumor suppressors. Mutations in Ras genes that inhibit GTPase activity, as well as overexpression of Ras proteins as a result of transcriptional activation, lead to continuous cell proliferation, a major step in the development of cancer in many organs, including the pancreas, colon, and lungs. In addition, mutations in and overexpression of GEFs, which facilitate exchange of GTP for GDP, and GTPase-activating proteins, which accelerate GTP hydrolysis, may also be oncogenic (see Fig. 3.8B). Guanylyl Cyclase Receptors ANP binds to the extracellular domain of the plasma membrane receptor guanylyl cyclase and induces a conformational change in the receptor that causes receptor dimerization and activation of guanylyl cyclase, which metabolizes GTP to cGMP. cGMP activates cGMP-dependent protein kinase, which phosphorylates proteins on specific serine and threonine residues. In the kidney, ANP inhibits reabsorption of sodium and water by the collecting duct. Nitric oxide activates a soluble receptor guanylyl cyclase that converts GTP to cGMP, which relaxes smooth muscle. Because nitroglycerin increases blood concentrations of nitric oxide, which increases cGMP and thereby relaxes smooth muscle in coronary arteries, it has long been used to treat angina pectoris (i.e., chest pain caused by inadequate blood flow to heart muscle; see Chapter 17). Threonine/Serine Kinase Receptors The TGF-β receptor is a threonine/serine kinase that has two subunits. Binding of TGF-β to the type II subunit induces it to phosphorylate the type I subunit on specific serine and threonine residues, which in turn phosphorylates other downstream effector proteins on serine and threonine residues and thereby elicits cellular responses, including cell growth, cell differentiation, and apoptosis. Tyrosine Kinase Receptors

There are two classes of tyrosine kinase receptors. Nerve growth factor (NGF) receptors are typical examples of one class. Ligand binding to two NGF receptors facilitates their dimerization and thus enables the cytoplasmic tyrosine kinase domain of each monomer to phosphorylate and activate the other monomer. Once the other monomer is phosphorylated, the cytoplasmic domains can recruit GEFs such as growth factor receptor–bound protein 2 to the plasma membrane, which in turn activates Ras and downstream kinases that regulate gene transcription programs important for cell survival and proliferation. Activation of the insulin receptor (which is tetrameric and composed of two α and two β subunits) by insulin is an example of the other type of tyrosine kinase receptor. Binding of insulin to the α subunits produces a conformational change that facilitates interaction between the two α and β pairs. Binding of insulin to its receptor causes autophosphorylation of tyrosine residues in the catalytic domains of the β subunits, and the activated receptor then phosphorylates cytoplasmic proteins to initiate its cellular effects, including stimulating the absorption of glucose from the blood into skeletal muscle and fat tissue. Tyrosine Kinase–Associated Receptors The tyrosine kinase–associated receptors have no intrinsic kinase activity but associate with proteins that do have tyrosine kinase activity, including tyrosine kinases of the Src family and Janus family. Receptors in this class bind several cytokines, including interleukin-6, a proinflammatory cytokine that is necessary for resistance to bacterial infections, and erythropoietin, which stimulates the production of red blood cells. Tyrosine kinase–associated receptor subunits assemble into homodimers (αα), heterodimers (αβ), or heterotrimers (αβγ) when ligands bind. Subunit assembly enhances the binding of tyrosine kinases, which induces kinase activity and thereby phosphorylates tyrosine residues on the kinases, as well as on the receptor. Most polypeptide growth factors bind to tyrosine kinase–associated receptors.

Regulation of Gene Expression by Signal Transduction Pathways Steroid and thyroid hormones, cAMP, and receptor tyrosine kinases are transcription factors that regulate gene expression and thereby participate in signal transduction pathways. This section discusses the regulation of gene expression by steroid and thyroid hormones, cAMP, and receptor tyrosine kinases.

Nuclear Receptor Signal Transduction Pathways The family of nuclear receptors includes more than 30 genes and has been divided into two subfamilies on the basis of structure and mechanism of action: (1) steroid hormone receptors and (2) receptors that bind retinoic acid, thyroid hormones (iodothyronines), and vitamin D. When ligands bind to these receptors, the ligand-receptor complex activates transcription factors that bind to DNA and regulate the expression of genes (see Figs. 3.2B, 3.5, and 3.7). The location of nuclear receptors varies. Glucocorticoid and mineralocorticoid receptors are located in the cytoplasm, where they interact with chaperones (i.e., heat shock proteins; see Fig. 3.2B). Binding of hormone to these receptors results in a conformational change that causes chaperones to dissociate from the receptor, thereby revealing a nuclear localization motif that facilitates translocation of the hormonebound receptor complex to the nucleus. Estrogen and progesterone receptors are located primarily in the nucleus, and thyroid hormone and retinoic acid receptors are located in the nucleus bound to DNA. When activated by hormone binding, nuclear receptors bind to specific DNA sequences in the

regulatory regions of responsive genes called hormone response elements. Ligand-receptor binding to DNA causes a conformational change in DNA that initiates transcription. Nuclear receptors also regulate gene expression by acting as transcriptional repressors. For example, glucocorticoids suppress the transcription activator protein-1 (AP-1) and nuclear factor κB, which stimulate the expression of genes that cause inflammation. By this mechanism glucocorticoids reduce inflammation.

Cell-Surface Signal Transduction Pathways Control Gene Expression As noted previously, cAMP is an important second messenger. In addition to its importance in activating PKA, which phosphorylates specific serine and threonine residues on proteins, cAMP stimulates the transcription of many genes, including those that code for hormones, including somatostatin, glucagon, and vasoactive intestinal polypeptide (see Fig. 3.10). Many genes activated by cAMP have a cAMP response element (CRE) in their DNA. Increases in cAMP stimulate PKA, which not only acts in the cytoplasm but also can translocate to the nucleus, where it phosphorylates CREB and thereby increases its affinity for CREB-binding protein (CBP). The CREB-CBP complex activates transcription. The response is terminated when PKA phosphorylates a phosphatase that dephosphorylates CREB (see Fig. 3.10). Many growth factors, including EGF, PDGF, NGF, and insulin, bind to and activate enzyme-linked receptors that have tyrosine kinase activity. Activation of tyrosine kinases initiates a cascade of events that enhance the activity of the small GTP-binding protein Ras, which in a series of steps and intermediary proteins phosphorylates the mitogen-activated protein kinase, which then translocates to the nucleus and stimulates transcription of genes that stimulate cell growth. Tyrosine kinase–associated receptors, as noted earlier, are activated by a variety of hormones, including cytokines, growth hormone, and interferon. Although these receptors do not have tyrosine kinase activity, they are associated with Janus family proteins, which do have tyrosine kinase activity. Once activated, hormone tyrosine kinase–associated receptors activate Janus family protein, which phosphorylates latent transcription factors called signal transducers and activators of transcription (STATs). When phosphorylated on tyrosine residues, STATs dimerize and then enter the nucleus and regulate transcription.

Key Points 1. The function of cells is tightly coordinated and integrated by external chemical signals, including hormones, neurotransmitters, growth factors, odorants, and products of cellular metabolism that serve as chemical messengers and provide cell-to-cell communication. Chemical and physical signals interact with receptors located in the plasma membrane, cytoplasm, and nucleus. Interaction of these signals with receptors initiates a cascade of events that mediate the response to each stimulus. These pathways ensure that the cellular response to external signals is specific, amplified, tightly regulated, and coordinated. 2. There are two classes of GTP-binding proteins: monomeric G proteins and heterotrimeric G proteins composed of α, β, and γ subunits. Monomeric G proteins regulate actin cytoskeleton organization, cell cycle progression, intracellular vesicular transport, and gene expression. Heterotrimeric G proteins regulate ion channels, adenylyl cyclase and the cAMP-PKA signaling pathway, phosphodiesterases (which also regulate cAMP and cGMP signaling pathways), and

phospholipases, which regulate the production of prostaglandins, prostacyclins, and thromboxanes. 3. There are four subtypes of enzyme-linked receptors that mediate the cellular response to a wide variety of signals, including ANP, nitric oxide, TGF-β, PDGF, insulin, and interleukins. 4. There are two types of nuclear receptors: (1) one type that in the absence of ligand is located in the cytoplasm and when bound to ligand translocates to the nucleus and (2) another type that permanently resides in the nucleus. Both classes of receptors regulate gene transcription. aThe acetylcholine receptor in skeletal muscle is termed nicotinic because nicotine can mimic this action of the neurotransmitter.

In contrast, the acetylcholine receptor in cardiac muscle is termed muscarinic because this effect is mimicked by muscarine, an alkaloid derived from the mushroom Amanita muscaria. b

See U.S. Food and Drug Administration. FDA Drug Safety Communication: FDA Strengthens Warning That Non-aspirin Nonsteroidal Anti-inflammatory Drugs (NSAIDs) Can Cause Heart Attacks or Strokes 2015. Accessed July 9, 2022. http://www.fda.gov/Drugs/DrugSafety/ucm451800.htm

S E CT I ON 2

Neurophysiology Mark Yeckel

Chapter 4 The Nervous System: Introduction to Cells and Systems Chapter 5 Generation and Conduction of Action Potentials Chapter 6 Synaptic Transmission Chapter 7 The Somatosensory System Chapter 8 The Special Senses Chapter 9 Organization of Motor Function Chapter 10 Integrative Functions of the Nervous System Chapter 11 The Autonomic Nervous System and Its Central Control

C H AP T E R 4

The Nervous System: Introduction to Cells and Systems LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. What are the major cell types of the central and peripheral nervous systems? 2. What are the major components of a neuron, and what are their functional roles? 3. What are the functional roles of the major glial cell types? 4. What are the main divisions of the central nervous system? 5. How and where is the cerebrospinal fluid formed, and how does it circulate and exit the ventricular system? 6. How is axon transport related to the response of the axon to transection?

The nervous system is a communication and control network that allows an organism to interact rapidly and adaptively with its environment, where environment includes both the external environment (exteroceptive; the world outside the body) and the internal environment (interoceptive; the components and cavities of the body). To carry out its function the nervous system takes in sensory information from a variety of sources using specialized sensors (receptors), integrates this information with previously obtained information stored as memories and with the intrinsic goals and drives of the organism that have been embedded in its nervous system through evolution, decides on a course of action, and then issues commands to the effector organs (muscles and glands) to execute the chosen behavioral response. Moreover, almost all behavioral responses require the coordination of many body parts. For example, even a simple reaching movement of the arm may require coactivation of axial muscles and possibly muscles in the lower extremity to maintain posture and balance, which themselves may be monitored by up to three different sensory systems (vision, vestibular, and proprioceptive) whose information has to be integrated. Furthermore, movements can alter the internal environment and thus can require compensatory changes in heart and breathing rates, blood vessel diameters, and other internal processes. All these variables are monitored and controlled by various specialized subsystems of the nervous system, all of which must work together for the organism to perform movements and more generally to survive. The succeeding chapters will describe these major subsystems individually; however, it should be remembered that in reality their activity is integrated to generate normal behavior. To begin, it is useful to divide the nervous system into central and peripheral parts. The central nervous system (CNS) consists of the brain and spinal cord. The peripheral nervous system (PNS)

consists of nerves and ganglia (small groups of neurons) that innervate all parts of the body and provide an interface between the environment and the CNS. The transition between the CNS and PNS occurs on the dorsal and ventral rootlets near to where they emerge from the spinal cord and on the cranial nerve fibers near to where they arise from the brain.

Cellular Components of the Nervous System The nervous system is made up of cells, connective tissue, and blood vessels. The major cell types are neurons (nerve cells) and glia (neuroglia = “nerve glue”). In its most general form a neuron’s function can be defined as generation of signals (to be sent to other neurons or effector cells [e.g., muscle cells]) based on an integration of its own electrical properties with electrochemical signals from other neurons. The points where specific neuron-to-neuron communication occurs are known as synapses, and the process of synaptic transmission is critical to neuronal function (see Chapter 6). Neuroglia, or just glia, traditionally have been characterized as supportive cells that sustain neurons both metabolically and physically, isolate individual neurons from each other, and help maintain the internal milieu of the nervous system; however, it is now known that they also have important roles in shaping the flow of activity through the nervous system.

Neurons The typical neuron consists of three main cellular compartments: a cell body (also referred to as a perikaryon or soma), a variable number of processes that extend from the soma called dendrites and an axon (Fig. 4.1). A tremendous number of morphological variants of this basic template exist, including cases where dendrites or an axon may be absent (Fig. 4.2). These variations do not occur randomly but rather relate to the distinct functional properties of each neuronal class. Indeed, neurons with similar morphologies often characterize specific regions of the CNS and reflect the distinct neuronal processing performed in each CNS region.

FIG. 4.1 Schematic diagram of an idealized neuron and its major components and connections. A, Afferent input from axons of other cells terminates in synapses on the dendrites and cell body. The initial segment of the axon attaches at the axon hillock. This axon is myelinated, as indicated by the blue structures that encapsulate segments of the axon. The axon terminates on two postsynaptic neurons by forming synaptic terminals. B, Nodes of Ranvier are the gaps between the myelin segments where the axon membrane is exposed to the extracellular space. C, Higher-magnification view of synapse. (Redrawn from Blumenfeld H. Neuroanatomy Through Clinical Cases. 2nd ed. Sunderland, MA: Sinauer Associates; 2010.)

FIG. 4.2 A, Purkinje cell. B, Pyramidal cell. C, Golgi cell. D, Granule cell. E, Inferior olive cells. F, Bipolar cells. (A, Courtesy of Boris Barbour. B, Courtesy of T.F. Fletcher, from http://vanat.cvm.umn.edu/neurHistAtls/pages/neuron3.html. C, Figure was provided by Court Hull and Wade Regehr, Department of Neurobiology, Harvard Medical School. D, From Delvendahl I, Straub I, Hallermann S. Front Cell Neurosci 2015;9:93, Fig. 1A. E, From Mathy A, Clark BA. In: Manto M, Schmahmann JD, Rossi F, Gruol DL, Koibuchi N, eds. Handbook of the Cerebellum and Cerebellar Disorders. Dordrecht, Netherlands: Springer Science+Business Media Dordrecht; 2013. F, From Li W, DeVries SH. Nat Neurosci 2006;9:669-675, Fig. 2.)

The cell body is the main genetic and metabolic center of the neuron. Correspondingly it contains the nucleus and nucleolus of the cell and also possesses a well-developed biosynthetic apparatus for manufacturing membrane constituents, synthetic enzymes, and other chemical substances needed for the specialized functions of nerve cells. The neuronal biosynthetic apparatus includes Nissl bodies, which are stacks of rough endoplasmic reticulum, and a prominent Golgi apparatus. The soma also contains numerous mitochondria and cytoskeletal elements, including neurofilaments and microtubules. The cell body is also a region in which the neuron receives synaptic input (i.e., electrical and chemical signals from other neurons). Although quantitatively the synaptic input to the soma is usually much less than that to dendrites, it often differs qualitatively from dendritic inputs, and by virtue of the closeness of the soma to the axon, inputs to the soma can override those to the dendrites (see Chapter 6). Dendrites are tapering and branching extensions of the soma and are the main direct recipients of signals from other neurons. They can be thought of as a way to expand and specialize the surface area of a neuron, and indeed, they may account for more than 90% of the surface area available for synaptic contact (soma plus dendrites). Dendrites can be divided into primary dendrites (those that extend directly from the soma) and higher-order dendrites (daughter branches extending from a more proximal branch, in which proximal refers to closeness to the soma). The main cytoplasmic organelles in dendrites are microtubules, neurofilaments, and smooth endoplasmic reticulum; the primary dendrites can also contain

Nissl bodies and parts of the Golgi apparatus. A neuron’s set of dendrites is termed its dendritic tree. Dendritic trees differ tremendously between different types of neurons in terms of the size, number, and spatial organization of the dendrites. A dendritic tree can consist of just a few unbranched dendrites or of many highly ramified dendrites. Individual dendrites can be longer than 1 mm or only 10 to 20 µm in length. Another major morphological variation is whether or not a dendrite has spines, which are small mushroom- or lollipop-shaped protrusions from the main dendrite. Spines are sites specialized for synaptic contact (usually but not always) from excitatory inputs. The shape and size of the dendritic tree, as well as the population and distribution of channels in the dendritic membrane, are all important determinants of how the synaptic input will affect the neuron (see Chapter 6). The axon is an extension of the cell that conveys the output of the cell to other neurons or, in the case of a motor neuron, to muscle cells as well. In general, each neuron has only one axon, and it is usually of uniform diameter. The length and diameter of axons vary with the neuronal type. Some axons do not extend much beyond the length of the dendrites, whereas others may be a meter or more long. Axons may have orthogonal branches en passant, but they often end in a spray of branches called a terminal arborization (represented by the four terminal branches and their synaptic terminals in Fig. 4.1A). The size, shape, and organization of the terminal arborization determine which other cells it will contact. The first part of the axon is known as the initial segment and arises from the soma (or sometimes from a proximal dendrite) in a specialized region called the axon hillock. The axon differs from the soma and proximal dendrites in that it lacks rough endoplasmic reticulum, free ribosomes, and a Golgi apparatus. The initial segment is usually the site where action potentials (spikes) that are propagated down the axon are initiated (see Chapter 5). An axon may terminate in a synapse and/or it may make synapses along its length. Synapses will be described in detail in Chapter 6. Neurons are special because of their ability to control and respond to electricity. Moreover, the response and control mechanisms of each part of a neuron are distinct from those in other parts. This intraneuronal specialization is a consequence of the particular morphology and the ion channel composition of each part of the neuron. For example, dendrites have ligand-gated ion channels that allow neurons to respond to chemicals released by other neurons, and their characteristic branching pattern allows for integration of multiple input signals. In contrast the axon typically has a long length and high concentration of voltage-gated channels that allow it to convey electrical signals (action potentials) rapidly over long distances without alteration. Axonal Transport Because the soma is the metabolic engine of the neuron, substances needed to support axonal and synaptic function are synthesized there. These substances must be distributed to replenish secreted or inactivated materials along the axon and especially to the presynaptic terminals. Most axons are too long to allow efficient movement of substances from the soma to the synaptic endings by simple diffusion. Thus special axonal transport mechanisms have evolved to accomplish this task (Fig. 4.3). A consequence of this metabolic dependency is that axons degenerate when disconnected from the cell body, a fact that has been used by scientists tracing out neuronal pathways; they would cut an axonal pathway and then determine where the degenerating axons distal to the cut projected to.

FIG. 4.3 Axonal transport. Schematic of neuron and enlargement of axonal transport mechanism. Axonal transport depends on movement of material along transport filaments such as microtubules. Transported components attach to transport filaments by means of cross-bridges. Different objects are transported anterogradely (from cell body to axon terminal) and others retrogradely (toward the cell body). The direction of transport—retrograde and anterograde—is determined by specific proteins such as dynein and kinesin, respectively.

Several types of axonal transport exist. Membrane-bound organelles and mitochondria are transported relatively rapidly by fast axonal transport. Substances that are dissolved in cytoplasm (e.g., proteins) are moved by slow axonal transport. In mammals, fast axonal transport proceeds as rapidly as 400 mm/day, whereas slow axonal transport occurs at about 1 mm/day. Synaptic vesicles, which travel by fast axonal transport, can travel from the soma of a motor neuron in the spinal cord to a neuromuscular junction in a person’s foot in about 2.5 days. In comparison the movement of some soluble proteins over the same distance can take nearly 3 years. Axonal transport requires metabolic energy and involves calcium ions. Microtubules provide a system of guidewires along which membrane-bound organelles move (see Fig. 4.3). Organelles attach to microtubules through a linkage similar to that between the thick and thin filaments of skeletal muscle fibers. Ca++ triggers movement of the organelles along the microtubules. Special microtubule-associated motor proteins called kinesin and dynein are required for axonal transport. Axonal transport occurs in both directions. Transport from the soma toward the axonal terminals is called anterograde axonal transport. This process involves kinesin, and it allows replenishment of synaptic vesicles and enzymes responsible for the synthesis of neurotransmitters in synaptic terminals. Transport in the opposite direction, which is driven by dynein, is called retrograde axonal transport. This process returns recycled synaptic vesicle membrane to the soma for lysosomal degradation.

IN THE C LIN IC Certain viruses and toxins can be conveyed by axonal transport along peripheral nerves. For example, herpes zoster, the virus of chickenpox, invades dorsal root ganglion cells. The virus may be harbored by these neurons for many years. However, eventually the virus may become active because of a change in immune status. The virus may then be transported along the sensory axons to the skin, causing shingles, a very painful disease. Another example is the axonal transport of tetanus toxin. Clostridium tetani bacteria may grow in a dirty wound, and if the person had not been vaccinated against tetanus toxin, the toxin can be transported retrogradely in the axons of motor neurons. The toxin can escape into the extracellular space of the spinal cord ventral horn and block the synaptic receptors for inhibitory amino acids. This process can result in tetanic convulsions.

Glia The major nonneuronal cellular elements of the nervous system are the glia (Fig. 4.4). Glial cells in the human CNS outnumber neurons by 4- to 10-fold; there are as many as 1013 glia and 1012 neurons. Glial cells in the CNS include astrocytes, oligodendrocytes, microglia, and ependymal cells (see Fig. 4.4); in the PNS the glial cells are Schwann cells and satellite cells. Traditionally, glial cells were thought of as supportive cells, and consistent with that conception, their functions include regulation of the microenvironment and myelination of axons. Glial cells are now also recognized to be important determinants of the flow of signals through neuronal circuits based on their ability to modulate synaptic and nonsynaptic transmission, and their role in synaptogenesis and maintenance.

FIG. 4.4 Schematic representation of cellular elements in the CNS. Two astrocytes are shown ending on a soma and dendrites of a neuron. Astrocytes also contact the pial surface or capillaries or both. An oligodendrocyte provides the myelin sheaths for axons. Also shown are microglia and ependymal cells. N, Neuron. (Redrawn from Williams PL, Warwick R. Functional Neuroanatomy of Man. Edinburgh: Churchill Livingstone; 1975.)

Astrocytes (named for their star shape) help regulate the microenvironment of the CNS, both under normal conditions and in response to damage to the nervous system. Astrocytes have a cell body from which several main branches arise. Through repeated branching these main processes give rise to hundreds to thousands of branchlets. Astrocyte processes contact neurons and surround synaptic endings, isolating them from adjacent synapses and the general extracellular space. Astrocytes also have foot processes that contact the capillaries and connective tissue at the surface of the CNS, the pia mater (see Fig. 4.4). These foot processes may help mediate the entry of substances into the CNS. Astrocytes can actively take up K+ ions and neurotransmitters, which they metabolize, biodegrade, or slowly recycle back into the extracellular environment; astrocytes serve to buffer the extracellular environment of neurons with respect to both ions and neurotransmitters. The cytoplasm of astrocytes contains glial filaments that provide mechanical support for CNS tissue. After injury the astrocytes undergo a variety of changes to become reactive astrocytes. One example is a class of reactive astrocytes that act to form a glial scar around an area of focal damage, which segregates the damaged tissue and thereby allows inflammatory processes to act selectively at the site of damage, minimizing the impact on surrounding normal tissue. Astrocytes can also affect the properties of synaptic transmission, which is discussed in Chapter 6.

AT THE C ELLU LAR LEVEL Astrocytes are coupled to each other by gap junctions such that they form a syncytium through which small molecules and ions can redistribute along their concentration gradients or by current flow. When

normal neural activity gives rise to a local increase in extracellular [K+], this coupled network can enable spatial redistribution of K+ over a wide area via current flow in many astrocytes. Under conditions of hypoxia, such as might be associated with ischemia secondary to blockage of an artery (i.e., a stroke), [K+] in the extracellular space of a brain region can increase by a factor of as much as 20. This will depolarize neurons and synaptic terminals and result in release of transmitters such as glutamate, which will cause further release of K+ from neurons. The additional release only exacerbates the problem and can lead to neuronal death. Under such conditions, local astroglia will probably take up the excess K+ by K+-Cl− symport rather than by spatial buffering, because the elevation in extracellular [K+] tends to be widespread rather than local. Oligodendrocytes and Schwann cells are critical for the function of axons. Many axons are surrounded by a myelin sheath, which is a spiral multilayered wrapping of glial cell membrane (Fig. 4.5A,B). In the CNS, myelin is formed by the oligodendrocytes, whereas in the PNS Schwann cells form myelin. Myelin increases the speed and fidelity of action potential conduction, in part by restricting the flow of ionic current to small unmyelinated portions of the axon between adjacent glial cells, called nodes of Ranvier (see Chapter 5). Although both act to increase the speed of conduction, there are several important differences in the relationship between axons and either oligodendrocytes or Schwann cells. One major difference is that a single oligodendrocyte typically helps myelinate multiple axons in the CNS, whereas each Schwann cell helps myelinate only a single axon in the PNS. A second difference is that in the CNS, unmyelinated axons are bare, whereas in the PNS, unmyelinated axons are not. Rather, they are surrounded by Schwann cell processes; the Schwann cell, however, does not form a multilayered covering (i.e., myelin), but instead extends processes that surround parts of several axons (the Schwann cell with its set of unmyelinated axons is called a Remak bundle) (see Fig. 4.5C).

FIG. 4.5 Axonal/glial associations. A, Myelinated axons in the CNS. A single oligodendrocyte (G) emits several processes, each of which winds in a spiral fashion around an axon to form a myelin segment. The axon is shown in cutaway. The myelin from a single oligodendrocyte ends before the next wrapping from another oligodendrocyte. The bare axon between the myelinated segments is the node of Ranvier (N). B, Electron micrograph of myelinated axon in the PNS shown in cross section. The axon (Ax) is seen at the center within a sheath consisting of multiple wrappings of the Schwann cell’s cytoplasmic membrane. The Schwann cell soma (SC) is at the upper right. C, Electron micrograph of unmyelinated axons in PNS. Nine axons (asterisks) cut in cross section are seen embedded in a Schwann cell whose nucleus is at the center (N). At lower right a portion of a myelinated axon is visible. (B, From Peters A, Palay S, Webster H. The Fine Structure of the Nervous System. New York: Oxford University Press; 1991, Fig. 6.5. C, From Pannese E. Neurocytology. 2nd ed. Basel, Switzerland: Springer International; 2015.)

Satellite cells encapsulate dorsal root and cranial nerve ganglion cells and regulate their microenvironment in a fashion similar to that of astrocytes. Microglia are derived from erythromyeloid stem cells that migrate into the CNS early in development. They play an important role in immune responses within the CNS. When the CNS is damaged, microglia

help remove the cellular products of the damage by phagocytosis. They are assisted by other glia and by other phagocytes that invade the CNS from the circulation. In addition to their role in immune responses, recent evidence suggests they are also active in healthy brain tissue and may have important roles in normal brain development and function, including pruning of excess synapses that are formed during development and synaptic plasticity. Ependymal cells form the epithelium lining the ventricular spaces of the brain, which contain cerebrospinal fluid (CSF). CSF is secreted by specialized ependymal cells of the choroid plexuses located in the ventricular system. Many substances diffuse readily across the ependyma, which lies between the extracellular space of the brain and the CSF.

IN THE C LIN IC Most neurons in the adult nervous system are postmitotic cells (although some stem cells may also remain in certain sites in the brain). Many glial precursor cells are present in the adult brain, and they can still divide and differentiate. Thus the cellular elements that give rise to most intrinsic brain tumors in the adult brain are the glial cells. For example, brain tumors can be derived from astrocytes (which vary in malignancy from the slowly growing astrocytoma to the rapidly fatal glioblastoma multiforme), from oligodendroglia (oligodendroglioma), or from ependymal cells (ependymoma). Meningeal cells can also give rise to slowly growing tumors (meningiomas) that compress brain tissue, as can Schwann cells (e.g., acoustic schwannomas, which are tumors formed by Schwann cells of the eighth cranial nerve). In the brain of infants, neurons that are still dividing can sometimes give rise to neuroblastomas (e.g., of the roof of the fourth ventricle) or retinoblastomas (in the eye).

The Peripheral Nervous System The PNS provides an interface between the environment and the CNS, both for sensory information flowing to the CNS and for motor commands issued from the CNS. It includes sensory (or primary afferent) neurons, somatic motor neurons, and autonomic motor neurons. Sensory pathways into the nervous system start with a receptor, which may simply be a specialized part of an axon in the PNS or may include additional cells. Each sensory receptor is organized so that it transduces a specific type of energy into an electrical signal, and can be classified in terms of the type of energy they transduce (e.g., photoreceptors transduce light, mechanoreceptors transduce displacement and force). They may also be classified according to the source of the input (e.g., exteroceptors signal external events, proprioceptors signal the state of a body part such as the angle of elbow, and interoceptors signal the distension of the gut). The transduction process leads to an electrical response in the primary afferent called a receptor potential, which triggers action potentials in the primary afferent fibers innervating the receptor. These action potentials contain information about the sensory stimulus that is conveyed to the CNS via the primary afferent. Somatic and autonomic motor neurons convey signals from the CNS to their respective effector targets. The somatic motor neurons innervate the skeletal muscles throughout the body. Their cell bodies lie in the ventral horn (or equivalent brainstem nuclei) and project out of the CNS via a ventral root or cranial nerve. The details of their relationship to muscles are covered in Chapter 9. The autonomic motor pathway is responsible for controlling the functioning of organs, smooth muscle, and glands. It is actually a two-neuron pathway, and its properties are covered in Chapter 11.

The Central Nervous System The CNS is built from the cellular elements just described and includes the spinal cord and brain (Fig. 4.6A). These cellular elements are connected in a variety of complex ways to form the subsystems that underlie the multitude of functions performed by the CNS. The physiology of these systems is covered in Chapters 7 through 11; however, a basic knowledge of CNS anatomy is needed to understand systems physiology and will be briefly discussed here.

FIG. 4.6 A, Schematic of the major components of the CNS as shown in a longitudinal midline view. B–F, Representative sections through the brain and spinal cord, with the major landmarks labeled. B, Cerebrum and thalamus; C, midbrain; D, pons; E, medulla; F, cervical spinal cord. Note that many pathways (e.g., corticospinal fibers) cross sides (decussate) as they travel through the CNS, but these decussations are not indicated in the figure (see Chapters 7 and 9 for details on the motor and sensory pathway crossings). (A, From Haines DE. Fundamental Neuroscience for Basic and Clinical Applications. 3rd ed. Philadelphia: Churchill Livingstone; 2006.)

Regions of the CNS containing high concentrations of axon pathways (and very few neurons) are called white matter because the axonal myelin sheaths of the axons are highly refractive to light. Regions containing high concentrations of neurons and dendrites are by contrast called gray matter. Note that axons are also present in gray matter. These axons may be related to local processing (i.e., either originating from local neurons or terminating on them) or may be fibers of passage. Thus effects of damage to an area may result in loss of local function and/or disconnection of remote regions that had been linked by fibers of passage through the area that was damaged. In the CNS, axons often travel in bundles or tracts. The names applied to tracts usually describe their origin and termination. For example, the spinocerebellar tracts convey information from the spinal cord to the cerebellum. The term pathway is similar to tract but is generally used to suggest a particular function (e.g., the auditory pathway: a series of neuron-to-neuron links across several synapses that convey and process auditory information). Gray matter exists in two main configurations in the CNS. A nucleus is a group of neurons in the CNS (in the PNS such a grouping is called a ganglion). Examples include the thalamic, cerebellar, and cranial nerve nuclei. A cortex is neurons that are organized into layers and usually found on the surface of the CNS. The most prominent are the cerebral and cerebellar cortices, which cover the surface of the cerebral hemispheres and the cerebellum, respectively (Fig. 4.7).

FIG. 4.7 Lateral view of the human brain showing the left cerebral hemisphere, cerebellum, pons, and medulla. Note the division of the lobes of the cerebrum (frontal, parietal, occipital, and temporal) and the two major fissures (lateral and central). (From Nolte J, Angevine J. The Human Brain in Photographs and Diagrams. 2nd ed. St Louis: Mosby; 2000.)

In most nuclei and cortices, one can classify neurons into two broad categories: projection cells and local interneurons. Projection cells are neurons that send their axon to another region and thus are the origins of the various tracts of the nervous system. In contrast, local interneurons have axons that terminate in the same neural structure as their cell of origin and are involved with local computations rather than conveying signals from one region to another. These categories are not exclusive; many neurons have axons that both give off local branches and project to one or more distant regions.

Regional Anatomy of the CNS The spinal cord can be subdivided into a series of regions (see Fig. 4.6A), each composed of a number of segments named for the vertebrae where their nerve roots enter or leave: 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal. Each portion maintains its tubular appearance. Within the gray matter the dorsal horn receives and processes sensory information from the dorsal roots, whereas the ventral horn is primarily a motor structure and contains the motor neurons whose axons project out via the ventral roots (Fig. 4.8).

FIG. 4.8 Diagram of the spinal cord, spinal roots, and spinal nerve. The spinal nerve begins where the dorsal and ventral roots fuse, and has multiple branches (rami), the first few of which are represented. A primary afferent neuron is shown with its cell body in the dorsal root ganglion and its central and peripheral processes distributed, respectively, to the spinal cord gray matter and to a sensory receptor in the skin. An α motor neuron is shown to have its cell body in the spinal cord gray matter and to project its axon out the ventral root to innervate a skeletal muscle fiber.

The surrounding white matter consists of many tracts interconnecting spinal cord levels and for communication with the brain. Three major ones are the lateral corticospinal tract (motor), spinothalamic tract/anterolateral system (sensory), and dorsal column-medial lemniscus pathway (sensory) (see Fig. 4.6F). The brainstem consists of the medulla, pons, and midbrain (Fig. 4.9; also see Fig. 4.6). In addition to the longitudinal pathways interconnecting with the spinal cord, the brainstem contains nuclei and many additional pathways that vary by level. These structures have many functions, some of which are analogous to those of the spinal cord (e.g., conveying basic sensory information and motor commands) and others related to a variety of other brain functions, such as cardiac control and state of consciousness. The brainstem also receives input and sends motor output via cranial nerves (Table 4.1).

FIG. 4.9 Midsagittal view of the brain showing the third and fourth ventricles, the cerebral aqueduct of the midbrain, and the choroid plexus. The CSF formed by the choroid plexus in the lateral ventricles enters this circulation via the interventricular foramen. Note also the location of the corpus callosum and other commissures. (From Haines DE, Fundamental Neuroscience for Basic and Clinical Applications. 3rd ed. Philadelphia: Churchill Livingstone; 2006.)

Table 4.1 Parts and Functions of the Central Nervous System Region

Nerves (Input/Output)

General Functions of the Region

Spinal cord

Dorsal/ventral roots

Sensory input, reflex circuits, somatic and autonomic motor output

Medulla

Cranial nerves VIII–XII

Cardiovascular and respiratory control, auditory and vestibular input, brainstem reflexes

Pons

Cranial nerves V–VIII

Respiratory/urinary control, control of eye movement, facial sensation/motor control

Cerebellum

Cranial nerve VIII

Motor coordination, motor learning, equilibrium

Midbrain

Cranial nerves III–IV

Acoustic relay and mapping, control of the eye (including movement, lens and pupillary reflexes), pain modulation

Thalamus

Cranial nerve II

Sensory and motor relay to the cerebral cortex, regulation of cortical activation, visual input

Hypothalamus

Autonomic and endocrine control, motivated behavior

Basal ganglia



Shape patterns of thalamocortical motor inhibition

Cerebral cortex

Cranial nerve I

Sensory perception, cognition, learning and memory, motor planning and voluntary movement, language

The cerebellum sits dorsal to the pons and medulla. It receives inputs from spinal cord, brainstem, and cerebral cortex and projects back to many of these same structures. The cerebellum is critical for motor coordination and motor learning, and is increasingly recognized as having key roles in other cognitive function and behavior. The thalamus sits at the upper end of the brainstem and is enclosed by the cerebrum with which it is highly interconnected (see Fig. 4.6B). With a few exceptions, ascending information first reaches the thalamus, which conveys it to the cerebral cortex. These structures play a major role in many functions, including conscious awareness, volition, memory, and language. In addition to the cortex, the cerebrum contains a group of deep nuclei, the basal ganglia, that are interconnected with the cortex and thalamus and whose function will be described in Chapter 9. The major functions of the different parts of the CNS are listed in Table 4.1.

Cerebrospinal Fluid CSF fills the ventricular system, a series of interconnected spaces within the brain, and the subarachnoid space directly surrounding the brain. The intraventricular CSF reflects the composition of the brain’s extracellular space via free exchange across the ependyma, and the brain “floats” in the subarachnoid CSF to minimize the effect of external mechanical forces. The volume of CSF within the cerebral ventricles is approximately 30 mL, and that in the subarachnoid space is about 125 mL. Because about 0.35 mL of CSF is produced each minute, CSF is turned over more than three times daily. CSF is a filtrate of capillary blood formed largely by the choroid plexuses, which comprise pia mater,

invaginating capillaries, and ependymal cells specialized for transport. The choroid plexuses are located in the lateral, third, and fourth ventricles (see Fig. 4.9). CSF flows through apertures or formina between the ventricles. The lateral ventricles are situated within the two cerebral hemispheres. They each connect with the third ventricle through the interventricular foramina (of Monro). The third ventricle lies in the midline between the diencephalon on the two sides. The cerebral aqueduct (of Sylvius) traverses the midbrain and connects the third ventricle with the fourth ventricle. The fourth ventricle is a space defined by the pons and medulla below and the cerebellum above. The central canal of the spinal cord continues caudally from the fourth ventricle, although in adult humans the canal is not fully patent and continues to close with age. CSF exits the ventricular system through three foramina (a medial foramen of Magendie and two lateral foramina of Luschka) located in the roof of the fourth ventricle. After leaving the ventricular system, CSF circulates through the subarachnoid space that surrounds the brain and spinal cord. Regions where these spaces are expanded are called subarachnoid cisterns. An example is the lumbar cistern, which surrounds the lumbar and sacral spinal roots below the level of termination of the spinal cord. The lumbar cistern is the target for lumbar puncture, a clinical procedure to sample CSF. A large part of CSF is removed by bulk flow through the valvular arachnoid granulations into the dural venous sinuses in the cranium. Because the extracellular fluid within the CNS communicates with the CSF, the composition of the CSF is a useful indicator of the composition of the extracellular environment of neurons in the brain and spinal cord. The main constituents of CSF in the lumbar cistern are listed in Table 4.2. For comparison, the concentrations of the same constituents in blood are also given. CSF has a lower concentration of K+, glucose, and protein but a greater concentration of Na+ and Cl− than blood does. Furthermore, CSF contains practically no blood cells. The increased concentration of Na+ and Cl− enables CSF to be isotonic to blood. Table 4.2 Constituents of Cerebrospinal Fluid and Blood Constituent

Lumbar CSF

Blood

Na (mEq/L)

148

136–145

K+ (mEq/L)

2.9

3.5–5

Cl− (mEq/L)

120–130

100–106

Glucose (mg/dL)

50–75

70–100

Protein (mg/dL)

15–45

6.8 × 103

pH

7.3

7.4

+

From Willis WD, Grossman RG. Medical Neurobiology. 3rd ed. St Louis: Mosby; 1981.

The pressure in the CSF column is about 120 to 180 mm H2O when a person is recumbent. The rate at which CSF is formed is relatively independent of the pressure in the ventricles and subarachnoid space, as well as systemic blood pressure. However, the absorption rate of CSF is a direct function of CSF pressure.

IN THE C LIN IC Obstruction of the circulation of CSF leads to increased CSF pressure and hydrocephalus, an abnormal accumulation of fluid in the cranium. In hydrocephalus the ventricles become distended, and if the increase in pressure is sustained, brain substance is lost. When the obstruction is within the ventricular system or in the foramina of the fourth ventricle, the condition is called a noncommunicating hydrocephalus. If the obstruction is in the subarachnoid space or the arachnoid villi, it is known as a communicating hydrocephalus.

The Blood-Brain Barrier The local environment of most CNS neurons is controlled such that neurons are normally protected from extreme variations in the composition of the extracellular fluid that bathes them. Part of this control is provided by the presence of a blood-brain barrier (other mechanisms are the buffering functions of glia, regulation of CNS circulation, and exchange of substances between the CSF and extracellular fluid of the CNS). Movement of large molecules and highly charged ions from blood into the brain and spinal cord is severely restricted. The restriction is at least partly due to the barrier action of the capillary endothelial cells of the CNS and the tight junctions between them. Astrocytes may also help limit the movement of certain substances. For example, astrocytes can take up potassium ions and thus regulate [K+] in the extracellular space. Some pharmaceutical agents, such as penicillin, are removed from the CNS by transport mechanisms.

IN THE C LIN IC The blood-brain barrier can be disrupted by pathology of the brain. For example, brain tumors may allow substances that are otherwise excluded to enter the brain from the circulation. Radiologists can exploit this by introducing a substance into the circulation that normally cannot penetrate the bloodbrain barrier. If the substance can be imaged, its leakage into the region occupied by the brain tumor can be used to demonstrate the distribution of the tumor.

Nervous Tissue Reactions to Injury Injury to nervous tissue elicits responses by neurons and glia. Severe injury causes cell death. Except in specific instances, once a neuron is lost, it cannot be replaced because, in general, neurons are postmitotic cells. In animals, two exceptions are olfactory bulb and hippocampal neurons; however, in humans, only for the hippocampus has evidence been found for significant levels of neurogenesis in the adult CNS.

Degeneration When an axon is transected, the soma of the neuron may show chromatolysis, or “axonal reaction.” Normally, Nissl bodies stain well with basic aniline dyes, which attach to the RNA of ribosomes (Fig. 4.10A). After injury to the axon (see Fig. 4.10B), the neuron attempts to repair the axon by making new structural proteins, and the cisterns of the rough endoplasmic reticulum become distended with the products of protein synthesis. The ribosomes appear to be disorganized, and the Nissl bodies are stained weakly by basic aniline dyes. This process, called chromatolysis, alters the staining pattern (see Fig.

4.10C). In addition, the soma may swell and become rounded, and the nucleus may assume an eccentric position. These morphological changes reflect the cytological processes that accompany increased protein synthesis.

FIG. 4.10 A, Normal motor neuron innervating a skeletal muscle fiber. B, A motor axon has been severed, and the motor neuron is undergoing chromatolysis. C, This is associated in time with sprouting and, in D, with regeneration of the axon. The excess sprouts degenerate. E, When the target cell is reinnervated, chromatolysis is no longer present.

Because it cannot synthesize new protein, the axon distal to the transection dies (see Fig. 4.10C). Within a few days the axon and all the associated synaptic endings disintegrate. If the axon had been a myelinated axon in the CNS, the myelin sheath would also fragment and eventually be removed by phagocytosis. However, in the PNS the Schwann cells that had formed the myelin sheath remain viable, and in fact they undergo cell division. This sequence of events was originally described by Waller and is called Wallerian degeneration. If the axons that provide the sole or predominant synaptic input to a neuron or to an effector cell are interrupted, the postsynaptic cell may undergo transneuronal degeneration and even death. The bestknown example of this is atrophy of skeletal muscle fibers after their innervation by motor neurons has been interrupted. However, if only one or a few of the innervating axons are removed, the other surviving axons may sprout additional terminals, thereby taking up the synaptic space of the damaged axons and increasing their influence on the postsynaptic cell.

Regeneration

In the PNS, after an axon is lost through injury, many neurons can regenerate a new axon. The proximal stump of the damaged axon develops sprouts (see Fig. 4.10C), these sprouts elongate, and they grow along the path of the original nerve if this route is available (see Fig. 4.10D). The Schwann cells in the distal stump of the nerve not only survive the Wallerian degeneration but also proliferate and form rows along the course previously taken by the axons. Growth cones of the sprouting axons find their way along these rows of Schwann cells, and they may eventually reinnervate the original peripheral target structures (see Fig. 4.10E). The Schwann cells then remyelinate the axons. The rate of regeneration is limited by the rate of slow axonal transport to about 1 mm/day. In the CNS, transected axons also sprout. However, proper guidance for the sprouts is lacking, in part because the oligodendroglia do not form a path along which the sprouts can grow. This limitation may be a consequence of the fact that a single oligodendrocyte myelinates many central axons, whereas a single Schwann cell provides myelin for only a single axon in the periphery. In addition, different chemical signals may affect peripheral and central attempts at regeneration differently. Other obstacles to successful CNS regeneration include formation of a glial scar by astrocytes and lack of trophic influences that guided axonal trajectories during development.

Key Points 1. The functions of the nervous system include excitability, sensory detection, information processing, and behavior. 2. The CNS includes the spinal cord and brain. The brain includes the medulla, pons, cerebellum, midbrain, thalamus, hypothalamus, basal ganglia, and cerebral cortex. 3. The neuron is the functional unit of the nervous system. Neurons have three major compartments: the dendrites, cell body, and axon. The first two receive and integrate signals, and the axon conveys the output signals of the neuron to other cells. 4. The PNS includes primary afferent neurons and the sensory receptors they innervate, the axons of somatic motor neurons, and autonomic neurons. 5. Information is conveyed through neural circuits by action potentials in the axons of neurons and by synaptic transmission between axons and the dendrites and somas of other neurons or between axons and effector cells. 6. Different types of neurons are specialized as a consequence of their individual morphology and the ion channel distribution in the cell membrane of their soma, dendrites, and axons. 7. Sensory receptors include exteroceptors, interoceptors, and proprioceptors. Stimuli are environmental events that excite sensory receptors, responses are the effects of stimuli, and sensory transduction is the process by which stimuli are detected by transforming their energy into electrical signals. 8. Sensory receptors can be classified in terms of the type of energy they transduce or by the source of the input. Central pathways are usually named by their origin and termination or for the type of information conveyed. 9. Chemical substances are distributed along the axons by fast or slow axonal transport. The direction of axonal transport may be anterograde or retrograde. 10. Glial cells include astrocytes (regulate the CNS microenvironment), oligodendroglia (form CNS myelin), Schwann cells (form PNS myelin), ependymal cells (line the ventricles), and microglia (CNS macrophages). Myelin sheaths increase the conduction velocity of axons.

11. The ependymal cells of choroid plexus produce CSF. CSF differs from blood in having a lower concentration of K+, glucose, and protein and a higher concentration of Na+ and Cl−; CSF normally lacks blood cells. 12. The extracellular fluid composition of the CNS is regulated by CSF, the blood-brain barrier, and astrocytes. 13. Damage to the axon of a neuron causes an axonal reaction (chromatolysis) in the cell body and Wallerian degeneration of the axon distal to the injury. Regeneration of PNS axons is more likely than regeneration of CNS axons.

C H AP T E R 5

Generation and Conduction of Action Potentials LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. How is a nerve membrane’s response to small-amplitude stimuli like a passive electric circuit comprising batteries, resistors, and capacitors? 2. What factors determine the time and length constants of a nerve membrane? How do these constants shape the electric responses of the nerve membrane? 3. How does an action potential differ from the subthreshold responses of a membrane (i.e., the passive and local responses)? 4. What is the sequence of conductances that underlies the action potential? 5. How are the responses of Na+ and K+ channels to membrane depolarization similar? How does the presence of the Na+ channel inactivation gate cause the responses to differ? 6. How do the gating properties of Na+ and K+ channels relate to the absolute and relative refractory periods of the action potential? 7. How is the action potential propagated without decrement? What are the factors that determine its propagation velocity? 8. What are the structural properties of myelin that underlie its ability to increase conduction velocity? 9. Given the all-or-none nature of action potentials, how are the characteristics of different stimuli distinguished by the central nervous system?

An action potential is a rapid, all-or-none change in the membrane potential, followed by a return to the resting membrane potential. This chapter describes how action potentials are generated by voltagedependent ion channels in the plasma membrane and propagated with the same shape and size along the length of an axon. The influences of axon geometry, ion channel distribution, and myelin on action potentials are discussed and explained. The ways in which information is encoded by the frequency and pattern of action potentials in individual cells and in groups of nerve cells are also described. Finally, because the nervous system provides important information about the external world through specific sensory receptors, general principles of sensory transduction and coding are introduced. More detailed information about these sensory mechanisms and systems is provided in other chapters.

Membrane Potentials

Observations on Membrane Potentials When a sharp microelectrode (tip diameter, 52



TRPV3

34–38

Activated by camphor

TRPV4

27–40



TRPM8

3000 kDa) that extends from the Z line to the center of the sarcomere (see Fig. 12.3C), and appears to be important for the organization and alignment of the thick filaments in the sarcomere. Some forms of muscular dystrophy have been attributed to defects in titin (i.e., titinopathies). Additional proteins found in the thick filaments (e.g., myomesin and C protein) may also participate in the bipolar organization or packing of the thick filament (or both). The cytoskeleton (including the intermediate filament protein desmin) participates in the highly organized alignment of sarcomeres. Desmin extends from the Z lines of adjacent sarcomeres to the

integrin protein complexes on the sarcolemma and thus participates in both the alignment of sarcomeres across muscles and the lateral transmission of force (described later in this section). Defects in desmin have been associated with myofibrillar myopathies. The force of contraction is transmitted both longitudinally to the tendon (via myotendinous junctions) and laterally to connective tissue adjacent to the muscle fibers (via costameres). The myotendinous junction represents a specialized region where the muscle fiber connects to the tendon (Fig. 12.4A,B). Folding of the sarcolemma at the myotendinous junction results in an interdigitation of the tendon with the end of the muscle fiber, which increases the contact area between the muscle fiber and the connective tissue and hence reduces the force per unit area at the end of the muscle fiber.

FIG. 12.4 The force of contraction of the muscle fiber is transmitted both longitudinally to the tendon (at the myotendinous junction) and laterally to adjacent extracellular connective tissue (at costameres). The force of contraction is transmitted from the end of the muscle fiber (M) to the tendon by connections with numerous collagen fibers (A, tip of arrow). Folds in the sarcolemma at the end of the muscle fiber (B) result in an interdigitation of the muscle fiber with the tendon, and represents the myotendinous junction. Costameres are located on the sides of the muscle fibers, and represent the bridges between the Z lines in the subsarcolemmal myofibrils and the extracellular connective tissue (C). Costameres facilitate the lateral transmission of force of contraction, which helps stabilize the sarcolemma. DGC, Dystrophinassociated glycoprotein complex. (A and B, From Tidball JG. Myotendinous junction: morphological changes and mechanical failure associated with muscle cell atrophy. Exp Molec Pathol 1984;40:1-12. C, From Hughes D, Wallace M, Baar K. Effects of aging, exercise, and disease on force transfer in skeletal muscle. Am J Physiol Endocrin Metab 2015;309:E1-E10.)

Lateral transmission of the force of contraction involves costameres, which link the Z lines of subsarcolemmal sarcomeres to extracellular matrix through a series of proteins (see Fig. 12.4C). The lateral transmission of force is thought to stabilize the sarcolemma and to protect it from damage during contraction. Defects in the myotendinous junction and/or costameres (which includes the dystrophinglycoprotein complex) have been associated with some forms of muscular dystrophy. The myotendinous junction and costameres also contain signaling molecules.

AT THE C ELLU LAR LEVEL The muscular dystrophies constitute a group of genetically determined degenerative disorders.

Duchenne’s muscular dystrophy (described by G.B. Duchenne in 1861) is the most common of the muscular dystrophies and affects 1 per 3500 boys (3–5 years of age). Severe muscle wasting occurs, and most affected patients are wheelchair bound by the age of 12; many die of respiratory failure in adulthood (30–40 years of age). Duchenne’s muscular dystrophy is an X-linked recessive disease that has been linked to a defect in the dystrophin gene that leads to a deficiency of the dystrophin protein in skeletal muscle, brain, retina, and smooth muscle. Dystrophin is a large (427-kDa) protein that is present at low levels (0.025%) in skeletal muscle. It is localized on the intracellular surface of the sarcolemma in association with several integral membrane glycoproteins (forming a dystrophinglycoprotein complex; Figs. 12.4C and 12.5A). This dystrophin-glycoprotein complex provides a structural link between the subsarcolemmal cytoskeleton of the muscle cell and the extracellular matrix and appears to stabilize the sarcolemma and hence prevent contraction-induced injury (rupture). The dystrophin-glycoprotein complex may also serve as a scaffold for cell signaling cascades. The enzyme nitric oxide synthase is present in the dystrophin-glycoprotein complex.

FIG. 12.5 A, Organization of the dystrophin-glycoprotein complex in skeletal muscle. The dystrophinglycoprotein complex provides a structural link between the cytoskeleton of the muscle cell and the extracellular matrix, which appears to stabilize the sarcolemma and hence prevents contractioninduced injury (rupture). Duchenne’s muscular dystrophy is associated with loss of dystrophin. Numbers in dystrophin indicate hinge regions (e.g., H1, H2) and spectrin-like repeat domains (e.g., 4, 8, 12). ABD, Actin-binding domain; C, carboxy terminus; CC, coiled-coil domain; DBD, dystroglycanbinding domain; N, amino terminus; nNOSµ, neuronal nitric oxide synthase µ; SBS, syntrophin-binding site; SSPN, sarcospan; Syn, syntrophin. Electron micrographs of a longitudinal view (B) and a crosssectional view (C) show the distribution of dystrophin in skeletal muscle of a normal patient (CTRL). Another cross-sectional view (D) shows the loss of dystrophin from skeletal muscle in a patient with Duchenne’s muscular dystrophy (DMD). (A, From Allen D, Whitehead N, Froehner S. Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy. Physiol Rev 2016;96:253-305. B, From Anastasi G, Cutroneo G, Santoro G, et al. Costameric proteins in human skeletal muscle during muscular inactivity. J Anat 2008;213:284-295. C and D, From Beekman C, Sipkens J, Testerink J, et al. A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with Duchenne muscular dystrophy. PLoS One 2014;9[9]:e107494.)

Although defects in the dystrophin-glycoprotein complex are involved in many forms of muscular dystrophy, some forms of muscular dystrophy that involve other mechanisms have been identified. Specifically, a defect in sarcolemma repair (attributed to loss/mutation of the protein dysferlin) appears to underlie at least one form of muscular dystrophy (limb-girdle muscular dystrophy 2B, associated with muscle wasting in the pelvic region). Defects in the protein titin (titinopathies) have been implicated in other forms of muscular dystrophy (e.g., limb-girdle muscular dystrophy 2J and tibial muscular dystrophy). Mutations in the protease calpain 3 (resulting in loss of protease activity) have also been implicated in some types of muscular dystrophy (e.g., limb-girdle muscular dystrophy 2A), apparently secondary to apoptosis.

Organization of the thick filament is shown in Fig. 12.6. Each myosin molecule (≈480 kDa) consists of two heavy chains (≈200 kDa) and four light chains (≈20 kDa). The heavy chains are wound together in an α-helical configuration to form a long rod-like segment (which forms the backbone of the thick filament), and an N-terminal globular head (which extends from each myosin heavy chain toward the actin filament).

FIG. 12.6 Organization of a thick filament. A thick filament is formed by the polymerization of myosin molecules in a tail-to-tail configuration extending from the center of the sarcomere (A). An individual myosin molecule has a tail region and a cross-bridge region. The cross-bridge region is composed of an arm and globular heads (B). The globular heads contain light chains that are important for the function of myosin ATPase activity. LMM, Light meromyosin; S-1 and S-2, myosin subfragments 1 and 2.

The globular head of each myosin molecule contains an essential light chain (which is crucial for the ATPase activity of myosin), and a regulatory light chain. The regulatory light chain can be phosphorylated by Ca++/calmodulin-dependent myosin light chain protein kinase, which can influence the interaction of myosin with actin (see the section “Skeletal Muscle Types”). Thus myosin ATPase activity occurs in the two globular heads of myosin and requires the presence of the “essential” light chain in each globular head. Myosin filaments form by a tail-to-tail association of myosin molecules, which results in a bipolar arrangement of the thick filament (see Fig. 12.6A). The thick filament then extends on either side of the central bare zone by a head-to-tail association of myosin molecules, thus maintaining the filament’s bipolar organization centered on the M line. Such a bipolar arrangement is critical for drawing the Z lines together (i.e., shortening the length of the sarcomere) during contraction.

Control of Skeletal Muscle Activity Motor Nerves and Motor Units

Skeletal muscle is controlled by the central nervous system. Specifically, each skeletal muscle is innervated by an α motor neuron. The cell bodies of α motor neurons are in the ventral horn of the spinal cord (Fig. 12.7; see also Chapter 9). The motor axons exit via the ventral roots and reach the muscle through mixed peripheral nerves. The α motor nerves branch in the muscle, and each branch innervates a single muscle fiber. The specialized cholinergic synapse that forms the neuromuscular junction and the neuromuscular transmission process that generates an action potential in the muscle fiber are described in Chapter 6.

FIG. 12.7 Skeletal muscle is voluntary muscle controlled by the central nervous system, with efferent signals (i.e., action potentials) passing through an α motor neuron to muscle fibers. Each motor neuron may innervate many muscle fibers within a muscle, although each muscle fiber is innervated by only one motor neuron (A). A scanning electron micrograph (B) shows innervation of several muscle fibers by a single motor neuron. (B, From Bloom W, Fawcett DW. A Textbook of Physiology. 12th ed. New York: Chapman & Hall; 1994.)

A motor unit consists of the α motor nerve and all the muscle fibers innervated by the nerve. The motor unit is the functional contractile unit because all the muscle cells within a motor unit contract synchronously when the motor nerve fires. The size of motor units within a muscle varies, depending on the function of the muscle. Activation of motor units with a small number of fibers facilitates fine motor control. Activation of varying numbers of motor units within a muscle is one way in which the tension developed by a muscle can be controlled (see “Recruitment” in the section “Modulation of the Force of Contraction”). The neuromuscular junction formed by the α motor neuron is called an end plate (see Chapter 6 for

details). Acetylcholine released from the α motor neuron at the neuromuscular junction initiates an action potential in the muscle fiber that rapidly spreads along its length. The duration of the action potential in skeletal muscle is less than 5 milliseconds. The short duration of the skeletal muscle action potential allows very rapid contractions of the fiber and provides yet another mechanism by which the force of contraction can be increased. Increasing tension by repetitive stimulation of the muscle is called tetany (see the section “Modulation of the Force of Contraction”).

Excitation-Contraction Coupling When an action potential is transmitted along the sarcolemma of the muscle fiber and then down the T tubules, Ca++ is released from the terminal cisternae of the SR into the myoplasm (Fig. 12.8A). This Ca++ release causes intracellular [Ca++] to rise, which in turn promotes actin-myosin interaction and hence contraction (see Fig. 12.8B). The action potential is extremely short-lived ( 1), and when perfusion exceeds ventilation, the ventilation/perfusion ratio is less than 1 (V̇/Q̇ < 1). Mismatching of pulmonary blood flow and ventilation results in impaired O2 and CO2 transfer. In individuals with cardiopulmonary disease, mismatching of pulmonary blood flow and alveolar ventilation is the most frequent cause of systemic arterial hypoxemia (reduced Pao2). In general, V̇/Q̇ ratios greater than 1 are not associated with hypoxemia. A normal ventilation/perfusion ratio does not mean that ventilation and perfusion of that lung unit are normal; it simply means that the relationship between ventilation and perfusion is normal. For example, in lobar pneumonia, ventilation to the affected lobe is decreased. If perfusion to this area remains unchanged, perfusion would exceed ventilation; that is, the ventilation/perfusion ratio would be less than 1 (V̇/Q̇ < 1). However, the decrease in ventilation to this area leads to hypoxic vasoconstriction in the pulmonary arterial bed supplying this lobe. This results in a decrease in perfusion to the affected area and a more “normal” ventilation/perfusion ratio. Nonetheless, neither the ventilation nor the perfusion to this area is normal (both are decreased), but the relationship between the two could approach the normal

range.

Regional Differences in Ventilation/Perfusion Ratios The ventilation/perfusion ratio varies in different areas of the lung. In an upright individual, although both ventilation and perfusion increase from the apex to the base of the lung, the increase in ventilation is less than the increase in blood flow. As a result, the normal V̇/Q̇ ratio at the apex of the lung is much greater than 1 (ventilation exceeds perfusion), whereas the V̇/Q̇ ratio at the base of the lung is much less than 1 (perfusion exceeds ventilation). The relationship between ventilation and perfusion from the apex to the base of the lung is depicted in Fig. 23.7.

FIG. 23.7 Ventilation/perfusion relationships in a normal lung in the upright position. Only the apical and basal values are shown for clarity. In each column, the number on top represents values at the apex of the lung, and the number on the bottom represents values at the base. Pco2, Partial pressure of carbon dioxide; Pn2, partial pressure of nitrogen; Po2, partial pressure of oxygen; Q̇, perfusion per minute; V̇A, alveolar ventilation per minute.

Alveolar-Arterial Difference for Oxygen PAco2 and Paco2 are equal because of the solubility properties of CO2 (see Chapter 24). The same is not true for alveolar and arterial O2. Even in individuals with normal lungs, PAo2 is slightly greater than Pao2. The difference between PAo2 and Pao2 is called the alveolar-arterial difference for oxygen

(AaDo2). An increase in the AaDo2 is a hallmark of abnormal O2 exchange. This small difference in healthy individuals is not caused by “imperfect” gas exchange, but by the small number of veins that bypass the lung and empty directly into the arterial circulation. The thebesian vessels of the left ventricular myocardium drain directly into the left ventricle (rather than into the coronary sinus in the right atrium), and some bronchial and mediastinal veins drain into the pulmonary veins. This results in venous admixture and a decrease in Pao2. (This is an example of an anatomical shunt; see the section “Anatomical Shunts.”) Approximately 2% to 3% of the cardiac output is shunted in this way. To measure the clinical effectiveness of gas exchange in the lung, Pao2 and Paco2 are measured. PAo2 is calculated from the alveolar air equation (Eq. 23.13). The difference between the calculated PAo2 and the measured Pao2 is the AaDo2. In individuals with normal lungs who are breathing room air, the AaDo2 is less than 15 mm Hg. The mean value rises approximately 3 mm Hg per decade of life after 30 years of age. Hence, an AaDo2 lower than 25 mm Hg is considered the upper limit of normal.

IN THE C LIN IC An individual with pneumonia is receiving 30% supplemental O2 by face mask. Arterial blood gas pH is 7.40, Paco2 is 44 mm Hg, and Pao2 is 70 mm Hg. What is the patient’s AaDo2? (Assume that the patient is at sea level and the patient’s respiratory quotient is 0.8.) According to the alveolar air equation (Eq. 23.13),

Therefore,

This high AaDo2 suggests that the patient has lung disease (in this case, pneumonia). Abnormalities in Pao2 can occur with or without an elevation in AaDo2. Hence, the relationship between Pao2 and AaDo2 is useful in determining the cause of an abnormal Pao2 and in predicting the response to therapy (particularly to supplemental O2 administration). Causes of a reduction in Pao2 (arterial hypoxemia) and their effect on AaDo2 are listed in Table 23.2. Each of these causes is discussed in greater detail in the following sections.

Table 23.2 Causes of Hypoxic Hypoxia Cause

Pao 2

AaDo 2

Pao 2 Response to 100% O2

Anatomical shunt

Decreased

Increased

No significant change

Physiological shunt

Decreased

Increased

Decreased

Decreased Fio2

Decreased

Normal

Increased

Low ventilation/perfusion ratio

Decreased

Increased

Increased

Diffusion abnormality

Decreased

Increased

Increased

Hypoventilation

Decreased

Normal

Increased

AaDo2, Alveolar-arterial difference for oxygen; Fio2, fraction of inspired oxygen; Pao2, partial pressure of arterial oxygen.

Arterial Blood Hypoxemia, Hypoxia, and Hypercarbia Arterial hypoxemia is defined as a Pao2 lower than 80 mm Hg in an adult who is breathing room air at sea level. Hypoxia is defined as insufficient O2 to carry out normal metabolic functions; hypoxia often occurs when the Pao2 is less than 60 mm Hg. There are four major categories of hypoxia. The first, hypoxic hypoxia, is the most common. The six main pulmonary conditions associated with hypoxic hypoxia—anatomical shunt, physiological shunt, decreased Fio2, V̇/Q̇ mismatching, diffusion abnormalities, and hypoventilation—are described in the following sections and in Table 23.2. A second category is anemic hypoxia, which is caused by a decrease in the amount of functioning hemoglobin as a result of too little hemoglobin, abnormal hemoglobin, or interference with the chemical combination of oxygen and hemoglobin (e.g., carbon monoxide poisoning; see the following “In the Clinic” box). The third category is hypoperfusion hypoxia, which results from low blood flow (e.g., decreased cardiac output) and reduced oxygen delivery to the tissues. Histotoxic hypoxia, the fourth category of hypoxia, occurs when the cellular machinery that uses oxygen to produce energy is poisoned, as in cyanide poisoning. In this situation, arterial and venous PO2 are normal or increased because oxygen is not being utilized.

IN THE C LIN IC Carbon monoxide is a combustion by-product, and can be generated from a variety of sources such as a fuel burning space heater, car exhaust, or from a burning building. Individuals exposed to increased levels of carbon monoxide experience headache, nausea, and dizziness. If the exposure is severe, exposed individuals may die. In carbon monoxide poisoning, the lips often have a cherry-red appearance, and oxygen saturation as measured by pulse oximeter can be falsely elevated. Even on an arterial blood gas, the PAo2 may be normal. However, as CO is tightly bound to the hemoglobin molecule, there is little available hemoglobin to bind to and transport oxygen. This results in tissue hypoxemia. Thus it is imperative that the clinician recognize a potential case of carbon monoxide poisoning and order an oxygen saturation measurement with the use of a carbon monoxide oximeter (CO-oximeter) or by arterial blood gas analysis. If a patient has carbon monoxide poisoning, there will

be a marked difference between the measurement of oxygen saturation by oximetry and that measured with a carbon monoxide oximeter. Arterial blood gas analysis will confirm elevation of COhemoglobin.

Ventilation/Perfusion Abnormalities and Shunts Anatomical Shunts A useful way to examine the relationship between ventilation and perfusion is with the hypothetical two– lung unit model (Fig. 23.8). Two alveoli “lung units” are ventilated, and each is supplied by blood from the heart. When ventilation is uniform, half the inspired gas goes to each alveolus, and when perfusion is uniform, half the cardiac output goes to each alveolus. In this normal unit, the ventilation/perfusion ratio in each of the alveoli is the same and is equal to 1. The alveoli are perfused by mixed venous blood that is deoxygenated and contains increased Paco2. PAo2 is higher than mixed venous O2, and this provides a gradient for movement of O2 into blood. In contrast, mixed venous CO2 is greater than PAco2, and this provides a gradient for movement of CO2 into the alveolus. Note that in this ideal model, alveolar-arterial O2 values do not differ.

FIG. 23.8 Simplified lung model of two normal parallel lung units. Both units receive equal volumes of air and blood flow for their size. The blood and alveolar gas partial pressures are normal values in a resting person at sea level. PAco2, Partial pressure of alveolar carbon dioxide; PAo2, partial pressure of alveolar oxygen; Pico2, partial pressure of inspired carbon dioxide; Pio2, partial pressure of inspired oxygen; Ppvco2, partial pressure of carbon dioxide in pulmonary venous blood; Ppvo2, partial pressure of oxygen in pulmonary venous blood; , partial pressure of carbon dioxide in mixed venous blood; partial pressure of oxygen in mixed venous blood.

,

An anatomical shunt occurs when mixed venous blood bypasses the gas-exchange unit and goes directly into the arterial circulation (Fig. 23.9). Alveolar ventilation, the distribution of alveolar gas, and the composition of alveolar gas are normal, but the distribution of cardiac output is changed. Some of the cardiac output goes through the pulmonary capillary bed that supplies the gas-exchange units, but the rest of it bypasses the gas-exchange units and goes directly into the arterial circulation. The blood that bypasses the gas-exchange unit is thus shunted, and because the blood is deoxygenated, this type of bypass is called a right-to-left shunt. Most anatomical shunts develop within the heart, and they develop when deoxygenated blood from the right atrium or ventricle crosses the septum and mixes with blood

from the left atrium or ventricle. The effect of this right-to-left shunt is to mix deoxygenated blood with oxygenated blood, and it results in varying degrees of arterial hypoxemia.

FIG. 23.9 Right-to-left shunt. Alveolar ventilation is normal, but a portion of the cardiac output bypasses the lung and mixes with oxygenated blood. Pao2 varies according to the size of the shunt. PAco2, Partial pressure of alveolar carbon dioxide; PAo2, partial pressure of alveolar oxygen; Pico2, partial pressure of inspired carbon dioxide; Pio2, partial pressure of inspired oxygen; Ppvco2, partial pressure of carbon dioxide in pulmonary venous blood; Ppvo2, partial pressure of oxygen in pulmonary venous blood; , partial pressure of carbon dioxide in mixed venous blood; venous blood.

, partial pressure of oxygen in mixed

An important feature of an anatomical shunt is that if an affected individual is given 100% O2 to breathe, there is only a minimal increase in oxygen saturation. The blood that bypasses the gas-exchanging units is never exposed to the enriched O2, and thus it continues to be deoxygenated. The PO2 in the blood that is not being shunted increases and it mixes with the deoxygenated blood. Thus the degree of persistent hypoxemia in response to 100% O2 varies with the volume of the shunted blood. Normally, the hemoglobin in the blood that perfuses the ventilated alveoli is almost fully saturated. Therefore, most of the added O2 is in the form of dissolved O2 (see Chapter 24). The Paco2 in an anatomical shunt is not usually increased, even though the shunted blood has an elevated level of CO2. The reason for this is that the central chemoreceptors (see Chapter 25) respond to any elevation in CO2 with an increase in ventilation and reduce Paco2 to the normal range. If the hypoxemia is severe, the increased respiratory drive secondary to the hypoxemia increases the ventilation and can decrease Paco2 to below the normal range. Physiological Shunts A physiological shunt (also known as venous admixture) can develop when ventilation to lung units is absent in the presence of continuing perfusion (Fig. 23.10). In this situation, in the two–lung unit model, all the ventilation goes to the other lung unit, whereas perfusion is equally distributed between both lung units. The lung unit without ventilation but with perfusion has a V̇/Q̇ ratio of 0. The blood perfusing this

unit is mixed venous blood; because there is no ventilation, no gas is exchanged in the unit, and the blood leaving this unit continues to resemble mixed venous blood. The effect of a physiological shunt on oxygenation is similar to the effect of an anatomical shunt; that is, deoxygenated blood bypasses a gasexchanging unit and admixes with arterial blood. Clinically, atelectasis (in which a portion of the lung becomes deflated or deaerated) is an example of a situation in which the lung region has a V̇/Q̇ of 0, as there is no ventilation to the atelectatic lung units. Causes of atelectasis include mucous plugs, airway edema, foreign bodies, and tumors in the airway. Decreased inspiratory effort following surgery is a common cause of atelectasis.

FIG. 23.10 Schema of a physiological shunt (venous admixture). Notice the marked decrease in Pao2 in comparison to Pco2. The alveolar-arterial difference for oxygen (AaDo2) in this example is 85 mm Hg. PAco2, Partial pressure of alveolar carbon dioxide; PAo2, partial pressure of alveolar oxygen; Pico2, partial pressure of inspired carbon dioxide; Pio2, partial pressure of inspired oxygen; Ppvco2, partial pressure of carbon dioxide in pulmonary venous blood; Ppvo2, partial pressure of oxygen in pulmonary venous blood; , partial pressure of carbon dioxide in mixed venous blood; , partial pressure of oxygen in mixed venous blood. Note differing Ppvo2 and Ppvco2 levels in pulmonary veins draining the unventilated lung unit (top) and fully ventilated lung unit (bottom.) The resulting mixed Ppvo2 is substantially lower than the Ppvo2 from fully ventilated lung unit.

Low Ventilation/Perfusion Mismatching between ventilation and perfusion is the most frequent cause of arterial hypoxemia in individuals with respiratory disorders. In the most common example, the composition of mixed venous blood, total blood flow (cardiac output), and the distribution of blood flow are normal. However, when alveolar ventilation is distributed unevenly between the two gas-exchange units (Fig. 23.11) and blood flow is equally distributed, the unit with decreased ventilation has a V̇/Q̇ ratio of less than 1, whereas the unit with the increased ventilation has a V̇/Q̇ of greater than 1. This causes the alveolar and end-capillary gas compositions to vary. Both the arterial O2 content and CO2 content are abnormal in the blood that has come from the unit with the decreased ventilation (V̇/Q̇, 1) has a lower CO2 content and a higher O2 content because it is being overventilated. The actual Pao2 and Paco2 vary, depending on the relative contribution of each of these units to arterial blood. The alveolar-arterial O2 gradient (AaDo2) is increased because the relative overventilation of one unit does

not fully compensate (either by the addition of extra O2 or by the removal of extra CO2) for underventilation of the other unit. The failure to compensate is greater for O2 than for CO2, as indicated by the flatness of the upper part of the oxyhemoglobin dissociation curve, in contrast to the slope of the CO2 dissociation curve (see Chapter 24). In other words, increased ventilation increases PAo2, but it adds little extra O2 content to the blood because hemoglobin is close to being 100% saturated in the overventilated areas. This is not the case for CO2, for which the steeper slope of the CO2 curve indicates removal of more CO2 when ventilation increases. Thus inasmuch as CO2 moves by diffusion, then as long as a CO2 gradient is maintained, CO2 diffusion will occur.

FIG. 23.11 Effects of ventilation/perfusion mismatching on gas exchange. The decrease in ventilation to the one lung unit could be due to mucus obstruction, airway edema, bronchospasm, a foreign body, or a tumor. PAco2, Partial pressure of alveolar carbon dioxide; PAo2, partial pressure of alveolar oxygen; Pico2, partial pressure of inspired carbon dioxide; Pio2, partial pressure of inspired oxygen; Ppvco2, partial pressure of carbon dioxide in pulmonary venous blood; Ppvo2, partial pressure of oxygen in pulmonary venous blood; , partial pressure of carbon dioxide in mixed venous blood; of oxygen in mixed venous blood.

, partial pressure

Alveolar Hypoventilation The PAo2 is determined by a balance between the rate of O2 uptake and the rate of O2 replenishment by ventilation. Oxygen uptake depends on blood flow through the lung and the metabolic demands of the tissues. If ventilation decreases, PAo2 decreases, and Pao2 subsequently decreases. In addition, Va and PAco2 are directly but inversely related. When ventilation is halved, the PAco2 doubles, and thus so does the Paco2 (see Eq. 23.16). Ventilation insufficient to maintain normal levels of CO2 is called hypoventilation. Hypoventilation always decreases Pao2 and increases Paco2. One of the hallmarks of hypoventilation is a normal AaDo2. Hypoventilation reduces PAo2, which in turn results in a decrease in Pao2. Because gas exchange is normal, the AaDo2 remains normal. Hypoventilation accompanies diseases associated with decreased central drive to breathe, weakness of the respiratory muscles, and is also associated with drugs that reduce respiratory drive. In the presence of hypoventilation, however, regions of the lung may become deaerated (atelectatic) and in these regions the V̇/Q̇ ratio is 0. When this occurs, the AaDo2 rises.

Diffusion Abnormalities Abnormalities in diffusion of O2 across the alveolar-capillary barrier could potentially result in arterial hypoxia. Equilibration between alveolar and capillary O2 and CO2 content occurs rapidly: in a fraction of the time that it takes for red blood cells to transit the pulmonary capillary network. Hence, diffusion equilibrium almost always occurs in normal people, even during exercise, when the transit time of red blood cells through the lung increases significantly. An increased AaDo2 attributable to incomplete diffusion (diffusion disequilibrium) has been observed in normal persons only during exercise at high altitude (≥10,000 feet). Even in individuals with an abnormal diffusion capacity, diffusion disequilibrium at rest is unusual but can occur during periods of increased metabolic demand such as exercise or illness, or when at high altitude. Alveolar-capillary block, or thickening of the air-blood barrier, is an uncommon cause of hypoxemia. Even when the alveolar wall is thickened, there is usually sufficient time for gas diffusion unless the red blood cell transit time is increased. Carbon dioxide diffuses nearly 20 times more rapidly than oxygen. Impaired CO2 diffusion rarely clinically relevant.

Mechanisms of Hypercapnia Two major mechanisms account for the development of hypercapnia (elevated Pco2): hypoventilation and increases in dead space ventilation. As noted previously, alveolar ventilation and alveolar CO2 are inversely related. When ventilation is halved, PAco2 and Paco2 double. Hypoventilation always decreases Pao2 and increases Paco2 and thereby results in a hypoxemia that responds to an enriched source of O2. Dead space ventilation increased when pulmonary blood flow is interrupted in the presence of normal ventilation. This is sometimes referred to as “wasted ventilation.” This is most often caused by a blood clot (pulmonary embolus) obstructing blood flow in a region of the pulmonary circulation. The embolus halts blood to pulmonary areas with normal ventilation (V̇/Q̇ = ∞). In this situation, the ventilation is wasted because it fails to oxygenate any of the mixed venous blood, and that region becomes physiologic dead space. The remaining perfused regions of the lung receive all of the blood flow (regional perfusion is increased) and normal ventilation (regional ventilation is unchanged.) As a result, there is relative “hypoventilation” as the V̇/Q̇ ratio is decreased. If compensation does not occur, Paco2 increases and Pao2 decreases. Compensation after a pulmonary embolus, however, begins almost immediately; local bronchoconstriction occurs, and the distribution of ventilation shifts to the areas being perfused. As a result, changes in arterial CO2 and O2 content are minimized.

Effect of 100% Oxygen on Arterial Blood Gas Abnormalities One of the ways that a right-to-left shunt can be distinguished from other causes of hypoxemia is for the individual to breathe 100% O2 through a nonrebreathing face mask for approximately 15 minutes. When the individual breathes 100% O2, all of the N2 in the alveolus is replaced by O2. Thus the PAo2, according to the alveolar air equation (Eq. 23.13), is calculated as follows:

Equation 23.20

In a normal lung, the PAo2 rapidly increases, and it provides the gradient for transfer of O2 into capillary blood. This is associated with a marked increase in Pao2 (see Table 23.2). Similarly, during the 15-minutes of breathing 100% O2, even areas with very low V̇/Q̇ ratios develop high alveolar O2 pressure as the N2 is replaced by O2. In the presence of normal perfusion to these areas, there is a gradient for gas exchange, and the end-capillary blood is highly enriched with O2. In contrast, in the presence of a right-to-left shunt, oxygenation is not corrected because mixed venous blood continues to flow through the shunt and mix with blood that has perfused normal units. The poorly oxygenated blood from the shunt lowers the arterial O2 content and maintains the AaDo2. An elevated AaDO2 during a properly conducted study with 100% O2 signifies the presence of a shunt (anatomical or physiological); the magnitude of the AaDo2 can be used to quantify the proportion of the cardiac output that is being shunted.

Regional Differences The regional differences in ventilation and perfusion and the relationship between ventilation and perfusion were discussed earlier in this chapter. The effects of various physiological abnormalities (e.g., shunt, V̇/Q̇ mismatch, and hypoventilation) on arterial O2 and CO2 levels were also described. In addition, however, it should be noted that because the V̇/Q̇ ratio varies in different regions of the lung, the end-capillary blood coming from these regions has different O2 and CO2 levels. These differences are shown in Fig. 23.7, and they demonstrate the complexity of the lung. First, recall that the volume of the lung at the apex is less than the volume at the base. As previously described, ventilation and perfusion are less at the apex than at the base, but the differences in perfusion are greater than the differences in ventilation. Thus the V̇/Q̇ ratio is high at the apex and low at the base. This difference in ventilation/perfusion ratios is associated with a difference in alveolar O2 and CO2 content between the apex and the base. The PAo2 is higher and the PAco2 is lower in the apex than in the base. This results in differences in end-capillary contents for these gases. End-capillary PO2 is lower, and, as a consequence, the O2 content is lower in end-capillary blood at the lung base than at the apex. In addition, there is significant variation in blood pH in the end capillaries in these regions because of the variation in CO2 content. During exercise, blood flow to the apex increases and becomes more uniform in the lung; as a result, the difference between the content of gases in the apex and in the base of the lung diminishes with exercise.

Key Points

1. The volume of air in the conducting airways is called the anatomical dead space. Dead space ventilation varies inversely with tidal volume. The total volume of gas in each breath that does not participate in gas exchange is called the physiological dead space. It includes both the anatomical dead space and the dead space secondary to ventilated but unperfused alveoli. 2. The sum of the partial pressures of a gas is equal to the total pressure. The partial pressure of a gas (Pgas) is equal to the fraction of the gas in the gas mixture (Fgas) multiplied by the total pressure (Ptotal). The conducting airways do not participate in gas exchange. Therefore, the partial pressures of O2, N2, and water vapor in humidified air remain unchanged in the airways until the gas reaches the alveolus. 3. The partial pressure of O2 in the alveolus is given by the alveolar air equation (Eq. 23.13). This equation is used to calculate the AaDo2, a useful measurement of abnormal arterial O2. 4. The relationship between CO2 production and alveolar ventilation is defined by the alveolar carbon dioxide equation (Eq. 23.14). There is an inverse relationship between the PAco2 and VA, regardless of the exhaled quantity of CO2. In normal lungs, Paco2 is tightly regulated by the brainstem respiratory center to remain constant at around 40 mm Hg. 5. Because of the effects of gravity, there are regional differences in ventilation and perfusion. The ventilation/perfusion (V̇/Q̇) ratio is defined as the ratio of ventilation to blood flow. In a normal lung, the overall ventilation/perfusion ratio is approximately 0.8. When ventilation exceeds perfusion, the ventilation/perfusion ratio is greater than 1 (V̇/Q̇ > 1), and when perfusion exceeds ventilation, the ventilation/perfusion ratio is less than 1 (V̇/Q̇ < 1). The V̇/Q̇ ratio at the apex of the lung is high (ventilation is increased in relation to very little blood flow), whereas the V̇/Q̇ ratio at the base of the lung is low. In individuals with normal lungs who are breathing room air, the AaDo2 is less than 15 mm Hg; the upper limit of normal is 25 mm Hg. 6. The pulmonary circulation is a low-pressure, low-resistance system. Recruitment of new capillaries and dilation of arterioles without an increase in pressure are unique features of the lung and allow for adjustments during stress, as in the case of exercise. Pulmonary vascular resistance is the change in pressure from the pulmonary artery (PPA) to the left atrium (PLA), divided by cardiac output (QT). This resistance is about 10 times less than in the systemic circulation. 7. There are four categories of hypoxia (hypoxic hypoxia, anemic hypoxia, diffusion hypoxia, and histotoxic hypoxia) and six mechanisms of hypoxic hypoxia and hypoxemia (anatomical shunt, physiological shunt, decreased Fio2, V̇/Q̇ mismatching, diffusion abnormalities, and hypoventilation.) 8. There are two mechanisms of the development of hypercapnia: increase in dead space ventilation and hypoventilation.

C H AP T E R 2 4

Oxygen and Carbon Dioxide Transport LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. What are the basic gas diffusion principles and how do they affect O2 and CO2 absorption and expiration? 2. What are the chemical transport mechanisms of O2 and CO2 in blood? 3. How do the O2 and CO2 dissociation curves differ? How does these differences promote tissue O2 delivery and CO2 removal? 4. What is the difference between perfusion limitation and diffusion limitation? Why is the diffusion of O2 and CO2 considered to be perfusion limited, and why is CO is considered to be diffusion limited? 5. What is meant by a leftward or rightward shift of the oxyhemoglobin dissociation curve? 6. How do the oxyhemoglobin and carboxyhemoglobin dissociation curves differ? What is the clinical significance of the differences? 7. What is the difference between the chloride shift and the Haldane effect on CO2 transport?

The respiratory and circulatory systems function together to transport oxygen (O2) from the lungs to the tissues to sustain normal cellular activity and to transport carbon dioxide (CO2) from the tissues to the lungs for expiration (Fig. 24.1). To enhance uptake and transport of these gases between the lungs and tissues, specialized mechanisms (e.g., binding of O2 and hemoglobin and HCO3− transport of CO2) have evolved that enable O2 uptake and CO2 expiration to occur simultaneously. To understand the mechanisms involved in the transport of these gases, gas diffusion properties, gas transport, and gas delivery mechanisms must be considered.

FIG. 24.1 Oxygen (O2) and carbon dioxide (CO2) transport in arterial and venous blood. Oxygen in arterial blood is transferred from arterial capillaries to tissues. The flow rates for O2 and CO2 are shown for 1 L of blood.

Gas Diffusion Gas movement throughout the respiratory system occurs predominantly via diffusion. The respiratory and circulatory systems contain several unique anatomical and physiological features to facilitate gas diffusion: (1) large surface areas for gas exchange (alveolar to pulmonary capillary bed and end organ capillary bed to tissue) with short distances to travel, (2) substantial partial pressure gradient differences, and (3) gases with advantageous diffusion properties. Transport and delivery of O2 from the lungs to the tissue and vice versa for CO2 are dependent on basic gas diffusion laws.

Diffusion of Gases From Regions of Higher to Lower Partial Pressure in the Lungs The process of gas diffusion is passive and similar whether diffusion occurs in a gaseous or liquid state. The rate of diffusion of a gas through a liquid is described by Graham's law, which states that the rate is directly proportional to the solubility coefficient of the gas and inversely proportional to the square root

of its molecular weight. Calculation of the diffusion properties for O2 and CO2 reveals that CO2 diffuses approximately 20 times faster than O2. Rates of O2 diffusion from the lungs into blood and from blood into tissue, and vice versa for CO2, are predicted by Fick's law of gas diffusion (Fig. 24.2). Fick's law states that gas diffusion across a permeable membrane ( ) is proportional to the diffusion coefficient for the gas (D), the surface area of the membrane (A), and the pressure gradient across the membrane (P1 − P2). It is inversely proportional to the thickness of the membrane (T). The ratio of surface area (A) x diffusion coefficient (D) to membrane thickness (T) (or A•D/T) represents the conductance of a gas from the alveolus to the blood. The diffusing capacity of the lung (DL) is its conductance (A•D/T) when considered for the entire lung; thus, with Fick's equation, DL can be calculated as follows:

Equation 24.1

FIG. 24.2 According to Fick's law, diffusion of a gas across a sheet of tissue (V˙gas) is directly related to the surface area (A) of the tissue, the diffusion constant (D) of the specific gas, and the partial pressure difference (P1 − P2) of the gas on each side of the tissue, and it is inversely related to tissue thickness (T).

where V˙gas = gas diffusion. Fick's law of diffusion could be used to assess the diffusion properties of O2 in the lungs, except that the capillary partial pressure of oxygen cannot be measured. This limitation can be overcome with the use of carbon monoxide (CO) rather than O2. Because CO has low solubility in the capillary membrane, the rate of CO equilibrium across the capillary is slow, and the partial pressure of CO in capillary blood remains close to 0. In contrast, the solubility of CO in blood is high. Thus, the only limitation for diffusion of CO is the alveolar-capillary membrane, and thus CO is a useful gas for calculating DL. The capillary partial pressure (P2 in Eq. 24.1) is essentially 0 for CO, and therefore DL can be measured from the diffusion of carbon monoxide (

) and the average partial pressure of Co in the alveolus (P1); that is,

Equation 24.2

where DLco = diffusion capacity of the lung for carbon monoxide. Assessment of DLco has become a classic measurement of the diffusion barrier of the alveolarcapillary membrane. It is useful in the differential diagnosis of certain obstructive lung diseases, such as emphysema. Although exposure to high levels of carbon monoxide gas can be toxic, in gas diffusion testing the total CO exposure is negligible.

IN THE C LIN IC A patient with interstitial pulmonary fibrosis (a restrictive lung disease) inhales a single breath of 0.3% CO from residual volume to total lung capacity. He holds his breath for 10 seconds and then exhales. After discarding the exhaled gas from the dead space, a representative sample of alveolar gas from late in exhalation is collected. The average alveolar CO pressure is 0.1 mm Hg, and 0.25 mL of CO has been taken up. The diffusion capacity for CO in this patient is

The normal range for DLco is 20 to 30 mL/minute/mm Hg. Patients with interstitial pulmonary fibrosis

have an initial alveolar inflammatory response with subsequent scar formation within the interstitial space. The inflammation and scar replace the alveoli and decrease the surface area for gas diffusion to occur, which results in decreased DLco. This is a classic characteristic of certain types of restrictive lung disease.

Oxygen and Carbon Dioxide Exchange in the Lung Is Perfusion Limited Different gases have different solubility factors. Gases that are insoluble in blood (i.e., anesthetic gases such as nitrous oxide and ether) do not chemically combine with proteins in blood and equilibrate rapidly between alveolar gas and blood. The equilibration occurs in less time than the 0.75 to 1.0 seconds that the red blood cell spends in the capillary bed (the capillary transit time). The diffusion of insoluble gases between alveolar gas and blood is considered perfusion limited because the partial pressure of gas in the blood leaving the capillary has reached equilibrium with alveolar gas and is limited only by the amount of blood perfusing the alveolus. In contrast, a gas that is diffusion limited, such as CO, has low solubility in the alveolar-capillary membrane but high solubility in blood because of its high affinity for hemoglobin (Hgb). These features prevent the equilibration of CO between alveolar gas and blood during the red blood cell transit time. The high affinity of CO for Hgb enables large amounts of CO to be taken up in blood with little or no appreciable increase in its partial pressure. Gases that are chemically bound to Hgb do not exert a partial pressure in blood. Like CO, both CO2 and O2 have relatively low solubility in the alveolar-capillary membrane but high solubility in blood because of their ability to bind to Hgb. However, their rate of equilibration is sufficiently rapid for complete equilibration to occur during the transit time of the red blood cell within the capillary. Equilibration for O2 and CO2 usually occurs within 0.25 seconds. Thus, O2 and CO2 transfer is normally perfusion limited. The partial pressure of a gas that is diffusion limited (i.e., CO) does not reach equilibrium with the alveolar pressure over the time that it spends in the capillary (Fig. 24.3). Although CO2 has a greater rate of diffusion in blood than O2 does, it has a lower membrane-blood solubility ratio and consequently takes approximately the same amount of time to reach equilibration in blood.

FIG. 24.3 Uptake of nitrous oxide (N2O), carbon monoxide (CO), and O2 in blood in relation to their partial pressures and the transit time of the red blood cell in the capillary. For gases that are perfusion limited (N2O and O2), their partial pressures have equilibrated with alveolar pressure before exiting the capillary. In contrast, the partial pressure of CO, a gas that is diffusion limited, does not reach equilibrium with alveolar pressure. In rare conditions, O2 uptake can become diffusion limited.

Diffusion limitation for O2 and CO2 would occur if red blood cells spent less than 0.25 seconds in the capillary bed. This is occasionally the case in very fit athletes during vigorous exercise and in healthy subjects who exercise at high altitude.

Oxygen Transport Oxygen is carried in blood in two forms: O2 dissolved in plasma and O2 bound to Hgb. The dissolved form is measured clinically in an arterial blood gas sample as the partial pressure of arterial oxygen (Pao2). Only a small percentage of O2 in blood is in the dissolved form, and its contribution to O2 transport under normal conditions is almost negligible. However, dissolved O2 can become a significant factor in conditions of severe hypoxemia. Binding of O2 to Hgb to form oxyhemoglobin within red blood cells is the primary transport mechanism of O2. Hgb not bound to O2 is referred to as deoxyhemoglobin or reduced Hgb. The O2-carrying capacity of blood is enhanced about 65 times by its ability to bind to Hgb.

Hemoglobin Hgb is the major transport molecule for O2. The Hgb molecule is a protein with two major components: four nonprotein heme groups, each containing iron in the reduced ferric (Fe+++) form, which is the site of O2 binding; and a globin portion consisting of four polypeptide chains. Normal adults have two α-globin chains and two β-globin chains (HgbA), whereas children younger than 6 months of age have

predominantly fetal Hgb (HgbF), which consists of two α chains and two γ chains. This difference in the structure of HgbF increases its affinity for O2 and aids in the transport of O2 across the placenta. In addition, HgbF is not inhibited by 2,3-diphosphoglycerate (2,3-DPG), a product of glycolysis; thus O2 uptake is further enhanced. Binding of O2 to Hgb alters the ability of Hgb to absorb light. This effect of O2 on Hgb is responsible for the change in color between oxygenated arterial blood (bright red) and deoxygenated venous blood (dark red-bluish). Binding and dissociation of O2 with Hgb occur in milliseconds, thus facilitating O2 transport because red blood cells spend only 0.75 seconds in the capillaries. There are approximately 280 million Hgb molecules per red blood cell, which provides an efficient mechanism to transport O2. Myoglobin, a protein in striated muscle similar in structure and function to Hgb, has only one subunit of the Hgb molecule. It aids in the transfer of O2 from blood to muscle cells and in the storage of O2, which is especially critical in O2-deprived conditions. Abnormalities of the Hgb molecule occur with mutations in the amino acid sequence (i.e., sickle cell disease) or in the spatial arrangement of the globin polypeptide chains and result in abnormal function. Compounds such as CO, nitrites (nitric oxide), and cyanides can oxidize the iron molecule in the heme group and change it from the reduced ferrous state (Fe++) to the ferric state (Fe+++), which reduces the ability of O2 to bind to Hgb.

Oxyhemoglobin Dissociation Curve In the alveoli, the majority of O2 in plasma quickly diffuses into red blood cells and chemically binds to Hgb. This process is reversible, so that Hgb quickly gives up its O2 to tissue through passive diffusion (the concentration of O2 in Hgb decreases). The oxyhemoglobin dissociation curve illustrates the relationship between Po2 in blood and the number of O2 molecules bound to Hgb (Fig. 24.4). The S shape of the curve demonstrates the dependence of Hgb saturation on Po2, especially at partial pressures lower than 60 mm Hg. The clinical significance of the flat portion of the oxyhemoglobin dissociation curve (>60 mm Hg) is that a drop in Po2 over a wide range of partial pressures (100–60 mm Hg) has a minimal effect on Hgb saturation, which remains at 90% to 100%, a level sufficient for normal O2 transport and delivery. The clinical significance of the steep portion (60 mm Hg), increasing or decreasing Po2 has only a minimal effect on Hgb saturation from 100% to 90%. This ensures adequate Hgb saturation over a large range of Po2 values. 5. The CO2 dissociation curve is linear and directly related to Pco2. Pco2 is solely dependent on CO2 production and alveolar ventilation. 6. The CO2 to HCO3− pathway plays a critical role in the regulation of H+ ions and in maintaining acid-base balance in the body. 7. Tissue oxygenation is dependent on Hgb within red blood cells and subsequently the number (and production) of red blood cells, which is controlled by the hormone erythropoietin. Low O2 delivery, low Hgb concentration, and low Pao2 stimulate the secretion of erythropoietin in the kidneys. 8. Tissue hypoxia occurs when insufficient amounts of O2 are supplied to the tissue to conduct normal levels of aerobic metabolism.

C H AP T E R 2 5

Control of Respiration LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. How is ventilation controlled by the central nervous system? 2. How do the central and peripheral chemoreceptors provide feedback for regulation of ventilation? 3. How are chemoreceptors and pulmonary mechanoreceptors similar and different in regulation of respiration? 4. How do circumstances such as exercise or high altitude exposure alter respiratory drive? 5. How does obstructive sleep apnea differ from central sleep apnea?

People breathe without thinking, and they can willingly modify their breathing pattern and even hold their breath. Control of ventilation includes the generation and regulation of rhythmic breathing by the respiratory center in the brainstem. The rhythmic breathing pattern can be altered in response to input from systemic receptors and from higher brain centers. The goals of breathing are, from a mechanical perspective, to minimize work and, from a physiological perspective, to maintain and regulate arterial blood O2 (Pao2) and CO2 (Paco2). Another goal of breathing is to maintain acid-base balance by regulating Paco2. Automatic respiration begins at birth. In utero, the placenta, not the lung, is the organ of gas exchange in the fetus. Its microvilli interdigitate with the maternal uterine circulation, and Pao2 transport and Paco2 removal from the fetus occur by passive diffusion across the maternal circulation.

Ventilatory Control: An Overview There are four major sites of ventilatory control: (1) the respiratory control center, (2) central chemoreceptors, (3) peripheral chemoreceptors, and (4) pulmonary mechanoreceptors/sensory nerves. The respiratory control center is located in the medulla oblongata of the brainstem and is composed of multiple nuclei that generate and modify the basic ventilatory rhythm. This center consists of two main parts: (1) a ventilatory pattern generator, which sets the rhythmic pattern; and (2) an integrator, which controls generation of the pattern, processes input from higher brain centers and chemoreceptors, and controls the rate and amplitude of the ventilatory pattern. Input to the integrator arises from higher brain centers, including the cerebral cortex, hypothalamus, limbic system including the amygdalae, and cerebellum.

Central chemoreceptors are located in the central nervous system just below the ventrolateral surface of the medulla. These central chemoreceptors detect changes in the Paco2 and pH of interstitial fluid in the brainstem, and they modulate ventilation. Peripheral chemoreceptors are located on specialized cells in the aortic arch (aortic bodies) and at the bifurcation of the internal and external carotid arteries (carotid bodies) in the neck. These peripheral chemoreceptors sense the Pao2, Paco2, and pH of arterial blood. They feed information back to the integrator nuclei in the medulla through the vagus nerves, and by the carotid sinus nerves that are branches of the glossopharyngeal nerves. Pulmonary mechanoreceptors and sensory nerve stimulation, in response to lung inflation or to stimulation by irritants or release of local mediators in the airways, modify the ventilatory pattern. The collective output of the respiratory control center to motor neurons located in the anterior horn of the spinal column controls the muscles of respiration, and this output determines the automatic rhythmic pattern of respiration. Motor neurons located in the cervical region of the spinal column control the activity of the diaphragm through the phrenic nerves, whereas other motor neurons located in the thoracic region of the spine control the intercostal muscles and the accessory muscles of respiration. In contrast to automatic respiration, voluntary respiration bypasses the respiratory control center in the medulla. The neural activity controlling voluntary respiration originates in the motor cortex and signaling passes directly to motor neurons in the spine through the corticospinal tracts. The motor neurons to the respiratory muscles act as the final site of integration of the voluntary (corticospinal tract) and automatic (ventrolateral tracts) control of ventilation. Voluntary control of these muscles competes with automatic influences at the level of the spinal motor neurons, and this competition can be demonstrated by breath holding. At the start of the breath hold, voluntary control dominates the spinal motor neurons. However, as the breath hold continues, the automatic ventilatory control eventually overpowers the voluntary effort and limits the duration of the breath hold. Motor neurons also innervate muscles of the upper airway. These neurons are located within the medulla near the respiratory control center. They innervate muscles in the upper airways through the cranial nerves. When activated, they dilate the pharynx and large airways at the initiation of inspiration.

Response to Carbon Dioxide Ventilation is also regulated by Paco2, Pao2, and pH in arterial blood. Paco2 is the most important of these regulators. Both the rate and depth of breathing are controlled to maintain Paco2 close to 40 mm Hg. In a normal awake individual, there is a linear rise in ventilation as Paco2 reaches and exceeds 40 mm Hg (Fig. 25.1). The ventilatory drive or response to changes in Paco2 can be reduced by hyperventilation and by drugs that depress the respiratory center and decrease the ventilatory response to both CO2 and O2. These drugs include opiates, benzodiazepines, barbiturates, and anesthetic agents. In these instances, the stimulus is inadequate to stimulate the motor neurons that innervate the muscles of respiration. It is also depressed during sleep. In addition, the ventilatory response to changes in Paco2 is reduced if the work of breathing is increased, which can occur in individuals with chronic obstructive pulmonary disease (COPD). This effect occurs primarily because the neural output of the respiratory center is less effective in promoting ventilation as a result of the mechanical limitation to ventilation.

FIG. 25.1 Relationship between partial pressure of arterial carbon dioxide (Paco2) and alveolar ventilation in awake normal states, during sleep, after narcotic ingestion and deep anesthesia, and in the presence of metabolic acidosis. Both the slopes of the response (sensitivity) and the position of the response curves (threshold, the point at which the curve crosses the x-axis [not shown]) are changed, which indicates differences in ventilatory responses and response thresholds. COPD, Chronic obstructive pulmonary disease.

Changes in Paco2 are sensed by central and peripheral chemoreceptors, and they transmit this information to the medullary respiratory centers. The respiratory control center then regulates minute ventilation and thereby maintains Paco2 within the normal range. In the presence of a normal Pao2, ventilation increases by approximately 3 L/minute for each 1 mm Hg rise in Paco2. The response to an increase in Paco2 is further increased when the Pao2 is low (Fig. 25.2A). With a low Pao2, ventilation is greater for any given Paco2, and the increase in ventilation for a given increment in Paco2 is enhanced. The slope of the minute ventilation response as a function of the inspired CO2 is termed the ventilatory response to CO2 and is a test of CO2 sensitivity. It is important to recognize that this relationship is amplified by low O2 (see Fig. 25.2B). The responsiveness to low O2 is enhanced because different mechanisms are responsible for sensing Pao2 and Paco2 in the peripheral chemoreceptors. Thus the presence of both hypercapnia-elevated CO2 and hypoxemia-low O2 (often called asphyxia when both changes are present) has an additive effect on chemoreceptor output and on the resulting ventilatory stimulation.

FIG. 25.2 The effects of hypoxia and hypercapnia on ventilation as the other respiratory gas partial pressures are varied. A, At a given partial pressure of arterial carbon dioxide (Paco2), ventilation increases more and more as partial pressure of arterial oxygen (Pao2) decreases. When Paco2 is allowed to decrease (the normal condition) during hypoxia, there is little stimulation of breathing until Pao2 falls below 60 mm Hg. The hypoxic response is mediated through the carotid body chemoreceptors. B, The sensitivity of the ventilatory response to CO2 is enhanced by hypoxia.

Control of Ventilation: The Details The Respiratory Control Center When the brain is transected experimentally between the medulla and the pons, periodic breathing is maintained, thus demonstrating that the inherent rhythmicity of breathing originates in the medulla. Although no single group of neurons in the medulla has been found to be the breathing “pacemaker,” two distinct nuclei within the medulla are involved in generation of the respiratory pattern (Fig. 25.3). One nucleus is the dorsal respiratory group (DRG), which is composed of cells in the nucleus tractus solitarius and is located in the dorsomedial region of the medulla. Cells in the DRG receive afferent input

from cranial nerves IX and X, which originate from airways and the lungs and constitute the initial intracranial processing station for this afferent input. The second group of medullary cells is the ventral respiratory group (VRG), located in the ventrolateral region of the medulla. The VRG is composed of three cell groups: the rostral nucleus retrofacialis, the caudal nucleus retroambiguus, and the nucleus para-ambiguus. The VRG contains both inspiratory and expiratory neurons. The nucleus retrofacialis and the caudally located cells of the nucleus retroambiguus are active during exhalation, whereas the rostrally located cells of the nucleus retroambiguus are active during inspiration. The nucleus para-ambiguus has inspiratory and expiratory neurons that travel in the vagus nerve to the laryngeal and pharyngeal muscles. Discharges from cells in these areas excite some cells and inhibit other cells.

FIG. 25.3 The respiratory control center is located in the medulla, the most primitive portion of the brain. The neurons are mainly in two areas: the dorsal respiratory group (DRG), which consists of the nucleus tractus solitarius; and the ventral respiratory group (VRG), which consists of the rostral nucleus retrofacialis, nucleus para-ambiguus, and the caudal nucleus retroambiguus. C1 refers to the first cervical signal segment to the caudal border of the pons. The fourth ventricle of the brain is located below the cerebellum and above, and between, the pons and the medulla.

At the level of the respiratory control center, inspiration and exhalation involve three phases: one inspiratory and two expiratory (Fig. 25.4). Inspiration begins with an abrupt increase in discharge from cells in the nucleus tractus solitarius, the nucleus retroambiguus, and the nucleus para-ambiguus, followed by a steady, ramp-like increase in firing rate throughout inspiration. This leads to progressive contraction of the respiratory muscles during automatic breathing. At the end of inspiration, an “off-switch” event causes neuron firing to decrease markedly, at which point exhalation begins. At the start of exhalation (phase I of expiration), a paradoxical increase in inspiratory neuron firing slows the expiratory phase down by increasing inspiratory muscle tone and expiratory neuron firing. This inspiratory neuron firing decreases and stops during phase II of exhalation. Although many different neurons in the DRG and VRG are involved in ventilation, each cell type appears to have a specific function. For example, the HeringBreuer reflex is an inspiratory-inhibitory reflex that arises from afferent stretch receptors located in the smooth muscles of the airways. Increasing lung inflation stimulates these stretch receptors and results in early exhalation by stimulating the neurons associated with the off-switch phase of inspiratory muscle control. Thus rhythmic breathing depends on a continuous (tonic) inspiratory drive from the DRG and an intermittent (phasic) expiratory drive from the cerebrum, thalamus, cranial nerves, and ascending sensory tracts in the spinal cord.

FIG. 25.4 Diagram of the basic wiring of the brainstem ventilatory controller. The signs of the main output (arrows) of the neuron pools indicate whether the output is excitatory (+) or inhibitory (−). Pool A provides tonic inspiratory stimuli to the muscles of breathing. Pool B is stimulated by pool A and provides additional stimulation to the muscles of breathing, and pool B stimulates pool C. Other brain centers feed into pool C (inspiratory cutoff switch), which sends inhibitory impulses to pool A. Afferent information (feedback) from various sensors acts at different locations: Chemoreceptors act on pool A, and intrapulmonary sensory fibers act via the vagus nerves on pool B. A pneumotaxic center in the anterior pons receives input from the cerebral cortex, and it acts on pool C.

Central Chemoreceptors A chemoreceptor is a receptor that responds to a change in the chemical composition of blood or other fluid around it. Central chemoreceptors are specialized cells on the ventrolateral surface of the medulla. Chemoreceptors are sensitive to the pH of the surrounding extracellular fluid. Because this extracellular fluid is in contact with cerebrospinal fluid (CSF), changes in the pH of CSF affect ventilation by acting on these chemoreceptors. CSF is an ultrafiltrate of plasma that is secreted continuously by the choroid plexus and is reabsorbed by the arachnoid villi. Because it is in contact with the extracellular fluid in the brain, the composition of CSF is influenced by the metabolic activity of the cells in the surrounding area and the composition of the blood. Although the origin of CSF is plasma, the composition of CSF is not the same as that of plasma because the blood-brain barrier exists between the two sites (Fig. 25.5). The blood-brain barrier is composed of endothelial cells, smooth muscle, and the pial and arachnoid membranes, and it regulates the movement of ions between blood and CSF. In addition, the choroid plexus also determines the ionic composition of CSF by transporting ions into and out of CSF. The blood-brain barrier is relatively impermeable to H+ and HCO3− ions, but it is very permeable by CO2. Thus the Pco2 in CSF parallels the arterial Pco2. CO2 is also produced by cells of the brain as a product of metabolism. As a consequence, the Pco2 in CSF is usually a few millimeters of mercury higher than that in arterial blood, and so the pH is slightly more acidic (7.33) in CSF than in plasma (Table 25.1).

FIG. 25.5 Carbon dioxide and the blood-brain barrier. Paco2 crosses the blood-brain barrier and rapidly equilibrates with CO2 in cerebrospinal fluid (CSF). H+ and HCO3− ions cross the barrier slowly. The partial pressure of arterial carbon dioxide (Paco2) combines with CO2 generated by metabolism to dilate the smooth muscle. In comparison with arterial blood, the pH of CSF is lower and the Pco2 is higher, with little protein buffering.

Table 25.1 Normal Values for the Composition of Cerebrospinal Fluid and Arterial Blood Parameter

Cerebrospinal Fluid

Arterial Blood

pH

7.33

7.40

Pco2 (mm Hg)

44

40

HCO3− (mEq/L)

22

24

Pco2, Partial pressure of carbon dioxide.

AT THE C ELLU LAR LEVEL The Henderson-Hasselbalch equation relates the pH of CSF to the concentration of bicarbonate ([HCO3−]) and Pco2:

where α is the solubility coefficient (0.03 mmol/L per mm Hg) and pK is the negative logarithm of the dissociation constant for carbonic acid (6.1). The Henderson-Hasselbalch equation demonstrates that an increase in CSF Pco2 causes the pH of CSF to decrease at any given [HCO3−]. The decrease in pH stimulates the central chemoreceptors and thereby increases ventilation. Thus CO2 in blood regulates ventilation by its effect on the pH of CSF. The resulting hyperventilation reduces the Paco2, and therefore the Pco2 of CSF, and returns the pH of CSF toward a normal value. Furthermore, cerebral vasodilation accompanies an increase in Paco2, and this enhances the diffusion of CO2 into CSF. In contrast, an increase in CSF [HCO3−] causes an increase in the pH of CSF at any given Paco2. Changes in Paco2 that result from alterations in pH activate homeostatic mechanisms that return the pH back toward a normal value. The blood-brain barrier regulates the pH of CSF by adjusting the ionic composition and [HCO3−] of CSF. These changes in CSF [HCO3−], however, occur slowly, over a period of several hours, whereas changes in CSF Pco2 can occur within minutes. Thus compensation for changes in the pH of CSF requires hours to develop fully.

Peripheral Chemoreceptors The carotid and aortic bodies are peripheral chemoreceptors that respond to changes in Pao2 (the O2 dissolved in plasma, not the total O2 content of blood), Paco2, and pH, and they transmit afferent information to the central respiratory control center. The peripheral chemoreceptors are the only chemoreceptors that respond to changes in Pao2. The peripheral chemoreceptors are also responsible for approximately 40% of the ventilatory response to Paco2. These chemoreceptors are small, highly vascularized structures. They consist of type I (glomus) cells that are rich in mitochondria and endoplasmic reticulum. They also have several types of cytoplasmic granules (synaptic vesicles) that contain various neurotransmitters, including dopamine, acetylcholine, norepinephrine, and neuropeptides. Afferent nerve fibers synapse with type I cells, and they transmit information to the brainstem through the carotid sinus nerve (carotid body) and vagus nerve (aortic body). Type I cells are the cells primarily responsible for sensing Pao2, Paco2, and pH. In response to even small decreases in Pao2, there is an increase in chemoreceptor discharge, which enhances respiration. The response is robust when Pao2 decreases below 75 mm Hg. Thus ventilation is regulated by changes in arterial and CSF pH through effects on peripheral and central chemoreceptors (Fig. 25.6).

FIG. 25.6 The ventilatory response to partial pressure of arterial carbon dioxide (Paco2) is affected by the concentration of hydrogen ([H+]) in cerebrospinal fluid (CSF) and brainstem interstitial fluid. During chronic metabolic acidosis (e.g., diabetic ketoacidosis), the [H+] in CSF is increased, and the ventilatory response to inspired Paco2 is increased (steeper slope). Conversely, during chronic metabolic alkalosis (a relatively uncommon condition), the [H+] in CSF is decreased and the ventilatory response to inspired Paco2 is decreased (reduced slope). The positions of the response lines are also shifted, which indicates altered thresholds.

IN THE C LIN IC Imagine flying from New York City to Denver. The barometric pressure in New York is approximately 760 mm Hg, whereas in the mountains surrounding Denver, Colorado, it is 600 mm Hg. At sea level, the Pao2 is approximately 95 mm Hg and Pao2 = [(760 − 47) × 0.21] − [40/0.8] = 100 mm Hg (according to the alveolar air equation; see Chapter 23). If the alveolar-arterial Po2 difference [AaDo2] is 5 mm Hg, then Pao2 = 100 mm Hg − 5 mm Hg = 95 mm Hg. In the CSF, pH would be approximately 7.33, Paco2 would be 44 mm Hg (Paco2 + CO2 produced by metabolism of brain cells), and HCO3− would be approximately 22 mEq/L. When you arrive in Denver, there is an abrupt decrease in the partial pressure of inspired O2 (Pio2): Pio2 = (600 − 47) × 0.21 = 116 mm Hg; there are also decreases in the partial pressures of alveolar and arterial O2: Pao2 = 116 − (40/0.8) = 66 mm Hg, and Pao2 = 61 mm Hg (if there is no change in AaDo2). This decrease in arterial O2 stimulates the peripheral chemoreceptors and thereby increases ventilation. The increase in ventilation decreases Paco2 and elevates arterial pH. The result of this increase in ventilation is to minimize the hypoxemia by increasing Pao2. For example, assume that Paco2 decreases to 30 mm Hg. Then Pao2 = [(600 − 47) × 0.21] − [30/0.8] = 78 mm Hg, a 12-mm Hg increase in Pao2. The decrease in Paco2 also causes a reduction in the Pco2 of CSF. Because [HCO3−] is unchanged, the pH of CSF increases. This increase in the pH of CSF attenuates the rate of discharge of the central chemoreceptors and decreases their contribution to the ventilatory drive. Over the next 12 to 36 hours, [HCO3−] in CSF decreases as acid-base transporter proteins in the blood-brain barrier reduce [HCO3−]. As a consequence, the pH of CSF returns toward normal. Central chemoreceptor discharge

increases, and minute ventilation is further increased. At the same time that [HCO3−] in CSF decreases, HCO3− is gradually excreted from plasma by the kidneys. This results in a gradual return of arterial pH toward normal values. Peripheral chemoreceptor stimulation increases further as arterial pH becomes normal (peripheral chemoreceptors are inhibited by the elevated arterial pH). Finally, within 36 hours of arriving at high altitude, minute ventilation increases significantly. This delayed response is greater than the immediate effect of the hypoxemia on ventilation. This further increase in ventilation is due to both central and peripheral chemoreceptor stimulation. Thus after 36 hours, both arterial pH and CSF pH are approaching normal values; minute ventilation is increased, Pao2 is decreased, and Paco2 is decreased. You now return home. When you land in New York, the Pio2 returns to a normal value, and the hypoxic stimulus to ventilation is removed. Pao2 returns to a normal value, and the peripheral chemoreceptor stimulation to ventilation decreases. This causes an increase in arterial [CO2] toward normal values, which in turn causes an increase in CSF [CO2]. This increase is associated with a decrease in the pH of CSF as [HCO3−] in CSF is reduced and ventilation is augmented. Over the next 12 to 36 hours, the acid-base transporters in the blood-brain barrier transport HCO3− back into CSF, and the pH of CSF gradually returns toward normal values. Similarly, the pH of blood decreases as Paco2 rises because arterial [HCO3−] falls. This stimulates the peripheral chemoreceptors, and minute ventilation remains augmented. Over the next 12 to 36 hours, [HCO3−] excretion by the kidneys increases (see Chapter 36), arterial pH returns to a normal value, and minute ventilation returns to a normal level.

Pulmonary Mechanoreceptors Chest Wall and Lung Reflexes Several reflexes that arise from the chest wall and lungs affect ventilation and ventilatory patterns (Table 25.2). The Hering-Breuer inspiratory-inhibitory reflex is stimulated by increases in lung volume, especially those associated with an increase in both ventilatory rate and tidal volume. This stretch reflex is mediated by vagal fibers, and when elicited, it results in cessation of inspiration by stimulating the offswitch neurons in the medulla. This reflex is inactive during quiet breathing and appears to be most important in newborns. Stimulation of nasal or facial receptors with cold water initiates the diving reflex. When this reflex is elicited, apnea, or cessation of breathing, and bradycardia occur. This reflex protects individuals from aspirating water in the initial stages of drowning. Activation of receptors in the nose is responsible for the sneeze reflex.

Table 25.2 Reflexes in the Respiratory Tract Reflex

Stimuli

Site of Activation

Receptor Type

Effect

HeringBreuer Inflation

Lung inflation (maximal)

Airway smooth muscles (bronchi, bronchioles)

Stretch receptor, vagal afferent

Inhibition of medullary and pontine apenustic center, inhibiting active inspiration

HeringLung deflation Breuer (maximal) Deflation

Airway smooth muscles (bronchi, bronchioles)

Stretch receptor or Inhibition of respiratory center ​proprioceptor, inhibatory signal vagal afferent

Diving

Cold water, possibly pressure

Face and anterior nasal mucosa

Chemesthetic Apnea, bradycardia, increased chemoreceptor, peripheral vascular resistance trigeminal afferent

Cough

Inhaled irritant (acid, dust, noxious gas, capscacin), mechanical irritant

Trachea, main carina, Chemical and brancing points of probably larger airways, mechanical proximal receptors, conducting vagal afferent airways

Sequence of inspiration, brief glottic closure with expiratory muscle activation, quick glottic opening releasing forceful exhalation

Sneeze

Chemical or mechanical irritant

Nasal cavity

Eye closing, deep inhalation, glottic closure during forced exhalation, abrupt glotic opening with forceful airflow through nose and mouth.

Chemical and probably mechanical receptors, trigeminal afferent

The aspiration or sniff reflex can be elicited by stimulation of mechanical receptors in the nasopharynx and pharynx. This is a strong, short-duration inspiratory effort that brings material from the nasopharynx to the pharynx, where it can be swallowed or expectorated. The mechanical receptors responsible for the sniff reflex are also important in swallowing by inhibiting respiration and causing laryngeal closure. For anatomical reasons, only newborns can breathe and swallow simultaneously, which allows more rapid ingestion of nutrients. The larynx contains both superficial and deep receptors. Activation of the superficial receptors results in apnea, cough, and expiratory movements that protect the lower respiratory tract from aspirating foreign material. The deep receptors are located in the skeletal muscles of the larynx, and they control muscle fiber activation, as in other skeletal muscles. Sensory Receptors and Reflexes Three major types of sensory receptors located in the tracheobronchial tree respond to a variety of different stimuli, and those responses result in changes in the lung’s mechanical properties, alterations in the respiratory pattern, and the development of respiratory symptoms. Inhaled dust, noxious gases, and cigarette smoke stimulate irritant receptors in the trachea and large airways that transmit information through myelinated vagal afferent fibers. Stimulation of these receptors results in an increase in airway resistance, reflex apnea, and coughing. These receptors are also known as rapidly adapting pulmonary

stretch receptors. Slowly adapting pulmonary stretch receptors respond to mechanical stimulation, and they are activated by lung inflation. They also transmit information through myelinated, vagal afferent fibers. The increase in lung volume in people with COPD stimulates these pulmonary stretch receptors and delays the onset of the next inspiratory effort. This explains the long, slow expiratory effort in affected individuals, and it is essential to minimize dynamic, expiratory airway compression. In addition, specialized sensory receptors located in the lung parenchyma respond to chemical or mechanical stimulation in the lung interstitium. These receptors are called juxta-alveolar, (or J) receptors. They transmit their afferent input through unmyelinated, vagal C fibers. They may be responsible for the sensation of dyspnea (abnormal shortness of breath) and the rapid, shallow ventilatory patterns that occur in interstitial lung edema and some inflammatory lung states. Somatic receptors are also located in the intercostal muscles, rib joints, accessory muscles of respiration, and tendons, and they respond to changes in the length and tension of the respiratory muscles. Although they do not directly control respiration, they do provide information about lung volume and play a role in terminating inspiration. They are especially important in individuals with increased airway resistance and decreased pulmonary compliance because they can augment muscle force within the same breath. Somatic receptors also help minimize the chest wall distortion during inspiration in newborns, who have very compliant rib cages.

Exercise The ability to exercise depends on the capacity of the cardiac and respiratory systems to increase delivery of O2 to tissues and remove CO2 from the body. Ventilation increases immediately when exercise begins, and this increase in minute ventilation closely matches the increases in O2 consumption and CO2 production that accompany exercise (Fig. 25.7). Ventilation is linearly related to both CO2 production and O2 consumption at low to moderate levels (see Fig. 25.7). During maximal exercise, a physically fit individual can achieve an O2 consumption of 4 L/minute with a minute ventilation volume of 120 L/minute, which is almost 15 times the resting level.

FIG. 25.7 Oxygen consumption (V̇̇o2) as a function of the metabolic changes that occur during exercise. The anaerobic threshold (arrow) is the point at which the illustrated variables change and is due to lactic acidosis. Paco2, Partial pressure of arterial carbon dioxide; Pao2, partial pressure of arterial oxygen; V̇c o2, carbon dioxide consumption.

Exercise is remarkable because of the lack of significant changes in blood gases. Except at maximal exertion, changes in Paco2 and Pao2 are minimal during exercise. Arterial pH remains within normal values during moderate exercise. During strenuous exercise, arterial pH begins to fall as lactic acid is liberated from muscles during anaerobic metabolism. This decrease in arterial pH stimulates ventilation that is out of proportion to the level of exercise. The level of exercise at which sustained metabolic (lactic) acidosis begins is called the anaerobic threshold (see Fig. 25.7).

Abnormalities in the Control of Breathing Changes in the ventilatory pattern can occur for both primary and secondary reasons. During sleep, approximately one-third of healthy individuals have brief episodes of apnea or hypoventilation that have no significant effects on Pao2 or Paco2. The apnea usually lasts less than 10 seconds, and it occurs in the lighter stages of slow-wave and rapid eye movement (REM) sleep. In sleep apnea syndromes, the duration of apnea is abnormally prolonged, and it changes Pao2 and Paco2. There are two major categories of sleep apnea (Fig. 25.8). The first, obstructive sleep apnea (OSA), is the most common of the sleep apnea syndromes, and it occurs when the upper airway (generally the hypopharynx) closes during inspiration. Although the process is similar to what happens during snoring, it is more severe, inasmuch as it obstructs the airway and causes cessation of airflow.

FIG. 25.8 The two main categories of sleep apnea. A, Obstructive sleep apnea, the pleural pressure oscillations increase as CO2 level rises. This indicates that resistance to airflow is very high as a result of upper airway obstruction. B, Central sleep apnea is characterized by no attempt to breathe, as demonstrated by no oscillations in pleural pressure.

IN THE C LIN IC The clinical histories of individuals with OSA are very similar. A spouse usually reports that the affected individual snores. The snoring becomes louder and louder and then stops while the individual continues to make vigorous respiratory efforts (see Fig. 25.8). The individual then awakens, falls back to sleep, and continues the same process repetitively throughout the night. Individuals with OSA awaken when the arterial hypoxemia and hypercapnia stimulate both peripheral and central chemoreceptors. Respiration is restored briefly before the next apneic event occurs. Individuals with OSA can have hundreds of these events each night that interrupt sleep. Complications of OSA include sleep deprivation, polycythemia, right-sided cardiac failure (cor pulmonale), and pulmonary hypertension secondary to the recurrent, hypoxic events. OSA is common in individuals with obesity and in those with excessive compliance of the hypopharynx, upper airway edema, and structural abnormalities of the upper airway. The second sleep apnea syndrome is central sleep apnea. This variant of apnea occurs when the ventilatory drive to the respiratory motor neurons decreases. Individuals with central sleep apnea have repeated episodes of apnea, during which time they make no respiratory effort, every night (see Fig. 25.8). The degree of hypercapnia and hypoxemia in individuals with central sleep apnea is less than that in individuals with OSA, but the same complications (e.g., polycythemia) can occur when central sleep apnea is recurrent and severe. Cheyne-Stokes ventilation is another abnormality of breathing that is characterized by varying tidal

volume and ventilatory frequency (Fig. 25.9). After a period of apnea, tidal volume and respiratory frequency increase progressively over several breaths, and then they progressively decrease until apnea recurs. This irregular breathing pattern is seen in some individuals with central nervous system diseases, head trauma, and increased intracranial pressure. It is also present on occasion in healthy individuals during sleep at high altitude. The mechanism underlying Cheyne-Stokes respiration is not known. In some individuals, it appears to be due to slow blood flow in the brain in association with periods of overshooting and undershooting ventilatory effort in response to changes in Pco2.

FIG. 25.9 In Cheyne-Stokes breathing, tidal volume and, as a consequence, arterial blood gas levels wax and wane. In general, Cheyne-Stokes breathing is a sign of vasomotor instability, particularly low cardiac output. Paco2, Partial pressure of arterial carbon dioxide; Pao2, partial pressure of arterial oxygen.

IN THE C LIN IC Central alveolar hypoventilation, also known as Ondine’s curse, is a rare disease in which voluntary breathing is intact but abnormalities in automaticity exist. It is the most severe of the central sleep apnea syndromes. As a result, people with central alveolar hypoventilation can breathe as long as they do not fall asleep. For these individuals, mechanical ventilation or, more recently, bilateral diaphragmatic pacing (similar to a cardiac pacemaker) can be lifesaving.

IN THE C LIN IC Sudden infant death syndrome (SIDS) is the most common cause of death in infants in the first year of life after the perinatal period. Although the cause of SIDS is not known, abnormalities in ventilatory control, particularly in CO2 responsiveness, have been implicated. Placing infants on their backs to sleep (which reduces the potential for CO2 rebreathing) has dramatically decreased (but not eliminated) the rate of death from this syndrome.

Apneustic breathing is another abnormal breathing pattern that is characterized by sustained periods of inspiration separated by brief periods of exhalation (Fig. 25.10C). The mechanism underlying this ventilatory pattern appears to be a loss of inspiratory-inhibitory activities that results in augmentation of the inspiratory drive. The pattern sometimes occurs in individuals with central nervous system injury.

FIG. 25.10 Some patterns of breathing. A, Normal rate of breathing is in the range of 12 to 20 breaths per minute. B, When sensory input is removed from various lung receptors (mainly stretch), each breathing cycle is lengthened and tidal volume is increased, so that alveolar ventilation is not significantly affected. C, When input from the cerebral cortex and thalamus is also eliminated, together with vagal blockade, the result is prolonged inspiratory activity broken after several seconds by brief expirations (apneusis).

Key Points 1. Ventilatory control is composed of the respiratory control center, central chemoreceptors, peripheral chemoreceptors, and pulmonary mechanoreceptors/sensory nerves. Paco2 is the major factor that influences ventilation. 2. The respiratory control center is composed of the dorsal respiratory group and the ventral respiratory group. Rhythmic breathing depends on a continuous (tonic) inspiratory drive from the dorsal respiratory group and on intermittent (phasic) expiratory input from the cerebrum, thalamus, cranial nerves, and ascending spinal cord sensory tracts. The peripheral and central chemoreceptors respond to changes in Paco2 and pH. The peripheral chemoreceptors (carotid and aortic bodies) are the only chemoreceptors that respond to changes in Pao2. 3. Acute hypoxia and chronic hypoxia affect breathing differently because the slow adjustments in CSF [H+] in chronic hypoxia alter sensitivity to CO2. 4. Irritant receptors protect the lower respiratory tract from particles, chemical vapors, and physical factors, primarily by inducing cough. C fiber J receptors in the terminal respiratory units are stimulated by distortion of the alveolar walls (by lung congestion or edema). 5. The two most important clinical abnormalities of breathing are obstructive and central sleep

apnea. 6. Pao2, Paco2, and pH remain within normal limits during moderate exercise; however, during strenuous exercise, pH falls, which stimulates ventilation, whereas Pao2 and Paco2 remain relatively normal.

C H AP T E R 2 6

Host Defense and Metabolism in the Lung LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. How do the components of the mucociliary clearance system function to remove xenobiotic substances and particulates? 2. How does particle shape and size influence their pattern of airway deposition and/or clearance? 3. What are the major mechanisms of particle deposition? 4. How are the mucosal and systemic immune responses similar? How are they different? 5. What unique features of immunoglobulin A make it well suited for immunologic protection in the mucosal environment? 6. What are the cellular components of the adaptive and innate immune cells in the respiratory system?

In addition to their primary function of gas exchange, the lungs act as a primary barrier between the outside world and the inside of the body, with host defense functions. They are also active organs in the metabolism of xenobiotic and endogenous compounds.

Host Defense To cope with the inhalation of foreign substances, the respiratory system and, in particular, the conducting airways have developed unique structural features: the mucociliary clearance system and specialized adaptive and innate immune response mechanisms.

Mucociliary Clearance System The mucociliary clearance system protects the conducting airways by trapping and removing inhaled pathogenic viruses and bacteria, in addition to nontoxic and toxic particulates (e.g., pollen, ash, mineral dust, mold spores, and organic particles), from the lungs. These particulates are inhaled with each breath and must be removed. The three major components of the mucociliary clearance system are two fluid layers, referred to as the sol (periciliary fluid) and gel (mucus) layers, and cilia, which are positioned on the surface of bronchial epithelial cells (Fig. 26.1). Inhaled material is trapped in the viscoelastic (sticky) mucus layer, whereas the watery periciliary fluid allows the cilia to move freely and establish an upward flow to clear particulates from the lung. Effective clearance requires both ciliary activity and the

appropriate balance of periciliary fluid and mucus.

FIG. 26.1 Overview of the epithelial lining and innervation of the tracheobronchial tree. The cilia of the epithelial cell reside in the periciliary fluid layer, and the mucus layer is on top. Interspersed between the ciliated epithelial cells are surface secretory (goblet) cells and submucosal glands. Sympathetic and parasympathetic nerve fibers descend into the submucosal glands and smooth muscles.

Periciliary Fluid Layer The periciliary fluid layer is composed of nonviscous serous fluid, which is produced by the pseudostratified ciliated columnar epithelial cells that line the airways. These cells have the ability to either secrete fluid, a process that is mediated by activation of cystic fibrosis transmembrane regulator (CFTR) chloride (Cl−) ion channels (Na+ secretion follows passively between cells across the tight junctions), or reabsorb fluid, a process that is mediated by activation of epithelial sodium channels (ENaC; Cl− absorption follows passively between cells across the tight junctions). NaCl secretion or reabsorption temporarily establishes an osmotic gradient across the pseudostratified epithelium, which provides the driving force for passive water movement. The balance between CFTR-mediated Cl− secretion and ENaC-mediated Na+ absorption is regulated by a variety of hormones and determines the volume of the periciliary fluid, which in the healthy lung is 5 to 6 µm deep, a level that is optimal for rhythmic beating of the cilia and mucociliary clearance.

IN THE C LIN IC Cystic fibrosis (CF) is the most common lethal inherited disease among white people. It is an autosomal recessive disease caused by mutations in the CFTR gene. It is characterized by chronic bacterial lung infection, progressive decline in lung function, and premature death at an average age of 48 years. More than 2000 mutations of the CFTR gene have been described, but 70% of affected individuals have a deletion of phenylalanine at codon 508 (F508del-CFTR) in at least one allele. This

mutation results in a lack of Cl− secretion and an increase in ENaC-mediated Na+ reabsorption, which in turn results in a reduction in the volume of the periciliary fluid. Detailed study of the different CFTR mutations has resulted in an understanding of various diseaserelated phenotypes, some of which are associated with milder disease and some with more severe disease. Since the 1980s, research findings have elucidated how many of the most common mutations in the CFTR gene cause CF, and this has led to the development of drugs that target specific mutations and reverse the progressive reduction in lung function. For example, in one mutation (G551D-CFTR, which affects ≈5% of patients with CF), the CFTR Cl− channel reaches the plasma membrane of airway epithelial cells but does not secrete Cl−. Through precision medicine, a drug, ivacaftor (Kalydeco), has been found to stimulate Cl− secretion via the G551D-CFTR, thereby improving lung function and decreasing the rate of disease progression. When the alleles are homozygous for the F508del-CFTR mutation (which affects ≈50% of patients with CF), the CFTR Cl− channel does not reach the plasma membrane of airway epithelial cells. In 2015 and 2018, the U.S. Food and Drug Administration approved combination drug therapies, lumacaftor/ivacaftor (Orkambi) and tezacaftor/ivacaftor (Symdeko), that have been shown to correct the gene defect, increase the amount of F508del-CFTR in the plasma membrane, and improve Cl− transport. More recently, the combination of elexacaftor/tezacaftor/ivacaftor (Trikafta) has been approved to treat additional mutations in the CFTR gene. Both Symdeko and Trikafta have been approved for use in people with two alleles of F508del-CFTR as well as people with one F508delCFTR allele and the other allele containing a variety of other mutations in the CFTR gene. Clinically, these medications have been shown to improve lung function and to decrease the rate of decline in lung function. Mucus Layer The mucus layer lies on top of the periciliary fluid layer and is composed of a complex mixture of macromolecules and electrolytes. Because the mucus layer is in direct contact with air, it entraps inhaled substances, including pathogens. The mucus layer is predominantly water (95%–97%), 5 to 10 µm thick, and exists as a discontinuous blanket (i.e., islands of mucus). Mucus has low viscosity and high elasticity and is composed of glycoproteins with groups of oligosaccharides attached to a protein backbone. Healthy individuals produce approximately 100 mL of mucus each day. Four cell types contribute to the quantity and composition of the mucus layer: goblet cells and Clara cells within the tracheobronchial epithelium, and mucous cells and serous cells within the tracheobronchial submucosal glands. Goblet cells, also referred to as surface secretory cells, represent approximately 15% to 20% of the tracheobronchial epithelium, and are found in the tracheobronchial tree up to the 12th division. In many respiratory diseases, goblet cells appear further down the tracheobronchial tree; thus the smaller airways are more susceptible to obstruction by mucus plugging. Goblet cells secrete neutral and acidic glycoproteins rich in sialic acid in response to chemical stimuli. In the presence of infection or cigarette smoke or in patients with chronic bronchitis, goblet cells can increase in size and number, extend above the 12th division of the tracheobronchial tree, and secrete copious amounts of mucus. Injury and infection increase the viscosity of the mucus secreted by goblet cells, which reduces mucociliary clearance of inhaled particles and pathogens. Submucosal tracheobronchial glands are present wherever there is cartilage in the upper regions of the conducting airways, and they secrete water, ions, and mucus into the airway lumen through a ciliated duct (Fig. 26.1). Although both mucous and serous cells secrete mucus, their cellular structure and mucus

composition are distinctly different (Table 26.1). In several lung diseases, including chronic bronchitis, the number and size of submucosal glands are increased, which leads to increases in mucus production, alterations in chemical composition of mucus (i.e., increased viscosity and decreased elasticity), and the formation of mucus plugs that cause airway obstruction. Mucus secretion from submucosal tracheobronchial glands is stimulated by parasympathetic (cholinergic) compounds such as acetylcholine and substance P and inhibited by sympathetic (adrenergic) compounds such as norepinephrine and vasoactive intestinal polypeptide. Local inflammatory mediators such as histamine and arachidonic acid metabolites also stimulate mucus production. Table 26.1 Properties of Serous and Mucous Cells in Submucosal Gland Property

Serous Cells

Mucous Cells

Location

Most distal

Middle to distal

Granules

Small, electron-dense

Large, electron-lucent

Glycoproteins

Acidic Neutral Lysozyme, lactoferrin

Hormones

α-Adrenergic > β-Adrenergic

β-Adrenergic > α-Adrenergic

Receptors

Muscarinic

Muscarinic

Degranulation α-Adrenergic Cholinergic Substance P

β-Adrenergic Cholinergic

Clara cells, located in the epithelium of bronchioles, also contribute to the composition of mucus through secretion of a nonmucinous material containing carbohydrates and proteins. These cells play a role in bronchial regeneration after injury.

AT THE C ELLU LAR LEVEL Sputum is expectorated mucus. However, in addition to mucus, sputum contains serum proteins, lipids, electrolytes, Ca++, DNA from degenerated white blood cells (collectively known as bronchial secretions), and extrabronchial secretions, including nasal, oral, lingual, pharyngeal, and salivary secretions. The color of sputum is more closely correlated with the amount of time that it has been present in the lower respiratory tract than with the presence of infection. Although not precisely identifiable with disease diagnosis, the color of sputum can be informative in helping lead to a diagnosis and stage of disease. Mucus has many colors: white, yellow, green, red, pink, brown, gray, and black. The coloration is commonly due to the type of cell present in the airways (inflammatory cells, such as neutrophils or eosinophils, or red blood cells) and how long they have been there. Clear or cloudy white thin mucus is considered normal; however, if amounts and thickness are increased, it may represent an early sign of infection. Thick white mucus can be the only identifiable feature of gastroesophageal reflux disease caused by gastric acid reflux into the airways. Yellow and green coloration of mucus is due to the presence and breakdown of neutrophils and eosinophils in infectious

and allergic diseases. Yellow is typically associated with more acute disease (infection, allergy), and green usually indicates a more chronic stage with the presence of bacteria (chronic bronchitis, bronchiectasis, cystic fibrosis, and lung abscess). Red mucus indicates the presence of red blood cells in the airways and is associated with pneumococcal pneumonia, lung cancer, tuberculosis, and pulmonary emboli. Pink mucus is typically associated with the breakdown of eosinophils in individuals with allergies. Gray, brown, and black mucus is often associated with cigarette or marijuana smoking, cocaine use, air pollution (workplace environment, such as coal mines), and old blood. Ciliated Cells and Cilia As noted previously, the respiratory tract to the level of the bronchioles is lined by a pseudostratified, ciliated columnar epithelium (see Fig. 26.1). These cells maintain the level of the periciliary fluid in which cilia and the mucociliary transport system function. Mucus and inhaled particles are removed from the airways by the rhythmic beating of the cilia. There are approximately 250 cilia per airway epithelial cell, and each is 2 to 5 µm in length. Cilia are composed of nine microtubular doublets that surround two central microtubules held together by dynein arms, nexin links, and spokes. The central microtubule doublet contains an adenosine triphosphatase (ATPase) that is responsible for the contractile beat of the cilium. Cilia beat with a coordinated oscillation in a characteristic, biphasic, and wave-like rhythm called metachronism. They beat at approximately 1000 strokes per minute, with a power forward stroke and a slow return or recovery stroke. During their power forward stroke, the tips of the cilia extend upward into the viscous mucus layer and thereby move it and the entrapped particles. On the reverse beat, the cilia release the mucus and withdraw completely into the sol layer. Cilia in the nasopharynx beat in the direction that propels the mucus into the pharynx, whereas cilia in the trachea propel mucus upward toward the pharynx, where it is swallowed.

Particle Deposition and Clearance In general, deposition of particles in the lung depends on the particle size, density, and shape; the distance over which it has to travel; airflow speed; and the relative humidity of the air. The four major mechanisms for deposition are impaction, sedimentation, interception, and Brownian movement. Particle characteristics and properties, which influence the mechanism of deposition, are listed in Table 26.2. In general, particles larger than 10 µm are deposited by impaction in the nasal passages and do not penetrate into the lower respiratory tract. Particles 2 to 10 µm in size are deposited in the lower respiratory tract predominantly by inertial impaction at points of turbulent airflow (i.e., nasopharynx, trachea, and bronchi) and at airway bifurcations because their tendency to move in a straight direction prevents them from changing directions rapidly. In more distal areas, where airflow is slower, smaller particles (0.2–2 µm) are deposited on the surface by sedimentation as a result of gravity. For substances with elongated shapes (i.e., asbestos, silica), the mechanism of deposition is interception. The elongated particle’s center of gravity is compatible with the flow of air; however, when the distal tip of the particulate comes in contact with a cell or mucus layer, deposition is facilitated. Particles smaller than 0.2 µm are deposited in the smaller airways and alveoli and are influenced mainly by their diffusion coefficient and Brownian motion. Unlike the deposition of larger particles in the upper airways, particle density does not influence diffusion of these smaller particles, and deposition is enhanced with decreased size. These smaller particles come in contact with the alveolar epithelium, where cilia and the mucociliary transport system do not exist; thus they are removed by the phagocytic activity of alveolar macrophages or absorption into

the interstitium with subsequent clearance by lymphatic drainage. Although most alveolar macrophages are adjacent to the epithelium of the alveolus, some are located in the terminal airways and interstitial space. Table 26.2 Particle Deposition Characteristics Method of Deposition

Particle Size (µm)

Deposition Site

Airflow

Determining Factors

Impaction

>10

Nasal passages

Fast

Size, density

Fast

Size, density

2–10 Nasal pharynx Trachea Bronchi

Sedimentation

0.2–2.0

Distal airways

Slow

Size, density, diameter

Interception

NA

NA

Slow

Shape (elongated)

Brownian movement

80, diabetes, obesity, hypertension, and male gender. The virus is spread primarily by respiratory droplets or fomites produced when an infected individual coughs or sneezes, although transmission may occur by other mechanisms as well. Infected individuals can transmit the virus days before onset of their own symptoms. SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2) receptor found on the surface of alveolar type II cells. This can cause diffuse alveolar damage and progress to acute respiratory distress syndrome and death. Extrapulmonary manifestations include vascular complications (pulmonary emboli, stroke, and other blood vessel damage). Damage to the alveolarcapillary network leads to impaired gas diffusion. This in turn can produce capillary leak introducing serum proteins into the alveoli and disrupting the surfactant system. Combined, these processes decrease lung compliance and increased work of breathing. SARS-CoV-2 may also trigger an acute hyperinflammatory response known as a cytokine storm leading to elevations in IL-1, IL-2, IL-6, TNFalpha, and interferon-gamma. Cytokine storm can worsen respiratory distress and lead to blood clotting events (including stroke and myocardial infarction), acute kidney injury, and inflammation of the vascular endothelium.

Clinical Manifestations Associated With Abnormalities in Mucosal Innate and Adaptive Immunity By far the most common pathological conditions associated with mucosal tissue are allergic responses (e.g., allergic asthma, allergic rhinitis, and food and skin allergies). Some common human allergens include components of inhaled house dust mites, cockroaches, and cat dander, as well as bee stings and ingestion of peanuts. One of the most common drug allergens is penicillin, which can bind to many endogenous proteins and also alter their antigenicity. As previously described, the predominant antibody response in MALT is IgA; however, in an allergic response, IgE is the predominant antibody. It is generated by a switchover mechanism induced by the synthesis of IL-4 from Th2-primed CD4 T cells. The IL-4 induces the antibody-producing B cells to switch over from synthesizing IgG antibodies to IgE. IgE binds to the surface of mast cells in the submucosa through its crystallizable fragment (constant) region of the antibody molecule. Upon

reexposure to the inhaled antigen and its subsequent migration from the airway lumen to the submucosa, the allergen then binds to the antigen-binding fragment region of the IgE molecule, which forms an immune complex (IgE-Ag) on the surface of the mast cell. The final step is that IgE-Ag complexes must crosslink with other IgE-Ag complexes on the surface of the mast cell, which induce intracellular signaling pathways to initiate degranulation and immediate release of preformed Th2 mediators (histamine, heparin, prostaglandins, leukotrienes, IL-4, IL-5, IL-13, proteases). These mediators induce the classical signs of asthma: smooth muscle constriction (bronchoconstriction), eosinophil recruitment and activation (inflammation), and connective tissue remodeling. Symptoms of wheezing, coughing, and shortness of breath occur within minutes, followed by a late response of eosinophilia and airway inflammation. The inflammatory response can resolve spontaneously or as a result of therapy (bronchodilator or antiinflammatory drugs, such as corticosteroids). Low-grade inflammation may persist and result in a process called airway remodeling, manifested by permanent, irreversible structural changes such as submucosal fibrosis and airway smooth muscle hypertrophy. The mechanisms responsible for airway remodeling in allergic diseases are not fully understood, but chemokines and cytokines such as TGF-β, a potent profibrotic cytokine, play important roles. Thus a very elegant, highly effective defensive system against infectious and parasitic organisms is “tricked” into responding to an innocuous substance, an allergen, as if it were harmful and initiates its defenses; the result is allergic airway disease.

Metabolic Functions of the Lung The lungs are exposed to and metabolize a wide variety of xenobiotic substances. The endothelial cells within the lung capillary bed have a large surface area that receives very high blood flow, and lung endothelial cells have developed various mechanisms and cell surface receptors to metabolize xenobiotics. Most of the metabolic processing of inhaled or ingested xenobiotic compounds occurs enzymatically within the liver and intestinal tract with members of the cytochrome P-450 (CYP) enzyme families (e.g., CYP1, CYP2, CYP3). The lungs and other organs also selectively participate in the processing of xenobiotics and typically have lower levels of cytochrome P-450 enzymes. Prominent cytochrome P-450 enzymes in the lung include CYP1B1, CYP2B6, CYP2E1, CYP2J2, CYP3A5, and CYP1A1, the last of which is present in high levels in people who smoke cigarettes. Drugs for the treatment of asthma and chronic obstructive pulmonary disease—such as corticosteroids, long-acting β2 receptor agonists, leukotriene receptor antagonists, and methylxanthines—are degraded enzymatically in the lungs. In addition, a wide array of endogenous substances are metabolized by endothelial cells within the pulmonary capillary bed, including vasoactive amines, cytokines, lipid mediators, and proteins. Box 26.1 provides a list of compounds metabolized in the lung. Metabolism can occur through either intracellular or extracellular processing of endogenous substances that pass though the capillaries or by direct synthesis and secretion by endothelial cells. For example, angiotensin I is activated by angiotensin-converting enzyme, which is on the surface of endothelial cells. Serotonin, a vasoconstrictor, binds to a specific receptor on the surface of endothelial cells and is internalized and metabolized inside cells. Approximately 80% of the serotonin entering the lungs is metabolized in a single pass through the pulmonary capillary bed. Box 26.1

C o mpo unds M e t a bo lize d in t he Lung s Enzymatic Degradation in Pulmonary Circulation • Corticosteroids • Long-acting beta agonists • Methylxanthines Endothelial Cells in Pulmonary Capillary Bed • Vasoactive amines • Cytokines • Lipid mediators • Proteins

Pulmonary vascular endothelial cells synthesize and secrete prostacyclin, endothelin, clotting factors, nitric oxide, prostaglandins, and cytokines. Vascular endothelial cells, however, lack 5-lipoxygenase and are not able to synthesize leukotrienes (smooth muscle constrictors). Compounds not metabolized by the pulmonary capillary bed include epinephrine, dopamine, histamine, isoproterenol, angiotensin II, and substance P.

AT THE C ELLU LAR LEVEL Angiotensin-converting enzyme (ACE) is present in small indentations (caveolae) on the surface of pulmonary endothelial cells and catalyzes the conversion of the physiological inactive angiotensin I to the active angiotensin II, a potent vasoconstrictor. This is a major mechanism in the body’s ability to supply systemic levels of angiotensin II and thus influence blood pressure. The therapeutic use of ACE inhibitors is important in the management of patients with high blood pressure.

IN THE C LIN IC The lungs play a key role in the metabolism of many prodrugs, which are inactive, into active drugs. Administration of prodrugs improves the amount of active drug delivered to their targets in the body. In many cases, inactive precursor prodrugs are delivered systemically or locally (through inhalation) to the lungs, where they are activated in situ. An example of this is beclomethasone dipropionate (Qvar, Beconase AQ), a medication inhaled by patients with asthma, which is activated by esterases within the lung to the active form 17-beclamethasone monopropionate.

Key Points 1. The respiratory system has developed unique structural (mucociliary transport system) and immunological (mucosal immune system) features to cope with the constant environmental exposure to foreign substances; these features limit or inhibit inflammation. 2. The three components of the mucociliary transport system are the sol phase (periciliary fluid), the

gel phase (mucus), and cilia. 3. The depth of the periciliary fluid layer is maintained by the balance between Cl− secretion and Na+ absorption and is essential to normal ciliary beating. 4. Mucus is a complex macromolecule composed of glycoproteins, proteins, electrolytes, and water. It has low viscosity and high elastic mechanical properties. 5. Goblet cells, Clara cells, and the mucous and serous cells residing in the tracheobronchial glands produce mucus. 6. Particle deposition in the lung is dependent on their size, density, and shape; the distance traveled; airflow speed; and relative humidity. The major mechanisms for particle deposition are impaction (particles larger than 10 µm, in nasal passages, and particles 2 to 10 µm, in the nasopharynx, trachea, and bronchi), sedimentation (particles 0.2–2 µm in size, in distal airways), interception (particles with elongated shape, in the lower airways), and Brownian movement (particles smaller than 0.2 µm, in the alveoli). 7. The respiratory system is part of the mucosal immune system, which is composed of the intestinal (GALT), the respiratory (BALT), and urinary tract systems. These systems do not contain true lymph nodes with afferent and efferent lymph flow; they are composed mainly of nonencapsulated lymph nodules without true lymphatic drainage. 8. The nonciliated lymphoepithelium of BALT establishes a break in the mucociliary blanket that acts as a drain to facilitate the collection and immune processing of foreign particulates throughout the conducting airways. 9. TCRγδ T cells, IgA synthesizing plasma cells, NK cells, and alveolar macrophages are highly specialized innate and adaptive immune cells unique to the anti-inflammatory defense system in the lung and other mucosal tissues.

S E CT I ON 6

Gastrointestinal Physiology Kim E. Barrett and Helen E. Raybould

Chapter 27 Functional Anatomy and General Principles of Regulation in the Gastrointestinal Tract Chapter 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal Chapter 29 The Gastric Phase of the Integrated Response to a Meal Chapter 30 The Small Intestinal Phase of the Integrated Response to a Meal Chapter 31 The Colonic Phase of the Integrated Response to a Meal Chapter 32 Transport and Metabolic Functions of the Liver

C H AP T E R 2 7

Functional Anatomy and General Principles of Regulation in the Gastrointestinal Tract LEARNING OBJECTIVES Upon completion of this chapter, you should be able to answer the following questions: 1. What is the neural innervation of the GI tract, and how is GI function regulated? 2. What are some examples of neural, paracrine, and humoral regulation of GI function?

The gastrointestinal (GI) tract consists of the alimentary tract from the mouth to the anus and includes the associated glandular organs that empty their contents into the tract. The overall function of the GI tract is to absorb nutrients and water into the circulation and eliminate waste products. The major physiological processes that occur in the GI tract are motility, secretion, digestion, and absorption. Most of the nutrients in the diet of mammals are taken in as solids and as macromolecules that are not readily transported across cell membranes to enter the circulation. Thus, digestion consists of physical and chemical modification of food such that absorption can occur across intestinal epithelial cells. Digestion and absorption require motility of the muscular wall of the GI tract to move the contents along the tract and to mix the food with secretions. Secretions from the GI tract and associated organs consist of enzymes, biological detergents, and ions that provide an intraluminal environment optimized for digestion and absorption. These physiological processes are highly regulated to maximize digestion and absorption, and the GI tract is endowed with complex regulatory systems to ensure this occurs. In addition, the GI tract absorbs drugs administered by the oral or rectal routes. The GI tract also serves as an important organ for excretion of substances. It stores and excretes waste substances from ingested food materials and excretes products from the liver such as cholesterol, steroids, and drug metabolites (all sharing the common property of being lipid-soluble molecules). When considering the physiology of the GI tract, it is important to remember that it is a long tube that is in contact with the body’s external environment. As such, it is vulnerable to infectious microorganisms that can enter along with food and water. To protect itself, the GI tract possesses a complex system of defenses consisting of immune cells and other nonspecific defense mechanisms. In fact, the GI tract represents the largest immune organ of the body. This chapter provides an overview of the functional anatomy and general principles of regulation in the GI system.

Functional Anatomy The structure of the GI tract varies greatly from region to region, but there are common features in the

overall organization of the tissue. Essentially the GI tract is a hollow tube divided into major functional segments; the major structures along the tube are the mouth, pharynx, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, and anus (Fig. 27.1). Together the duodenum, jejunum, and ileum make up the small intestine, and the colon, rectum and anus are referred to as the large intestine. Associated with the tube are blind-ending glandular structures that are invaginations of the lining of the tube; these glands empty their secretions into the gut lumen (e.g., Brunner’s glands in the duodenum, which secrete copious amounts of HCO3−). Additionally, there are glandular organs attached to the tube via ducts through which secretions empty into the gut lumen for example, the salivary glands and the exocrine pancreas.

FIG. 27.1 General anatomy of the GI system and its division into functional segments.

The major structures along the GI tract have many functions. One important function is storage; the stomach and colon are important storage organs for processed food (also referred to as chyme) and exhibit specialization in terms of both their functional anatomy (e.g., shape and size) and control mechanisms (characteristics of smooth muscle to produce tonic contractions) that enable them to perform this function efficiently. The predominant function of the small intestine is digestion and absorption; the major specialization of this region of the GI tract is a large surface area over which absorption can occur. The colon reabsorbs water and ions to ensure they do not get eliminated from the body. Ingested food is moved along the GI tract by the action of muscle in its walls. Separating the regions of the GI tract are also specialized muscle structures called sphincters. These function to isolate one region from the next and provide selective retention of contents or prevent backflow, or both. The blood supply to the intestine is important for carrying absorbed nutrients to the rest of the body. Unlike other organ systems of the body, venous drainage from the GI tract does not return directly to the heart but first enters the portal circulation leading to the liver. Thus, the liver is unusual in receiving a considerable part of its blood supply from other than the arterial circulation. GI blood flow is also notable for its dynamic regulation. Splanchnic blood flow receives about 25% of cardiac output, an amount disproportionate to the mass of the GI tract it supplies. After a meal, blood can also be diverted

from muscle to the GI tract to serve the metabolic needs of the gut wall and also to remove absorbed nutrients. The lymphatic drainage of the GI tract is important for the transport of lipid-soluble substances that are absorbed across the GI tract wall. As we will see in Chapter 30, lipids and other lipid-soluble molecules (including some vitamins and drugs) are packaged into particles that are too large to pass into the capillaries and instead pass into lymph vessels in the intestinal wall. These lymph vessels drain into larger lymph ducts, which finally drain into the thoracic duct and thus into the systemic circulation on the arterial side. This has major physiological implications in lipid metabolism and also in the ability of drugs to be delivered straight into the systemic circulation.

Cellular Specialization The wall of the tubular gut is made up of layers consisting of specialized cells (Fig. 27.2).

FIG. 27.2 General organization of the layers composing the wall of the GI tract.

Mucosa The mucosa is the innermost layer of the GI tract. It consists of the epithelium, the lamina propria, and the muscularis mucosae. The epithelium is a single layer of specialized cells that line the lumen of the GI tract. It forms a continuous layer along the tube and with the glands and organs that drain into the lumen of the tube. Within this cell layer are a number of specialized epithelial cells; the most abundant are cells termed absorptive enterocytes, which express many proteins important for digestion and absorption of macronutrients. Enteroendocrine cells contain secretory granules that release regulatory peptides and amines to help regulate GI function. In addition, cells in the gastric mucosa are specialized for production of protons, and mucin-producing cells throughout the GI tract produce a glycoprotein (mucin) that helps protect the GI tract and lubricate the luminal contents. The columnar epithelial cells are linked together by intercellular connections called tight junctions.

These junctions are complexes of intracellular and transmembrane proteins, and the tightness of these junctions is regulated throughout the postprandial period. The nature of the epithelium varies greatly from one part of the digestive tract to another, depending on the predominant function of that region. For example, the intestinal epithelium is designed for absorption; these cells mediate selective uptake of nutrients, ions, and water. In contrast, the esophagus has a squamous epithelium that has no absorptive role. It is a conduit for transportation of swallowed food and thus needs some protection, provided by the squamous epithelium, from rough food such as fiber. The surface area of the epithelium is arranged into villi and crypts (Fig. 27.3). Villi are finger-like projections that serve to increase the surface area of the mucosa. Crypts are invaginations or folds in the epithelium. The epithelium lining the GI tract is continuously renewed and replaced by dividing cells; in humans this process takes about 3 days. These proliferating cells are localized to the crypts, where there is a proliferative zone of intestinal stem cells.

FIG. 27.3 Comparison of the morphology of the epithelium of the small intestine and colon.

The lamina propria immediately below the epithelium consists largely of loose connective tissue that contains collagen and elastin fibrils (Fig. 27.2). The lamina propria is rich in several types of glands and contains lymph vessels and nodules, capillaries, and nerve fibers. The muscularis mucosae is the thin innermost layer of intestinal smooth muscle. When seen through an endoscope, the mucosa has folds and ridges that are caused by contractions of the muscularis mucosae. Submucosa The next layer is the submucosa (Fig. 27.2), which consists largely of loose connective tissue with collagen and elastin fibrils. In some regions of the GI tract, glands (invaginations or folds of the mucosa) are present in the submucosa. The larger nerve trunks, blood vessels, and lymph vessels of the intestinal wall lie in the submucosa, together with one of the plexuses of the enteric nervous system (ENS), the

submucosal plexus. Muscle Layers The muscularis externa, or muscularis propria, typically consists of two substantial layers of smooth muscle cells: an inner circular layer and an outer longitudinal layer (Fig. 27.2). Muscle fibers in the circular muscle layer are oriented circumferentially, whereas muscle fibers in the longitudinal muscle layer are oriented along the longitudinal axis of the tube. Between the circular and longitudinal layers of muscle lies the other plexus of the ENS, the myenteric plexus. Contractions of the muscularis externa mix and circulate the contents of the lumen and propel them along the GI tract. The wall of the GI tract contains many interconnected neurons. The submucosa contains a dense network of nerve cells called the submucosal plexus (also referred to as Meissner’s plexus). The prominent myenteric plexus (Auerbach’s plexus) is located between the circular and longitudinal smooth muscle layers. These intramural plexuses constitute the ENS. The ENS helps integrate the motor and secretory activities of the GI system. If the sympathetic and parasympathetic nerves to the gut are cut, many motor and secretory activities continue because the ENS directly controls these processes. Serosa The serosa, or adventitia, is the outermost layer of the GI tract and consists of a layer of squamous mesothelial cells (Fig. 27.2). It is part of the mesentery that lines the surface of the abdominal wall and suspends the organs within the abdominal cavity. The mesenteric membranes secrete a thin viscous fluid that helps lubricate the abdominal organs so movement of the organs can occur as the muscle layers contract and relax.

Regulatory Mechanisms in the Gastrointestinal Tract Unlike the cardiovascular or respiratory systems, the GI tract undergoes periods of relative quiescence (intermeal period) and periods of intense activity after the intake of food (postprandial period). Consequently, the GI tract has to detect and respond appropriately to food intake. In addition, the macronutrient content of a meal can vary considerably and there are mechanisms that can detect this and mount appropriate physiological responses. Thus, the GI tract has to communicate with associated organs such as the pancreas. Finally, because the GI tract is essentially a long tube, there have to be mechanisms by which events occurring in the proximal portion of the GI tract are signaled to the more distal parts, and vice versa. There are three principal control mechanisms involved in the regulation of GI function: endocrine, paracrine, and neurocrine (Fig. 27.4).

FIG. 27.4 The three mechanisms by which function in the GI tract is regulated in the integrated response to a meal.

Endocrine Regulation Endocrine regulation describes the process whereby the sensing cell in the GI tract, an enteroendocrine cell (EEC), responds to a stimulus by secreting a regulatory peptide or hormone that travels via the bloodstream to target cells removed from the point of secretion. Cells responding to a GI hormone express specific receptors for the hormone. Hormones released from the GI tract have effects on cells located in other regions of the GI tract and also on glandular structures associated with the GI tract, such as the pancreas. In addition, GI hormones have effects on other tissues, including cells in liver, muscle, and brain. EECs are packed with secretory granules, the products of which are released from the cell in response

to chemical and mechanical stimuli to the wall of the GI tract (Fig. 27.5). In addition, EECs can be stimulated by neural input or other factors not associated with a meal. The most common EECs in the gut wall are referred to as the “open” type; these cells have an apical membrane that is in contact with the lumen of the GI tract (generally regarded as the location where sensing occurs) and a basolateral membrane through which secretion occurs. There are also “closed”-type EECs that do not have part of their membrane in contact with the luminal surface of the gut; an example is the enterochromaffin-like (ECL) cell in the gastric epithelium, which secretes histamine.

FIG. 27.5 Electron micrograph of an open-type endocrine cell in the GI tract. Note the microvilli at the apical projection and the secretory granules in the basolateral portion of the cell. (From Barrett K. Gastrointestinal Physiology [Lange Physiology Series]. New York: McGraw-Hill; 2005.) (Courtesy of Leonard R. Johnson, Ph.D.)

There are many examples of hormones secreted by the GI tract (Table 27.1); it is worth remembering that the first hormone ever identified was the GI hormone secretin. One of the most well-characterized GI hormones is gastrin, which is released from endocrine cells located in the wall of the distal part of the stomach. Release of gastrin is stimulated by activation of parasympathetic outflow to the GI tract and gastrin potently stimulates gastric acid secretion in the postprandial period.

Table 27.1 Hormonal and Paracrine Mediators in the GI Tract Stimulus for Release

Pathway of Action

Gastric antrum (G cells)

Oligopeptides

Endocrine

Cholecystokinin

Duodenum (I cells)

Fatty acids, Paracrine, hydrolyzed endocrine protein

Vagal afferent Inhibition of gastric terminals, emptying and H+ pancreatic secretion; stimulation of acinar pancreatic enzyme cells secretion, gallbladder contraction, inhibition of food intake

Secretin

Duodenum (S cells)

Protons

Paracrine, endocrine

Vagal afferent Stimulation of pancreatic terminals, duct secretion (H2O and pancreatic HCO3−) duct cell

GlucoIntestine (K insulinotropic cells) peptide (GIP)

Fatty acids, glucose

Endocrine

Beta cells of the pancreas

Stimulation of insulin secretion

Peptide YY (PYY)

Intestine (L cells)

Fatty acids, Endocrine, glucose, paracrine hydrolyzed protein

Neurons, smooth muscle

Inhibition of gastric emptying, pancreatic secretion, gastric acid secretion, intestinal motility, food intake

Proglucagonderived peptides 1/2 (GLP-1/2)

Intestine (L cells)

Fatty acids, Endocrine, glucose, paracrine hydrolyzed protein

Neurons, epithelial cells

Glucose homeostasis, epithelial cell proliferation

GI Hormone

Source

Gastrin

Targets

Effect

ECL cells Stimulation of parietal cells and to secrete H+ and ECL parietal cells to secrete histamine cells of the gastric corpus

ECL, Enterochromaffin-like; GLP, glucagon-like peptide.

Paracrine Regulation Paracrine regulation describes the process whereby a chemical messenger or regulatory peptide is released from a sensing cell (often an EEC) in the intestinal wall that acts on a nearby target cell by diffusion through the interstitial space. Paracrine agents exert their actions on several different cell types in the wall of the GI tract, including smooth muscle cells, absorptive enterocytes, secretory cells in glands, and even on other EECs. There are several important paracrine agents, and they are listed in Table 27.1 along with their site of production, site of action, and function. An important paracrine

mediator in the gut wall is histamine. In the stomach, histamine is stored and released by ECL cells located in the gastric glands. Histamine diffuses through the interstitial space in the lamina propria to neighboring parietal cells and stimulates production of acid. Serotonin (5-hydroxytryptamine [5-HT]), released from enteric neurons, mucosal mast cells, and specialized cells called enterochromaffin cells, regulates smooth muscle function and water absorption across the intestinal wall. There are other paracrine mediators in the gut wall, including prostaglandins, adenosine, and nitric oxide (NO); the functions of these mediators are not well described, but they are capable of producing changes in GI function. Many substances can be both paracrine and endocrine regulators of GI function. For example, cholecystokinin, which is released from the duodenum in response to dietary protein and lipid, acts locally on nerve terminals in a paracrine fashion and also affects the pancreas. This will be discussed in more detail in Chapter 30.

Neural Regulation of Gastrointestinal Function Nerves and neurotransmitters play an important role in regulating the function of the GI tract. In its simplest form, neural regulation occurs when a neurotransmitter is released from a nerve terminal located in the GI tract and the neurotransmitter has an effect on the cell that is innervated. However, in some cases there are no synapses between motor nerves and effector cells in the GI tract. Neural regulation of GI function is very important within an organ, as well as between distant parts of the GI tract.

AT THE C ELLU LAR LEVEL There are multiple receptor subtypes for the regulatory peptide hormones released from endocrine cells in the wall of the gut. The selectivity of receptors to peptide hormones is determined by posttranslational modifications, which then confers receptor selectivity. An example of this is peptide YY (PYY). There are multiple receptor subtypes for PYY, classified as Y1 to Y7. PYY is released from endocrine cells in the wall of the gut, mainly in response to fatty acids. It is released as a 36-amino acid peptide and binds to the Y1, Y2, and Y5 receptors; however, it can be cleaved to PYY3-36 by the enzyme dipeptidyl peptidase IV, a membrane peptidase. This form of the peptide is more selective for the Y2 receptor. Thus, the presence of the enzyme that cleaves the peptide can alter the biological response to PYY secretion.

IN THE C LIN IC Glucagon-like peptide 1 (GLP-1) is a regulatory peptide released from EECs in the gut wall in response to the presence of luminal carbohydrate and lipids. GLP-1 arises from differential processing of the glucagon gene, the same gene that is expressed in the pancreas and that gives rise to glucagon. GLP-1 is involved in glucose homeostasis via its action to stimulate the release of insulin from pancreatic islets and to inhibit the release of glucagon, resulting in lowering of postprandial blood glucose. Two therapies based on these actions of GLP-1 have been developed and used in the treatment Type 2 diabetes: long-acting GLP-1 agonists (e.g., exenatide) and, inhibitors of dipeptidyl peptidase 4 inhibitors, the enzyme that rapidly degrades GLP-1 in plasma and tissue. These therapies have gained widespread use in patients, based on effects on insulin secretion but also because of the extrapancreatic effects to reduce gastric emptying and appetite, improve insulin sensitivity, and improve lipid profiles. Indeed, GLP-1-based therapies are also used in irritable bowel syndrome,

short bowel syndrome, and nonalcoholic fatty liver disease. Actions at the relevant extrapancreatic sites are likely based on the widespread distribution of GLP-1 receptors. Neural regulation of the GI tract is surprisingly complex. The gut is innervated by two sets of nerves, the extrinsic and intrinsic nervous systems. The extrinsic nervous system is defined as nerves that innervate the gut, with cell bodies located outside the gut wall; these extrinsic nerves are part of the autonomic nervous system (ANS). The intrinsic nervous system, also referred to as the enteric nervous system (ENS), has cell bodies that are contained within the wall of the gut (submucosal and myenteric plexuses). Some GI functions are highly dependent on the extrinsic nervous system, yet others can take place independently of the extrinsic nervous system and are mediated entirely by the ENS. However, extrinsic nerves can often modulate intrinsic nervous system function (Fig. 27.6).

FIG. 27.6 Hierarchical neural control of GI function. Stimuli to the GI tract from a meal (e.g., chemical, mechanical, osmotic) will activate both the intrinsic and extrinsic sensory (afferent) pathways, which in turn will activate the extrinsic and intrinsic neural reflex pathways.

Extrinsic Neural Innervation Extrinsic neural innervation to the gut is via the two major subdivisions of the ANS, namely, parasympathetic and sympathetic innervation (Fig. 27.7). Parasympathetic innervation to the gut is via the vagus and pelvic nerves. The vagus nerve, the 10th cranial nerve, innervates the esophagus, stomach, gallbladder, pancreas, first part of the intestine, cecum, and the proximal part of the colon. The pelvic nerves innervate the distal part of the colon and the anorectal region, in addition to the other pelvic organs that are not part of the GI tract.

FIG. 27.7 Extrinsic innervation of the GI tract, consisting of the parasympathetic (A) and sympathetic (B) subdivisions of the autonomic nervous system.

Consistent with the typical organization of the parasympathetic nervous system, the preganglionic nerve cell bodies lie in the brainstem (vagus) or the sacral spinal cord (pelvic). Axons from these neurons run in the nerves to the gut (vagus and pelvic nerves, respectively), where they synapse with postganglionic neurons in the wall of the organ, which in this case are enteric neurons in the gut wall. There is no direct innervation of these efferent nerves to effector cells within the wall of the gut; the transmission pathway is always via a neuron in the ENS. Consistent with transmission in the ANS, the synapse between preganglionic and postganglionic neurons is an obligatory nicotinic synapse. That is, the synapse between preganglionic and postganglionic neurons is mediated via acetylcholine released from the nerve terminal and acting at nicotinic receptors localized on the postganglionic neuron, which in this case is an intrinsic neuron. Sympathetic innervation is supplied by cell bodies in the spinal cord and fibers that terminate in the prevertebral ganglia (celiac, superior, and inferior mesenteric ganglia); these are the preganglionic neurons. These nerve fibers synapse with postganglionic neurons in the ganglia, and the fibers leave the

ganglia and reach the end organ along the major blood vessels and their branches. Rarely there is a synapse in the paravertebral (chain) ganglia, as seen with sympathetic innervation of other organ systems. Some vasoconstrictor sympathetic fibers directly innervate blood vessels of the GI tract, and other sympathetic fibers innervate glandular structures in the wall of the gut. The ANS, both parasympathetic and sympathetic, also carries the fibers of afferent (toward the central nervous system [CNS]) neurons; these are sensory in nature. The cell bodies for the vagal afferents are in the nodose ganglion. These neurons have a central projection terminating in the nucleus of the tractus solitarius in the brainstem and the other terminal in the gut wall. The cell bodies of the spinal afferent neurons that run with the sympathetic pathway are segmentally organized and are found in the dorsal root ganglia. Peripheral terminals of the spinal and vagal afferents are located in all layers of the gut wall, where they detect information about the state of the gut. Afferent neurons send this information to the CNS. Information sent to the CNS relays the nature of the luminal contents (e.g., acidity, nutrient content, osmolality of the luminal contents), as well as the degree of stretch or contraction in smooth muscle. Afferent innervation is also responsible for transmitting painful stimuli to the CNS. The components of a reflex pathway—afferents, interneurons, and efferent neurons—exist within the extrinsic innervation to the GI tract. These reflexes can be mediated entirely via the vagus nerve (termed a vagovagal reflex), which has both afferent and efferent fibers. The vagal afferents send sensory information to the CNS, where they synapse with an interneuron, which then drives activity in the efferent motor neuron. These extrinsic reflexes are very important in regulating GI function after ingestion of a meal. An example of an important vagovagal reflex is the gastric receptive relaxation reflex, in which distention of the stomach results in relaxation of the smooth muscle in the stomach; this allows filling of the stomach to occur without an increase in intraluminal pressure. In general, as with other visceral organ systems, the parasympathetic and sympathetic nervous systems tend to work in opposition. However, this is not as simple as in the cardiovascular system, for example. Activation of the parasympathetic nervous system is important in the integrative response to a meal and is discussed in the following chapters. The parasympathetic nervous system generally results in activation of physiological processes in the gut wall, although there are notable exceptions. In contrast, the sympathetic nervous system tends to be inhibitory to GI function and is more frequently activated in pathophysiological circumstances. Overall, sympathetic activation inhibits smooth muscle function. The exception to this is the sympathetic innervation of GI sphincters, in which sympathetic activation tends to induce contraction of smooth muscle. Moreover, the sympathetic nervous system is notably important in regulation of blood flow in the GI tract. Intrinsic Neural Innervation The ENS is made up of two major plexuses, which are collections of nerve cell bodies (ganglia) and their fibers, all originating in the wall of the gut (Fig. 27.8). The myenteric plexus lies between the longitudinal and circular muscle layers, and the submucosal plexus lies in the submucosa. Interganglionic strands link neurons in the two plexuses.

FIG. 27.8 The enteric nervous system in the wall of the GI tract.

Neurons in the ENS are characterized functionally as afferent neurons, interneurons, or efferent neurons, similar to neurons in the extrinsic part of the ANS. Thus, all components of a reflex pathway can be contained within the ENS. Stimuli in the wall of the gut are detected by afferent neurons, which activate interneurons and then efferent neurons to alter function. In this way the ENS can act autonomously from extrinsic innervation. However, neurons in the ENS, as we have already seen, are innervated by extrinsic neurons, and thus the function of these reflex pathways can be modulated by the extrinsic nervous system. Because the ENS is capable of performing its own integrative functions and complex reflex pathways, it is sometimes referred to as the “little brain in the gut” as a result of its importance and complexity. It is estimated that there are as many neurons in the ENS as in the spinal cord. In addition,

many GI hormones also act as neurotransmitters in the ENS and in the brain in regions involved in autonomic outflow. These mediators and regulatory peptides are thus referred to as brain-gut peptides, and the extrinsic and intrinsic components innervating the gut are sometimes referred to as the brain-gut axis.

Response of the GI Tract to a Meal This introductory chapter provides a broad overview of the anatomy and regulatory mechanisms in the GI tract. In the following chapters there will be discussion of the integrated response to a meal to provide the details of GI physiology. The response to a meal is classically divided into phases: cephalic, oral, esophageal, gastric, duodenal, and intestinal. In each phase the meal presents certain stimuli (e.g., chemical, mechanical, and osmotic) that activate different pathways (e.g., neural, paracrine, and humoral reflexes) that result in changes in effector function (e.g., secretion and motility). There is considerable crosstalk between the regulatory mechanisms that have been outlined, and this will be discussed in the next chapters. As with maintenance of homeostasis in other systems of the body, control of GI function requires complex regulatory mechanisms to sense and act in a dynamic fashion.

IN THE C LIN IC Hirschsprung’s disease is a congenital disorder of the enteric nervous system characterized by failure to pass meconium at birth, severe chronic constipation in infancy, swelling of the abdomen, and vomiting. The typical features are absence of neural crest-derived myenteric and submucosal neurons in the distal part of the colon and rectum. It is a polygenic disorder with characteristic mutations in at least three different classes of genes involved in neuronal development and differentiation.

Key Concepts 1. The GI tract is a tube subdivided into regions that serve different functions associated with digestion and absorption. 2. The lining of the GI tract is subdivided into layers—the mucosal, submucosal, muscle layers, and serosa/adventitia. 3. There are three major control mechanisms: hormonal, paracrine, and neurocrine. 4. The innervation of the GI tract is particularly interesting because it consists of two interacting components, extrinsic and intrinsic. 5. Extrinsic innervation (cell bodies outside the wall of the GI tract) consists of the two subdivisions of the ANS: parasympathetic and sympathetic. Both have an important sensory (afferent) component. 6. The intrinsic or enteric nervous system (cell bodies in the wall of the GI tract) can act independently of extrinsic neural innervation. 7. When a meal is in different regions of the tract, sensory mechanisms detect the presence of the nutrients and mount appropriate physiological responses in that region of the tract, as well as in more distal regions. These responses are mediated by endocrine, paracrine, and neurocrine pathways.

C H AP T E R 2 8

The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal LEARNING OBJECTIVES Upon completion of this chapter, you should be able to answer the following questions: 1. What constitutes the functional anatomy of salivary glands, including their secretory elements? 2. What are the cephalic and oral phases (what, why, how it happens) of the response to a meal? 3. What are the general principles of secretion along the gastrointestinal (GI) tract (where do secretions come from, what are the components)? 4. How do the components of secretion vary with the gland or region of the GI tract? 5. What is the correlation between the composition and functions of salivary secretion? 6. How are primary and secondary secretions within salivary glands generated and regulated? 7. What is the sequence of events in swallowing? 8. What are the stimulus and neural pathways generating primary and secondary esophageal peristalsis? 9. What changes in gastric motility take place during swallowing, and what is the significance? 10. What are the major functions of the esophagus and associated structures in terms of protection and propulsion?

This chapter will describe the processes that occur in the gastrointestinal (GI) tract in the early stages of the integrated response to a meal. There are changes in GI tract physiology (1) before food is ingested (the cephalic phase), (2) when ingested food is in the mouth (the oral phase), and (3) when food is transferred from the mouth to the esophagus (the esophageal phase). The responses of the GI tract to the presence of food are mainly associated with preparing the GI tract for digestion and absorption.

Cephalic and Oral Phases The main feature of the cephalic phase is activation of the GI tract in readiness for the meal. The stimuli involved are cognitive and include anticipation or thinking about the consumption of food, olfactory input, visual input (seeing or smelling appetizing food when hungry), and auditory input. The latter may be an unexpected link but was clearly demonstrated in the classic conditioning experiments of Pavlov, in which he paired an auditory stimulus to the presentation of food to dogs; eventually the auditory stimulus alone could stimulate secretion. A real-life analogy is presumably being told that dinner is ready. All these stimuli result in an increase in excitatory parasympathetic neural outflow to the gut. Sensory input (e.g.,

smell) stimulates sensory nerves that activate parasympathetic outflow from the brainstem. Higher brain sites (e.g., limbic system, hypothalamus, cortex) are also involved in the cognitive components of this response. The response can be both positive and negative; thus, anticipation of palatable food and a person's psychological status, such as anxiety, can alter the cognitive response to a meal. However, the final common pathway is activation of the dorsal motor nucleus in the brainstem, the region where the cell bodies of the vagal preganglionic neurons arise. Activation of the nucleus leads to increased activity in efferent fibers passing to the GI tract in the vagus nerve. In turn the efferent fibers activate the postganglionic motor neurons (referred to as motor because their activation results in change of function of an effector cell). Increased parasympathetic outflow enhances salivary secretion, gastric acid secretion, pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi (the sphincter between the common bile duct and duodenum). All these responses enhance the ability of the GI tract to receive and digest the incoming food. The salivary response is mediated via the ninth cranial nerve; the remaining responses are mediated via the vagus nerve. Many of the features of the oral phase are indistinguishable from the cephalic phase. The only difference is that food is in contact with the surface of the GI tract. Thus, there are additional stimuli generated from the mouth, both mechanical and chemical (taste). However, many of the responses initiated by the presence of food in the oral cavity are identical to those initiated in the cephalic phase, because the efferent pathway is the same. The responses specifically initiated in the mouth, which consist mainly of the stimulation of salivary secretion, will be discussed next. The mouth is important for the mechanical disruption of food and for initiation of digestion. Chewing subdivides and mixes the food with the enzymes salivary amylase and lingual lipase and with the glycoprotein mucin, which lubricates food for chewing and swallowing. Minimal absorption occurs in the mouth, although alcohol and some drugs are absorbed from the oral cavity, and this can be clinically important. However, as with the cephalic phase, it is important to realize that stimulation of the oral cavity initiates responses in the more distal GI tract, including increased gastric acid secretion, increased pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi, mediated via the efferent vagal pathway.

Properties of Secretion General Considerations Secretions in the GI tract come from glands associated with the tract (salivary glands, pancreas, and liver), from glands formed by the gut wall itself (e.g., submucosal glands in esophagus and duodenum), and from the intestinal mucosa itself. The exact nature of the secretory products can vary tremendously, depending on the function of that region of the GI tract. However, these secretions have several characteristics in common. Secretions from the GI tract and associated glands include water, electrolytes, protein, and humoral agents. Water is essential for generating an aqueous environment for efficient enzyme action. Secretion of electrolytes is important for generation of osmotic gradients to drive the movement of water. Digestive enzymes in secreted fluid catalyze the breakdown of macronutrients in ingested food. Moreover, many additional proteins secreted along the GI tract have specialized functions, some of which are fairly well understood, such as those of mucin and immunoglobulins, and others that are only just beginning to be understood, such as those of trefoil peptides. Secretion is initiated by multiple signals associated with the meal, including chemical, osmotic, and mechanical components. Secretion is elicited by the action of specific effector substances called

secretagogues acting on secretory cells. Secretagogues work in one of the three ways that have already been described in Chapter 27—endocrine, paracrine, and neural. Constituents of Secretions Inorganic secretory components are region or gland specific, depending on the particular conditions required in that part of the GI tract. The inorganic components are electrolytes, including H+ and HCO3−. Two examples of different secretions include acid (HCl) in the stomach, which is important to activate pepsin and start protein digestion, and HCO3− in the duodenum, which neutralizes gastric acid and provides optimal conditions for the action of digestive enzymes in the small intestine. Organic secretory components are also gland or organ specific and depend on the function of that region of the gut. The organic constituents are enzymes (for digestion), mucin (for lubrication and mucosal protection), and other factors such as growth factors, immunoglobulins, bile acids, and absorptive factors.

Salivary Secretion During the cephalic and oral phases of the meal, considerable stimulation of salivary secretion takes place. Saliva has a variety of functions, including those important for the integrative responses to a meal and for other physiological processes (Box 28.1). The main functions of saliva in digestion include lubrication and moistening of food for swallowing, solubilization of material for taste, initiation of carbohydrate digestion, and clearance and neutralization of refluxed gastric secretions in the esophagus. Saliva also has antibacterial actions that are important for overall health of the oral cavity and teeth. Box 28.1

F unc t io ns o f Sa liva a nd C he w ing Disruption of food to produce smaller particles Formation of a bolus for swallowing Initiation of starch and lipid digestion Facilitation of taste Production of intraluminal stimuli in the stomach Regulation of food intake and ingestive behavior Cleansing of the mouth and selective antibacterial action Neutralization of refluxed gastric contents Mucosal growth and protection in the rest of the GI tract Aid in speech

Functional Anatomy of the Salivary Glands There are three pairs of major salivary glands: parotid, submandibular, and sublingual. In addition, many smaller glands are found on the tongue, lips, and palate. These glands are the typical tubuloalveolar structure of glands located in the GI tract (Fig. 28.1). The acinar portion of the gland is classified

according to its major secretion: serous (“watery”), mucous, or mixed. The parotid gland produces mainly serous secretion, the sublingual gland secretes mainly mucous, and the submandibular gland produces a mixed secretion.

FIG. 28.1 General structure of tubuloalveolar secretory glands (e.g., salivary glands, pancreas) associated with the digestive tract.

Cells in the secretory end pieces, or acini, are called acinar cells and are characterized by basally located nuclei, abundant rough endoplasmic reticulum, and apically located secretory granules that contain the enzyme amylase and other secreted proteins. There are also mucous cells in the acinus; the granules in these cells are larger and contain the specialized glycoprotein mucin. There are three kinds of ducts in the gland that transport secretions from the acinus to the opening in the mouth and also modify the secretion: intercalated ducts drain acinar fluid into larger ducts, the striated ducts, which then empty into even larger excretory ducts. In addition, a single large duct from each gland drains saliva to the mouth. The ductal cells lining the striated ducts, in particular, modify the ionic composition and osmolarity of saliva. Composition of Saliva The important properties of saliva are a large flow rate relative to the mass of gland, low osmolarity, high K+ concentration, and organic constituents, including enzymes (amylase, lipase), mucin, and growth factors. The latter are not important in the integrated response to a meal but are essential for long-term maintenance of the lining of the GI tract. The inorganic composition is entirely dependent on the stimulus and the rate of salivary flow. In humans, salivary secretion is always hypotonic. The major components are Na+, K+, HCO3−, Ca++, Mg++, and Cl−. Fluoride can be secreted in saliva, and fluoride secretion forms the basis of oral fluoride

treatment for prevention of dental caries. The concentration of ions varies with the rate of secretion; the flow rate of salivary secretion is stimulated during the postprandial period. The primary secretion is produced by acinar cells in the secretory end pieces (or acini) and is modified by duct cells as saliva passes through the ducts. The primary secretion is isotonic, and the concentration of the major ions is similar to that in plasma. Secretion is driven predominantly by Ca++dependent signaling, which opens apical Cl− channels in the acinar cells. Cl− therefore flows out into the duct lumen and establishes an osmotic and electrical gradient. Because the epithelium of the acinus is relatively leaky, Na+ and water then follow across the epithelium via the tight junctions (i.e., via paracellular transport). Transcellular water movement may also occur, mediated by aquaporin 5 water channels. The amylase content and rate of fluid secretion vary with the type and level of stimulus. As the fluid passes along the ducts, the excretory and striated duct cells modify the ionic composition of the primary secretion to produce the secondary secretion. The duct cells reabsorb Na+ and Cl− and secrete K+ and HCO3− into the lumen. Na+ is exchanged for protons, but some of the secreted protons are then reabsorbed in exchange for K+. HCO3− on the other hand is secreted only in exchange for Cl−, thereby alkalinizing salivary secretion. At rest, final salivary secretion is hypotonic and slightly alkaline. The alkalinity of saliva is important in restricting microbial growth in the mouth, as well as in neutralizing refluxed gastric acid once the saliva is swallowed. When salivary secretion is stimulated, there is a small decrease in the K+ concentration (but it always remains above plasma concentrations), the Na+ concentration increases toward plasma levels, and Cl− and HCO3− concentrations increase, thus the secreted fluid becomes even more alkaline (Fig. 28.2). Note that HCO3− secretion can be directly stimulated by the action of secretagogues on duct cells. The duct epithelium is relatively tight and lacks expression of aquaporin, and therefore water cannot follow the ions rapidly enough to maintain isotonicity at moderate or high flow rates during stimulated salivary secretion. Thus, with an increase in secretion rate, there is less time for ionic modification by the duct cells, and the resulting saliva more closely resembles the primary secretion and therefore plasma. However, [HCO3−] remains high because secretion from duct cells and possibly acinar cells is stimulated (Fig. 28.2).

FIG. 28.2 A, The composition of salivary secretion as a function of the salivary flow rate compared with the concentration of ions in plasma. Saliva is hypotonic to plasma at all flow rates. [HCO3−] in saliva exceeds that in plasma except at very low flow rates. B, Schematic representation of the two-stage model of salivary secretion. The primary secretion containing amylase and electrolytes is produced in the acinar cell. The concentration of electrolytes in plasma is similar to that in the primary secretion, but it is modified as it passes through ducts that absorb Na+ and Cl− and secrete K+ and HCO3−.

The organic constituents of saliva—proteins and glycoproteins—are synthesized, stored, and secreted by the acinar cells. The major products are amylase (an enzyme that initiates starch digestion), lipase (important for lipid digestion), glycoprotein (mucin, which forms mucus when hydrated), and lysozyme (attacks bacterial cell walls to limit colonization of bacteria in the mouth). Although salivary amylase begins the process of digestion of carbohydrates, it is not required in healthy adults because of the excess of pancreatic amylase. Similarly, the importance of lingual lipase is unclear.

Metabolism and Blood Flow of Salivary Glands The salivary glands produce a prodigious flow of saliva. The maximal rate of saliva production in humans is about 1 mL/min/g of gland; thus, at this rate, the glands are producing their own weight in saliva each minute. Salivary glands have a high rate of metabolism and high blood flow; both are proportional to the rate of saliva formation. Blood flow to maximally secreting salivary glands is approximately 10 times that of an equal mass of actively contracting skeletal muscle. Stimulation of the parasympathetic nerves to salivary glands increases blood flow by dilating the vasculature of the glands. Vasoactive intestinal polypeptide (VIP) and acetylcholine are released from parasympathetic nerve terminals in the salivary glands and are vasodilatory during secretion. Regulation of Salivary Secretion Control of salivary secretion is exclusively neural. Salivary secretion is stimulated by both the sympathetic and parasympathetic subdivisions of the autonomic nervous system. Primary physiological control of the salivary glands during the response to a meal is via the parasympathetic nervous system. Acinar cells and duct cells are innervated by parasympathetic nerve endings. Parasympathetic stimulation increases synthesis and secretion of salivary amylase and mucins, enhances the transport activities of the ductular epithelium, greatly increases blood flow to the glands, and stimulates glandular metabolism and growth. If the parasympathetic supply is interrupted, salivation is severely impaired and the salivary glands atrophy. Sympathetic fibers to the salivary glands stem from the superior cervical ganglion. Preganglionic parasympathetic fibers travel via branches of the facial and glossopharyngeal nerves (cranial nerves VII and IX, respectively). These fibers form synapses with postganglionic neurons in ganglia in or near the salivary glands. Ionic Mechanisms of Salivary Secretion Ion Transport in Acinar Cells Fig. 28.3 shows a simplified view of the mechanisms of ion secretion by serous acinar cells. The basolateral membrane of the cell contains Na+, K+-ATPase and an Na+-K+-2Cl− symporter. The concentration gradient for Na+ across the basolateral membrane, which is dependent on Na+,K+-ATPase, provides the driving force for entry of Na+, K+, and Cl− into the cell. Cl− and HCO3− leave the acinar cell and enter the lumen via an anion channel located in the apical membrane of the acinar cell. This secretion of anions drives the entry of Na+ and thus water into the acinar lumen across the relatively leaky tight junctions.

FIG. 28.3 Ionic transport mechanism involved in the secretion of amylase and electrolytes in salivary acinar cells.

Acinar cell fluid secretion is strongly enhanced in response to elevations in intracellular [Ca++] as a result of activation of the muscarinic receptor for acetylcholine. Ion Transport in Ductular Cells Fig. 28.4 shows a simplified model of ion transport processes in epithelial cells of the excretory and striated ducts. Na+,K+-ATPase located in the basolateral membrane maintains the electrochemical gradients for Na+ and K+ that drive most of the other ionic transport processes of the cell. In the apical membrane the parallel operation of the Na+/H+ antiporter, the Cl−/HCO3− antiporter, and the H+/K+ antiporter results in absorption of Na+ and Cl− from the lumen and secretion of K+ and HCO3− into the lumen. The relative impermeability of the ductular epithelium to water prevents the ducts from absorbing too much water by osmosis.

FIG. 28.4 Ionic transport mechanism involved in secretion and absorption in epithelial cells of the striated and excretory duct of the salivary gland.

Swallowing Swallowing can be initiated voluntarily, but thereafter it is almost entirely under reflex control. The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach. This reflex also inhibits respiration and prevents entrance of food into the trachea during swallowing. The afferent limb of the swallowing reflex begins when touch receptors, most notably those near the opening of the pharynx, are stimulated. Sensory impulses from these receptors are transmitted to an area in the medulla and lower pons called the swallowing center. Motor impulses travel from the swallowing center to the musculature of the pharynx and upper esophagus via various cranial nerves and to the remainder of the esophagus by vagal motor neurons.

AT THE C ELLU LAR LEVEL The acinar cells and duct cells of the salivary glands respond to both cholinergic and adrenergic agonists. Nerves stimulate the release of acetylcholine, norepinephrine, substance P, and vasoactive intestinal polypeptide (VIP) by salivary glands, and these hormones increase the secretion of amylase and the flow of saliva. These neurotransmitters act mainly by elevating the intracellular concentration of cyclic adenosine monophosphate (cAMP) and by increasing the concentration of Ca++ in the cytosol. Acetylcholine and substance P, acting on muscarinic and tachykinin receptors, respectively, increase the cytosolic concentration of Ca++ in serous acinar cells. In contrast, norepinephrine, acting on β

receptors, and VIP, binding to its receptor, elevate the cAMP concentration in acinar cells. Agonists that elevate the cAMP concentration in serous acinar cells elicit a secretion that is rich in amylase; agonists that mobilize Ca++ elicit a secretion that is more voluminous but has a lower concentration of amylase. Ca++-mobilizing agonists may also elevate the concentration of cyclic guanosine monophosphate (cGMP), which may mediate the trophic effects evoked by these agonists.

IN THE C LIN IC People with dysphagia have difficulty in swallowing, often with accompanying pain (odynophagia). Swallowing is a complex process, involving coordination of voluntary and involuntary reflex control of many different muscle groups in the oral cavity (tongue and jaw muscles), the pharynx, and the esophagus. Dysphagia occurs when there is a defect in the neural control or coordination of any or all of these structures. If serious, dysphagia can result in difficulty in swallowing even liquids (including a patient's own saliva) and a patient may not be able to maintain adequate nutrition. Dysphagia is most common in the elderly population and can be associated with a stroke or head injury, or neurodegenerative diseases such as Parkinson's disease, amyotrophic lateral sclerosis (ALS), or cognitive decline. Some treatments for cancers in the head, neck, or esophagus can cause dysphagia. Treatment options for dysphagia are limited, and involve physical therapy to help patients change behavior, muscle strength, and posture.

IN THE C LIN IC The ability to measure and monitor a wide range of molecular components that are indicative of overall health is useful in diagnosis and monitoring. Saliva is easy to access, and collection of it is noninvasive. It is used to identify individuals with disease (presence of biomarkers) and to monitor the progress of affected individuals under treatment. In endocrinology, levels of steroids can be measured in the free form rather than as the free and bound form, as in plasma (e.g., the stress hormone cortisol and the sex hormones estradiol, progesterone, and testosterone). Viral infections such as human immunodeficiency virus (HIV), herpes, hepatitis C, SARS-CoV-2, and Epstein-Barr virus infection can be detected by polymerase chain reaction (PCR) techniques. Bacterial infections, such as Helicobacter pylori, can likewise be detected in saliva, and saliva is also used for monitoring drug levels. The timing of events in swallowing is shown in Fig. 28.5. The voluntary phase of swallowing is initiated when the tip of the tongue separates a bolus of food from the mass of food in the mouth. First the tip of the tongue and later the more posterior portions of the tongue press against the hard palate. The action of the tongue moves the bolus upward and then backward into the mouth. The bolus is forced into the pharynx, where it stimulates the touch receptors that initiate the swallowing reflex. The pharyngeal phase of swallowing involves the following sequence of events, which occur in less than 1 second: 1. The soft palate is pulled upward and the palatopharyngeal folds move inward toward one another; these movements prevent reflux of food into the nasopharynx and open a narrow passage through which food moves into the pharynx. 2. The vocal cords are pulled together, and the larynx is moved forward and upward against the epiglottis; these actions prevent food from entering the trachea and help open the upper esophageal sphincter (UES). 3. The UES relaxes to receive the bolus of food.

4. The superior constrictor muscles of the pharynx then contract strongly to force the bolus deeply into the pharynx.

FIG. 28.5 Timing of motor events in the pharynx and UES during a swallow. UES, Upper esophageal sphincter.

A peristaltic wave is initiated with contraction of the pharyngeal superior constrictor muscles, and the wave moves toward the esophagus. This wave forces the bolus of food through the relaxed UES. During the pharyngeal stage of swallowing, respiration is also reflexively inhibited. After the bolus of food passes the UES, a reflex action causes the sphincter to constrict.

IN THE C LIN IC Gastroesophageal reflux disease (GERD) is commonly referred to as heartburn or indigestion and is a common cause of noncardiac chest pain. It is primarily a condition of the lower esophageal sphincter (LES) and occurs when the LES allows the acidic contents of the stomach to reflux back into the distal part of the esophagus. This is thought to be caused by transient relaxations of the LES that occur independently of a swallow. This is not an uncommon event, even in healthy individuals, and only a small percentage of reflex events are symptomatic. This region of the esophagus, unlike the stomach, does not have a robust system to protect the mucosal lining. Acid will activate pain fibers resulting in discomfort and pain and in the long term, continual reflux can result in damage to the esophageal mucosa. GERD can be treated by therapies that reduce gastric acid secretion, for example, H2 receptor antagonists (e.g., ranitidine [Zantac]) or by proton pump inhibitors (e.g., omeprazole [Prilosec]). In GERD patients refractory to treatment, antireflex surgery called fundoplication may be used, which involves reinforcement of the LES by wrapping the upper part of the stomach around it, but the use of this surgery is controversial.

Esophageal Phase The esophagus, the UES, and the lower esophageal sphincter (LES) serve two main functions (Fig.

28.6). First, they propel food from the mouth to the stomach. Second, the sphincters protect the airway during swallowing and protect the esophagus from acidic gastric secretions.

FIG. 28.6 The esophagus and associated sphincters have multiple functions involved in movement of food from the mouth to the stomach and also in protection of the airway and esophagus. LES, Lower esophageal sphincter; UES, upper esophageal sphincter.

The stimuli that initiate the changes in smooth muscle activity that result in these propulsive and protective functions are mechanical and consist of pharyngeal stimulation during swallowing and distention of the esophageal wall itself. The pathways are exclusively neural and involve both extrinsic and intrinsic reflexes. Mechanosensitive afferents in both the extrinsic (vagus) nerves and intrinsic neural pathways respond to esophageal distention. These pathways include activated reflex pathways via the brainstem (extrinsic, vagus) or solely intrinsic pathways. The striated muscle is regulated from the nucleus ambiguus in the brainstem, and the smooth muscle is regulated by parasympathetic outflow via the vagus nerve. The changes in function resulting from mechanical stimuli and activation of reflex pathways are peristalsis of striated and smooth muscle, relaxation of the LES, and relaxation of the proximal portion of the stomach.

Functional Anatomy of the Esophagus and Associated Structures The esophagus, like the rest of the GI tract, has two muscle layers—circular and longitudinal—but the esophagus is one of two places in the gut where striated muscle occurs, the other being the external anal sphincter. The type of muscle (striated or smooth) in the esophagus varies along its length. The UES and LES are formed by thickening of striated or circular smooth muscle, respectively.

Motor Activity During the Esophageal Phase The UES, esophagus, and LES act in a coordinated manner to propel material from the pharynx to the stomach. At the end of a swallow, a bolus passes through the UES, and the presence of the bolus, via stimulation of mechanoreceptors and reflex pathways, initiates a peristaltic wave (alternating contraction and relaxation of the muscle) along the esophagus that is called primary peristalsis (Fig. 28.7). This

wave moves down the esophagus slowly (3–5 cm/s). Distention of the esophagus by the moving bolus initiates another wave called secondary peristalsis. Frequently, repetitive secondary peristalsis is required to clear the esophagus of the bolus. Stimulation of the pharynx by the swallowed bolus also produces reflex relaxation of the LES and the most proximal region of the stomach. Thus, when the bolus reaches the LES, it is already relaxed to allow passage of the bolus into the stomach. Similarly, the portion of the stomach that receives the bolus is relaxed. In addition, esophageal distention produces further receptive relaxation of the stomach. The proximal part of the stomach relaxes at the same time as the LES; this occurs with each swallow, and its function is to allow the stomach to accommodate large volumes with a minimal rise in intragastric pressure. This process is called receptive relaxation (Fig. 28.8).

FIG. 28.7 Changes in pressure in the different regions of the pharynx, esophagus, and associated sphincters initiated during a swallow. The pressure trace is a diagrammatic representation from that obtained during manometry in an awake human. Stimulation of the pharynx by the presence of a bolus initiates a decrease in pressure (= opening) of the UES and a peristaltic wave of contraction along the esophagus. Stimulation of the pharynx also relaxes the smooth muscle of the LES to prepare for entry of food.

FIG. 28.8 Swallowing in the form of pharyngeal stimulation induces neural reflex relaxation of the LES and the proximal part of the stomach to allow entry of food.

The LES also has important protective functions. It is involved in preventing acid reflux from the stomach back into the esophagus. An insufficient tonic contraction of the LES is associated with reflux disease, a gradual erosion of the esophageal mucosa, which is not as well protected as the gastric and duodenal mucosa. There is also some evidence that peristalsis in the absence of swallowing (secondary peristalsis) is important for clearing refluxed gastric contents.

Key Concepts 1. The cephalic and oral phases of the meal share many characteristics and prepare the remainder of the GI tract for the meal; these responses are neurally mediated, predominantly by the efferent vagus nerve. 2. Salivary secretion has important functions and, together with chewing of the food, allows the formation of a bolus that can be swallowed and passed along the esophagus to the stomach. 3. The ionic composition of salivary secretion varies with the flow rate, which is stimulated during a meal. The primary secretion comes from cells in the acini and is modified by epithelial cells as it passes through the ducts. 4. Regulation of salivary secretion is exclusively neural; parasympathetic innervation is most important in the response to food. 5. The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach. 6. The major function of the esophagus is to propel food from the mouth to the stomach. The esophagus has sphincters at either end that are involved in protective functions important in swallowing and preserving the integrity of the esophageal mucosa. 7. Esophageal peristalsis (primary) is stimulated by mechanical stimulation of the pharynx, and secondary peristalsis is stimulated by distention of the esophageal wall. 8. Esophageal function and the associated sphincters are regulated by extrinsic and intrinsic neural pathways.

C H AP T E R 2 9

The Gastric Phase of the Integrated Response to a Meal LEARNING OBJECTIVES Upon completion of this chapter, you should be able to answer the following questions: 1. What are the major functions of the stomach? 2. What are the gross functional regions of the stomach? 3. What is the role of the gastric epithelium in digestion and absorption? 4. What is the role of the proton pump in parietal cell function? 5. What are some examples of how gastric acid secretion is regulated during the postprandial period? 6. What are the differences between gastric mucosal protection and defense? 7. What is the functional anatomy of GI smooth muscle? 8. What is the significance of gap junctions, interstitial cells of Cajal, and pacemaker cells in the functioning of GI smooth muscle? 9. How is the basic electrical rhythm (slow wave) generated, how is it regulated by chemical messengers (hormones, paracrine, neurotransmitters), and what causes contractions associated with the slow wave to occur? 10. What physiological events in gastric motility occur in the gastric phase?

In this chapter, gastrointestinal (GI) tract physiology when food is in the stomach (i.e., the gastric phase of digestion) will be discussed. This includes gastric function and its regulation, in addition to changes in function that occur in more distal regions of the GI tract. The main functions of the stomach are to act as a temporary reservoir for the meal and to initiate protein digestion through secretion of acid and the enzyme precursor pepsinogen. Other functions are listed in Box 29.1. Box 29.1

F unc t io ns o f t he St o ma c h Storage—acts as temporary reservoir for the meal Secretion of H+ to kill microorganisms and convert pepsinogen to its active form Secretion of intrinsic factor to absorb vitamin B12 (cobalamin)

Secretion of mucus and HCO3− to protect the gastric mucosa Secretion of water for lubrication and to provide aqueous suspension of nutrients Motor activity for mixing secretions (H+ and pepsin) with ingested food Coordinated motor activity to regulate the emptying of contents into the duodenum

Food entering the stomach from the esophagus causes mechanical stimulation of the gastric wall via distention and stretching of smooth muscle. Food, predominantly oligopeptides and amino acids, also provides chemical stimulation when present in the gastric lumen. Regulation of gastric function during the gastric phase is dependent on endocrine, paracrine, and neural pathways. These pathways are activated by mechanical and chemical stimuli, which result in intrinsic and extrinsic neural reflex pathways that are important for regulation of gastric function. Afferent neurons that pass from the GI tract to the central nervous system via the vagus nerve (and to a lesser extent to the spinal cord) respond to these mechanical and chemical stimuli and activate parasympathetic outflow. The endocrine pathways include the release of gastrin, which stimulates gastric acid secretion, and the release of somatostatin, which inhibits gastric secretion. Important paracrine pathways include histamine release, which stimulates gastric acid secretion. The responses elicited by activation of these pathways include both secretory and motor responses; secretory responses include secretion of acid, pepsinogen, mucus, intrinsic factor, gastrin, lipase, and HCO3−. Overall, these secretions initiate protein digestion and protect the gastric mucosa. Motor responses (changes in activity of smooth muscle) include inhibition of motility of the proximal part of the stomach (receptive relaxation) and stimulation of motility of the distal part of the stomach, which causes antral peristalsis. These changes in motility play important roles in storage and mixing of the meal with secretions and are also involved in regulating the flow of contents out of the stomach.

Functional Anatomy of the Stomach The stomach is divided into three regions: the cardia, the corpus (also referred to as the fundus or body), and the antrum (Fig. 29.1). However, when discussing the physiology of the stomach, it is helpful to think of it as subdivided into two functional regions: the proximal and distal parts of the stomach. The proximal portion of the stomach (proximal because it is the most cranial) and the distal portion of the stomach (furthest away from the mouth) have quite different functions in the postprandial response to a meal, which will be discussed later.

FIG. 29.1 The three functional regions of the stomach. The regions have different luminal secretions and patterns of smooth muscle activity indicative of their unique functions in response to food. LES, Lower esophageal sphincter.

The lining of the stomach is covered with a columnar epithelium folded into gastric pits; each pit is the opening of a duct into which one or more gastric glands empty (Fig. 29.2). The gastric pits account for a significant fraction of the total surface area of the gastric mucosa. The gastric mucosa is divided into three distinct regions based on the structure of the glands. The small cardiac glandular region, located just below the lower esophageal sphincter (LES), primarily contains mucus-secreting gland cells. The remainder of the gastric mucosa is divided into the oxyntic or parietal (acid-secreting) gland region, located above the gastric notch (equivalent to the proximal part of the stomach), and the pyloric gland region, located below the notch (equivalent to the distal part of the stomach).

FIG. 29.2 Representation of the structure of the gastric mucosa showing a section through the wall of the stomach (A) and detail of the structure of gastric glands and cell types in the mucosa (B).

The structure of a gastric gland from the oxyntic glandular region is illustrated in Fig. 29.2. Surface epithelial cells extend slightly into the duct opening. The opening of the gland is called the isthmus and is lined with surface mucous cells and a few parietal cells. Mucous neck cells are located in the narrow neck of the gland. Parietal or oxyntic cells, which secrete HCl and intrinsic factor (involved in absorption of vitamin B12), and chief or peptic cells, which secrete pepsinogens, are located deeper in the gland. Oxyntic glands also contain enterochromaffin-like (ECL) cells that secrete histamine, and D cells that secrete somatostatin. Parietal cells are particularly numerous in glands in the fundus, whereas mucus-secreting cells are more numerous in glands of the pyloric (antral) glandular region. In addition, the pyloric glands contain G cells that secrete the hormone gastrin. The parietal glands are also divided into regions: the neck (mucous neck cells and parietal cells) and the base (peptic/chief and parietal cells). Endocrine cells are scattered throughout the glands.

Gastric Secretion Gastric secretion is a mixture of secretions from the surface epithelial cells and cells in the gastric glands. One of the most important components is H+, which is secreted against a very large concentration gradient. Thus H+ secretion by the parietal mucosa is an energy-intensive process. The cytoplasm of the parietal cell is densely packed with mitochondria, which have been estimated to fill 30% to 40% of the cell’s volume. One major function of H+ is conversion of inactive pepsinogen (the major enzyme product of the stomach) to pepsins, which initiate protein digestion in the stomach. Additionally, H+ ions are important for preventing invasion and colonization of the gut by bacteria and other pathogens that may be ingested with food. The stomach also secretes significant amounts of HCO3− and mucus, which are important for protection of the gastric mucosa against the acidic and peptic luminal environment. The

gastric epithelium also secretes intrinsic factor, which is necessary for absorption of vitamin B12 (cobalamin).

Composition of Gastric Secretions Gastric secretion consists of inorganic and organic constituents together with water. Among the important components of gastric juice are HCl, salts, pepsins, intrinsic factor, mucus, and HCO3−. Secretion of all these components increases after a meal. Inorganic Constituents of Gastric Secretion The ionic composition of gastric secretions depends on the rate of secretion. The higher the secretory rate, the higher the concentration of H+ ions. At lower secretory rates, [H+] decreases and [Na+] increases. [K+] is always higher in gastric juice than in plasma. Consequently, prolonged vomiting may lead to hypokalemia. At all rates of secretion, Cl− is the major anion of gastric juice. Gastric HCl converts pepsinogens to active pepsins and provides the acid pH at which pepsins are active. The rate of gastric H+ secretion varies considerably among individuals. In humans, basal (unstimulated) rates of gastric H+ production typically range from about 1 to 5 mEq/hr. During maximal stimulation, HCl production rises to 6 to 40 mEq/hr. The basal rate is greater at night and lowest in the early morning. The total number of parietal cells in the stomach of normal individuals varies greatly, and this variation is partly responsible for the wide range in basal and stimulated rates of HCl secretion. Organic Constituents of Gastric Secretions The predominant organic constituent of gastric secretions is pepsinogen, the inactive proenzyme of pepsin. Pepsins, often collectively called “pepsin,” are a group of proteases secreted by the chief cells of the gastric glands. Pepsinogens are contained in membrane-bound zymogen granules in the chief cells. Zymogen granules release their contents by exocytosis when chief cells are stimulated to secrete (Table 29.1). Pepsinogens are converted to active pepsins by the cleavage of acid-labile linkages. Pepsins also act proteolytically on pepsinogens to form more pepsin. Pepsins are most proteolytically active at pH 3 and below. Pepsins may digest as much as 20% of the protein in a typical meal but are not required for digestion, because their function can be replaced by that of pancreatic proteases. When the pH of the duodenal lumen is neutralized, pepsins are inactivated by the neutral pH. Table 29.1 Stimulation of Chief Cells in the Integrated Response to a Meal Stimulant

Source

Acetylcholine (ACh)

Enteric neurons

Gastrin

G cells in the gastric antrum

Histamine

ECL cells in the gastric corpus

Cholecystokinin (CCK)

I cells in the duodenum

Secretin

S cells in the duodenum

Intrinsic factor, a glycoprotein secreted by parietal cells of the stomach, is required for normal

absorption of vitamin B12. Intrinsic factor is released in response to the same stimuli that elicit secretion of HCl by parietal cells.

Cellular Mechanisms of Gastric Acid Secretion Parietal cells have a distinctive ultrastructure (Fig. 29.3). Branching secretory canaliculi course through the cytoplasm and are connected by a common outlet to the cell’s luminal surface. Microvilli line the surfaces of the secretory canaliculi. The cytoplasm of unstimulated parietal cells contains numerous tubules and vesicles called the tubulovesicular system. The membranes of tubulovesicles contain the transport proteins responsible for secretion of H+ and Cl− into the lumen of the gland. When parietal cells are stimulated to secrete HCl (see Fig. 29.3), tubulovesicular membranes fuse with the plasma membrane of the secretory canaliculi. This extensive membrane fusion greatly increases the number of H+/K+ antiporters in the plasma membrane of the secretory canaliculi. When parietal cells secrete gastric acid at the maximal rate, H+ is pumped against a concentration gradient that is about 1 million–fold. Thus the pH is 7 in the parietal cell cytosol and 1 in the lumen of the gastric gland.

FIG. 29.3 Parietal cell ultrastructure. A, A resting parietal cell showing the tubulovesicular apparatus in the cytoplasm and the intracellular canaliculus. B, An activated parietal cell that is secreting acid. The tubulovesicles have fused with the membranes of the intracellular canaliculus, which is now open to the lumen of the gland and lined with abundant long microvilli.

The cellular mechanism of H+ secretion by the parietal cell is depicted in Fig. 29.4. Cl− enters the cell across the basolateral membrane in exchange for HCO3− generated in the cell by the action of carbonic anhydrase, which produces HCO3− and H+. H+ is secreted across the luminal membrane by H+,K+ATPase in exchange for K+. K+ recycles across the luminal membrane via a K+ channel. Cl− enters the lumen via an ion channel (a chloride channel [CLC] family Cl− channel) located in the luminal membrane. Increased intracellular Ca++ and cyclic adenosine monophosphate (cAMP) stimulate luminal membrane conduction of Cl− and K+. Increased K+ conductance hyperpolarizes the luminal membrane potential, which increases the driving force for efflux of Cl− across the luminal membrane. The K+ channel in the basolateral membrane also mediates the efflux of K+ that accumulates in the parietal cell via the activity

of H+,K+-ATPase. In addition, cAMP and Ca++ promote trafficking of Cl− channels into the luminal membrane, as well as fusion of cytosolic tubulovesicles containing H+,K+-ATPase with the membrane of the secretory canaliculi (see Figs. 29.3 and 29.4). Parietal cell secretion of H+ is also accompanied by transport of HCO3− into the bloodstream to maintain intracellular pH.

FIG. 29.4 Mechanism of H+ and Cl− secretion by an activated parietal cell in the gastric mucosa. ATP, Adenosine triphosphate.

Secretion of HCO3− The surface epithelial cells also secrete a watery fluid that contains Na+ and Cl− in concentrations similar to those in plasma but with higher K+ and HCO3− concentrations. HCO3− is entrapped by the viscous mucus that coats the surface of the stomach; thus, the mucus secreted by the resting mucosa lines the stomach with a sticky alkaline coat. In the postprandial period, rates of secretion of both mucus and HCO3− increase.

Secretion of Mucus Secretions that contain mucins are viscous and sticky and are collectively termed mucus. Mucins are secreted by mucous neck cells located in the necks of gastric glands and by the surface epithelial cells of the stomach. Mucus is stored in large granules in the apical cytoplasm of mucous neck cells and surface epithelial cells and is released by exocytosis. Gastric mucins are about 80% carbohydrate by weight and consist of four similar monomers of about 500,000 Da each that are linked together by disulfide bonds (Fig. 29.5). These tetrameric mucins form a sticky gel that adheres to the surface of the stomach. This gel is subject to proteolysis by pepsins to release fragments that do not form gels and thus dissolves the protective mucous layer. Maintenance of the protective mucous layer requires continuous synthesis of new tetrameric mucins to replace the mucins cleaved by pepsins.

FIG. 29.5 Schematic representation of the structure of gastric mucins before and after hydrolysis by pepsin. Intact mucins are tetramers of four similar monomers of about 500,000 Da. Each monomer is largely covered by carbohydrate side chains that protect it from proteolytic degradation. The central portion of the mucin tetramer, near the disulfide cross-links, is more susceptible to proteolytic digestion. Pepsins cleave bonds near the center of the tetramers to release fragments about the size of monomers.

Mucus is secreted at a significant rate in the resting stomach. Secretion of mucus is stimulated by some of the same stimuli that enhance acid and pepsinogen secretion, especially acetylcholine released from parasympathetic nerve endings.

Regulation of Gastric Secretion Parasympathetic innervation via the vagus nerve is the strongest stimulant of gastric H+ secretion. Extrinsic efferent fibers terminate on intrinsic neurons that innervate parietal cells, ECL cells that secrete the paracrine mediator histamine, and endocrine cells that secrete the hormone gastrin. In addition, vagal stimulation results in secretion of pepsinogen, mucus, HCO3−, and intrinsic factor. Stimulation of the parasympathetic nervous system also occurs during the cephalic and oral phase of the meal. However, the gastric phase produces the largest stimulation of gastric secretion of the postprandial period (Fig. 29.6).

FIG. 29.6 Neural regulation of gastric acid secretion in the gastric phase of the meal is mediated by the vagus nerve. The stimulation that occurs in the cephalic and oral phases (before food reaches the stomach) results in stimulation of parietal cells to secrete acid and chief cells to secrete pepsinogen. Thus, when food reaches the stomach, protein digestion is initiated by generating protein hydrolysate, which further stimulates secretion of gastrin from the mucosa of the gastric antrum. In addition, gastric distention activates a vagovagal reflex that further stimulates gastric acid and pepsinogen secretion.

Stimulation of gastric acid secretion is an excellent example of a “feed-forward” (or cascade) response that uses endocrine, paracrine, and neural pathways. Activation of intrinsic neurons by vagal efferent activity results in release of acetylcholine from nerve terminals, which activates cells in the gastric epithelium. Parietal cells express muscarinic receptors and are activated to secrete H+ in response to vagal efferent nerve activity. In addition, parasympathetic activation, via gastrin-releasing peptide from intrinsic neurons, releases gastrin from G cells located in the gastric glands in the gastric antrum (see Fig. 29.6). Gastrin enters the bloodstream and, via an endocrine mechanism, further stimulates the parietal cell to secrete H+. Parietal cells express cholecystokinin type B (CCKB) receptors for gastrin. Histamine is also secreted in response to vagal nerve stimulation, and ECL cells express muscarinic and gastrin receptors. Thus, gastrin and vagal efferent activity induce release of histamine, which potentiates the effects of both gastrin and acetylcholine on the parietal cell. Hence activation of parasympathetic (vagal) outflow to the stomach is very efficient at stimulating the parietal cell to secrete acid (Figs. 29.7 and 29.8).

FIG. 29.7 The parietal cell is regulated by neural, hormonal, and paracrine pathways. Activation of vagal parasympathetic preganglionic outflow to the stomach acts in three ways to stimulate gastric acid secretion. There is direct neural innervation and activation of the parietal cell via release of acetylcholine (A) from enteric neurons, which acts on the parietal cell via muscarinic receptors. In addition, neural activation of the ECL cell stimulates release of histamine (H), which acts via a paracrine pathway to stimulate the parietal cell. Finally, G cells located in gastric glands in the gastric antrum are activated by release of gastrin-releasing peptide (GRP) from enteric neurons, which acts on the G cell to stimulate release of gastrin (G). Gastrin thereafter acts via a humoral pathway to stimulate the parietal cell.

FIG. 29.8 Feedback regulation of gastric acid secretion by release of somatostatin and its action on G cells in the gastric antrum. Endocrine cells in the mucosa of the gastric antrum sense the presence of H+ and secrete somatostatin. This in turn acts on specific receptors on G cells to inhibit release of gastrin and thus bring about inhibition of gastric acid secretion.

In the gastric phase, the presence of food in the stomach is detected and activates vagovagal reflexes to stimulate secretion. Food in the stomach results in distention and stretch, which are detected by afferent (or sensory) nerve endings in the gastric wall. These are the peripheral terminals of vagal afferent nerves that transmit information to the brainstem and thereby drive activity in vagal efferent fibers, a vagovagal reflex (see Fig. 29.6). In addition, digestion of proteins increases the concentration of oligopeptides and free amino acids in the lumen, which are detected by chemosensors in the gastric mucosa. Oligopeptides and amino acids also stimulate vagal afferent activity. The exact nature of the chemosensors is not clear but may involve endocrine cells that release their contents to activate nerve endings. This topic will be discussed in more detail in Chapter 30. There is also an important negative feedback mechanism whereby the presence of acid in the distal part

of the stomach (antrum) induces a feedback loop to inhibit the parietal cell such that meal-stimulated H+ secretion does not go unchecked. When the concentration of H+ in the lumen reaches a certain threshold ( 60 mL/minute is considered normal. Values < 60 mL/minute may indicate impaired kidney function. It is important to note that a fall in GFR may be the first and only clinical sign of kidney disease. Thus determining the GFR is important when kidney disease is suspected. However, a 50% loss of functioning nephrons reduces GFR only by about 25%. The decline in GFR is not 50% because the remaining nephrons compensate. Kidney function is usually assessed by measuring the plasma concentration of creatinine (PCr), which is inversely related to GFR (Fig. 33.14). However, as Fig. 33.14 shows, GFR must decline substantially before an increase in PCr can be detected. For example, a fall in GFR from 120 to 100 mL/minute is accompanied by an increase in PCr from 1.0 to 1.2 mg/dL. This does not appear to be a significant change in PCr, but GFR has actually fallen by almost 20%.

FIG. 33.14 Relationship between GFR and plasma [creatinine] (Pcr). The amount of creatinine filtered is equal to the amount excreted; thus GFR × PCr = UCr × V˙. Because the production of creatinine is constant, excretion must be constant to maintain creatinine balance. Therefore if GFR falls from 120 to 60 mL/minute, PCr must increase from 1 to 2 mg/dL to keep filtration of creatinine and its excretion equal to the production rate. GFR, Glomerular filtration rate.

Glomerular Filtration The first step in the formation of urine is ultrafiltration of plasma by the glomerulus. In normal adults, GFR ranges from 90 to 140 mL/minute in males and from 80 to 125 mL/minute in females. Thus in 24 hours as much as 180 L of plasma is filtered by the glomeruli. The plasma ultrafiltrate is devoid of cellular elements (i.e., red and white blood cells and platelets) and is essentially protein free. The concentration of salts and organic molecules (e.g., glucose, amino acids) is similar in plasma and the ultrafiltrate. Starling forces drive ultrafiltration across the glomerular capillaries, and changes in these forces alter GFR. GFR and RPF are normally held within very narrow ranges by a phenomenon called autoregulation. The next sections of this chapter review the composition of the glomerular filtrate, the dynamics of its formation, and the relationship between RPF and GFR. In addition, factors that contribute to autoregulation and regulation of GFR and RBF are discussed.

Determinants of Ultrafiltrate Composition The glomerular filtration barrier determines the composition of the plasma ultrafiltrate. It restricts filtration of molecules on the basis of both size and electric charge (Fig. 33.15). In general, neutral molecules with a radius smaller than about 18 Å are freely filtered, molecules larger than about 42 Å are not filtered, and molecules between about 18 and 42 Å are filtered to varying degrees. Fig. 33.15 shows how electric charge affects filtration of macromolecules (e.g., dextrans) by the glomerulus. Dextrans are a family of exogenous polysaccharides manufactured in various molecular weights. They can be electrically neutral or have either negative (polyanionic) or positive (polycationic) charges. As the size (i.e., effective molecular radius) of a dextran molecule increases, the rate at which it is filtered decreases. For any given

molecular radius, cationic molecules are more readily filtered than anionic molecules. The reduced filtration rate for anionic molecules is explained by the presence of negatively charged glycoproteins on the surface of all components of the glomerular filtration barrier. These charged glycoproteins repel similarly charged molecules. Because most plasma proteins are negatively charged, the negative charge on the filtration barrier restricts filtration of anionic proteins more than the filtration of neutral and polyanionic proteins with a molecular radius between approximately 18 to 42 Å. For example, serum albumin, an anionic protein that has an effective molecular radius of 35.5 Å, is poorly filtered. Because the small amount of filtered albumin is normally reabsorbed avidly by the proximal tubule, almost no albumin appears in urine.

FIG. 33.15 Influence of the size and electric charge of dextran on its filterability. A value of 1 indicates that it is filtered freely, whereas a value of zero indicates that it is not filtered. The filterability of dextrans between approximately 18 and 42 Å depends on charge. Dextrans larger than 42 Å are not filtered regardless of charge, and polycationic dextrans and neutral dextrans smaller than 18 Å are freely filtered. The major proteins in plasma are albumin and immunoglobulins. Because the effective molecular radii of immunoglobulin (Ig)G (53 Å) and IgM (>100 Å) are greater than 42 Å, they are not filtered. Although the effective molecular radius of albumin is 35 Å, it is a polyanionic protein, so it does not cross the filtration barrier to a significant degree.

IN THE C LIN IC The importance of the negative charges on the filtration barrier in restricting filtration of plasma proteins is shown in Figs. 33.15 and 33.16. Removal of the negative charges from the filtration barrier causes proteins to be filtered solely on the basis of their effective molecular radius (Fig. 33.16). Hence at any molecular radius between approximately 18 and 42 Å, filtration of polyanionic proteins will exceed the filtration that prevails in the normal state (in which the filtration barrier has anionic charges). In a number of glomerular diseases the negative charges on the filtration barrier are reduced because of immunological damage and inflammation. As a result, filtration of anionic proteins between approximately 18 and 42 Å in radius is increased. When the filtered proteins exceed the ability of the proximal tubule to reabsorb and catabolize them, anionic proteins begin to appear in urine (proteinuria), which is a marker of kidney disease.

FIG. 33.16 Reduction of the negative charges on the glomerular wall results in filtration of proteins on the basis of size only. In this situation the relative filterability of proteins depends only on the molecular radius. Accordingly, excretion of polyanionic proteins (18–42 Å) in urine increases because more proteins of this size are filtered.

Dynamics of Ultrafiltration The forces responsible for glomerular filtration of plasma are the same as those in other capillary beds. Ultrafiltration occurs because the Starling forces (i.e., hydrostatic and oncotic pressures) combine to drive fluid from the lumen of glomerular capillaries across the filtration barrier and into Bowman’s space (Fig. 33.17). The hydrostatic pressure inside the glomerular capillary (PGC) is oriented to promote movement of fluid from the glomerular capillary into Bowman’s space. Because the glomerular ultrafiltrate is essentially protein free under normal conditions, owing in large part to the paucity of proteins in serum smaller than 18 Å in radius that can be effectively filtered, the reflection coefficient (σ) for proteins across the glomerular capillary is essentially 1. Thus the oncotic pressure in Bowman’s space (πBS) is near zero. Therefore PGC is the principal force favoring filtration. In contrast, the hydrostatic pressure in Bowman’s space (PBS) and the oncotic pressure in the glomerular capillary (πGC) both oppose filtration.

FIG. 33.17 Idealized glomerular capillary and the Starling forces across it. The reflection coefficient (σ) for protein across the glomerular capillary is approximately 1. PBS, Hydraulic pressure in Bowman’s space; PGC, hydraulic pressure in the glomerular capillary; PUF, net ultrafiltration pressure; πBS, oncotic pressure in Bowman’s space; πGC, oncotic pressure in the glomerular capillary. The negative signs for PBS and πGC indicate that these forces oppose formation of the glomerular filtrate.

As shown in Fig. 33.17, a net ultrafiltration pressure (PUF) of 17 mm Hg exists at the afferent end of the glomerulus, whereas at the efferent end it is 8 mm Hg (where PUF = PGC − PBS − πGC). Two additional points concerning Starling forces and this pressure change are important to note. First, PGC decreases slightly along the length of the capillary because of the resistance to flow along the length of the capillary. Second, πGC increases as plasma is filtered while protein is retained within the glomerular capillary, thereby progressively increasing the protein concentration along the length of the capillary. GFR is proportional to the sum of the Starling forces that exist across the capillaries [(PGC − PBS) − σ(πGC − πBS)] multiplied by the ultrafiltration coefficient (Kf) of the capillary. That is,

Equation 33.10 Kf is the product of the intrinsic permeability of the glomerular capillary and the glomerular surface area available for filtration. The rate of glomerular filtration is considerably greater in glomerular capillaries than in systemic capillaries, mainly because Kf is approximately 100 times greater in glomerular capillaries. Furthermore PGC is approximately twice as great as the hydrostatic pressure in systemic capillaries. GFR can be altered by changing Kf or by changing any of the Starling forces. In normal individuals,

GFR is regulated by alterations in PGC that are mediated mainly by changes in afferent or efferent arteriolar resistance. PGC is affected in three ways: 1. Changes in afferent arteriolar resistance: A decrease in resistance increases PGC and GFR, whereas an increase in resistance decreases PGC and GFR. 2. Changes in efferent arteriolar resistance: A decrease in resistance reduces PGC and GFR, whereas an increase in resistance elevates PGC and GFR. 3. Changes in renal arteriolar pressure: An increase in blood pressure transiently increases PGC (which enhances GFR), whereas a decrease in blood pressure transiently decreases PGC (which reduces GFR).

IN THE C LIN IC A reduction in GFR in disease states is most often due to decreases in Kf because of the loss of filtration surface area. GFR also changes in pathophysiological conditions because of changes in PGC, πGC, and PBS. 1. Changes in Kf: Increased Kf enhances GFR, whereas decreased Kf reduces GFR. Some kidney diseases reduce Kf by decreasing the number of filtering glomeruli (i.e., diminished surface area). Some drugs and hormones that dilate the glomerular arterioles also increase Kf. Similarly, drugs and hormones that constrict the glomerular arterioles also decrease Kf. 2. Changes in PGC: With decreased renal perfusion, GFR declines because PGC falls. As previously discussed, a reduction in PGC is caused by a decline in renal arterial pressure, an increase in afferent arteriolar resistance, or a decrease in efferent arteriolar resistance. 3. Changes in πGC: An inverse relationship exists between πGC and GFR. Alterations in πGC result from changes in protein synthesis outside the kidneys. In addition the protein loss in urine caused by some renal diseases can lead to a decrease in the plasma protein concentration and thus in πGC. 4. Changes in PBS: Increased PBS reduces GFR, whereas decreased PBS enhances GFR. Acute obstruction of the urinary tract (e.g., a kidney stone occluding the ureter) increases PBS.

Renal Blood Flow Blood flow through the kidneys serves several important functions: 1. indirectly determines GFR 2. modifies the rate of solute and water reabsorption by the proximal tubule 3. participates in concentration and dilution of urine 4. delivers O2, nutrients, and hormones to cells along the nephron and returns CO2, reabsorbed fluid, and solutes to the general circulation 5. delivers substrates for excretion in urine

Blood flow through any organ may be represented by the following equation:

Equation 33.11 where Q = blood flow ΔP = mean arterial pressure minus venous pressure for that organ R = resistance to flow through that organ Accordingly, RBF is equal to the pressure difference between the renal artery and the renal vein divided by renal vascular resistance:

Equation 33.12

The afferent arteriole, efferent arteriole, and interlobular arteries are the major resistance vessels in the kidneys and thereby determine renal vascular resistance. Like most other organs, the kidneys regulate their blood flow by adjusting vascular resistance in response to changes in arterial pressure. As shown in Fig. 33.18 these adjustments are so precise that blood flow remains relatively constant as arterial blood pressure changes between 90 and 180 mm Hg. GFR is also regulated over the same range of arterial pressures. The phenomenon whereby RBF and GFR are maintained relatively constant between blood pressures of 90 and 180 mm Hg, namely autoregulation, is achieved by changes in vascular resistance, mainly through the afferent arterioles of the kidneys. Because both RBF and GFR are regulated over the same range of pressures and because RBF is an important determinant of GFR, it is not surprising that the same mechanisms regulate both flows.

FIG. 33.18 Relationship between arterial blood pressure and RBF and between arterial blood pressure and GFR. Autoregulation maintains GFR and RBF relatively constant as blood pressure changes from 90 to 180 mm Hg. GFR, Glomerular filtration rate; RBF, renal blood flow.

Two mechanisms are responsible for autoregulation of RBF and GFR: one mechanism that responds to changes in arterial pressure and another that responds to changes in [NaCl] in tubular fluid. Both regulate the tone of the afferent arteriole. The pressure-sensitive mechanism, the so-called myogenic mechanism, is related to an intrinsic property of vascular smooth muscle: the tendency to contract when stretched. Accordingly, when arterial pressure rises and the renal afferent arteriole is stretched, the smooth muscle contracts in response. Because the increase in resistance of the arteriole offsets the increase in pressure, RBF, and therefore GFR, remains constant. (That is, RBF is constant if ΔP/R is kept constant [see Eq. 33.11].) The second mechanism responsible for autoregulation of GFR and RBF is the [NaCl]-dependent mechanism known as tubuloglomerular feedback. This mechanism involves a feedback loop in which a change in GFR leads to alteration in the concentration of NaCl in tubular fluid, which is sensed by the macula densa of the juxtaglomerular apparatus and converted into signals that affect afferent arteriolar resistance and thus the GFR (Fig. 33.19). For example, when the GFR increases and causes [NaCl] in tubular fluid in the loop of Henle to rise, more NaCl enters the macula densa cells in this segment (Fig. 33.20). This leads to an increase in formation and release of adenosine triphosphate (ATP) and adenosine (a metabolite of ATP) by macula densa cells, which causes vasoconstriction of the afferent arteriole and normalization of GFR. In contrast, when GFR and [NaCl] in tubule fluid decrease, less NaCl enters the macula densa cells, and both ATP and adenosine production and release decline. The fall in [ATP] and [adenosine] results in afferent arteriolar vasodilation, which returns GFR to normal. NO, a vasodilator produced by the macula densa, attenuates tubuloglomerular feedback, whereas angiotensin II enhances tubuloglomerular feedback. Thus the macula densa may release both vasoconstrictors (e.g., ATP and adenosine) and a vasodilator (e.g., NO) that oppose each other’s action at the level of the afferent arteriole. Production plus release of either vasoconstrictors or vasodilators ensures exquisite control over tubuloglomerular feedback.

FIG. 33.19 Tubuloglomerular feedback. An increase in glomerular filtration rate (GFR) (1) increases [NaCl] in tubule fluid in the loop of Henle (2). The increase in [NaCl] is sensed by the macula densa and converted to a signal (3) that increases the resistance of the afferent arteriole (RA) (4), which decreases GFR. A decrease in GFR has the opposite effects. JGA, juxtaglomerular apparatus. (Modified from Cogan MG. Fluid and Electrolytes: Physiology and Pathophysiology. Norwalk, CT: Appleton & Lange; 1991.)

FIG. 33.20 Cellular mechanism whereby an increase in delivery of NaCl to the macula densa causes vasoconstriction of the afferent arteriole of the same nephron (i.e., tubuloglomerular feedback). An increase in GFR elevates [NaCl] in tubule fluid at the macula densa. This in turn enhances uptake of NaCl across the apical cell membrane of macula densa cells via the 1Na+-1K+-2Cl− (NKCC2) symporter, which leads to an increase in [ATP] and [adenosine] (ADO) release. ATP binds to P2X receptors and adenosine binds to adenosine A1 receptors in the plasma membrane of smooth muscle cells surrounding the afferent arteriole, both of which increase intracellular [Ca+ +]. The rise in [Ca+ +] induces vasoconstriction of the afferent arteriole, thereby returning GFR to normal levels. Note that ATP and adenosine also inhibit renin release by granular cells in the afferent arteriole. This too results from an increase in intracellular [Ca+ +] as a reflection of electrical coupling of the granular and vascular smooth muscle (VSM) cells. When GFR is reduced, [NaCl] in tubule fluid falls, as does uptake of NaCl into macula densa cells. This in turn decreases release of ATP and adenosine by the macula densa, which decreases intracellular [Ca++] in smooth muscle cells and thereby increases GFR and stimulates renin release by granular cells. In addition a decrease in entry of NaCl into macula densa cells enhances production of PGE2, which also stimulates renin secretion by granular cells. As discussed in detail in Chapter 34, renin increases plasma [angiotensin II], a hormone that enhances NaCl and water retention by the kidneys. (Modified from Persson AEG et al. Acta Physiol Scand. 2004;181:471.)

Fig. 33.20 also illustrates the role of the macula densa in controlling renin secretion by granular cells of the afferent arteriole. This aspect of function of the juxtaglomerular apparatus is considered in detail in Chapter 35. Because animals engage in many activities that can change arterial blood pressure, mechanisms that maintain RBF and GFR relatively constant despite changes in arterial pressure are highly desirable. If GFR and RBF were to rise or fall suddenly in proportion to changes in blood pressure, urinary excretion of fluid and solute would also change suddenly. Such changes in excretion of water and solutes without comparable changes in intake would alter fluid and electrolyte balance (the reason for which is discussed in Chapter 35). Accordingly, autoregulation of GFR and RBF provides an effective means for uncoupling renal function from arterial pressure, and it ensures that fluid and solute excretion remain relatively

constant. Three points concerning autoregulation should be noted: 1. Autoregulation is absent when arterial pressure is less than 90 mm Hg. 2. Autoregulation is not perfect; RBF and GFR do change slightly as arterial blood pressure varies. 3. Despite autoregulation, RBF and GFR can be changed by several hormones and by alterations in sympathetic nerve activity that change in response to alterations in the extracellular fluid volume (ECFV) (Table 33.1). Table 33.1 Major Hormones That Influence Glomerular Filtration Rate and Renal Blood Flow

Stimulus

Effect on GFR

Effect on RBF

Vasoconstrictors







Sympathetic nerves

↓ ECFV

↓

↓

Angiotensin II

↓ ECFV

↓

↓

Endothelin

↑ Stretch, A-II, bradykinin, epinephrine; ↓ ECFV

↓

↓

Vasodilators







Prostaglandins (PGE1, PGE2, PGI2)

↓ ECFV; ↑ shear stress, A-II

No change/↑ ↑

Nitric oxide (NO)

↑ Shear stress, acetylcholine, histamine, bradykinin, ATP

↑

↑

Bradykinin

↑ Prostaglandins, ↓ ACE

↑

↑

Natriuretic peptides (ANP, BNP)

↑ ECFV

↑

No change

A-II, Angiotensin II; ACE, angiotensin-converting enzyme; ECFV, extracellular fluid volume.

AT THE C ELLU LAR LEVEL Tubuloglomerular feedback (TGF) is absent in mice that do not express the adenosine receptor (A1). This underscores the importance of adenosine signaling in TGF. Studies have shown that when GFR increases and causes the concentration of NaCl in tubular fluid at the macula densa to rise, more NaCl enters cells via the 1Na+-1K+-2Cl− symporter (NKCC2) located in the apical plasma membrane (see Fig. 33.20). Increased intracellular [NaCl] in turn stimulates release of ATP via ATP-conducting ion channels located in the basolateral membrane of macula densa cells. In addition, adenosine production is also enhanced. Adenosine binds to A1 receptors and ATP binds to P2X receptors located on the plasma membrane of smooth muscle cells in the afferent arteriole. Both hormones increase intracellular [Ca++], which causes vasoconstriction of the afferent artery and therefore a fall in GFR. Although adenosine is a vasodilator in most other vascular beds, it constricts the afferent arteriole in the kidney.

Regulation of Renal Blood Flow and Glomerular Filtration Rate Several factors and hormones affect both RBF and GFR (see Table 33.1). As already discussed, the myogenic mechanism and tubuloglomerular feedback play key roles in maintaining RBF and GFR constant when blood pressure is greater than 90 mm Hg and ECFV is in the normal range. However, when the ECFV changes sympathetic nerves, angiotensin II, prostaglandins, NO, endothelin, bradykinin, ATP, and adenosine exert major control over RBF and GFR. Fig. 33.21 shows how changes in efferent and afferent arteriolar resistance, mediated by changes in the hormones listed in Table 33.1, modulate both RBF and GFR.

FIG. 33.21 Relationship between selective changes in resistance of either the afferent arteriole or the efferent arteriole on RBF and GFR. Constriction of either the afferent or efferent arteriole increases resistance, and according to Eq. 33.11 (Q = ΔP/R), an increase in resistance (R) decreases flow (Q) (i.e., RBF). Dilation of either the afferent or afferent arteriole increases flow (i.e., RBF). Constriction of the afferent arteriole (A) decreases PGC because less of the arterial pressure is transmitted to the glomerulus, thereby reducing GFR. In contrast, constriction of the efferent arteriole (B) elevates PGC and thus increases GFR. Dilation of the efferent arteriole (C) decreases PGC and thus decreases GFR. Dilation of the afferent arteriole (D) increases PGC because more of the arterial pressure is transmitted to the glomerulus, thereby increasing GFR. GFR, Glomerular filtration rate; RBF, renal blood flow. (Modified from Rose BD, Rennke KG. Renal Pathophysiology: The Essentials. Baltimore: Williams & Wilkins; 1994.)

Sympathetic Nerves The afferent and efferent arterioles are innervated by sympathetic neurons; however, sympathetic tone is minimal when ECFV is normal (see Chapter 35). When ECFV is reduced, sympathetic nerves release norepinephrine and dopamine, and circulating epinephrine (a catecholamine-like norepinephrine and dopamine) is secreted by the adrenal medulla. Norepinephrine and epinephrine cause vasoconstriction by binding to α1-adrenoceptors, which are located mainly in afferent arterioles. Activation of α1adrenoceptors decreases RBF and GFR. Dehydration or strong emotional stimuli (e.g., fear, pain) also activate sympathetic nerves and reduce RBF and GFR.

IN THE C LIN IC Individuals with renal artery stenosis (narrowing of the lumen of the artery) caused by atherosclerosis, for example, often have elevated systemic arterial blood pressure mediated by the renin-angiotensin system. Pressure in the renal artery proximal to the stenosis is increased, but pressure distal to the stenosis is normal or reduced. Autoregulation is important in maintaining RBF, PGC, and GFR in the presence of this stenosis. Administration of drugs to lower systemic blood pressure also lowers the pressure distal to the stenosis; accordingly, RBF, PGC, and GFR fall.

IN THE C LIN IC Significant hemorrhage decreases ECF volume and arterial blood pressure and therefore activates sympathetic innervation of the kidneys via the baroreceptor reflex (Fig. 33.22). Norepinephrine causes intense vasoconstriction of the afferent and efferent glomerular arterioles and thereby decreases both RBF and GFR. The rise in sympathetic activity also increases release of epinephrine and angiotensin II, which cause further vasoconstriction and a fall in RBF. The rise in vascular resistance of the kidneys and other vascular beds increases total peripheral resistance. The resulting tendency for blood pressure to increase (blood pressure = cardiac output × total peripheral resistance) offsets the tendency of blood pressure to decrease in response to hemorrhage. Hence this system works to preserve arterial pressure at the expense of maintaining normal RBF and GFR.

FIG. 33.22 Pathway by which hemorrhage activates renal sympathetic nerve activity and stimulates production of angiotensin II. GFR, Glomerular filtration rate; RBF, renal blood flow. (Modified from Vander AJ. Renal Physiology. 2nd ed. New York: McGraw-Hill; 1980.)

Angiotensin II Angiotensin II is produced systemically as well as locally within the kidneys. It constricts the afferent and efferent arteriolesd and decreases both RBF and GFR. Fig. 33.22 shows how norepinephrine, epinephrine, and angiotensin II act together to decrease RBF and GFR and thereby increase blood pressure and ECF volume (e.g., as would occur with hemorrhage).

Prostaglandins Prostaglandins do not play a major role in regulating RBF in healthy resting individuals. However, during pathophysiological conditions such as hemorrhage and reduced ECFV, prostaglandins (PGI2, PGE1, and PGE2) are produced locally within the kidneys and serve to increase RBF without changing GFR. Prostaglandins increase RBF by dampening the vasoconstrictor effects of both sympathetic activation and angiotensin II. These effects are important because they prevent severe and potentially harmful vasoconstriction and renal ischemia. Synthesis of prostaglandins is stimulated by ECFV depletion and stress (e.g., surgery, anesthesia), angiotensin II, and sympathetic nerves. Nonsteroidal anti-inflammatory

drugs (NSAIDs), such as ibuprofen and naproxen, potently inhibit prostaglandin synthesis. Thus administration of these drugs during renal ischemia and hemorrhagic shock is contraindicated because, by blocking the production of prostaglandins, they decrease RBF and increase renal ischemia. Prostaglandins also play an increasingly important role in maintaining RBF and GFR as individuals age. Accordingly, NSAIDs can significantly reduce RBF and GFR in the elderly.

Nitric Oxide NO, an endothelium-derived relaxing factor, is an important vasodilator under basal conditions, and it counteracts the vasoconstriction produced by angiotensin II and catecholamines. When blood flow increases, greater shear force acts on endothelial cells in the arterioles and increases production of NO. In addition a number of vasoactive hormones, including acetylcholine, histamine, bradykinin, and ATP, facilitate release of NO from endothelial cells. Increased production of NO causes dilation of the afferent and efferent arterioles in the kidneys. Whereas increased levels of NO decrease total peripheral resistance, inhibition of NO production increases total peripheral resistance.

IN THE C LIN IC Abnormal production of NO is observed in individuals with diabetes mellitus and hypertension. Excess renal NO production in diabetes may be responsible for glomerular hyperfiltration (i.e., increased GFR) and damage to the glomerulus, problems characteristic of this disease. Elevated NO levels increase glomerular capillary pressure secondary to a fall in resistance of the afferent arteriole. The ensuing hyperfiltration is thought to cause glomerular damage. The normal response to an increase in dietary salt intake includes stimulation of renal NO production, which prevents an increase in blood pressure. In some individuals, however, NO production may not increase appropriately in response to an elevation in salt intake, so blood pressure rises.

Endothelin Endothelin is a potent vasoconstrictor secreted by endothelial cells of the renal vessels, mesangial cells, and distal tubular cells in response to angiotensin II, bradykinin, epinephrine, and endothelial shear stress. Endothelin causes profound vasoconstriction of the afferent and efferent arterioles and decreases GFR and RBF. Although this potent vasoconstrictor may not influence GFR and RBF in resting subjects, production of endothelin is elevated in a number of glomerular disease states (e.g., renal disease associated with diabetes mellitus).

Bradykinin Kallikrein is a proteolytic enzyme produced in the kidneys. Kallikrein cleaves circulating kininogen to bradykinin, which is a vasodilator that acts by stimulating the release of NO and prostaglandins. Bradykinin increases RBF and GFR.

Adenosine Adenosine is produced within the kidneys and causes vasoconstriction of the afferent arteriole, thereby reducing RBF and GFR. As previously mentioned, adenosine plays an important role in tubuloglomerular

feedback.

Natriuretic Peptides Secretion of atrial natriuretic peptide (ANP) by the cardiac atria and brain natriuretic peptide (BNP) by the cardiac ventricle increases when ECFV is expanded and myocardial wall tension is increased. Both ANP and BNP dilate the afferent arteriole and constrict the efferent arteriole. Therefore ANP and BNP produce a modest increase in GFR with little change in RBF.

Adenosine Triphosphate Cells release ATP into the renal interstitial fluid. ATP can have bidirectional effects on both RBF and GFR. Under some conditions, ATP constricts the afferent arteriole, reduces RBF and GFR, and may play a role in tubuloglomerular feedback. Under other conditions, ATP may stimulate NO production and have directionally opposite effects, increasing both RBF and GFR.

Glucocorticoids Administration of therapeutic doses of glucocorticoids increases GFR and RBF.

Histamine Local release of histamine modulates RBF during the resting state and during inflammation and injury. Histamine decreases the resistance of the afferent and efferent arterioles and thereby increases RBF without elevating GFR.

Dopamine The proximal tubule produces the vasodilator substance dopamine. Dopamine has several actions within the kidney, such as increasing RBF and inhibiting renin secretion.

Hormones Finally, as illustrated in Fig. 33.23, endothelial cells play an important role in regulating the resistance of the renal afferent and efferent arterioles by producing a number of paracrine hormones, including NO, prostacyclin (PGI2), endothelin, and angiotensin II. These hormones regulate contraction or relaxation of smooth muscle cells in afferent and efferent arterioles and mesangial cells. Shear stress, acetylcholine, histamine, bradykinin, and ATP stimulate production of NO, which increases GFR and RBF. Angiotensinconverting enzyme (ACE), located on the surface of endothelial cells lining the afferent arteriole and glomerular capillaries, converts angiotensin I to angiotensin II, which decreases GFR and RBF. Angiotensin II is also produced locally by granular cells in the afferent arteriole and by proximal tubular cells. PGI2 and PGE2 release by endothelial cells is stimulated by both sympathetic nerve activity and angiotensin II, resulting in increased GFR and RBF. Finally, endothelin release from endothelial cells decreases both RBF and GFR.

FIG. 33.23 Examples of the interactions of endothelial cells with smooth muscle and mesangial cells. ACE, Angiotensin-converting enzyme; AI, angiotensin I; AII, angiotensin II. (Modified from Navar LG et al. Physiol Rev. 1996;76:425.)

IN THE C LIN IC ACE proteolytically inactivates the vasodilatory hormone bradykinin and converts angiotensin I, an inactive hormone, to angiotensin II, an active vasoconstrictive hormone. Thus ACE increases angiotensin II levels and decreases bradykinin levels. ACE inhibitors (e.g., lisinopril, enalapril, and captopril) are used clinically to reduce systemic blood pressure in patients with hypertension by decreasing angiotensin II levels and elevating bradykinin levels. Both effects lower systemic vascular resistance, reduce blood pressure, and decrease renal vascular resistance, thereby increasing GFR and RBF. Angiotensin II receptor antagonists (e.g., losartan) are also used to treat hypertension. As their name suggests, they block the binding of angiotensin II to the angiotensin II receptor (AT1). These antagonists block the vasoconstrictor effects of angiotensin II on the afferent arteriole; thus they increase RBF and GFR. In contrast to ACE inhibitors, angiotensin II receptor antagonists do not inhibit kinin metabolism (e.g., bradykinin).

Key Points 1. The first step in urine formation is passive movement of a plasma ultrafiltrate from the glomerular capillaries into Bowman’s space. The term ultrafiltration refers to passive movement of a plasma-like fluid that has a very low concentration of proteins from the glomerular capillaries into Bowman’s space. The endothelial cells of glomerular capillaries are covered by a basement membrane that is surrounded by podocytes. The capillary endothelium, basement membrane, and foot processes of podocytes form the so-called filtration barrier. 2. The juxtaglomerular apparatus is one component of an important feedback mechanism (i.e., tubuloglomerular feedback) that regulates RBF and GFR. The structures that make up the juxtaglomerular apparatus include the macula densa, extraglomerular mesangial cells, and reninand angiotensin II–producing granular cells. 3. Clinically, GFR is frequently estimated using measures of plasma [creatinine] or by the renal clearance of creatinine. 4. Autoregulation allows GFR and RBF to remain constant despite changes in arterial blood

pressure between 90 and 180 mm Hg. When ECFV is altered, sympathetic nerves, catecholamines, angiotensin II, prostaglandins, NO, endothelin, natriuretic peptides, bradykinin, and adenosine exert substantial control over GFR and RBF. a

The organization of the nephron is actually more complicated than presented here. However, for simplicity and clarity of presentation in subsequent chapters, the nephron is divided into five segments. The collecting duct system is not actually part of the nephron. However, again for simplicity, we consider the collecting duct system part of the nephron. b

Although the renal corpuscle is composed of glomerular capillaries and Bowman’s capsule, the term glomerulus is commonly used to described the renal corpuscle. c

For most substances cleared from plasma by the kidneys, only a portion is actually removed and excreted in a single pass through the kidneys. d

The efferent arteriole is more sensitive than the afferent arteriole to angiotensin II. Therefore with low concentrations of angiotensin II, constriction of the efferent arteriole predominates, and GFR increases and RBF decreases. However, with high concentrations of angiotensin II, constriction of both afferent and efferent arterioles occurs, and GFR and RBF both decrease (see Fig. 33.21).

C H AP T E R 3 4

Solute and Water Transport Along the Nephron: Tubular Function LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. What three processes are involved in the production of urine? 2. What is the composition of “normal” urine? 3. What transport mechanisms are responsible for NaCl reabsorption by the nephron? Where are they located along the nephron? 4. How is water reabsorption “coupled” to NaCl reabsorption in the proximal tubule? 5. Why are solutes but not water reabsorbed by the thick ascending limb of Henle’s loop? 6. What transport mechanisms are involved in secretion of organic anions and cations? What is the physiological relevance of these transport processes? 7. What is glomerulotubular balance, and what is its physiological importance? 8. What are the major hormones that regulate NaCl and water reabsorption by the kidneys? What is the nephron site of action of each hormone? 9. What is the aldosterone paradox?

The formation of urine involves three basic processes: (1) ultrafiltration of plasma by the glomerulus, (2) reabsorption of water and solutes from the ultrafiltrate, and (3) secretion of selected solutes into tubular fluid. Although an average of 115 to 180 L/day in women and 130 to 200 L/day in men of essentially protein-free fluid is filtered by the human glomeruli each day,a less than 1% of the filtered water and sodium chloride (NaCl) and variable amounts of other solutes are typically excreted in urine (Table 34.1). By the processes of reabsorption and secretion, the renal tubules determine the volume and composition of urine (Table 34.2), which in turn allows the kidneys to precisely control the volume, osmolality, composition, and pH of the extracellular and intracellular fluid compartments. Transport proteins in cell membranes of the nephron mediate reabsorption and secretion of solutes and water in the kidneys. Approximately 5% to 10% of all human genes code for transport proteins, and genetic and acquired defects in transport proteins are the cause of many kidney diseases (Table 34.3). In addition, numerous transport proteins are important drug targets. This chapter discusses NaCl and water reabsorption, transport of organic anions and cations, the transport proteins involved in solute and water transport, and some of the factors and hormones that regulate NaCl transport. Details on acid-base transport and on K+, Ca++, and inorganic phosphate (Pi) transport and their regulation are provided in

Chapters 35 through 37. Table 34.1 Filtration, Excretion, and Reabsorption of Water, Electrolytes, and Solutes by the Kidneys

a

Substance

Measure

Filtereda

Excreted

Reabsorbed

% Filtered Load Reabsorbed

Water

L/day

180

1.5

178.5

99.2

Na+

mEq/day

25,200

150

25,050

99.4

K+

mEq/day

720

100

620

86.1

Ca++

mEq/day

540

10

530

98.2

HCO3−

mEq/day

4320

2

4318

99.9+

Cl−

mEq/day

18,000

150

17,850

99.2

Glucose

mmol/day

800

0

800

100.0

Urea

g/day

56

28

28

50.0

The filtered amount of any substance is calculated by multiplying the concentration of that substance in the ultrafiltrate by the glomerular filtration rate (GFR); for example, the filtered amount of Na+ is calculated as [Na+]ultrafiltrate (140 mEq/L) × GFR (180 L/day) = 25,200 mEq/day.

Table 34.2 Composition of Urine Substance

Concentration

Na+

50–130 mEq/L

K+

20–70 mEq/L

Ammonium (NH4+)

30–50 mEq/L

Ca++

5–12 mEq/L

Mg++

2–18 mEq/L

Cl−

50–130 mEq/L

Inorganic phosphate (Pi)

20–40 mEq/L

Urea

200–400 mmol/L

Creatinine

6–20 mmol/L

pH

5.0–7.0

Osmolality

500–800 mOsm/kg H2O

Glucose

0

Amino acids

0

Protein

0

Blood

0

Ketones

0

Leukocytes

0

Bilirubin

0

The composition and volume of urine can vary widely in the healthy state. These values represent average ranges. Normal water excretion typically ranges between 0.5 and 1.5 L/day. Data from Valtin HV. Renal Physiology. 2nd ed. Boston: Little, Brown; 1983.

Table 34.3 Selected Monogenic Renal Diseases Involving Transport Proteins Diseases

Mode of Gene Inheritance

Transport Protein

Nephron Segment

Cystinuria type I

AR

SLC3A1, SLC7A9

Amino acid symporters

Proximal tubule

Increased excreti basic amino a nephrolithias (kidney stone

Proximal renal tubular acidosis (RTA)

AR

SLC4A4

Na+/HCO3− symporter

Proximal tubule

Hyperchloremic metabolic aci

Phenotype

X-linked nephrolithiasis (Dent’s disease)

XLR

CLCN, OCRL1

Chloride channel

Distal tubule

Hypercalciuria, nephrolithias

Bartter syndrome

AR-type I

SLC12A1

Na+/K+/2Cl− symporter

TAL

Hypokalemia, metabolic alk hyperaldoste



AR-type II

KCNJ1

ROMK potassium channel

TAL

Hypokalemia, metabolic alk hyperaldoste



AR-type III

CLCNKB

Chloride channel (basolateral membrane)

TAL

Hypokalemia, metabolic alk hyperaldoste



AR-type IV

BSND, Subunit of chloride CLCNKA channel, chloride CLCNKB channels

TAL

Hypokalemia, metabolic alk hyperaldoste

Hypomagnesemiahypercalciuria syndrome

AR

CLDN16

Claudin-16, also known as paracellin 1

TAL

Hypomagnesemi hypercalciuri nephrolithias

Gitelman syndrome

AR

SLC12A3

Thiazide-sensitive Na+/Cl− symporter

Distal tubule

Hypomagnesemi hypokalemic metabolic alk hypocalciuria hypotension

Pseudohypoaldosteronism type I

AR

SCNN1A, α, β, and γ subunits SCNN1B, of ENaC and SCNN1G

Collecting duct

Increased excreti Na+, hyperka hypotension

Pseudohypoaldosteronism type II

AD

MLR

Collecting duct

Increased excreti Na+ hyperkal hypotension

Liddle syndrome

AD

SCNN1B, β and γ subunits of SCNN1G ENaC

Collecting duct

Decreased excre Na+, hyperte

Nephrogenic diabetes insipidus (NDI) type II

AR/AD

AQP2

Aquaporin 2 water channel

Collecting duct

Polyuria, polydip plasma hyperosmola

Distal renal tubular acidosis

AD/AR

SLC4A1

Cl−/HCO3− antiporter Collecting duct

Metabolic acidos hypokalemia hypercalciuri nephrolithias

Distal renal tubular acidosis

AR

ATP6N1B

Subunit of H+ATPase

Metabolic acidos hypokalemia hypercalciuri nephrolithias

Mineralocorticoid receptor

Collecting duct

There are over 300 different solute transporter genes that form the so-called SLC (solute carrier) family of genes. AD, Autosomal dominant; AR, autosomal recessive; ENaC, epithelial Na+ channel; TAL, thick ascending limb of Henle’s loop;

XLR, X-linked recessive. Modified from Nachman RH, Glassock RJ. NephSAP. 2010;9(3).

Solute and Water Reabsorption Along the Nephron The general principles of solute and water transport across epithelial cells were discussed in Chapter 2. Quantitatively, reabsorption of NaCl and water represent the major function of nephrons. Approximately 25,000 mEq/day of Na+ and 179 L/day of water are reabsorbed by the renal tubules (see Table 34.1). In addition, renal transport of many other important solutes is linked either directly or indirectly to reabsorption of Na+. In the following sections, the NaCl and water transport processes of each nephron segment and their regulation by hormones and other factors are presented.

Proximal Tubule The proximal tubule reabsorbs approximately 67% of water, Na+, Cl−, K+, and most other solutes filtered by the glomerulus. In addition the proximal tubule reabsorbs virtually all the glucose, proteins and amino acids filtered by the glomerulus, as well as most of the HCO3−. The key element in proximal tubule reabsorption is Na+,K+-ATPase in the basolateral membrane. Reabsorption of every substance, including water, is linked in some manner to the operation of Na+,K+-ATPase.

Na+ Reabsorption Na+ is reabsorbed by different mechanisms in the first and the second halves of the proximal tubule. In the first half of the proximal tubule, Na+ is reabsorbed primarily with bicarbonate (HCO3−) and a number of other solutes (e.g., glucose, amino acids, Pi, lactate). In contrast, in the second half, Na+ is reabsorbed mainly with Cl−. This disparity is mediated by differences in the Na+ transport systems in the first and second halves of the proximal tubule and by differences in the composition of tubular fluid at these sites. In absolute terms the first half of the proximal tubule reabsorbs significantly more Na+ than the second half. In the first half of the proximal tubule, Na+ uptake into the cell is coupled with either H+ or organic solutes, including glucose (Fig. 34.1). Specific transport proteins mediate entry of Na+ into the cell across the apical membrane. For example, the Na+/H+ antiporter, NHE3 (see Fig. 34.1A), couples entry of Na+ with extrusion of H+ from the cell. H+ secretion results in reabsorption of sodium bicarbonate (NaHCO3) (see Chapter 37). Na+ also enters proximal tubule cells via several symporter mechanisms, including Na+/glucose (SGLT2), Na+/amino acid, Na+/Pi, and Na+/lactate (see Fig. 34.1B). The glucose and other organic solutes that enter the cell with Na+ leave the cell across the basolateral membrane via passive transport mechanisms (e.g., GLUT2, a passive glucose transporter). Any Na+ that enters the cell across the apical membrane leaves the cell and enters the blood via Na+,K+-ATPase. Thus reabsorption of Na+ in the first half of the proximal tubule is coupled to that of HCO3− and a number of organic molecules, and this generates a negative transepithelial voltage across the proximal tubule that provides the driving force for the paracellular reabsorption of Cl−. Reabsorption of many organic molecules, including glucose and lactate, is so avid they are almost completely removed from the tubular fluid in the first half of the

proximal tubule (Fig. 34.2). Reabsorption of NaHCO3 and Na+–organic solutes across the proximal tubule establishes a transtubular osmotic gradient (i.e., the osmolality of the interstitial fluid bathing the basolateral side of the cells is a few mOsm/L higher than the osmolality of tubule fluid) that provides the driving force for the passive reabsorption of water by osmosis. Because more water than Cl− is reabsorbed in the first half of the proximal tubule, the [Cl−] in tubular fluid rises along the length of the proximal tubule (see Fig. 34.2).

FIG. 34.1 Na+ transport processes in the first half of the proximal tubule. These transport mechanisms are present in all cells in the first half of the proximal tubule but are separated into different cells to simplify the discussion. A, Operation of the Na+/H+ antiporter (NHE3) in the apical membrane and the Na+,K+ATPase and HCO3− transporters, including the Na+/HCO3− symporter (NBCe1; see also Chapter 37) in the basolateral membrane, mediates reabsorption of NaHCO3. Carbon dioxide and water combine inside the cells to form H+ and HCO3− in a reaction facilitated by the enzyme carbonic anhydrase (CA). B, Operation of the Na+/glucose symporter (SGLT2) in the apical membrane, in conjunction with Na+,K+ATPase and the glucose transporter (GLUT2) in the basolateral membrane, mediates Na+-glucose reabsorption. Inactivating mutations in the GLUT2 gene lead to decreased glucose reabsorption in the proximal tubule and glucosuria (i.e., glucose in the urine). Though not shown, Na+ reabsorption is also coupled with other solutes, including amino acids, Pi, and lactate. Reabsorption of these solutes is mediated by the Na+/amino acid, Na+/Pi, and Na+/lactate symporters, respectively, located in the apical membrane and the Na+,K+-ATPase, amino acid, Pi, and lactate transporters, respectively, located in the basolateral membrane. Three classes of amino acid transporters have been identified in the proximal tubule: two that transport Na+ in conjunction with either acidic or basic amino acids and one that does not require Na+ and transports basic amino acids.

FIG. 34.2 Concentration of solutes in tubule fluid as a function of length along the proximal tubule. [TF] is the concentration of the substance in tubular fluid; [P] is the concentration of the substance in plasma. Values above 100 indicate that relatively less of the solute than water is reabsorbed, and values below 100 indicate that relatively more of the substance than water is reabsorbed.

In the second half of the proximal tubule, Na+ reabsorption is largely accompanied by Cl− reabsorption via both transcellular and paracellular pathways (Fig. 34.3). Na+ is primarily reabsorbed with Cl− rather than organic solutes or HCO3− as the accompanying anion because the Na+ transport mechanisms in the second half of the proximal tubule differ from those in the first half, and because the tubular fluid that enters the second half contains very little glucose or amino acids. In addition the high [Cl−] (140 mEq/L) in tubule fluid, which is due to preferential reabsorption of Na+ with HCO3− and organic solutes in the

first half of the proximal tubule, facilitates reabsorption of Cl− with Na+.

FIG. 34.3 Na+ transport processes in the second half of the proximal tubule. Na+ and Cl− enter the cell across the apical membrane through the operation of parallel Na+/H+(NHE3) and Cl−-base (e.g., formate, oxalate, and bicarbonate) antiporters (SLC26A6). More than one Cl−-base antiporter is involved in this process, but only one is depicted. The secreted H+ and base combine in the tubular fluid to form an Hbase complex that can recycle across the plasma membrane. Accumulation of the H-base complex in tubular fluid establishes an H-base concentration gradient that favors H-base recycling across the apical plasma membrane into the cell. Inside the cell, H+ and the base dissociate and recycle back across the apical plasma membrane. The net result is uptake of NaCl across the apical membrane. The base may be hydroxide ions (OH−), formate (HCO2−), oxalate, HCO3−, or sulfate. The positive transepithelial voltage in the lumen, indicated by the plus sign inside the circle in the tubular lumen, is generated by diffusion of Cl − (lumen to blood) across the tight junction. The high [Cl−] of tubular fluid provides the driving force for diffusion of Cl−. Some glucose is also reabsorbed in the second half of the proximal tubule (not shown) by a mechanism similar to that described in the first half of the proximal tubule, except that the Na+/glucose symporter (SGLT1 gene) transports 2Na+ with one glucose and has higher affinity and lower capacity than the Na+/glucose symporter in the first part of the proximal tubule, depicted in Fig. 34.1. In addition, glucose exits the cell across the basolateral membrane via GLUT1 rather than via GLUT2 as in the first part of the proximal tubule (not shown). KCC, KCl symporter.

IN THE C LIN IC Fanconi syndrome, a renal disease that is either hereditary or acquired, results from an impaired ability of the proximal tubule to reabsorb HCO3−, Pi, amino acids, glucose, and low-molecular-weight proteins. Because other downstream nephron segments cannot reabsorb these solutes and protein, Fanconi syndrome results in increased urinary excretion of HCO3−, amino acids, glucose, Pi, and lowmolecular-weight proteins.

The mechanism of transcellular Na+ reabsorption in the second half of the proximal tubule is shown in Fig. 34.3. Na+ enters the cell across the luminal membrane primarily via the parallel operation of a Na+/H+ antiporter (NHE3) and one or more Cl−-base antiporters (e.g., SLC26A6). Because the secreted H+ and base combine in the tubular fluid and reenter the cell, operation of the Na+/H+ and Cl−-base antiporters is equivalent to uptake of NaCl from tubular fluid into the cell. Na+ leaves the cell via Na+,K+-ATPase, and Cl− leaves the cell and enters the blood via a K+/Cl− symporter (KCC) and a Cl− channel in the basolateral membrane. Some NaCl is also reabsorbed across the second half of the proximal tubule via a paracellular route. Paracellular NaCl reabsorption occurs because the rise in [Cl−] in tubule fluid in the first half of the proximal tubule creates a [Cl−] gradient (140 mEq/L in the tubule lumen and 105 mEq/L in the interstitium). This concentration gradient favors diffusion of Cl− from the tubular lumen across the tight junctions into the lateral intercellular space. Movement of the negatively charged Cl− results in the tubular fluid becoming positively charged relative to blood. This positive transepithelial voltage causes diffusion of positively charged Na+ out of the tubular fluid across the tight junction into blood. Thus in the second half of the proximal tubule, some Na+ and Cl− are reabsorbed across the tight junctions via passive diffusion. In summary, reabsorption of Na+ and Cl− in the proximal tubule occurs via both paracellular and transcellular pathways. Approximately 67% of the NaCl filtered each day by the glomerulus is reabsorbed in the proximal tubule. Of this amount, two-thirds move across the transcellular pathway, whereas the remaining one-third moves across the paracellular pathway (Table 34.4). Table 34.4 NaCl Transport Along the Nephron Segment

% Filtered NaCl Reabsorbed

Mechanism of Na+ Entry Across Apical Membrane

Proximal tubule

67

Na+/H+ antiporter (NHE3), Na+ symporter with amino acids and organic solutes, paracellular

Angiotensin II Norepinephrine Epinephrine Dopamine

Loop of Henle

25

1 Na+/1K+/2Cl− symporter

Aldosterone Angiotensin II

Distal tubule

≈5

NaCl symporter

Aldosterone Angiotensin II

Late distal tubule and collecting duct

≈3

ENaC

Aldosterone, ANP, BNP, urodilatin, uroguanylin, guanylin, angiotensin II

Major Regulatory Hormones

ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide, ENaC, epithelial Na+ channel.

Water Reabsorption The proximal tubule reabsorbs 67% of the filtered water (Table 34.5). The driving force for water

reabsorption is a transtubular osmotic gradient established by reabsorption of solute (e.g., NaCl, Na+glucose). Reabsorption of Na+ along with organic solutes, HCO3−, and Cl− from tubular fluid into the lateral intercellular spaces reduces the osmolality of the tubular fluid and increases the osmolality of the lateral intercellular space. The osmotic gradient across the proximal tubule established by these transport processes is only a few mOsm/L (Fig. 34.4). Because the proximal tubule is highly permeable to water, primarily owing to expression of aquaporin water channels (AQP1) in the apical and basolateral membranes, water is reabsorbed across cells by osmosis. In addition the tight junctions in the proximal tubule are also water permeable, so some water is also reabsorbed across the paracellular pathway between proximal tubular cells. Accumulation of fluid and solutes within the lateral intercellular space increases hydrostatic pressure in this compartment. The increased hydrostatic pressure forces fluid and solutes into the capillaries.b Thus water reabsorption follows solute reabsorption in the proximal tubule. The reabsorbed fluid is slightly hyperosmotic relative to plasma. However, this difference in osmolality is so small; it is commonly said that proximal tubule reabsorption is isosmotic (i.e., ≈67% of both the filtered solute and water are reabsorbed). Indeed, there is little difference in the osmolality of tubular fluid at the start and end of the proximal tubule. An important consequence of osmotic water flow across the proximal tubule is that some solutes, especially K+ and Ca++, are entrained in the reabsorbed fluid and thereby reabsorbed by the process of solvent drag (see Fig. 34.4). Reabsorption of virtually all organic solutes, Cl− and other ions, and water is coupled to Na+ reabsorption. Therefore changes in Na+ reabsorption influence reabsorption of water and other solutes by the proximal tubule. This point will be discussed later, notably in Chapter 35, and is especially relevant during volume depletion when increased Na+ reabsorption by the proximal tubule is accompanied by a parallel increase in HCO3− reabsorption, which can contribute to metabolic alkalosis (i.e., volume contraction alkalosis). Table 34.5 Water Transport Along the Nephron Segment

% Filtrate Reabsorbed

Mechanism of Water Reabsorption

Hormones That Regulate Water Permeability

Proximal tubule

67

Passive

None

Loop of Henle

15

Descending thin limb only; passive

None

Distal tubule

0

No water reabsorption

None

Late distal tubule and collecting duct

≈8–17

Passive

AVP, ANP a, BNP a

aAtrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) inhibit vasopressin (AVP)-stimulated water permeability.

FIG. 34.4 Routes of reabsorption of water and solute across the proximal tubule. Transport of solutes, including Na+, Cl−, and organic solutes, into the lateral intercellular space increases the osmolality of this compartment, which establishes the driving force for osmotic reabsorption of water across the proximal tubule. This occurs because some Na+,K+-ATPase and some transporters of organic solutes, HCO3−, and Cl− are located on the lateral cell membranes and deposit these solutes between cells. Furthermore, some NaCl also enters the lateral intercellular space via diffusion across the tight junction (i.e., paracellular pathway). An important consequence of osmotic water flow across the transcellular and paracellular pathways in the proximal tubule is that some solutes, especially K+ and Ca++, are entrained in the reabsorbed fluid and thereby reabsorbed by the process of solvent drag.

Protein Reabsorption Proteins filtered by the glomerulus are reabsorbed in the proximal tubule. As mentioned previously, peptide hormones, small proteins, and small amounts of larger proteins such as albumin are filtered by the glomerulus. Overall, only a small percentage of proteins cross the glomerulus and enter Bowman’s space (i.e., the concentration of proteins in the glomerular ultrafiltrate is only ≈ 40 mg/L). However, the total amount of protein filtered per day is significant because the glomerular filtration rate (GFR) is so high:

Equation 34.1

Filtered proteins are reabsorbed in the proximal tubule by endocytosis either as intact proteins or after being partially degraded by enzymes on the surface of proximal tubule cells. Once the proteins and

peptides are inside the cell, enzymes digest them into their constituent amino acids, which then leave the cell across the basolateral membrane by transport proteins and are returned to the blood. Normally this mechanism reabsorbs virtually all the proteins filtered, and hence the urine is essentially protein free. However, because the mechanism is easily saturated, an increase in filtered proteins can result in proteinuria (appearance of protein in urine). Disruption of the glomerular filtration barrier to proteins increases the filtration of proteins and results in proteinuria, which is frequently seen with kidney disease. Secretion of Organic Anions and Organic Cations Cells of the proximal tubule also secrete organic anions and organic cations into the tubule fluid. Secretion of organic anions and cations by the proximal tubule plays a key role in regulating the plasma levels of xenobiotics (e.g., a variety of antibiotics, diuretics, statins, antivirals, antineoplastics, immunosuppressants, neurotransmitters, and nonsteroidal anti-inflammatory drugs [NSAIDs]) and toxic compounds derived from endogenous and exogenous sources. Many of the organic anions and cations (Boxes 34.1 and 34.2) secreted by the proximal tubule are end products of metabolism that circulate in plasma. Many of these organic compounds are bound to plasma proteins and thus are not readily filtered. Therefore only a small fraction of these potentially toxic substances are eliminated from the body by excretion resulting from filtration alone. Thus secretion of organic anions and cations, including many toxins from the peritubular capillary into the tubular fluid, promotes elimination of these compounds from plasma entering the kidneys. Hence these substances are removed from plasma by both filtration and secretion. It is important to note that when kidney function is reduced by disease, urinary excretion of organic anions and cations is severely reduced, which can lead to increased plasma levels of xenobiotics and potentially toxic accumulation of organic anions and cations. Box 34.1

So me Org a nic Anio ns Se c re t e d by P ro xima l Tubule Endogenous Anions cAMP, cGMP Bile salts Hippurates Oxalate Prostaglandins: PGE2, PGF2α Urate Vitamins: ascorbate, folate Drugs Acetazolamide Acyclovir Amoxicillin Captopril

Chlorothiazide Furosemide Losartan Penicillin Probenecid Salicylate (aspirin) Hydrochlorothiazide Simvastatin Bumetanide Nonsteroidal anti-inflammatory drugs (NSAIDs): indomethacin cAMP, Cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate.

Box 34.2

So me Org a nic C a t io ns Se c re t e d by P ro xima l Tubule Endogenous Creatinine Dopamine Epinephrine Norepinephrine Drugs Atropine Isoproterenol Cimetidine Morphine Quinine Amiloride Procainamide

AT THE C ELLU LAR LEVEL Water channels called aquaporins (AQPs) mediate transcellular reabsorption of water across many nephron segments. To date, 13 aquaporins have been identified. The AQP family is divided into two groups based on their permeability characteristics. One group (aquaporins) is permeable to water (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP8, AQP11, and AQP12). The other group (aquaglyceroporins) is permeable to water and small solutes, especially glycerol (AQP3, AQP7,

AQP9, AQP10). Aquaporins form tetramers in the plasma membrane of cells, with each subunit forming a water channel. In the kidneys, AQP1 is expressed in the apical and basolateral membranes of the proximal tubule and in portions of the descending thin limb of Henle’s loop. The importance of AQP1 in renal water reabsorption is underscored by studies in which the AQP1 gene was “knocked out” in mice. These mice exhibit increased urine output (polyuria) and reduced ability to concentrate urine. In addition the osmotic water permeability of the proximal tubule is fivefold less in mice lacking APQ1 than in normal mice. AQP7 and AQP8 are also expressed in the proximal tubule. AQP2 is expressed in the apical plasma membrane of principal cells in the collecting duct, and its abundance in the membrane is regulated by arginine vasopressin (AVP) (see Chapter 35). AQP3 and AQP4 are expressed in the basolateral membrane of principal cells in the collecting duct, and mice deficient in these AQPs (i.e., AQP3 and AQP4 knockout mice) have defects in the ability to concentrate urine (see Chapter 35).

AT THE C ELLU LAR LEVEL The endocytosis of proteins by the proximal tubule is mediated by apical membrane proteins that specifically bind proteins and peptides in tubule fluid. These receptors, called multiligand endocytic receptors, can bind a wide range of peptides and proteins and thereby mediate their endocytosis. Megalin and cubilin mediate protein and peptide endocytosis in the proximal tubule. Both are glycoproteins, with megalin being a member of the low-density lipoprotein receptor gene family.

IN THE C LIN IC Urinalysis is an important and routine tool for detection of kidney disease. A thorough analysis of urine includes macroscopic, microscopic, and biochemical assessments. This is performed by visual assessment of the urine, microscopic examination of urinary sediment, and biochemical evaluation of urinary composition using dipstick reagent strips. The dipstick test is both inexpensive and fast (i.e., 4 hours) secondary increase in NaCl and K+ transport by the collecting duct. Activating polymorphisms in Sgk1 cause an increase in blood pressure, presumably by enhancing NaCl reabsorption by the collecting duct, which increases the ECFV and thereby blood pressure.

IN THE C LIN IC Liddle syndrome is a rare genetic disorder characterized by an increase in blood pressure (i.e., hypertension) secondary to an increase in ECFV. Liddle syndrome is caused by activating mutations in either the β or γ subunit of the epithelial Na+ channel (ENaC). These mutations increase the number of Na+ channels in the apical cell membrane of principal cells and thereby the amount of Na+ reabsorbed. In Liddle syndrome, the rate of renal Na+ reabsorption is inappropriately high, which leads to an increase in ECFV and hypertension. There are two different forms of pseudohypoaldosteronism (PHA) (i.e., the kidneys reabsorb NaCl as they do when aldosterone levels are low; however, in PHA, aldosterone levels are elevated). The autosomal recessive form is caused by inactivating mutations in the α, β, or γ subunit of ENaC. The cause of the autosomal dominant form is an inactivating mutation in the mineralocorticoid receptor. Pseudohypoaldosteronism is characterized by an increase in Na+ excretion, a reduction in ECFV, hyperkalemia, and hypotension. Some individuals with expanded ECFV and elevated blood pressure are treated with drugs that inhibit angiotensin-converting enzyme (ACE) (e.g., Captopril, Enalapril, Lisinopril) and thereby lower fluid volume and blood pressure. Inhibition of ACE blocks degradation of angiotensin I to angiotensin II and thereby lowers plasma angiotensin II levels. The decline in plasma angiotensin II concentration has three effects. First, NaCl and water reabsorption by the nephron (especially the proximal tubule) falls. Second, aldosterone secretion decreases, thus reducing NaCl reabsorption in the thick ascending limb, distal tubule, and collecting duct. Third, because angiotensin is a potent vasoconstrictor, a reduction in its concentration permits the systemic arterioles to dilate and thereby lower arterial blood pressure. ACE also degrades the vasodilator hormone bradykinin; thus ACE inhibitors increase the concentration of bradykinin, a vasodilatory hormone. ACE inhibitors decrease ECFV and the arterial blood pressure by promoting renal NaCl and water excretion and by reducing total peripheral resistance. Uroguanylin and guanylin are produced by neuroendocrine cells in the intestine in response to oral ingestion of NaCl. These hormones enter the circulation and inhibit NaCl and water reabsorption by the kidneys via activation of membrane-bound guanylyl cyclase receptors, which increase intracellular [cGMP]. The involvement of these gut-derived hormones helps explain why the natriuretic response of the kidneys to an oral NaCl load is more pronounced than when delivered intravenously. Catecholamines stimulate reabsorption of NaCl. Catecholamines released from the sympathetic nerves (norepinephrine) and the adrenal medulla (epinephrine) stimulate reabsorption of NaCl and water by the proximal tubule, thick ascending limb of the loop of Henle, distal tubule, and collecting duct. Although

sympathetic nerves are not active when ECFV is normal, when ECFV declines (e.g., after hemorrhage), sympathetic nerve activity rises and dramatically stimulates reabsorption of NaCl and water by these four nephron segments. Dopamine, a catecholamine, is released from dopaminergic nerves in the kidneys and is also synthesized by cells of the proximal tubule. The action of dopamine is opposite to that of norepinephrine and epinephrine. Secretion of dopamine is stimulated by an increase in ECFV, and its secretion directly inhibits reabsorption of NaCl and water in the proximal tubule. Adrenomedullin is a 52–amino acid peptide hormone that is produced by a variety of organs, including the kidneys. Adrenomedullin induces a marked diuresis and natriuresis, and its secretion is stimulated by congestive heart failure and hypertension. The major effect of adrenomedullin on the kidneys is to increase GFR and RBF and thereby indirectly stimulate excretion of NaCl and water (see earlier discussion about ANP and BNP). Arginine vasopressin (AVP) is the most important hormone that regulates the reabsorption of water in the kidneys (see Chapter 35). This hormone is secreted by the posterior pituitary gland in response to an increase in plasma osmolality (1% or more) or a decrease in ECFV (>5%–10% from steady-state). AVP increases the permeability of the collecting duct to water. It increases reabsorption of water by the collecting duct because of the osmotic gradient that exists across the wall of the collecting duct (see Chapter 35). AVP has little effect on urinary NaCl excretion. Starling forces regulate reabsorption of NaCl and water across the proximal tubule. As previously described, Na+, Cl−, HCO3−, amino acids, glucose, and water are transported into the intercellular space of the proximal tubule. Starling forces between this space and the peritubular capillaries facilitate movement of the reabsorbed fluid into the capillaries. Starling forces across the wall of peritubular capillaries consist of hydrostatic pressure in the peritubular capillary (Ppc) and lateral intercellular space (Pi) and oncotic pressure in the peritubular capillary (πpc) and lateral intercellular space (πi). Thus reabsorption of water as a result of transport of Na+ from tubular fluid into the lateral intercellular space is modified by the Starling forces. Accordingly:

Equation 34.2 where J is flow (positive numbers indicate flow from the intercellular space into blood). Starling forces that favor movement from the interstitium into the peritubular capillaries are πpc and Pi (Fig. 34.10). The opposing Starling forces are πi and Ppc. Normally the sum of the Starling forces favors movement of solute and water from the interstitial space into the capillary. However, some of the solutes and fluid that enter the lateral intercellular space leak back into the proximal tubular fluid. Starling forces do not affect transport by the loop of Henle, distal tubule, and collecting duct because these segments are less permeable to water than the proximal tubule.

FIG. 34.10 Starling forces modify proximal tubule solute and water reabsorption. (1) Solute and water are reabsorbed across the apical membrane. This solute and water then cross the lateral cell membrane. Some solute and water reenters the tubule fluid (3), and the remainder enters the interstitial space and then flows into the capillary (2). The width of the arrows is directly proportional to the amount of solute and water moving by pathways 1 to 3. Starling forces across the capillary wall determine the amount of fluid flowing through pathway 2 versus pathway 3. Transport mechanisms in the apical cell membranes determine the amount of solute and water entering the cell (pathway 1). Pi, Interstitial hydrostatic pressure; Ppc, peritubular capillary hydrostatic pressure; πi, interstitial fluid oncotic pressure; πpc, peritubular capillary oncotic pressure. Thin arrows across the capillary wall indicate the direction of water movement in response to each force.

A number of factors can alter the Starling forces across the peritubular capillaries surrounding the proximal tubule. For example, dilation of the efferent arteriole increases Ppc, whereas constriction of the efferent arteriole decreases it. An increase in Ppc inhibits solute and water reabsorption by increasing back-leak of NaCl and water across the tight junction, whereas a decrease stimulates reabsorption by decreasing back-leak across the tight junction. Peritubular capillary oncotic pressure (πpc) is partially determined by the rate of formation of the glomerular ultrafiltrate. For example, if one assumes a constant plasma flow in the afferent arteriole, the plasma proteins become less concentrated in the plasma that enters the efferent arteriole and peritubular capillary as less ultrafiltrate is formed (i.e., as GFR decreases). Hence, πpc decreases. Thus πpc is directly related to the filtration fraction (FF = GFR/renal plasma flow [RPF]). A fall in the FF resulting from a decrease in GFR, at constant RPF, decreases πpc. This in turn increases the backflow of NaCl and water from the lateral intercellular space into tubular fluid and thereby decreases net reabsorption of solute and water across the proximal tubule. An increase in FF has the opposite effect. The importance of Starling forces in regulating solute and water reabsorption by the proximal tubule is underscored by the phenomenon of glomerulotubular (G-T) balance. Spontaneous changes in GFR markedly alter the filtered amount of Na+ (filtered Na+ = GFR × [Na+] in the filtered fluid). Without rapid adjustments in Na+ reabsorption to counter the changes in filtration of Na+, urinary excretion of Na+

would fluctuate widely and disturb the Na+ balance of the body and thus alter ECFV and blood pressure (see Chapter 35 for more details). However, spontaneous changes in GFR do not alter Na+ excretion in urine or Na+ balance when ECFV is normal because of the phenomenon of G-T balance. When body Na+ balance is normal (i.e., ECFV is normal), G-T balance refers to the fact that reabsorption of Na+ and water increases in proportion to the increase in GFR and filtered amount of Na+. Thus a constant fraction of the filtered Na+ and water is reabsorbed from the proximal tubule despite variations in GFR. The net result of G-T balance is to reduce the impact of changes in GFR on the amount of Na+ and water excreted in urine when ECFV is normal. Two mechanisms are responsible for G-T balance. One is related to the oncotic and hydrostatic pressure differences between the peritubular capillaries and the lateral intercellular space (i.e., Starling forces). For example, an increase in the GFR (at constant RPF) raises the protein concentration in glomerular capillary plasma above normal. This protein-rich plasma leaves the glomerular capillaries, flows through the efferent arterioles, and enters the peritubular capillaries. The increased πpc augments the movement of solute and fluid from the lateral intercellular space into the peritubular capillaries. This action increases net solute and water reabsorption by the proximal tubule. The second mechanism responsible for G-T balance is initiated by an increase in the filtered amount of glucose and amino acids. As discussed earlier, reabsorption of Na+ in the first half of the proximal tubule is coupled to that of glucose and amino acids. The rate of Na+ reabsorption therefore partially depends on the filtered amount of glucose and amino acids. As the GFR and filtered amount of glucose and amino acids increase, reabsorption of Na+ and water also rises. In addition to G-T balance, another mechanism minimizes changes in the filtered amount of Na+. As discussed in Chapter 33, an increase in GFR (and thus in the amount of Na+ filtered by the glomerulus) activates the tubuloglomerular feedback mechanism. This action returns the GFR and filtration of Na+ to normal values. Thus spontaneous changes in GFR (e.g., caused by changes in posture and blood pressure) increase the amount of Na+ filtered for only a few minutes. The mechanisms that underlie G-T balance maintain urinary Na+ excretion constant and thereby maintain Na+ homeostasis (and ECFV and blood pressure) until the GFR returns to normal.

Key Points 1. The four major segments of the nephron (proximal tubule, Henle’s loop, distal tubule, and collecting duct) determine the composition and volume of urine by the processes of selective reabsorption of solutes and water and secretion of some solutes. 2. Tubular reabsorption of substances filtered by the glomerulus allows the kidneys to retain substances that are essential and regulate their levels in plasma by altering the degree to which they are reabsorbed. Reabsorption of Na+, Cl−, other anions, and organic anions and cations together with water constitutes the major function of the nephron. Approximately 25,200 mEq of Na+ and 179 L of water are reabsorbed each day. Proximal tubule cells reabsorb 67% of the glomerular ultrafiltrate, and cells of Henle’s loop reabsorb about 25% of the NaCl that was filtered and about 15% of the water that was filtered. The distal segments of the nephron (distal tubule and collecting duct system) have a more limited reabsorptive capacity. However, although the proximal tubule reabsorbs the largest fraction of the filtered solutes and water (i.e., 67%), final adjustments in the composition and volume of urine and most of the regulation by hormones

and other factors occur primarily in the distal tubule and collecting duct. 3. Secretion of substances from the blood into tubular fluid is a means for excreting various byproducts of metabolism, and it also serves to eliminate exogenous organic anions and cations (e.g., drugs) and toxins from the body. Many organic anions and cations are bound to plasma proteins and are therefore unavailable for filtration. Thus secretion is their major route of excretion in urine. 4. Various hormones (including angiotensin II, aldosterone, AVP, ANP, BNP, urodilatin, uroguanylin, guanylin, and dopamine), sympathetic nerves, and Starling forces regulate reabsorption of NaCl by the kidneys. AVP is the major hormone that regulates water reabsorption. a

Normal glomerular filtration rate (GFR) averages 115 to 180 L/day in women and 130 to 200 L/day in men. Thus the volume of the ultrafiltrate represents a volume that is approximately 10 times that of the extracellular fluid volume (ECFV). For simplicity, we assume throughout the remainder of this section that GFR is 180 L/day. b

In addition, protein oncotic pressure in the peritubular capillaries (πpc) is elevated because of the process of glomerular filtration (see Chapter 33). The elevated πpc facilitates uptake of fluid and solute into the capillary.

C H AP T E R 3 5

Control of Body Fluid Osmolality and Volume LEARNING OBJECTIVES Upon completion of this chapter the student should be able to answer the following questions: 1. Why do changes in water balance alter [Na+] of extracellular fluid (ECF)? 2. How is the secretion of arginine vasopressin (AVP) controlled by changes in the body fluid osmolality, blood volume, and systemic blood pressure? 3. What cellular events are associated with the AVP action on the collecting duct and how do they increase the water permeability of this nephron segment? 4. What is the role of Henle’s loop in the production of both dilute and concentrated urine? 5. What is the composition of the medullary interstitial fluid, and how does it contribute to urinary concentration? 6. What are the roles of vasa recta in the process of diluting and concentrating urine? 7. How is the diluting and concentrating ability of the kidneys quantitated? 8. Why do changes in Na+ balance alter the volume of extracellular fluid? 9. What is the effective circulating volume, how is it influenced by changes in Na+ balance, and how does it influence renal Na+ excretion? 10. What mechanisms regulate the effective circulating volume? 11. What are the major signals for altering renal Na+ excretion? 12. How do changes in extracellular fluid volume alter Na+ transport in the different nephron segments and how these changes regulate renal Na+ excretion? 13. What mechanisms contribute to edema formation and what roles do the kidneys play in this process?

Body fluid osmolality represents one of the most highly regulated parameters of human physiology. The kidney controls the osmolality and volume of body fluid by regulating excretion of water and NaCl, respectively. This chapter discusses the renal mechanisms of water and NaCl excretion. The composition and volumes of the various body fluid compartments are reviewed in Chapter 2.

Control of Body Fluid Osmolality: Urine Concentration and Dilution As described in Chapter 2, water constitutes approximately 60% of the healthy adult human body. Water

is distributed between two major compartments in the body—the intracellular fluid (ICF) and extracellular fluid (ECF)—that exist in osmotic equilibrium because aquaporins (e.g., AQP1) make the cellular membranes permeable to water. The major source of body water is oral intake of liquids and solid foods containing a liquid component. Water is also generated during metabolism of ingested foods (e.g., carbohydrates). Intravenous fluids are an important route of water supply during disease states. The kidneys are responsible for regulating water balance and under most conditions are the major route for eliminating water from the body (Table 35.1). Water is also lost through the gastrointestinal tract. Fecal water loss is normally small (≈100 mL/day) but can increase dramatically with diarrhea (e.g., 20 L/day with cholera). Vomiting can also cause gastrointestinal water losses. The production of sweat is an active process of water and electrolyte elimination. The water loss through kidneys, gastrointestinal tract, and sweat glands is termed sensible water loss because the person is aware of its occurrence. Other routes of water elimination from the body are evaporation from cells of the skin and respiratory passages; collectively, the loss is termed insensible water loss because the individual is unaware of its occurrence. Sweating and insensible water loss can increase dramatically in a hot environment, during exercise, or in the presence of fever (Table 35.2). Table 35.1 Sources of Water Gain and Loss in Adults at Room Temperature (23°C) Gain

(mL/day)

Fluida

1200

Food

1000

Metabolically produced from food

300

Total

2500

Loss (mL/day)



Insensible

700

Sweat

100

Feces

100

Urine

1600

Total

2500

aFluid intake may vary widely for social and cultural reasons.

Table 35.2 Effect of Environmental Temperature and Exercise on Water Loss and Intake in Adults

Normal Temperature

Hot Weathera

Prolonged Heavy Exercisea

Water Loss







Insensible loss







Skin

350

350

350

Lungs

350

250

650

Sweat

100

1400

5000

Feces

100

100

100

Urinea

1600

1200

500

Total loss

2500

3300

6600

a

In hot weather and during prolonged heavy exercise, water balance is maintained by increased water ingestion. Decreased excretion of water by the kidneys alone is insufficient to maintain water balance.

Although water loss from sweating, defecation, and evaporation from the lungs and skin can vary depending on the environmental conditions or during disease states, loss of water by these routes cannot be regulated. In contrast, renal excretion of water is tightly regulated to maintain water balance. Maintenance of water balance requires that water intake and loss are precisely matched. If intake exceeds loss, positive water balance exists. Conversely, when intake is lower than loss, negative water balance exists (see Chapter 2 for review of steady-state balance). During states of decreased water intake or excessive water losses, the kidneys conserve water by producing low-volume, concentrated urine that is hyperosmotic with respect to plasma. Conversely, when water intake is high, a large volume of hypoosmotic urine is produced. In a healthy individual, urine osmolality (Uosm) can vary from approximately 50 to 1200 mOsm/kg H2O, and the corresponding urine volume can vary from approximately 18 L/day to 0.5 L/day. Importantly the kidneys can regulate excretion of water separately from excretion of total solute (e.g., Na+, K+, urea, etc.) (Fig. 35.1). The ability to regulate water excretion separate from excretion of solutes is essential for survival because it allows water balance to be achieved without upsetting other homeostatic functions of the kidneys.

FIG. 35.1 Relationships between plasma AVP levels, and urine osmolality, urine flow rate, and total solute excretion. Max, Maximum; Min, minimum. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

It is important to recognize that disorders of water balance are manifested by alterations in body fluid osmolality, which are usually measured by changes in plasma osmolality (Posm). Because the major determinant of plasma osmolality is Na+ (with its anions Cl− and HCO3−), these disorders also result in alterations in plasma or serum [Na+] (Fig. 35.2). One of the most common fluid and electrolyte disorders seen in clinical practice is an alteration in serum [Na+]. When an abnormal serum [Na+] is found in an individual, it is tempting to suspect a problem in Na+ balance. However, the problem most often relates to water balance, not Na+ balance. As described later, changes in Na+ balance result in alterations in the ECF volume (ECFV), not its osmolality.

FIG. 35.2 Response to changes in water balance. Illustrated are the effects of adding or removing 1 L of water from the ECF of a 70-kg individual. Positive Water Balance:(1) Addition of 1 L of water increases the ECFV and reduces its osmolality. The [Na+] is also decreased (hyponatremia). (2) The normal renal response is to excrete 1 L of water as hypoosmotic urine. (3) As a result of the renal excretion of water, the ECFV, osmolality, and [Na+] are returned to normal. Negative Water Balance:(4) The loss of 1 L of water from the ECF decreases its volume and increases its osmolality. The [Na+] is also increased (hypernatremia). (5) The renal response is to conserve water by excreting a small volume of hyperosmotic urine. (6) With ingestion of water, stimulated by thirst, and conservation of water by the kidneys, the ECF volume, osmolality, and [Na+] are returned to normal. Size of the boxes indicates relative ECF volume. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

IN THE C LIN IC In the clinical setting, hypoosmolality (a reduction in plasma osmolality) shifts water into cells, and this process results in cell swelling (see Chapter 2). Symptoms associated with hypoosmolality are related primarily to swelling of brain cells. For example, a rapid fall in Posm can alter neurological function and thereby cause nausea, malaise, headache, confusion, lethargy, seizures, and coma. When Posm is increased (i.e., hyperosmolality), water is lost from cells. Symptoms of an increase in Posm are also primarily neurological and include lethargy, weakness, seizures, coma, and even death. Symptoms associated with changes in body fluid osmolality vary depending on how quickly osmolality changes. The rapid osmolality changes (i.e., over hours) are less well tolerated than changes that occur more gradually (i.e., over days to weeks). Indeed, individuals who have developed alterations in their body fluid osmolality over an extended period of time may be entirely asymptomatic. This reflects the compensatory mechanisms in neurons to minimize changes in cell volume over time. For example, cells eliminate intracellular osmoles in response to hypoosmolality while they generate new intracellular osmoles in response to hyperosmolality (see Chapter 2). The following sections discuss the mechanisms by which the kidneys excrete either hypoosmotic (dilute) or hyperosmotic (concentrated) urine. Control of arginine vasopressin secretion and its essential role in regulating water excretion by the kidneys are also explained (see also Chapter 41).

Arginine Vasopressin Arginine vasopressin (AVP), a nonapeptide, is synthesized in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei.a AVP acts though vasopressin (V) receptors. Several nephron segments express the type-2 receptor (V2) that mediates the kidneys’ ability to regulate the urine volume and osmolality. When the plasma AVP level is low, a large volume of urine is excreted (diuretic effect), and the urine osmolality is lower than that of plasma (i.e., dilute). When the plasma AVP level is high, a small volume of urine is excreted (antidiuretic effect), and the urine osmolality is greater than that of plasma (i.e., concentrated). Hence, AVP is also known as the antidiuretic hormone (ADH).

FIG. 35.3 Anatomy of the hypothalamus and pituitary gland (midsagittal section) depicting the pathways for AVP section. Also shown are pathways involved in regulating AVP secretion. Afferent fibers from the baroreceptors are carried in the vagus and glossopharyngeal nerves. The inset box illustrates an expanded view of the hypothalamus and pituitary gland.

AT THE C ELLU LAR LEVEL The gene for AVP is located on chromosome 20. It contains approximately 2000 base pairs with three exons and two introns. The gene codes for a preprohormone that consists of a signal polypeptide, the AVP molecule, neurophysin, and a glycopeptide (copeptin). As the cell processes the preprohormone the signal peptide is cleaved off in the rough endoplasmic reticulum. Once packaged in neurosecretory granules, the preprohormone is further cleaved into AVP, neurophysin, and copeptin molecules. The neurosecretory granules are then transported down the axon to the posterior pituitary and stored in the nerve endings until released. When the neurons are stimulated to secrete AVP, the action potential opens Ca++ channels in the nerve terminal, which raises the intracellular [Ca++] and causes exocytosis of the neurosecretory granules. All three peptides are secreted in this process. Neurophysin and copeptin do not have an identified physiological function.

Secretion of AVP by the posterior pituitary can be influenced by several factors. Under physiological conditions AVP secretion is controlled by two major mechanisms: osmotic (changes in plasma osmolality) and hemodynamic (changes in blood pressure or volume). Other factors that alter AVP secretion include nausea, acute hypoglycemia, angiotensin II (stimulate), and atrial natriuretic peptide (inhibits). A number of medications and recreational drugs affect AVP secretion. For example, nicotine stimulates secretion, whereas ethanol and anti-emetics inhibit it. Osmotic Control of AVP Secretion Changes in the body fluid osmolality play the most important role in regulating AVP secretion. Specialized neurons termed osmoreceptors, located in the organum vasculosum of the lamina terminalis (OVLT) of the hypothalamus, modulate osmolality changes within a normal range between 275 and 295 mOsm/kg H2O. The osmoreceptor cells sense changes in body fluid osmolality in response to small changes in the concentration of effective solutes, such as Na+ and its anions, and are insensitive to ineffective solutes, such as urea and glucose (see Chapter 1). Effective solutes are those that penetrate cells slowly or not at all, thereby creating an osmotic gradient resulting in efflux of water across the cell membrane. When the effective plasma osmolality (tonicity) increases, osmoreceptor cell shrinkage activates membrane nonselective cationic channels that generate inward current depolarizing the cells. In turn, the osmotically evoked action potential in the OVLT neurons synaptically propagates the electrical activity to downstream effector neurons in the SON and PVN leading to AVP release. Conversely, when the effective plasma osmolality decreases, AVP synthesis and secretion are inhibited. Because AVP is rapidly degraded in the plasma, circulating levels can be reduced to zero within minutes. Recent data demonstrate that cell membrane stretch rather than cell volume determines osmoreceptor activity. The transient receptor potential vanilloid (TRPV) family of cation channels, including TRPV1, TRPV2, and TRPV4, mediate osmotic stimuli in mammals. The channels are activated by cell membrane stretch and mediate inactivation of the osmoreceptors in hypoosmolal states. The mediators of stretch-inactivated cationic channels that respond to cell shrinkage in hyperosmolal states are currently unknown. The coordinated action of the stimulatory and inhibitor components of the osmoreceptors creates a threshold, or set point. Fig. 35.4A illustrates the effect of changes in plasma osmolality on circulating AVP levels. The slope of the relationship is quite steep and accounts for the sensitivity of the system. The set point is the plasma osmolality value at which AVP secretion begins to increase. Below the set point, virtually no AVP is released. The absolute level of the effective plasma osmolality at which minimally and maximally effective levels of plasma AVP occur, varies appreciably from person to person, due to genetic influences on the set and sensitivity of the system. However, the average set point for AVP release corresponds to a plasma osmolality of 280 mOsmol/kg H2O and levels only 2% to 4% higher normally result in maximum antidiuretic effect. The set point is relatively stable in healthy individuals but can be decreased by pregnancy, menstrual cycle, estrogen, or a significant drop in blood pressure or blood loss. The mechanism responsible for the set point shift during pregnancy is likely due to increased levels of certain hormones (e.g., relaxin and chorionic gonadotropin).

FIG. 35.4 Osmotic and hemodynamic (nonosmotic) control of AVP secretion. A, Effect of changes in plasma osmolality (constant blood volume and pressure) on plasma AVP levels. B, Effect of changes in blood volume or pressure (constant plasma osmolality) on plasma AVP levels. C, Interactions between osmolar and blood volume and pressure stimuli on plasma AVP levels.

Hemodynamic (Nonosmotic) Control of AVP Secretion

A decrease in blood volume or pressure also stimulates AVP secretion. The receptors responsible for this response are located in both the low-pressure (left atrium and large pulmonary vessels) and the highpressure (aortic arch and carotid sinus) sides of the circulatory system. Because the low-pressure receptors are located in the high-compliance side of the circulatory system (i.e., venous), and because the majority of blood is on the venous side, these low-pressure receptors can be viewed as responding to the overall vascular volume. The high-pressure receptors respond to arterial pressure. Both groups of receptors are sensitive to stretch of the wall of the structure in which they are located (e.g., cardiac atrial and aortic arch) and are termed baroreceptors. Signals from these receptors are carried in afferent fibers of the vagus and glossopharyngeal nerves to the brainstem (solitary tract nucleus of the medulla oblongata), which is part of the center that regulates heart rate and blood pressure (see also Chapter 18). Signals are then relayed from the brainstem to the AVP secretory cells of the supraoptic and paraventricular hypothalamic nuclei. The sensitivity of the baroreceptor system is less than that of the central osmoreceptors, and a 5% to 10% decrease in blood volume or pressure is required before AVP secretion is stimulated. This is illustrated in Fig. 35.4B. A number of substances have been shown to alter the secretion of AVP through their effects on blood pressure. These include bradykinin and histamine, which lower pressure and thus stimulate AVP secretion, and norepinephrine, which increases blood pressure and inhibits AVP secretion. Alterations in blood volume and pressure also affect the response to changes in body fluid osmolality (see Fig. 35.4C). With a decrease in blood volume or pressure, the set point is shifted to lower osmolality values and the slope of the relationship is steeper. In terms of survival of the individual this means that faced with circulatory collapse, the kidneys will continue to conserve water, even though by doing so they reduce the osmolality of the body fluids. With an increase in blood volume or pressure, the opposite occurs. The set point is shifted to higher osmolality values and the slope is decreased.

IN THE C LIN IC Inadequate release of AVP from the posterior pituitary results in excretion of a large volume of dilute urine (polyuria). To compensate for this loss of water the individual must ingest a large volume of water (polydipsia) to maintain constant body fluid osmolality. If the individual is deprived of water, body fluid will become hyperosmotic. This condition is called central (pituitary) diabetes insipidus (CDI). It can be inherited, although this is rare. CDI occurs more commonly after head trauma and with brain neoplasms or infections. Individuals with CDI have a urine-concentrating defect that can be corrected by administration of exogenous AVP. The inherited (autosomal dominant) form of CDI is due to mutations in different regions of the AVP gene (i.e., AVP, copeptin, and neurophysin). The human placenta produces a cysteine aminopeptidase that degrades AVP. In some women the levels of this vasopressinase result in diabetes insipidus. The associated polyuria can be treated by administration of the synthetic AVP analog desmopressin (DDAVP). The syndrome of inappropriate AVP (ADH) secretion (SIADH) is a relatively common clinical problem characterized by plasma AVP levels that are elevated above what would be expected on the basis of body fluid osmolality, blood volume, or blood pressure—hence the term inappropriate AVP (ADH) secretion. The AVP action in the kidney collecting duct causes recruitment of water channels (see below), thus augmenting the effect of AVP to stimulate renal water retention. Individuals with SIADH retain water, and their body fluids become progressively hypoosmotic. In addition, their urine is more hyperosmotic than expected based on the low body-fluid osmolality. SIADH can be caused by drugs, central nervous system infection or tumors, pulmonary diseases, or lung carcinoma. These conditions either stimulate AVP secretion by altering neural input to the AVP secretory cells or secrete

AVP (small cell carcinoma). Drug-related SIADH is increasingly common and can be associated with many classes of over the counter and prescription medications, including proton pump inhibitors, nonsteroidal anti-inflammatory, antidepressants, antiseizure, antipsychotic, and antitumor drugs. AVP receptor antagonists bind to V1A and V2 receptors and induce water diuresis (aquaresis) to treat SIADH and other conditions resulting from AVP-dependent water retention by the kidneys (e.g., congestive heart failure and hepatic cirrhosis). AVP Actions on the Kidneys The primary action of AVP on the kidneys is to enhance absorption of water from the tubular fluid by increasing the water permeability of the latter portion of the distal tubule and collecting duct. In addition, and importantly, AVP increases the permeability of the medullary portion of the collecting duct to urea. Finally, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop, distal tubule, and collecting duct. In the absence of AVP, the apical membrane of principal cells (see Chapter 34), located in the latter portion of the distal tubule and along the collecting duct, is relatively impermeable to water. This reflects the fact that in the absence of AVP the apical membrane of these cells contains few water channels (aquaporins), since they are stored inside cells. Thus, in the absence of AVP, little water is reabsorbed by these nephron segments. Binding of AVP to the V2 receptor located in the basolateral membrane of principal cells results in the recruitment of aquaporin (AQP2) water channels to the apical membrane, allowing water to enter the cell from the tubular lumen. This water then exits the cell across the basolateral membrane, which is always freely permeable to water owing to the presence of AQP3 and AQP4 water channels. Thus in the presence of AVP, water is reabsorbed from the renal tubules.

AT THE C ELLU LAR LEVEL The gene for the V2 receptor is located on the X chromosome and codes for a 371–amino acid protein that belongs to the family of receptors with seven membrane spanning domains coupled to heterotrimeric G proteins. As shown in Fig. 35.5, binding of AVP to its receptor on the basolateral membrane activates adenyl cyclase. The increase in intracellular cyclic adenosine monophosphate (cAMP) then activates protein kinase A (PKA), which results in the phosphorylation of AQP2 water channels and increased transcription of the AQP2 gene via activation of a cAMP-response element (CRE). Intracellular vesicles containing phosphorylated AQP2 move toward the apical membrane along microtubules driven by the molecular motor dynein. Once near the apical membrane, proteins called SNAREs interact with the AQP2 containing vesicles and facilitate their fusion with the plasma membrane. Insertion of AQP2 to the membrane allows water to enter the cell driven by the osmotic gradient (lumen osmolality < cell osmolality). The water then exits the cell across the basolateral membrane through AQP3 and AQP4 water channels, which are constitutively present in the basolateral membrane. When the V2 receptor is not occupied by AVP, the AQP2 water channels are removed from the apical membrane by clathrin-mediated endocytosis, thus rendering the apical membrane impermeable to water. The endocytosed AQP2 molecules may be either stored in intracellular vesicles, ready for reinsertion into the apical membrane when AVP levels in the plasma increase, or degraded.

FIG. 35.5 Action of AVP via the V2 receptor on the principal cell of the late distal tubule and collecting duct. See text for details. AC, Adenyl cyclase; AP2, aquaporin 2 gene; AQP2, aquaporin 2; cAMP, cyclic adenosine monophosphate; CRE, cAMP response element; CREB-P, phosphorylated cAMP response element–binding protein; P, phosphorylated proteins. (Adapted from Brown D, Nielsen S. The cell biology of vasopressin action. In: Brenner BM, ed. The Kidney. 7th ed. Philadelphia: Saunders; 2004.)

AVP also regulates long-term expression of AQP2 and AQP3. When large volumes of water are ingested over an extended period of time (e.g., psychogenic polydipsia), the abundance of AQP2 and AQP3 in principal cells is reduced. As a consequence, when water ingestion is restricted, these individuals cannot maximally concentrate urine. Conversely, in states of restricted water ingestion, AQP2 and AQP3 protein expression in principal cells increases, thereby facilitating excretion of maximally concentrated urine. It is also clear that expression of AQP2 (and in some instances also AQP3) varies in pathological conditions associated with disturbances in urine concentration and dilution. AQP2 expression is reduced in a number of conditions associated with impaired urine-concentrating ability (e.g., hypercalcemia, hypokalemia). By contrast, in conditions associated with water retention (e.g., congestive heart failure, hepatic cirrhosis, pregnancy) AQP2 expression is increased. AVP also increases the permeability of the terminal portion of the inner medullary collecting duct to urea leading to increased reabsorption of urea and increased osmolality of the medullary interstitial fluid necessary for maximal urine concentration. The cells of the collecting duct express two types of urea transporters (UT), UT-A1 localized to the apical membrane and UT-A3 localized to the basolateral membrane. AVP, acting through the cAMP/PKA cascade, increases expression of UT-A1 and UT-A3. Increasing the osmolality of the interstitial fluid of the renal medulla also increases the permeability of the inner medullary collecting duct to urea. This effect is mediated by the phospholipase C/protein kinase C (PKC) pathway, which increases UT-A1 and UT-A3 expression. AVP also stimulates reabsorption of NaCl by the thick ascending limb of Henle’s loop and by the distal

tubule and cortical segment of the collecting duct. This increase in Na+ reabsorption is associated with increased abundance of three Na+ transporters: the Na+/K+/2Cl− symporter (thick ascending limb of Henle’s loop), the Na+/Cl− symporter (distal tubule), and the Na+ channel ENaC (the latter portion of the distal tubule and collecting duct). Stimulation of thick ascending limb NaCl transport may help maintain the hyperosmotic medullary interstitium necessary for absorption of water from the medullary portion of the collecting duct (see below).

IN THE C LIN IC When the collecting ducts do not respond normally to AVP, urine cannot be maximally concentrated leading to polyuria and polydipsia. This clinical entity is termed nephrogenic diabetes insipidus (NDI) to distinguish it from central diabetes insipidus. NDI can result from a number of systemic disorders and rarely can be inherited. Acquired NDI is caused by decreased expression of AQP2 in the collecting duct. Decreased expression of AQP2 impairs the urine-concentrating ability during hypokalemia, lithium ingestion (35% of individuals who take lithium for bipolar disorder develop some degree of NDI), urinary tract obstruction, low-protein diet, and hypercalcemia. Mutations in the AVP V2 receptor AVPR2 gene or the AQP2 gene lead to inherited NDI. Approximately 90% of the hereditary forms result from mutations in the AVPR2 gene and the remaining 10% result from AQP2 gene mutations. Since the AVPR2 gene is located on the X chromosome, its mutations lead to X-linked NDI. The AQP2 gene is located on chromosome 12, and its mutations can lead to autosomal recessive and very rarely autosomal dominant NDI. The AQP2 channel functions at the cell membrane as homotetramers. Mutations leading to recessive NDI affect the region of AQP2 gene associated with the formation of the homotetramer water channel pore. Heterozygous carriers produce both normal and defective AQP2 monomers. Since the defective AQP2 monomers are retained in the endoplasmic reticulum, the water channel forms only from normal monomers and the carriers remain asymptomatic. By contrast, mutations leading to the dominant NDI affect the region of AQP2 gene associated with post-translational modifications, such as AQP2 phosphorylation and not the water channel pore. Activating (gain-of-function) mutations in the AVPR2 gene lead to nephrogenic syndrome of inappropriate antidiuresis (NSIAD). In this X-linked disorder, V2 receptors are constitutively activated. These individuals have laboratory findings similar to those seen in SIADH, including reduced plasma osmolality, hyponatremia (reduced plasma [Na+]), and urine more concentrated than would be expected from the reduced body fluid osmolality. However, unlike SIADH where circulating levels of AVP are elevated and thus responsible for water retention by the kidneys, these individuals have undetectable levels of AVP in their plasma.

Thirst The perception of thirst is affected by changes in plasma osmolality, blood volume, or blood pressure. Increased plasma osmolality and reduced blood volume or pressure increase thirst. Of these stimuli, hyperosmolality is more potent and its increase by only 2% to 3% produces a strong desire to drink, whereas loss of blood volume or decrease in blood pressure in the range of 10% to 15% is required to produce the same response in thirst. As already discussed, there is a genetically determined threshold for AVP secretion (i.e., a body fluid osmolality above which AVP secretion increases). Similarly there is a genetically determined threshold for triggering the sensation of thirst. However, the thirst threshold is higher than the threshold for AVP

secretion. On average the threshold for AVP secretion is approximately 280 mOsm/kg H2O, whereas the thirst threshold is approximately 295 mOsm/kg H2O. Because of this difference, thirst is stimulated at a body fluid osmolality when AVP secretion is almost maximal. The center involved in regulating water intake (the thirst center) is located in the same region of the hypothalamus involved with regulating AVP secretion. However, it is not certain whether the same cells serve both functions. Indeed, the thirst response, like the regulation of AVP secretion, only occurs in response to effective solute (e.g., NaCl). Even less is known about the pathways involved in the thirst response to decreased blood volume or pressure, but it is believed that the pathways are the same as those involved in the volume- and pressure-related regulation of AVP secretion. Angiotensin II, acting on cells of the thirst center, also evokes the sensation of thirst. Because angiotensin II levels are increased when blood volume and pressure are reduced, this effect of angiotensin II contributes to the homeostatic response that restores and maintains body fluids at their normal volume. The sensation of thirst is satisfied by the act of drinking, even before sufficient water is absorbed from the gastrointestinal tract to correct the plasma osmolality. It is interesting to note that cold water is more effective in reducing the thirst sensation. Oropharyngeal and upper gastrointestinal receptors appear to be involved in this response. However, relief of the thirst sensation via these receptors is short lived, and thirst is only completely satisfied when the plasma osmolality or blood volume or pressure is corrected. It should be apparent that the AVP and thirst systems work in concert to maintain water homeostasis. An increase in plasma osmolality evokes drinking and, via AVP action in the kidneys, conservation of water. Conversely, when plasma osmolality is decreased, thirst is suppressed and, in the absence of AVP, renal water excretion is enhanced. When the fluid intake is dictated by cultural and social determinants rather than thirst, maintaining normal body fluid osmolality relies solely on the ability of the kidneys to excrete water. How the kidneys accomplishes this is discussed in detail in the following sections of this chapter.

IN THE C LIN IC With adequate access to water, the thirst mechanism can prevent development of hyperosmolality. This mechanism is responsible for the polydipsia seen in response to the polyuria of both CDI and NDI. Most individuals ingest water/beverages even in the absence of the thirst sensation. Normally the kidneys are able to excrete this excess water because they can excrete up to 18 L/day of urine. However, in some instances, the volume of water ingested exceeds the kidneys’ capacity to excrete water, especially over short periods of time. When this occurs, body fluids become hypoosmotic. An example of how water intake can exceed the capacity of the kidneys to excrete water is longdistance running. A study of participants in the Boston Marathon found that 13% of the runners developed hyponatremia during the race.b This reflected the practice of some runners to ingest water or other hypotonic drinks during the race to remain “well hydrated.” In addition, water is produced from the metabolism of glycogen and triglycerides used as fuels by the exercising muscle. Hyponatremia developed because, over the course of the race, the runners achieved a positive water balance resulting from higher ingestion and generation of water compared to its excretion by the kidneys and loss with sweat. In some racers the hyponatremia was severe enough to elicit the neurological symptoms. The maximum amount of water that can be excreted by the kidneys depends on the amount of solute excreted, which in turn depends on food intake. For example, with maximally dilute urine (Uosm = 50 mOsm/kg H2O), the maximum urine output of 18 L/day will be achieved only if the solute excretion rate is 900 mmol/day:

If solute excretion is reduced, as commonly occurs in the elderly with reduced food intake, the maximum urine output will decrease. For example, if solute excretion is only 400 mmol/day, a maximum urine output (at Uosm = 50 mOsm/kg H2O) of only 8 L/day can be achieved. Thus individuals with reduced food intake have a reduced capacity to excrete water.

Renal Mechanisms for Dilution and Concentration of Urine As already noted, water excretion is regulated separately from solute excretion. For this to occur, the kidneys must be able to excrete urine that is either hypoosmotic or hyperosmotic with respect to body fluid. This ability to excrete urine of varying osmolality in turn requires that solute be separated from water at some point along the nephron. As discussed in Chapter 34, reabsorption of solute in the proximal tubule results in reabsorption of a proportional amount of water. Hence solute and water are not separated in this portion of the nephron. Moreover, this proportionality between proximal tubule water and solute reabsorption occurs regardless of whether the kidneys excrete dilute or concentrated urine. Thus, the proximal tubule reabsorbs a large portion of the filtered amount of solute and water, but it does not produce dilute or concentrated tubular fluid. The loop of Henle, in particular the thick ascending limb, is the major site where solute and water are separated. Thus excretion of both dilute and concentrated urine requires normal function of the Henle’s loop. Excretion of hypoosmotic urine is relatively easy to understand. The nephron must simply reabsorb solute from the tubular fluid and not allow water reabsorption to also occur. As just noted, and as described in greater detail later, reabsorption of solute without concomitant water reabsorption occurs in the ascending limb of Henle’s loop. Under appropriate conditions (i.e., in the absence of AVP) the distal tubule and collecting duct also dilute the tubular fluid by reabsorbing solute but not water. Excretion of hyperosmotic urine (or urinary concentration) is more complex and in essence involves removing water from the tubular fluid without solute. Because water movement is passive, driven by an osmotic gradient, the kidney must generate a hyperosmotic compartment into which water is reabsorbed, without solute, osmotically from the tubular fluid. The hyperosmotic compartment that serves this function is the interstitium of the renal medulla. Henle’s loop is critical for generating the hyperosmotic medullary interstitium. Once established, this hyperosmotic compartment drives water reabsorption from the collecting duct and thereby concentrates urine. Fig. 35.6 summarizes tubular fluid osmolality at several points along the nephron, in both the absence and presence of AVP. Note that tubular fluid entering the loop of Henle from the proximal tubule is isosmotic with respect to plasma and is so regardless of the absence or presence of AVP. Also, tubular fluid leaving the thick ascending limb is hypoosmotic with respect to plasma, in both the absence and presence of AVP. The osmolality of tubular fluid along the collecting duct is hypoosmotic with respect to

plasma in the absence of AVP and becomes progressively hyperosmotic (i.e., from the cortex to inner medulla) in the presence of AVP.

FIG. 35.6 Tubular fluid osmolality along the nephron in the presence (+AVP) and in the absence (−AVP) of arginine vasopressin. See text for details. (Adapted from Sands JM, et al. Urine concentration and dilution. In: Taal MW, et al, eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.)

Establishment and maintenance of the hyperosmotic medullary interstitium has been a subject of study since the 1940s and the model remains incomplete. While it is generally accepted that the outer medulla contributes to the osmotic gradient by means of an active process termed countercurrent multiplication, the source of the gradient in the inner medulla is still incompletely understood. With the caveat that the current model needs refinement, it is presented here because it embodies some fundamental concepts that underlie the process. Countercurrent multiplication involves reabsorption of solute (principally NaCl) without water from the ascending limb of Henle’s loop into the surrounding medullary interstitium. This decreases the osmolality in the tubular fluid and raises the osmolality of the interstitium at this point. The increased osmolality of the interstitium then causes water to be reabsorbed from the descending limb of Henle’s loop, thus increasing the tubular fluid osmolality in this segment. Thus at any point along the loop of Henle the fluid in the ascending limb has an osmolality less than fluid in the adjacent descending limb. This osmotic difference is termed the single effect. Because of the countercurrent flow of tubular fluid in the descending limb (fluid flowing into the medulla) and ascending limb (fluid flow out of the medulla), this single effect could be multiplied, resulting in an osmotic gradient within the medullary interstitium, where the tip of the papilla has an osmolality of 1200 mOsm/kg H2O compared to 300 mOsm/kg H2O at the corticomedullary junction. Fig. 35.7 schematically depicts the processes for diluting and concentrating urine. Three key concepts

underlie these processes: 1. Urine is concentrated by AVP-dependent reabsorption of water from the collecting duct. 2. Reabsorption of NaCl from the ascending limb of Henle’s loop dilutes the tubular fluid and at the same time generates a high [NaCl] in the medullary interstitium (up to 600 mmol/L at the tip of the papilla), which then drives water reabsorption from the collecting duct. 3. Urea accumulates in the medullary interstitium (up to 600 mmol/L), which allows the kidneys to excrete urine with the same high urea concentration. This allows large amounts of urea to be excreted with relatively little water.

FIG. 35.7 Schematic of nephron segments involved in urine dilution and concentration. Henle’s loops of juxtamedullary nephrons are shown. A, Mechanism for excretion of dilute urine (water diuresis). AVP is absent and the collecting duct is essentially impermeable to water. Note also that during a water diuresis the osmolality of the medullary interstitium is reduced as a result of increased vasa recta blood flow and entry of some urea into the medullary collecting duct. B, Mechanism for excretion of a concentrated urine (antidiuresis). Plasma AVP levels are maximal and the collecting duct is highly permeable to water. Under this condition the medullary interstitial gradient is maximal. See text for details.

First, how the kidneys excrete dilute urine (water diuresis or aquaresis) when AVP levels are low or zero is considered. The following numbers refer to those encircled in Fig. 35.7A: 1. Fluid entering the descending thin limb of the loop of Henle from the proximal tubule is isosmotic with respect to plasma. This reflects the essentially isosmotic nature of solute and water reabsorption in the proximal tubule (see Chapter 34). (Note: Water is reabsorbed from the segments of the proximal tubule via AQP1.) 2. Water is reabsorbed from the thin descending limb of Henle’s loop. Most of this water is reabsorbed in the outer medulla, thereby limiting the amount of water added to the deepest part of the inner medullary interstitial space and thus preserving the hyperosmolality of this region of the medulla. (Note: Water is reabsorbed via AQP1.) 3. In the inner medulla the terminal portion of the descending thin limb and all of the thin ascending limb is impermeable to water. (Note: AQP1 is not expressed.) These same nephron segments express the Cl− channel CLC-K1, which mediates Cl− reabsorption, with Na+ following via the paracellular pathway. This passive reabsorption of NaCl without concomitant water reabsorption begins the process of diluting the tubular fluid. 4. The thick ascending limb of the loop of Henle is also impermeable to water and actively reabsorbs NaCl from the tubular fluid and thereby dilutes it further (see Chapter 34). Dilution occurs to such a degree that this segment is often referred to as the diluting segment of the kidney. Fluid leaving the thick ascending limb is hypoosmotic with respect to plasma (see Fig. 35.6). 5. The distal tubule and cortical portion of the collecting duct actively reabsorb NaCl. In the absence of AVP these segments are not permeable to water (i.e., AQP2 is not present in the apical membrane of the cells). Thus, when AVP is absent or present at low levels (i.e., decreased plasma osmolality), the osmolality of tubule fluid in these segments is reduced further because NaCl is reabsorbed without water. Under this condition, fluid leaving the cortical portion of the collecting duct is hypoosmotic with respect to plasma (see Fig. 35.6). 6. The medullary collecting duct actively reabsorbs NaCl. Even in the absence of AVP, this segment is slightly permeable to water and some water is reabsorbed. 7. The urine has an osmolality as low as approximately 50 mOsm/kg H2O and contains low concentrations of NaCl. The volume of urine excreted can be as much as 18 L/day, or approximately 10% of the glomerular filtration rate (GFR). Next, how the kidneys excrete concentrated urine (antidiuresis) when plasma osmolality and plasma AVP levels are high is considered. The following numbers refer to those encircled in Fig. 35.7B: 1–4. These steps are similar to those for production of dilute urine. An important point in understanding how a concentrated urine is produced is to recognize that while reabsorption of NaCl by the ascending thin and thick limbs of the loop of Henle dilutes the tubular fluid, the reabsorbed NaCl accumulates in the medullary interstitium and raises the osmolality of this compartment. Accumulation of NaCl in the medullary interstitium is crucial for production of urine hyperosmotic to plasma because it provides the osmotic driving force for water reabsorption by the medullary collecting duct. As already noted, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop. This is thought to maintain the medullary interstitial gradient at a time when water is being added to this compartment from the

medullary collecting duct, which would tend to dissipate the gradient. 5. Because of NaCl reabsorption by the ascending limb of the loop of Henle, the fluid reaching the collecting duct is hypoosmotic with respect to the surrounding interstitial fluid. Thus an osmotic gradient is established across the collecting duct. In the presence of AVP, which increases the water permeability of the latter portion of the distal tubule and the collecting duct by causing insertion of AQP2 into the luminal membrane of the cells, water diffuses out of the tubule lumen and the tubule fluid osmolality increases. This diffusion of water out of the lumen of the collecting duct begins the process of urine concentration. The maximum osmolality the fluid in the distal tubule and cortical portion of the collecting duct can attain is approximately 290 mOsm/kg H2O (i.e., the same as plasma), which is the osmolality of the interstitial fluid and plasma within the cortex of the kidney. 6. As the tubular fluid descends deeper into the medulla, water continues to be reabsorbed from the collecting duct, increasing the tubular fluid osmolality to 1200 mOsm/kg H2O at the tip of the papilla. 7. The urine produced when AVP levels are elevated has an osmolality of 1200 mOsm/kg H2O and contains high concentrations of urea and other nonreabsorbed solutes. Urine volume under this condition can be as low as 0.5 L/day. In comparing the two conditions just described, it should be apparent that a relatively constant volume of dilute tubular fluid is delivered to the AVP-sensitive portions of the nephron (latter portion of the distal tubule and collecting duct). Plasma AVP levels then determine the amount of water reabsorbed by these segments. When AVP levels are low, a relatively small volume of water is reabsorbed by these segments and a large volume of hypoosmotic urine is excreted (up to 10% of the filtered water). When AVP levels are high, a large volume of water is reabsorbed by these same segments and a small volume of hyperosmotic urine is excreted ( outer medulla > inner medulla) allows for maintenance of a hyperosmotic interstitial environment in the inner medulla by minimizing the amount of water entering this compartment. Medullary Interstitium As noted earlier, the interstitial fluid of the renal medulla is critically important in concentrating urine. The osmotic pressure of the interstitial fluid provides the driving force for reabsorbing water from both the descending thin limb of the loop of Henle and the collecting duct. The principal solutes of the medullary interstitial fluid are NaCl and urea, but the concentration of these solutes is not uniform throughout the medulla (i.e., a gradient exists from cortex to papilla). Other solutes also accumulate in the medullary interstitium (e.g., NH4+ and K+), but the most abundant solutes are NaCl and urea. For simplicity, this discussion assumes that NaCl and urea are the only solutes. As depicted in Fig. 35.8, NaCl and urea accumulate in the renal medulla, and the interstitial fluid at the tip of the papilla of the inner medulla reaches a maximum osmolality of 1200 mOsm/kg H2O, with approximately 600 mOsm/kg H2O attributable to NaCl (300 mmol/L) and 600 mOsm/kg H2O attributable to urea (600 mmol/L). Establishment of the NaCl gradient is essentially complete at the transition between the outer and inner medulla.

FIG. 35.8 The medullary interstitial gradient comprises primarily NaCl and urea. The concentrations for NaCl and urea depicted reflect those found in the antidiuretic state (i.e., excretion of hyperosmotic urine). See text for details. (Adapted from Sands JM, et al. Urine concentration and dilution. In: Taal MW, et al, eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Elsevier; 2012.)

The medullary gradient for NaCl results from accumulation of NaCl reabsorbed by the nephron segments in the medulla during countercurrent multiplication. The most important segment in this regard is the ascending limb of the loop of Henle. Urea accumulation within the medullary interstitium is more complex and occurs most effectively when hyperosmotic urine is excreted (i.e., antidiuresis). When dilute urine is produced, especially over extended periods, the osmolality of the medullary interstitium declines (see Fig. 35.7A). This reduced osmolality is almost entirely caused by a decrease in the concentration of urea. This decrease reflects washout by the vasa recta (discussed later) and diffusion of urea from the interstitium into the tubular fluid within the medullary portion of the collecting duct, which is permeable to urea even in the absence of AVP. (Note: The cortical and outer medullary portions of the collecting duct have a low permeability to urea, whereas the inner medullary portion has a relatively high permeability because of the presence of the urea transporters UT-A1 and UT-A3, the expression of which is increased by AVP.) Some of this reabsorbed urea is secreted into the thin descending limbs of Henle’s loops via the urea transporter UT-A2, and some enters the vasa recta via the UT-B transporter. The urea that is secreted into the descending thin limbs of Henle’s loops is then trapped in the nephron until it again reaches the medullary collecting duct, where it can reenter the medullary interstitium. Thus, urea recycles from the interstitium to the nephron and back into the interstitium. This process of urea recycling facilitates accumulation of urea in the medullary interstitium, where it can attain a concentration at the tip of the papilla of 600 mmol/L. As described, the hyperosmotic medulla is essential for concentrating the tubular fluid within the collecting duct. Because water reabsorption from the collecting duct is driven by the osmotic gradient established in the medullary interstitium, urine can never be more concentrated than that of the interstitial fluid in the papilla. Thus any condition that reduces the medullary interstitial osmolality impairs the ability of the kidneys to maximally concentrate urine. Urea within the medullary interstitium contributes to the total osmolality of the urine. However, because the inner medullary collecting duct is highly permeable to urea, especially in the presence of AVP, urea cannot drive water reabsorption across this

nephron segment. Instead, urea in the tubular fluid and medullary interstitium equilibrate, and a small volume of urine with a high concentration of urea is excreted.c It is the medullary interstitial NaCl concentration that is responsible for reabsorbing water from the medullary collecting duct and thereby concentrating the nonurea solutes (e.g., NH4+ salts, K+ salts, creatinine) in the urine. Vasa Recta Function The vasa recta, the capillary networks that supply blood to the medulla, are highly permeable to solute and water. As with the loop of Henle, the vasa recta form a parallel set of hairpin loops within the medulla (see Chapter 33). Not only do the vasa recta bring nutrients and oxygen to the medullary nephron segments, but more importantly they also remove the excess water and solute that is continuously added to the medullary interstitium by these nephron segments. The ability of the vasa recta to maintain the medullary interstitial gradient is flow dependent. A substantial increase in vasa recta blood flow dissipates the medullary gradient (i.e., washout of osmoles from the medullary interstitium). Alternatively, reduced blood flow reduces oxygen delivery to the nephron segments within the medulla. Because transport of salt and other solutes requires oxygen and ATP, reduced medullary blood flow decreases salt and solute transport by nephron segments in the medulla. As a result, the medullary interstitial osmotic gradient cannot be maintained. In summary, the kidneys maintain an osmotic gradient from the corticomedullary junction to the inner medullary tip. The cortical tissue is isotonic to plasma while the medullary tip is hypertonic. This gradient becomes steeper during antidiuresis and decreases in magnitude during diuresis. Recent studies have been focusing on the detailed understanding of the renal functional architecture, including threedimensional reconstruction and mathematical modeling to develop a more complete understanding how the permeability properties of nephron segments and their three-dimensional arrangements may contribute to the generation and maintenance of osmotic gradient necessary for urinary concentration.d

Assessment of Renal Diluting and Concentrating Ability Assessment of renal water handling includes measurements of urine osmolality and the volume of urine excreted. The range of urine osmolality is from 50 to 1200 mOsm/kg H2O. The corresponding range in urine volume is 18 L to as little as 0.5 L/day. The ranges are not fixed and vary from individual to individual and can be affected by disease processes. The ability of the kidneys to dilute or concentrate urine requires the separation of solute and water (i.e., the single effect of the countercurrent multiplication process). This separation of solute and water in essence generates a volume of water that is “free of solute.” When urine is dilute, solute-free water is excreted from the body. When urine is concentrated, solute-free water is returned to the body (i.e., conserved). For the kidneys to maximally excrete solute-free water (i.e., 18 L/day) the following conditions must be met: 1. AVP must be absent; without it the collecting duct does not reabsorb a significant amount of water. 2. The tubular structures that separate solute from water (i.e., dilute the tubule fluid) must function normally. In the absence of AVP the following nephron segments can dilute the renal tubular fluid: • ascending thin limb of Henle’s loop • thick ascending limb of Henle’s loop • distal tubule

• collecting duct Because of its high transport rate, the thick ascending limb is quantitatively the most important nephron segment involved in the separation of solute and water.

3. An adequate amount of tubular fluid must be delivered to the aforementioned nephron sites for maximal separation of solute and water. Factors that reduce delivery (e.g., decreased GFR or enhanced proximal tubule reabsorption) impair the ability to maximally excrete solute-free water. Similar requirements also apply to conservation of water by the kidneys. For the kidneys to conserve water maximally (6–8 L/day) the following conditions must be met: 1. An adequate amount of tubular fluid must be delivered to those nephron segments that separate solute from water; the most important segment in this regard is the thick ascending limb of Henle’s loop. Delivery of tubular fluid to Henle’s loop depends on GFR and proximal tubule reabsorption. 2. Reabsorption of NaCl by the nephron segments must be normal; again, the most important segment is the thick ascending limb of Henle’s loop. 3. A hyperosmotic medullary interstitium must be present. The interstitial fluid osmolality is maintained by NaCl reabsorption by Henle’s loop (conditions 1 and 2) and by effective accumulation of urea. Urea accumulation in turn depends on adequate dietary protein intake. 4. Maximum levels of AVP must be present and the collecting duct must respond normally to AVP.

Control of Extracellular Fluid Volume and Regulation of Renal NaCl Excretion The major solutes of ECF are the salts of Na+ (see Chapter 2). Of these, NaCl is the most abundant. Because NaCl is also the major determinant of ECF osmolality, alterations in Na+ balance are commonly assumed to disturb ECF osmolality. However, under normal circumstances this is not the case because the AVP and thirst systems maintain body fluid osmolality within a very narrow range (discussed earlier). As illustrated in Fig. 35.9, adding or removing NaCl from ECF changes its volume and not the [Na+] (compare initial condition and final conditions). For example, addition of NaCl to ECF (without water) increases the [Na+] and osmolality of this compartment (ICF osmolality also increases because of osmotic equilibration with ECF.) In response, AVP secretion and thirst are stimulated, and as a result water is ingested and renal water loss is reduced. This restores plasma osmolality (and serum [Na+]) to their initial values, but the volume of ECF is now increased. The opposite occurs when NaCl is lost from ECF. Changes in ECFV can be monitored by measuring body weight, because 1 L of ECF equals 1 kg of body weight.

FIG. 35.9 Impact of altered Na+ balance on ECFV. (1) Addition of NaCl (without water) to the ECF increases the [Na+] and osmolality. (2) The increase in ECF osmolality stimulates secretion of AVP from the posterior pituitary, which then acts on the kidneys to conserve water. (3) Decreased renal excretion of water together with water ingestion restore plasma osmolality and plasma [Na+] to normal. However, the ECF volume is now increased by 1 L. (4) Removal of NaCl (without water) from the ECF decreases plasma [Na+] and plasma osmolality. (5) Decreased ECF osmolality inhibits AVP secretion. In response to the decrease in plasma AVP the kidneys excrete water. (6) Increased renal excretion of water returns the plasma [Na+] and plasma osmolality to normal. However, ECF volume is now decreased by 1 L. As illustrated, changes in Na+ balance alter ECFV because of the efficiency of the AVP system to maintain normal body fluid osmolality. (Adapted from Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

The kidneys are the major route for excretion of NaCl from the body. Only about 10% of the total daily Na+ loss occurs by nonrenal routes (e.g., in perspiration and feces). As such, the kidneys are critically important in regulating ECFV. Under normal conditions the kidneys keep ECFV constant (a state termed euvolemia) by adjusting the excretion of NaCl to match the amount ingested from food and drink. If ingestion exceeds excretion, ECFV increases above normal (volume expansion), whereas the opposite occurs if excretion exceeds ingestion (volume contraction). The typical diet contains approximately 140 mEq/day of Na+ (8 g of NaCl), and thus Na+ excretion in urine is also about 140 mEq/day. However, the kidneys can vary excretion of Na+ over a wide range. Excretion rates as low as 10 mEq/day can be attained when individuals are placed on a low-salt diet. Conversely, the kidneys can increase their excretion rate to more than 1000 mEq/day when challenged by ingestion of a high-salt diet. These changes in Na+ excretion can occur with only modest changes in the ECFV and Na+ content of the body. The renal response to abrupt changes in NaCl intake typically takes several hours to days, depending

on the magnitude of the change. During this transition period the intake and excretion of Na+ are not matched as in the steady state. Thus the individual experiences either positive Na+ balance (intake > excretion) or negative Na+ balance (intake < excretion). However, by the end of the transition period, a new steady state is established and intake once again equals excretion. This section focuses on the receptors that control the ECFV and explains various signals that act on the kidneys to regulate NaCl excretion. In addition, responses of the nephron segments to these signals are considered.

Concept of Effective Circulating Volume As described in Chapter 2, the ECF is subdivided into two compartments: intravascular (plasma) and extravascular (interstitial fluid). Na+ balance, and thus ECFV, involves a complex system of sensors and effector signals that act primarily on the kidneys to regulate NaCl excretion. Plasma volume determines vascular volume, blood pressure, and cardiac output. Because the primary sensors of this system are located in the large vessels of the vascular system, changes in vascular volume, blood pressure, and cardiac output are the principal factors regulating renal NaCl excretion (discussed later). In a healthy individual, changes in ECFV lead to changes in vascular volume, blood pressure, and cardiac output. A decrease in ECFV results in reduced vascular volume, blood pressure, and cardiac output. Conversely, an increase in ECFV results in increased vascular volume, blood pressure, and cardiac output. The degree to which these cardiovascular parameters change is dependent upon the degree of ECF contraction or expansion and the effectiveness of cardiovascular reflex mechanisms (see Chapters 18 and 19). When a person is in negative Na+ balance, ECF contracts and renal NaCl excretion decreases. Conversely, with positive Na+ balance, ECF expands and renal NaCl excretion increases (i.e., natriuresis). However, in some pathological conditions (e.g., congestive heart failure, hepatic cirrhosis), the renal handling of Na+ does not correlate with the ECFV. Paradoxically, the ECFV increases and renal excretion of NaCl decreases. To explain renal Na+ handling in these two pathological states, it is necessary to understand the concept of effective circulating volume (ECV). Unlike the ECFV, ECV is not a measurable and distinct body fluid compartment. Effective circulating volume refers to that portion of the ECF that is contained within the vascular system and is “effectively” perfusing the tissues. More specifically the ECV reflects the activity of volume sensors located in the vascular system (discussed later).

IN THE C LIN IC Patients with congestive heart failure frequently have an increased ECF volume that manifests as increased plasma volume and interstitial fluid accumulating in the lungs (pulmonary edema) and peripheral tissues (generalized edema). This excess ECFV is the result of NaCl and water retention by the kidneys. The renal response (i.e., retention of NaCl) is paradoxical because the ECFV is increased. In spite of the increased ECF volume, these patients experience decreased ECV resulting from decreased cardiac output, low blood pressure, or capillary leak of fluid into the interstitial compartment. Therefore, the sensors located in the vascular system respond as they do in ECFV contraction and cause NaCl and water retention by the kidneys. In healthy individuals, ECV changes directly with ECFV and is determined by the volume of the vascular system (arterial and venous), arterial blood pressure, and cardiac output. However, as noted it is

not the case in certain disease states. In the remaining sections of this chapter we examine the relationship between ECFV and renal NaCl excretion in healthy adults.

Volume-Sensing Systems The volume-sensing sensors are called vascular volume receptors or baroreceptorse because they respond to pressure-induced stretch of the walls of the structure in which they are located (e.g., blood vessels or the cardiac atria and ventricles). Volume Sensors in the Low-Pressure Cardiopulmonary Circuit Baroreceptors located within the walls of the left and right atria, right ventricle, and large pulmonary vessels respond to distention of these structures (see Chapters 18 and 19). Because the low-pressure side of the circulatory system has a high compliance, these sensors respond mainly to the “fullness” of the vascular system. These baroreceptors send signals to the brainstem via afferent fibers in the glossopharyngeal and vagus nerves (cranial nerves IX and X). Activity of the sensors modulates both sympathetic nerve outflow and AVP secretion. For example, a decrease in filling of the pulmonary vessels and cardiac atria increases sympathetic nerve activity and stimulates AVP secretion. Conversely, distention of these structures decreases sympathetic nerve activity. Generally, at least a 5% change in blood volume and pressure is needed to evoke the response. The cardiac atria possess an additional mechanism related to control of renal NaCl excretion. The myocytes of the atria synthesize and store a peptide hormone, atrial natriuretic peptide (ANP). It is released when the atria are distended and, via mechanisms outlined later in this chapter, reduces blood pressure and increases excretion of NaCl and water by the kidneys. The ventricles of the heart also produce a natriuretic peptide, brain natriuretic peptide (BNP), so named because it was first isolated from the brain. Like ANP, BNP is also released from myocytes by distention of the ventricles. Its actions are similar to those of ANP. Volume Sensors in the High-Pressure Arterial Circuit Baroreceptors are also present in the arterial side of the circulatory system, located in the wall of the aortic arch, carotid sinus, and the renal afferent arterioles. The aortic arch and carotid baroreceptors send input to the brainstem via afferent fibers in the glossopharyngeal and vagus nerves to alter sympathetic outflow and AVP secretion. Decreased blood pressure increases sympathetic nerve activity and AVP secretion while increased pressure tends to reduce sympathetic nerve activity (and activates parasympathetic nerve activity). The sensitivity of the high-pressure baroreceptors is similar to that in the low-pressure side of the vascular system. The juxtaglomerular apparatus (JGA) of the kidneys (see Chapter 33), particularly the afferent arteriole, responds directly to changes in pressure. If perfusion pressure in the afferent arteriole is reduced, renin is released from the juxtaglomerular cells. By contrast, renin secretion is suppressed when perfusion pressure is increased. As described later in this chapter, renin determines blood levels of angiotensin II and aldosterone, both of which reduce renal NaCl excretion.

IN THE C LIN IC Constriction of a renal artery (e.g., by an atherosclerotic plaque) reduces renal perfusion pressure, which stimulates renin secretion by the afferent arteriole of the JGA. Renin increases the production of the potent vasoconstrictor angiotensin II that affects arterioles throughout the vascular system and

increases systemic blood pressure. Increased blood pressure is sensed by the JGA of the contralateral kidney (i.e., the kidney without stenosis of its renal artery), and renin secretion from that kidney is suppressed. In addition, increased angiotensin II levels also inhibit renin secretion by the contralateral kidney (negative feedback). Treatment strategies for patients with stenotic renal arteries include administration of angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, or surgical repair of renal arterial stenosis.

Volume Sensor Signals When the vascular volume sensors have detected a change in ECFV, they send signals to the kidneys to adjust NaCl and water excretion. Accordingly, when ECF expands, renal NaCl and water excretion increase. Conversely, when ECF contracts, renal NaCl and water excretion decrease. The signals involved in coupling the volume sensors to the kidneys are neural and hormonal and are summarized in Box 35.1. Box 35.1

Sig na ls Invo lve d in C o nt ro l o f R e na l N a C l a nd Wa t e r Exc re t io n Renal Sympathetic Nerves (↑Activity: ↓NaCl Excretion) ↓GFR ↑Renin secretion ↑Na+ reabsorption along the nephron Renin-Angiotensin-Aldosterone (↑Secretion: ↓NaCl Excretion) ↑Angiotensin II stimulates Na+ reabsorption along the nephron ↑Aldosterone stimulates Na+ reabsorption in the distal tubule and collecting duct and to a lesser degree in the thick ascending limb of Henle’s loop ↑Angiotensin II stimulates AVP secretion Natriuretic Peptides: ANP, BNP & Urodilatin (↑Secretion: ↑NaCl Excretion) ↑GFR ↓Renin secretion ↓Aldosterone secretion (indirect via ↓angiotensin II and direct on adrenal gland) ↓NaCl and water reabsorption by the collecting duct ↓AVP secretion and inhibition of AVP action on the distal tubule and collecting duct AVP (↑Secretion: ↓H2O Excretion) ↑H2O reabsorption by the distal tubule and collecting duct

Renal Sympathetic Nerves As described in Chapter 33, sympathetic nerve fibers innervate the glomerular afferent and efferent arterioles as well as the nephron cells. ECF contraction activates the low- and high-pressure vascular baroreceptors leading to stimulation of sympathetic nerve activity, including fibers innervating the kidneys. The stimulation has the following effects: 1. Constriction of the afferent and efferent arterioles (mediated by α-adrenergic receptors). This vasoconstriction is greater on the afferent arteriole and decreases the hydrostatic pressure within the glomerular capillary lumen, which decreases GFR. Reduction of GFR decreases Na+ filtration. 2. Stimulation of renin secretion by juxtaglomerular cells (mediated by β-adrenergic receptors). As described later, renin ultimately increases the circulating levels of angiotensin II and aldosterone, both of which stimulate Na+ reabsorption by the nephron. 3. Direct stimulation of NaCl reabsorption along the nephron (mediated by α-adrenergic receptors). Because of the large amount of Na+ reabsorbed by the proximal tubule, the effect of increased sympathetic nerve activity is quantitatively most important for this segment. As a result, increased renal sympathetic nerve activity decreases NaCl excretion, an adaptive response that works to restore ECFV to normal. With ECF expansion, renal sympathetic nerve activity decreases. This generally reverses the effects just described. Renin-Angiotensin-Aldosterone System Renin is an aspartyl protease that initiates a cascade leading to the production of angiotensin II, a potent vasoconstrictor that increases blood pressures and ECFV. Renin-expressing cells localize to the walls of the afferent arterioles at the entrance to the glomeruli and thus are termed juxtaglomerular (JG) cells. Renin synthesis and secretion are stimulated by renal baroreceptors, β-adrenergic receptors, and the macula densa. 1. Renal baroreceptors. The afferent arteriole behaves as a high-pressure baroreceptor. When perfusion pressure to the kidneys is reduced, renin secretion is stimulated. Conversely, an increase in perfusion pressure inhibits renin release. 2. β-Adrenergic receptors. The sympathetic nerve fibers from the main renal nerve densely innervate the afferent arterioles. Activation of β-adrenergic receptors stimulates renin secretion. 3. Macula densa. Delivery of NaCl to the macula densa regulates GFR by a process termed tubuloglomerular feedback (see Chapter 33). In addition, the macula densa plays a role in renin secretion. When NaCl delivery to the macula densa decreases, renin secretion is enhanced. Conversely, an increase in NaCl delivery inhibits renin secretion. It is likely that macula densa– mediated renin secretion helps maintain systemic arterial pressure under conditions of a reduced vascular volume. For example, when vascular volume is reduced, tissue perfusion (including the kidneys) decreases. This in turn decreases GFR and the filtered amount of NaCl. The reduced delivery of NaCl to the macula densa stimulates renin secretion, which acts through angiotensin II (a potent vasoconstrictor) to increase blood pressure and thereby maintain tissue perfusion.

AT THE C ELLU LAR LEVEL The adult kidney contains only a small number of JG cells producing sufficient amounts of renin to maintain the blood pressure and fluid and electrolyte balance during homeostasis. By contrast, situations that threaten homeostasis such as hypotension, dehydration, or sodium depletion require much higher renin levels to restore blood pressure as well as fluid and electrolyte balance. The higher demand for renin is met by recruitment of additional cells along the renal arterioles to produce renin. Recruitment occurs from the renin progenitors that differentiated after completion of renal morphogenesis to become arteriolar smooth muscle cells. The recruited smooth muscle cells dedifferentiate to become renin-producing again. Renin synthesis and secretion is stimulated by a decrease in intracellular [Ca++], a response opposite that of most secretory cells, where secretion is stimulated by an increase in intracellular [Ca++]. Conversely, signals that increase intracellular [Ca++] inhibit renin secretion. Renin secretion and release is mainly controlled by the cAMP pathway. βAdrenergic receptors linked to G-protein subunit alpha and JG cell–specific adenylyl cyclase V and VI are essential in cAMP generation in JG cells. cAMP availability is the net result of positive adenylyl cyclase activity and competing degradative activity of calcium calmodulin-activated phosphodiesterase 1C. Acutely increasing intracellular [Ca++] decreases net cAMP generation by dampening adenylyl cyclase and enhancing phosphodiesterase activities. Extracellular [Ca++] affects intracellular [Ca++] via Ca++-sensing receptor (CaSR). Acute stimulation of CaSR results in a marked decrease in cAMP levels and inhibition of renin release. By contrast, chronic CaSR stimulation leads to elevated renin levels. The specific subcellular mechanisms of these effects are an area of intense study. Stretch of the afferent arteriole, angiotensin-II, and endothelin increase intracellular [Ca++] and thus inhibit renin secretion. The stimulatory effect of sympathetic nerve activity on renin secretion is mediated by norepinephrine, which increases intracellular cAMP via β-adrenergic receptors. Prostaglandin E2 also increases JG cell cAMP levels and therefore stimulates renin secretion. Natriuretic peptides and nitric oxide (NO) inhibit renin secretion by increasing intracellular cyclic guanosine monophosphate (cGMP). Control of renin secretion by the macula densa is complex and appears to involve several paracrine factors, including ATP, adenosine, and prostaglandin E2 (see Chapter 33). Fig. 35.10 summarizes the essential components of the renin-angiotensin-aldosterone system (RAAS). Renin alone does not have a physiological function; it functions solely as a proteolytic enzyme. Its substrate is a circulating protein, angiotensinogen, which is produced by the liver. Angiotensinogen is cleaved by renin to yield a 10–amino acid peptide, angiotensin I. Angiotensin I also has no known physiological function, and it is cleaved to an 8–amino acid peptide, angiotensin II, by angiotensinconverting enzyme (ACE) found on the surface of vascular endothelial cells. Lung and renal endothelial cells are important sites for the conversion of angiotensin I to angiotensin II. ACE also degrades bradykinin, a potent vasodilator. Angiotensin II has several important physiological functions: 1. Stimulation of aldosterone secretion by the adrenal cortex. 2. Vasoconstriction of arterioles, which increases blood pressure. 3. Stimulation of AVP secretion and thirst. 4. Increasing NaCl reabsorption by the proximal tubule, thick ascending limb of Henle’s loop, distal tubule, and collecting duct. Quantitatively, the proximal tubule effect is the largest.

FIG. 35.10 Schematic representation of the essential components of the renin-angiotensin-aldosterone system (RAAS). Activation of RAAS decreases renal Na+ and water excretion. Note: Angiotensin I is converted to angiotensin II by an angiotensin-converting enzyme that is present on all vascular endothelial cells. As shown, the endothelial cells within the lungs play a significant role in this conversion process. See text for details.

Angiotensin II is an important secretagogue for aldosterone, a major mineralocorticoid produced by the glomerulosa cells of the adrenal cortex. Aldosterone sensitivity is conferred by the expression of mineralocorticoid receptor and an enzyme 11β-hydroxysteroid dehydrogenase 2 in the latter portion of the distal tubule and collecting duct and to a lesser extent in the thick ascending limb of Henle’s loop and early portion of the distal tubule. Aldosterone binds to the mineralocorticoid receptor while the enzyme increases aldosterone specificity by metabolizing another class of hormones named glucocorticoids and thus prevents them from occupying the mineralocorticoid receptor. Aldosterone has many effects in the kidneys (see also Chapters 34, 36, and 37). With regard to regulation of the ECFV, aldosterone stimulates NaCl reabsorption. It stimulates Na+ entry by increasing the abundance and activity of ENaC in the apical membrane of principal cells. Extrusion of Na+ from cells across the basolateral membrane occurs by Na+,K+-ATPase, the abundance of which is also increased by aldosterone. Thus, aldosterone increases reabsorption of NaCl from the tubular fluid in the distal nephron, whereas reduced levels of aldosterone decrease the amount of NaCl reabsorbed.

IN THE C LIN IC Diseases of the adrenal cortex can alter aldosterone levels and thereby impair the ability of the kidneys to maintain Na+ balance and euvolemia. With decreased secretion of aldosterone (hypoaldosteronism), the reabsorption of NaCl, mainly by the aldosterone-sensitive distal nephron, is reduced and NaCl is lost in the urine. When urinary NaCl loss exceeds the dietary NaCl intake,

negative Na+ balance ensues and ECFV decreases. In response to ECF contraction, sympathetic tone increases leading to elevated levels of renin, angiotensin II, and AVP. The opposite effects result from increased aldosterone secretion (hyperaldosteronism); NaCl reabsorption by the aldosteronesensitive distal nephron increases and excretion of NaCl falls, leading to increased ECFV. These effects lead to reduction of the sympathetic tone and levels of renin, angiotensin II, and AVP. As described later, ANP and BNP levels are elevated in this setting. As summarized in Box 35.1, RAAS activation occurs during ECFV contraction and leads to decreased renal excretion of NaCl. RAAS suppression results from ECFV expansion and leads to increased renal NaCl excretion. Natriuretic Peptides A number of endogenous substances act on the kidneys to increase NaCl excretion (see Chapter 34). The natriuretic peptides produced by the heart and kidneys are best understood and will be the focus of the following discussion. The heart produces two natriuretic peptides. Atrial natriuretic peptide (ANP) is produced and stored in atrial myocytes while brain natriuretic peptide (BNP) is produced and stored in ventricular myocytes. Both peptides are secreted when the heart dilates in states of volume expansion or heart failure. BNP and ANP relax vascular smooth muscle tone and promote renal NaCl and water excretion. Urodilatin is a natriuretic peptide produced by the kidneys and promotes renal NaCl excretion. The natriuretic peptides antagonize the effects of RAAS on renal NaCl and water excretion by the following mechanisms: 1. Increase of GFR and the filtered amount of NaCl by vasodilation of the afferent and constriction of the efferent glomerular arterioles. 2. Inhibition of renin secretion by the afferent arterioles. 3. Inhibition of aldosterone secretion: (a) indirectly by decreasing renin secretion, thereby reducing angiotensin II–induced aldosterone levels; and (b) directly by inhibiting aldosterone secretion from the glomerulosa cells of the adrenal cortex. 4. Inhibition of NaCl reabsorption from the collecting duct, which is also caused in part by reduced levels of aldosterone. However, natriuretic peptides increase cGMP, which inhibits cation channels in the apical membrane of cells in the medullary collecting duct cells and thereby decreases NaCl reabsorption. 5. Inhibition of AVP secretion by the posterior pituitary and AVP action on the collecting duct. These effects decrease water reabsorption by the collecting duct and thus increase excretion of water in the urine. The net effect of natriuretic peptides is to increase excretion of NaCl and water by the kidneys. Arginine Vasopressin As discussed previously, ECF contraction stimulates AVP secretion by the posterior pituitary. Elevated AVP levels decrease renal water excretion, and reestablishes euvolemia.

Control of NaCl Excretion During Euvolemia Maintenance of Na+ balance and therefore euvolemia requires precise balance between the amount

ingested and excreted. As already noted, kidneys are the major route for Na+ excretion. Accordingly, in a euvolemic individual we can equate daily urine Na+ excretion with Na+ intake. Under conditions of salt restriction (i.e., low-salt diet), virtually no Na+ appears in the urine. Conversely, in individuals who ingest large salt quantities, renal Na+ excretion can exceed 1000 mEq/day. The time course for adjustment of renal Na+ excretion varies (hours to days) and depends on the magnitude of the change in salt intake. Acclimation to large changes in salt intake requires a longer time than acclimation to small changes in intake. The general features of Na+ transport along the nephron are illustrated in Fig. 35.11. Most (67%) of the Na+ filtered by the glomerulus is reabsorbed by the proximal tubule. An additional 25% is reabsorbed by the thick ascending limb of the loop of Henle, and the remainder by the distal tubule and collecting duct.

FIG. 35.11 Segmental Na+ reabsorption. The percentage of the filtered load of Na+ reabsorbed by each nephron segment is indicated. CD, Cortical collecting duct; DT, distal tubule; PT, proximal tubule; TAL, thick ascending limb.

In a normal adult the filtered amount (load) of Na+ is approximately 25,000 mEq/day:

Equation 35.1

With a typical diet, less than 1% of this filtered load is excreted in urine (≈140 mEq/day).f Because of the large filtered load of Na+, small changes in its reabsorption by the nephron can profoundly affect Na+ balance and thus ECFV. For example, an increase in Na+ excretion from 1% to 3% of the filtered load represents an additional loss of approximately 500 mEq/day of Na+. Because the ECF [Na+] is

140 mEq/L, such Na+ loss would decrease the ECFV by more than 3 L (i.e., water excretion would parallel the loss of Na+ to maintain body fluid osmolality constant: 500 mEq/day ÷ 140 mEq/L = 3.6 L/day of fluid loss). Such fluid loss in a 70-kg individual would represent a 26% decrease in ECFV. In euvolemic individuals the nephron segments distal to the loop of Henle (distal tubule and collecting duct) are the main nephron segments where Na+ reabsorption is adjusted to maintain excretion at a level appropriate for dietary intake. However, this does not mean that other portions of the nephron are not involved in this process. Because the reabsorptive capacity of the distal tubule and collecting duct is limited, these other nephron segments (i.e., proximal tubule and loop of Henle) must reabsorb the bulk of the filtered Na+ load. During euvolemia, Na+ handling by the nephron occurs in two sequential events: 1. Na+ handling by the proximal tubule and loop of Henle delivers a relatively constant portion of the filtered Na+ load to the distal tubule. The combined action of these nephron segments reabsorbs approximately 92% of the filtered Na+ and delivers 8% to the distal tubule. 2. Na+ handling by the distal tubule and collecting duct leads to urinary excretion of Na+ that is equivalent to the amount of Na+ that is ingested in the diet.

Mechanisms for Maintaining Constant Delivery of NaCl to the Distal Tubule A number of mechanisms maintain a constant delivery of Na+ to the beginning of the distal tubule. These processes are autoregulation of the GFR (and thus the filtered Na+ load) glomerulotubular balance, and load dependency of Na+ reabsorption by the loop of Henle. Autoregulation of the GFR (see Chapter 33) allows maintenance of a relatively constant filtration rate over a wide range of perfusion pressures. Because the filtration rate is constant, the filtered Na+ load is also constant. Despite the autoregulatory control of GFR, small variations occur. If changes were not compensated by adjusting Na+ reabsorption from the nephron, Na+ excretion would change markedly. Fortunately, Na+ reabsorption in the euvolemic state, especially by the proximal tubule, changes in parallel with changes in GFR. This phenomenon is termed glomerulotubular balance. Thus if GFR increases, the amount of Na+ reabsorbed by the proximal tubule also increases. The opposite occurs if GFR decreases (see Chapter 34 for more details). The final mechanism that helps maintain constant delivery of Na+ to the distal tubule and collecting duct involves the ability of the loop of Henle to increase its reabsorptive rate in response to increased delivery of Na+.

Regulation of Distal Tubule and Collecting Duct NaCl Reabsorption When delivery of Na+ is constant, small adjustments in Na+ reabsorption, primarily by the aldosteronesensitive distal nephron, are sufficient to balance excretion with intake. Aldosterone is the primary regulator of Na+ reabsorption and thus of NaCl excretion. When aldosterone levels are elevated, Na+ reabsorption by these segments is increased (Na+ excretion is decreased). When aldosterone levels are

decreased, Na+ reabsorption is decreased (NaCl excretion is increased). Other factors have been shown to alter Na+ reabsorption (e.g., angiotensin II, natriuretic peptides), but their role during euvolemia is unclear. As long as variations in dietary salt intake are minor, the described mechanisms can maintain euvolemia. However, these mechanisms cannot effectively handle significant changes in, Na+ intake, leading to ECFV expansion or contraction. In extreme situations, additional factors act on the kidneys to adjust Na+ excretion and thereby reestablish the euvolemic state.

Control of NaCl Excretion During Volume Expansion During ECFV expansion, the high-pressure and low-pressure vascular volume sensors send signals to the kidneys to increase excretion of NaCl and water. The signals include: 1. Decreased activity of renal sympathetic nerves. 2. Decreased release of ANP and BNP from the heart and urodilatin from the kidneys. 3. Inhibition of AVP secretion from the posterior pituitary and its action on the collecting duct. 4. Decreased renin secretion and thus decreased production of angiotensin II. 5. Decreased aldosterone secretion as a result of reduced angiotensin II levels, and elevated natriuretic peptide levels. The integrated response of the nephron to the signals is illustrated in Fig. 35.12. Three general responses to ECFV expansion occur (the numbers match those encircled in Fig. 35.12): 1. GFR increases. GFR increases mainly as a result of decreased sympathetic nerve activity. Sympathetic fibers innervate the afferent and efferent arterioles of the glomerulus and control their diameter. Decreased sympathetic nerve activity leads to arteriolar dilation and elevation of renal plasma flow (RPF). Because the effect appears to be greater on the afferent arterioles, the hydrostatic pressure within the glomerular capillary increases, thereby increasing the GFR. Because RPF increases to a greater degree than GFR, the filtration fraction (GFR/RPF) decreases. Natriuretic peptides also increase GFR by dilating the afferent arterioles and constricting the efferent arterioles. Thus increased natriuretic peptide levels present during ECFV expansion contribute to this response. With the increase in GFR, the filtered load of Na+ increases. 2. The reabsorption of Na+ decreases in the proximal tubule and loop of Henle. Several mechanisms reduce Na+ reabsorption by the proximal tubule. Because activation of the sympathetic nerve fibers that innervate this nephron segment stimulates proximal tubule Na+ reabsorption, the decreased sympathetic nerve activity that results from ECF expansion decreases Na+ reabsorption. In addition, angiotensin II directly stimulates Na+ reabsorption by the proximal tubule. Because angiotensin II levels are also reduced under this condition, proximal tubule Na+ reabsorption decreases as a result. Starling forces across the proximal tubule also change. The elevated hydrostatic pressure within the glomerular capillaries also tends to increase the hydrostatic pressure within the peritubular capillaries. In addition, decreased filtration fraction reduces the peritubular oncotic pressure. These alterations in the capillary Starling forces (i.e., hydrostatic and oncotic) reduce the absorption of solute (e.g., NaCl) and water from the lateral

intercellular space and thus reduce tubular reabsorption of NaCl (see Chapter 34 for a complete description of this mechanism). Increased filtered load and decreased NaCl reabsorption by the proximal tubule result in delivery of more NaCl to the loop of Henle. Because activation of sympathetic nerves and angiotensin II and aldosterone stimulate NaCl reabsorption by the thick ascending limb of the loop of Henle, the reduced nerve activity and low angiotensin II and aldosterone levels that occur with ECF expansion reduce NaCl reabsorption by the thick ascending limb. Thus the fraction of the filtered load delivered to the distal tubule increases. 3. Na+ reabsorption decreases in the distal tubule and collecting duct. As noted, the amount of Na+ delivered to the distal tubule exceeds that observed in the euvolemic state (i.e., the amount of Na+ delivered to the distal tubule varies in proportion to the degree of ECF expansion). The increased Na+ load overwhelms the reabsorptive capacity of the distal tubule and collecting duct. NaCl reabsorption is also impaired due to reduced levels of angiotensin II and aldosterone, as well as increased levels of natriuretic peptides.

FIG. 35.12 Integrated response to ECF expansion. Numbers refer to the description of the response in the text. ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; GFR, glomerular filtration rate; PNa+, plasma [Na+]; R, tubular reabsorption of Na+; UNa+V̇, Na+ excretion rate.

The final component in the response to ECFV expansion is increased excretion of water. As Na+ excretion increases, plasma osmolality begins to fall. This decreases secretion of AVP. Its secretion is

also inhibited in response to elevated natriuretic peptide levels. In addition, natriuretic peptides inhibit the action of AVP on the collecting duct. Together, these effects decrease water reabsorption by the collecting duct and thereby increase renal water excretion. Excretion of Na+ and water occurs in concert; euvolemia can be restored and body fluid osmolality return to normal. The time course of the response (hours to days) depends on the magnitude of the ECFV expansion and the ongoing Na+ and water intake. If the degree of ECFV expansion is small, the mechanisms generally restore euvolemia within 24 hours. With large degrees of ECFV expansion, the response may take days. In summary, renal response to ECFV expansion involves an integrated action of the entire nephron: (1) the amount of filtered Na+ is increased; (2) Na+ reabsorption by the proximal tubule and loop of Henle is reduced (glomerulotubular balance does not occur under this condition); (3) reabsorption of Na+ by the distal tubule and collecting duct is decreased secondary to reduced aldosterone levels; and (4) excretion of a larger fraction of filtered Na+ restores euvolemia.

Control of NaCl Excretion During Volume Contraction During ECFV contraction, the high- and low-pressure vascular volume sensors signal to the kidneys to reduce NaCl and water excretion and thereby restore euvolemia. The signals include: 1. Increased renal sympathetic nerve activity. 2. Increased renin secretion, which increases angiotensin II and aldosterone levels. 3. Inhibition of ANP and BNP secretion by the heart and urodilatin secretion by the kidneys. 4. Stimulation of AVP secretion by the posterior pituitary. The integrated response of the nephron to the signals is described next and illustrated in Fig. 35.13. The numbers below correlate with those in the figure: 1. GFR decreases. Afferent and efferent arteriolar constriction occurs as a result of increased renal sympathetic nerve activity. Because the effect is greater on the afferent arteriole, the hydrostatic pressure in the glomerular capillaries falls, which decreases GFR. Because RPF decreases more than GFR, filtration fraction increases. The decrease in GFR reduces the filtered amount of Na+. 2. Na+ reabsorption by the proximal tubule and loop of Henle is increased. Several mechanisms augment Na+ reabsorption in the proximal tubule. For example, increased sympathetic nerve activity and angiotensin II levels directly stimulate Na+ reabsorption. Decreased hydrostatic pressure within the glomerular capillaries also reduces the hydrostatic pressure within the peritubular capillaries. In addition, as just noted, the increased filtration fraction results in an increase in the peritubular oncotic pressure. These alterations in the capillary Starling forces facilitate movement of fluid from the lateral intercellular space into the capillary and thereby stimulate reabsorption of NaCl and water by the proximal tubule (see Chapter 34 for a complete description of this mechanism). Increased sympathetic nerve activity as well as elevated levels of angiotensin II and aldosterone stimulate Na+ reabsorption by the thick ascending limb. 3. Na+ reabsorption by the distal tubule and collecting duct is enhanced. The small amount of Na+ that is delivered to the aldosterone-sensitive distal nephron, owing to decreased filtration and increased reabsorption by the proximal tubule and loop of Henle, is almost completely reabsorbed. Stimulation of Na+ reabsorption from this segment is enhanced by increased

aldosterone levels, although increased sympathetic nerve activity and increased angiotensin II levels may also contribute to this response.

FIG. 35.13 Integrated response to ECFV contraction. Numbers refer to the description of the response in the text. ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; GFR, glomerular filtration rate; PNa+, plasma [Na+]; R, tubular reabsorption of Na+; UNa+V̇, Na+ excretion rate.

Finally, water reabsorption by the latter portion of the distal tubule and the collecting duct is enhanced by AVP, the levels of which are elevated through activation of the low- and high-pressure vascular volume sensors as well as angiotensin II. As a result, water excretion is reduced. Because both water and Na+ are retained by the kidneys in equal proportions, euvolemia is reestablished and body fluid osmolality returns to normal. The time course (hours to days) and the degree to which euvolemia can be restored depend on the magnitude of ECF contraction as well as the Na+ and water intake (enteral or parenteral) and ongoing Na+ and water losses (sensible and insensible). In brief, the nephron’s response to ECFV contraction involves the integrated action of all its segments: (1) the filtered amount of Na+ is decreased; (2) Na+ reabsorption by the proximal tubule and loop of Henle is enhanced (GFR is decreased, whereas proximal reabsorption is increased, and thus glomerulotubular balance does not occur under this condition); (3) delivery of Na+ to the aldosteronesensitive distal nephron is reduced in addition to enhanced Na+ and water reabsorption from this segment, virtually eliminating urinary Na+ and water excretion.

Key Concepts 1. Regulation of body fluid osmolality (i.e., steady-state balance) requires that the amount of water added to the body exactly matches the amount lost. Water is lost from the body by sensible and insensible mechanisms. Excretion of water by the kidney is regulated by AVP secreted from the posterior pituitary. When AVP levels are high, urine volume decreases and it becomes hyperosmotic. When AVP levels are low, urine volume increases and its osmolality falls. 2. Disorders of water balance alter body fluid osmolality. Because Na+ with its anions is the major determinant of ECF osmolality, disorders of water balance manifest as changes in ECF [Na+]. Positive water balance (intake > excretion) leads to hyponatremia and decreases body fluid osmolality. Negative water balance (intake < excretion) leads to hypernatremia and increase body fluid osmolality. 3. The ECFV is determined by the amount of Na+ in the compartment. To maintain normal ECFV (i.e., euvolemia) Na+ excretion must match Na+ intake. The kidneys are the major site for regulating excretion of NaCl from the body. Volume sensors located primarily in the vascular system monitor volume and pressure. When ECFV expansion occurs, neural and hormonal signals increase renal excretion of NaCl and water and thereby restore euvolemia. When ECFV contraction occurs, neural and hormonal signals decrease renal Na+ and water excretion and euvolemia can be restored. The sympathetic nervous system, RAAS, and natriuretic peptides are important components of the system that maintain steady-state Na+ balance. a

The SON and PVN synthesize either AVP or oxytocin. AVP-secreting cells predominate in the SON, whereas oxytocin-secreting neurons are primarily found in the PVN. The synthesized hormone is packaged in granules that are transported down the axon of the cell and stored in nerve terminals located in the neurohypophysis (posterior pituitary). The anatomy of the hypothalamus and pituitary gland is shown in Fig. 35.3 (see also Chapter 41). b

Almond CS, et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med; 2005;352:11501556. c

On a typical diet the kidneys must excrete 450 mmol/day of urea. At a maximal urine [urea] of 600 mmol/L this amount of urea can be excreted in less than 1 L of urine. However, if the maximal urine [urea] is reduced because of a decrease in the medullary interstitial fluid [urea], a larger urine volume would be needed to excrete the 450 mmol/day of urea (e.g., 2.25 L of urine would be required if the maximal urine [urea] was only 200 mM). d

Nawata, CM, and Pannabecker, TL. Mammalian urine concentration: a review of renal medullary architecture and membrane transporters. J Comp Physiol B; 2018;188:899–918. e

The liver and central nervous system also have sensors that respond to changes in blood pressure and [Na+] and then signal the kidneys to alter NaCl excretion. These systems do not appear to be as important as vascular receptors in monitoring changes in ECFV and effecting changes in renal NaCl excretion and are not considered here. f

The percentage of the filtered load excreted in urine is termed fractional excretion. In this example the fractional excretion of Na+ is 140 mEq/day ÷ 25,200 mEq/day = 0.005, or 0.5%.

C H AP T E R 3 6

Potassium, Calcium, and Phosphate Homeostasis LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. How does the body maintain K+ homeostasis? 2. What is the distribution of K+ within the body compartments? Why is this distribution important? 3. What are the hormones and factors that regulate plasma K+ levels? Why is this regulation important? 4. How do the various nephron segments transport K+ and what mechanisms determine how much K+ is excreted in the urine? 5. Why is the distal tubule and collecting duct important in regulating K+ excretion? 6. How do plasma K+ levels, aldosterone, vasopressin, tubular fluid flow rate, and acid-base balance influence K+ excretion? 7. What is the physiological importance of calcium (Ca++) and inorganic phosphate (Pi)? 8. How does the body maintain Ca++ and Pi homeostasis? 9. What are the roles of kidneys, gastrointestinal tract, and bone in maintaining plasma Ca++ and Pi levels? 10. What hormones and factors regulate plasma Ca++ and Pi levels? 11. What are the cellular mechanisms responsible for Ca++ and Pi reabsorption along the nephron? 12. What hormones regulate renal Ca++ and Pi excretion by the kidneys? 13. What is the role of the calcium-sensing receptor? 14. What are the common clinical disorders of Ca++ and Pi homeostasis? 15. What is the role of the kidneys in the vitamin D metabolism? 16. What effects do loop and thiazide diuretics have on Ca++ excretion? 17. What is the effect of chronic dietary potassium deficiency on blood pressure? 18. What are the effects of chronic total body K+ depletion on kidney function?

K+ Homeostasis Potassium (K+) is the most abundant cation in the body. The vast majority of total body K+ is located intracellularly (98%) where the [K+] is 150 mEq/L. Only 2% of total body K+ exists in the ECF at a

concentration of approximately 4 mEq/L. The large [K+] difference across cell membranes (≈146 mEq/L) is maintained by the Na+,K+-ATPase. The [K+] gradient is important in maintaining the potential difference across cell membranes and is critical for the excitability of nerve and muscle cells, as well as for the contractility of cardiac, skeletal, and smooth muscle cells (Fig. 36.1). Skeletal muscles contain the largest single pool of K+ in the body. In an adult, the skeletal muscles contain approximately 225 times more K+ than all extracellular compartments in the body. Moreover, due to the large number of Na+,K+ATPase pumps and K+ channels, skeletal muscles possess a huge capacity for K+ exchange. Despite wide fluctuations of the dietary K+ load, [K+] remains remarkably constant in the intracellular fluid (ICF) and extracellular fluid (ECF). A [K+] in ECF that exceeds 5.0 mEq/L constitutes hyperkalemia. Conversely, a [K+] in ECF of less than 3.5 mEq/L constitutes hypokalemia. During hypokalemia, skeletal muscle cells release K+ to preserve [K+] in the ECF leading to total body K+ depletion.

FIG. 36.1 Effects of variations in plasma [K+] on resting membrane potential of skeletal muscle. Hyperkalemia causes membrane potential to become less negative, which decreases excitability by inactivating the fast Na+ channels responsible for the depolarizing phase of the action potential. Hypokalemia hyperpolarizes the membrane potential and thereby reduces excitability because a larger stimulus is required to depolarize the membrane potential to the threshold potential. Resting indicates “normal” resting membrane potential. Normal threshold indicates the membrane threshold potential.

IN THE C LIN IC The K+ level is usually determined from a venous blood sample. K+ levels were traditionally measured in serum from coagulated blood, but are now more frequently measured in plasma from heparinized blood. Serum levels may generally be 0.2 to 0.4 mEq/L higher than plasma levels. Inappropriate blood sampling technique may affect the results. K+ levels rise in the ECF after physical activity (see later). Thus blood sampling to measure K+ should be done after several minutes of rest. Hemolysis of red blood cells during or after phlebotomy will release K+ into plasma, thereby artificially elevating [K+] in the collected blood sample. Only needles, tubes, and tube adaptors approved for K+ measurements should be used to prevent hemolysis. A large vein should be used (e.g., the cubital vein) without fist clenching and without prolonged application of a tourniquet. Pseudohyperkalemia refers to potassium >5 mmol/L in the collection tube and normal K+ level in patient’s blood. In addition to causing

pseudohyperkalemia, errors of K+ determination may conceal hypokalemia. Hypokalemia may develop in people with chronic administration of diuretic, excessive use of laxatives, vomiting, eating disorders, or diarrheal illness. Gitelman syndrome (a genetic defect in the Na+/Cl− cotransporter in the apical membrane of distal renal tubule cells) also causes hypokalemia (see Chapter 34). Hyperkalemia may occur in patients with renal failure, or as a side effect of medications such as angiotensin-converting enzyme (ACE) inhibitors and K+-sparing diuretics in patients with underlying kidney disease (decreased ability to renally excrete K+), or in patients with diabetes mellitus (decreased ability to shift K+ intracellularly).

IN THE C LIN IC Cardiac arrhythmias can result from hyperkalemia and hypokalemia. The electrocardiogram (ECG; Fig. 36.2) (also see Chapter 16) monitors the electrical activity of the heart and is a fast and reliable method to determine whether changes in plasma [K+] influence the heart function. The first sign of hyperkalemia is the appearance of tall, thin T waves on the ECG. Further increases in plasma [K+] prolong the PR interval, depress the ST segment, and lengthen the QRS interval of the ECG. As plasma [K+] approaches 10 mEq/L, the P wave disappears, the QRS interval broadens, the ECG appears as a sine wave, and the ventricles fibrillate (i.e., manifest rapid, uncoordinated contractions of muscle fibers). Hypokalemia prolongs the QT interval, inverts the T wave, and lowers the ST segment of the ECG.

FIG. 36.2 Electrocardiographs from individuals with varying plasma [K+]. See text for details. (Modified from Barker LR, Burton JR, Zieve PD. Principles of Ambulatory Medicine. 5th ed. Baltimore: Williams & Wilkins; 1999.)

K+ absorbed from the gastrointestinal (GI) tract enters the ECF within minutes (Fig. 36.3). If the K+ ingested during a normal meal (≈33 mEq) were to remain in the ECF compartment (14 L) plasma [K+] would increase by 2.4 mEq/L (33 mEq added to 14 L of ECF):

Equation 36.1

FIG. 36.3 Overview of K+ homeostasis. An increase in plasma insulin, epinephrine, or aldosterone stimulates movement of K+ into cells and decreases plasma [K+], whereas a fall in plasma concentration of these hormones has the opposite effect and increases plasma [K+]. The amount of K+ in the body is determined by the kidneys. An individual is in K+ balance when dietary intake and urinary output (plus output by the GI tract) are equal. Excretion of K+ by the kidneys is regulated by plasma [K+], aldosterone, and arginine vasopressin (AVP).

Rapid (seconds to minutes) intracellular uptake of K+ is essential to prevent life-threatening hyperkalemia. Excretion of K+ by the kidneys is relatively slow (hours). Maintaining total body [K+] constant requires that almost all the K+ absorbed from the GI tract is eventually excreted by the kidneys. The colon is responsible for the remaining small fraction of K+ excretion, and in patients with end-stage kidney disease the colon may increase fecal K+ excretion.

Regulation of Plasma [K+]

Several hormones, including epinephrine, insulin, and aldosterone, increase uptake of K+ into skeletal muscle, liver, bone, and red blood cells (Box 36.1; see Fig. 36.3) by stimulating Na+,K+-ATPase and the Na+/K+/2Cl− and Na+/Cl− cotransporters in these cells. Acute stimulation of K+ uptake (i.e., within minutes) is mediated by increased activity of existing Na+,K+-ATPase and the Na+/K+/2Cl− and Na+/Cl− cotransporters, whereas a chronic increase in K+ uptake (i.e., within hours to days) is mediated by increased abundance of Na+,K+-ATPase. The rise in plasma [K+] that follows K+ absorption by the GI tract stimulates secretion of insulin from the pancreas, release of aldosterone from the adrenal cortex, and secretion of epinephrine from the adrenal medulla (see Fig. 36.3). In contrast, a decrease in plasma [K+] inhibits release of these hormones. Whereas insulin and epinephrine act within a few minutes, aldosterone requires about an hour to stimulate uptake of K+ into cells. Box 36.1

M a jo r F a c t o rs, Ho rmo ne s, a nd D rug s Inf lue nc ing D ist ribut io n o f K+ B e t w e e n Int ra c e llula r a nd Ext ra c e llula r F luid C o mpa rt me nt s Physiological: Keep Plasma [K+] Constant Epinephrine Insulin Aldosterone Pathophysiological: Displace Plasma [K+] From Normal Acid-base disorders Plasma osmolality Cell lysis Vigorous exercise Drugs That Induce Hyperkalemia Dietary K+ supplements ACE inhibitors K+-sparing diuretics Heparin

Epinephrine Catecholamines affect the distribution of K+ across cell membranes by activating α- and β2-adrenergic receptors. Stimulation of α-adrenoceptors releases K+ from cells, especially in the liver, whereas stimulation of β2-adrenoceptors promotes K+ uptake by cells.

For example, activation of β2-adrenoceptors after exercise is important in preventing hyperkalemia. The rise in plasma [K+] after a K+-rich meal is greater if the patient has been pretreated with a βadrenoceptor antagonist (e.g., propranolol). Furthermore, release of epinephrine during stress (e.g., myocardial ischemia) can rapidly lower plasma [K+].

Insulin Insulin is the most important hormone that shifts K+ into cells after ingestion of dietary K+. Insulin and glucose infusion can be used to correct life-threatening hyperkalemia. In patients with diabetes mellitus (i.e., insulin deficiency), the rise in plasma [K+] after a K+-rich meal is greater than in healthy people. In patients with chronic kidney disease, although insulin-stimulated glucose uptake into cells is impaired, insulin stimulation of K+ uptake into cells is preserved.

Aldosterone Aldosterone, like catecholamines and insulin, also promotes uptake of K+ into cells. A rise in aldosterone levels (e.g., primary aldosteronism) causes hypokalemia, whereas a fall in aldosterone levels (e.g., Addison’s disease) causes hyperkalemia. As discussed later and as illustrated in Fig. 36.3, aldosterone also stimulates urinary K+ excretion. Thus aldosterone alters plasma [K+] by acting on uptake of K+ into cells and altering urinary K+ excretion.

Alterations in Plasma [K+] Hyperkalemia usually develops when the amount of K+, either enteral (dietary or bleeding into the GI tract) or parenteral (intravenous administration or hemolysis), exceeds the ability of intracellular uptake and the kidneys to excrete K+ (see Box 36.1). Hypokalemia usually develops when intracellular K+ uptake and renal K+ loss exceeds K+ intake (dietary or intravenous) (see Box 36.1). In some situations (see later), changes in the distribution of K+ between the ECF and ICF alone can result in acute and clinically relevant disturbances of plasma [K+].

Acid-Base Balance Metabolic acidosis increases plasma [K+], whereas metabolic alkalosis decreases it. Respiratory alkalosis causes hypokalemia. In contrast, respiratory acidosis has little or no effect on plasma [K+]. Metabolic acidosis produced by addition of inorganic acids (e.g., HCl, H2SO4) increases plasma [K+] much more than an equivalent acidosis produced by accumulation of organic acids (e.g., lactic acid, acetic acid, ketoacids). The reduced pH (i.e., increased [H+]) promotes movement of H+ into cells and the reciprocal movement of K+ out of cells to maintain electroneutrality. This effect of acidosis occurs in part because acidosis inhibits the transporters that accumulate K+ inside cells, including Na+,K+-ATPase and the Na+/K+/2Cl− cotransporter. In addition, movement of H+ into cells occurs as the cells buffer changes in [H+] of the ECF (see Chapter 37). As H+ moves across cell membranes, K+ moves in the opposite direction, and thus cations are neither gained nor lost across cell membranes. Metabolic alkalosis has the opposite effect; plasma [K+] decreases as K+ moves into cells and H+ exits.

Although organic acids produce a metabolic acidosis, they do not cause significant hyperkalemia. Two explanations have been suggested for the reduced ability of organic acids to cause hyperkalemia. First, the organic anion may enter the cell with H+ and thereby eliminate the need for K+-H+ exchange across the membrane. Second, organic anions may stimulate insulin secretion, which moves K+ into cells. This movement may counteract the direct effect of the acidosis, which moves K+ out of cells.

Plasma Osmolality The osmolality of plasma also influences the distribution of K+ across cell membranes. An increase in the osmolality of ECF enhances the release of K+ by cells and thus increases extracellular [K+]. Plasma [K+] may increase by 0.4 to 0.8 mEq/L with a 10 mOsm/kg H2O elevation in plasma osmolality. In patients with diabetes mellitus who do not take insulin, plasma [K+] is often elevated, in part because of the lack of insulin and in part because of the increase in plasma [glucose] (i.e., from a normal value of ≈100 mg/dL to as high as ≈1200 mg/dL in some cases), which increases plasma osmolality. Hypoosmolality has the opposite action. The alterations in plasma [K+] associated with changes in osmolality are related to changes in cell volume. For example, as plasma osmolality increases, water leaves cells because of the osmotic gradient across the plasma membrane (see Chapter 1). Water leaves cells until the intracellular osmolality equals that of ECF. This loss of water shrinks cells and causes [K+] in cells to rise. The rise in intracellular [K+] provides a driving force for the exit of K+ from cells. This sequence increases plasma [K+]. A fall in plasma osmolality has the opposite effect.

Cell Lysis Cell lysis causes hyperkalemia as a result of release of intracellular K+ into the ECF. Severe trauma (e.g., burns), tumor lysis (i.e., destruction of tumor cells by chemotherapy or natural processes), and rhabdomyolysis (i.e., destruction of skeletal muscle cells) destroy cells and release K+ and other cellular contents into the ECF.

Exercise During high-intensity exercise or physical exertion, repetitive action potentials in skeletal muscles lead to K+ loss from the muscle cells followed by redistribution between plasma and the interstitial fluid (ECF compartment). Because skeletal muscles contain the major pool of K+ in the body, plasma K+ level may increase up to 8 mEq/L and the level may be sustained during exercise. Physical conditioning or training reduces exercise-induced hyperkalemia by increasing in the number of Na,K-ATPase pumps in the skeletal muscle cells. Upon cessation of exercise, recovering muscle cells regain lost K+ by the Na,KATPase–mediated K+ uptake followed by normalization of plasma K+ level within minutes, which may be preceded by a temporary undershoot of K+ level and transient hypokalemia. Changes in the K+ level during exercise are accompanied by changes in the volume of muscle cells. The contracting muscle cells swell as they lose K+. At cessation of exercise, water moves quickly out of the muscle cells into the interstitial space from where it is slowly redistributed into intravascular space. However, movement of K+ does not seem be important for control of the muscle cell volume.

K+ Excretion by the Kidneys The kidneys play a major role in maintaining K+ balance. As illustrated in Fig. 36.3 the kidneys excrete 90% to 95% of the K+ ingested from the diet. Excretion equals intake even when intake increases by as much as 10-fold. This balance in urinary excretion and dietary intake underscores the importance of the kidneys in maintaining K+ homeostasis. Although small amounts of K+ are lost each day in feces and sweat (≈5%–10% of the K+ ingested in the diet), except during severe diarrhea, this amount is essentially constant, is not regulated, and therefore is relatively less important than the K+ excreted by the kidneys. K+ secretion from blood into tubular fluid by cells of the distal tubule (DT) and collecting duct system is the key factor in determining urinary K+ excretion (Fig. 36.4).

FIG. 36.4 K+ transport along the nephron. Excretion of K+ depends on the rate and direction of K+ transport by the late segment of the distal tubule and collecting duct. Percentages refer to the amount of filtered K+ reabsorbed or secreted by each nephron segment. Arrows indicate direction of transport. Left, Dietary K+ depletion. An amount of K+ equal to 1% of the filtered load of K+ is excreted. Right, Normal and increased dietary K+ intake. An amount of K+ equal to 15% to 80% of the filtered load is excreted. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

IN THE C LIN IC Exercise-induced changes in plasma [K+] do not usually produce symptoms and are reversed after several minutes of rest. However, vigorous exercise can lead to life-threatening hyperkalemia in individuals (1) who have endocrine disorders that affect release of insulin, epinephrine (a β-adrenergic agonist), or aldosterone; (2) whose ability to excrete K+ is impaired (e.g., renal failure); or (3) who take certain medications, such as β1-adrenergic blockers. For example, during vigorous exercise, plasma [K+] may increase by at least 2 to 4 mEq/L in individuals who take β1-adrenergic receptor

antagonists for hypertension. The heart may be also exposed to a major drop in K+ level at cessation of exercise (see earlier). This drop seems to be associated with impaired cardiac repolarization, which could potentially induce arrhythmia and sudden cardiac death in individuals with pre-existing hypokalemia, ischemic heart disease, heart failure, ventricular arrhythmia, or inherited or acquired long QT-syndrome. Because K+ is not bound to plasma proteins, it is freely filtered at the glomerulus and is nearly completely reabsorbed in the proximal tubule (through the paracellular pathway in proportion to Na+ and water) and ascending limb of Henle (where transcellular K+ transport is mediated by the apical membrane Na+/K+/2Cl− cotransporter). The reabsorptive component of K+ is independent of K+ intake. Urinary K+ excretion results primarily from secretion along the aldosterone-sensitive distal nephron (ASDN), which comprises the last portion of the distal tubule (DT), connecting tubule, and cortical collecting duct (CCD). A rise in dietary K+ intake increases K+ secretion (see Fig. 36.4, right panel). In contrast, a low-K+ diet activates K+ reabsorption along the ASDN (see Fig. 36.4, left panel).

IN THE C LIN IC In individuals with end-stage kidney disease, the kidneys are unable to excrete ingested K+ and plasma [K+] rises. The resulting hyperkalemia reduces the resting membrane potential (i.e., the voltage becomes less negative). The reduced membrane potential decreases the excitability of neurons, cardiac cells, and muscle cells by inactivating fast Na+ channels, which are critical for the depolarization phase of the action potential (see Fig. 36.1). Severe rapid increases in plasma [K+] can lead to cardiac arrest and death. In contrast, in patients taking diuretics, urinary K+ excretion often exceeds dietary K+ intake. Accordingly, K+ balance is negative and hypokalemia develops. This decline in extracellular [K+] hyperpolarizes the resting cell membrane (i.e., the voltage becomes more negative) and reduces the excitability of neurons, cardiac cells, and muscle cells. Severe hypokalemia can lead to cardiac arrhythmias, paralysis, and death. Hypokalemia can also impair the ability of the kidneys to concentrate urine and can stimulate renal production of NH4+, which affects acid-base balance (see Chapter 37). Therefore, maintenance of high intracellular [K+], low extracellular [K+], and a high [K+] gradient across cell membranes is essential for cellular functions.

Cellular Mechanism of K+ Transport by Principal and Intercalated Cells in the DT and CCD Fig. 36.5A illustrates the cellular mechanisms of K+ secretion by principal cells. Secretion from blood into the tubular lumen is a two-step process: (1) uptake of K+ from blood across the basolateral membrane by Na+,K+-ATPase and (2) transport of K+ from the cell into tubular fluid via renal outer medullary K+ (ROMK) channel and Big K+ (BK) channel. A K+/Cl− cotransporter (KCC1) in the apical plasma membrane also secretes K+. The Na+, K+-ATPase creates a high intracellular [K+] that provides the chemical driving force for exit of K+ across the apical membrane through K+ channels. Although K+ channels are also present in the basolateral membrane, K+ preferentially leaves the cell across the apical membrane and enters the tubular fluid. K+ transport follows this route for two reasons. First, the

electrochemical gradient of K+ across the apical membrane favors its downhill movement into tubular fluid. Second, the permeability of the apical membrane to K+ is greater than that of the basolateral membrane. Therefore K+ preferentially diffuses across the apical membrane into tubular fluid. K+ secretion across the apical membrane via the K+/Cl− cotransporter is driven by the favorable concentration gradient of K+ between the cell and tubular fluid. The three major factors that control the rate of K+ secretion by principal cells in DT and CCD include: (1) the activity of Na+,K+-ATPase, (2) the driving forces (electrochemical gradient for K+ channels and the chemical concentration gradient for the K+/Cl− cotransporter) for movement of K+ across the apical membrane, and (3) the permeability of apical membrane K+ channels to K+. In the DT and CCD α-intercalated cells reabsorb K+ by a H+,K+-ATPase (HKA) transport mechanism located in the apical membrane (see Fig. 36.5B). This transporter mediates K+ uptake across the apical plasma membrane in exchange for H+. The exit of K+ from intercalated cells into the blood is mediated by a K+ channel. Reabsorption of K+ is activated by a low-K+ diet.

FIG. 36.5 Cellular mechanism of K+ secretion by principal cells (A) and α-intercalated cells (B) in the late segment of the distal tubule (DT) and cortical collecting duct (CCD). α-Intercalated cells contain very low levels of Na+,K+-ATPase in the basolateral membrane (not shown). K+ depletion increases K+ reabsorption by α-intercalated cells by stimulating the H+,K+-ATPase (HKA). AE1, Anion exchanger 1; CA, carbonic anhydrase.

K+ Excretion by the DT and CCD K+ excretion by the ASDN is determined by plasma [K+], Na+ delivery and tubular fluid flow (i.e., K+ sensing), aldosterone, arginine vasopressin (AVP), glucocorticoid levels, and acid-base status.

Plasma [K+] Plasma [K+] is an important determinant of K+ secretion by the DT and CCD. Hyperkalemia stimulates secretion of K+ within minutes by several mechanisms. First, hyperkalemia stimulates Na+, K+-ATPase

and thereby increases K+ uptake across the basolateral membrane. This uptake raises intracellular [K+] and increases the electrochemical driving force for exit of K+ across the apical membrane. Second, hyperkalemia also increases the permeability of the apical membrane to K+. Third, hyperkalemia stimulates secretion of aldosterone by the adrenal cortex, which as discussed later, acts synergistically with plasma [K+] to stimulate its secretion. Fourth, hyperkalemia also increases the flow rate of tubular fluid, which as discussed later, stimulates secretion of K+ by the DT and CCD. Hypokalemia decreases K+ secretion via actions opposite to those described for hyperkalemia. Hence hypokalemia inhibits Na+, K+-ATPase, decreases the electrochemical driving force for efflux of K+ across the apical membrane, reduces permeability of the apical membrane to K+, and decreases plasma aldosterone levels.

AT THE C ELLU LAR LEVEL ROMK is the primary channel in the apical membrane of principal cells that mediates constitutive (as opposed flow-stimulated) K+ secretion. ROMK has a low conductance and high probability of being open under physiologic conditions. In addition, the Ca++-activated BK channel is also expressed in the apical membrane. The BK channel has large single channel conductance and is quiescent in the basal state and mediates K+ secretion under conditions of increased flow discussed earlier. Interestingly, knockout of the KCNJ1 gene encoding ROMK channel causes increased excretion of NaCl and K+ by the kidneys, thereby leading to reduced ECFV and hypokalemia. Although this effect is somewhat perplexing, it should be noted that ROMK is also expressed in the apical membrane of the thick ascending limb (TAL) of Henle’s loop, where it plays a very important role in recycling of K+ across the apical membrane, an effect that is critical for operation of the Na+/K+/2Cl− cotransporter. In the absence of ROMK, reabsorption of NaCl by the TAL is reduced, which leads to loss of NaCl in urine. Reduction of NaCl reabsorption by the TAL also reduces the positive transepithelial luminal voltage, which is the driving force for reabsorption of K+ by this nephron segment. Thus the reduction in paracellular K+ reabsorption by the TAL increases urinary K+ excretion even when the cortical collecting duct is unable to secrete the normal amount of K+ because of a lack of ROMK channels. The CCD, however, does secrete K+ even in ROMK knockout mice via the flow and Ca++-dependent BK channel expressed in the apical membrane of principal cells.

IN THE C LIN IC Chronic hypokalemia ([K+] < 3.5 mEq/L) may occur in patients who receive diuretics, abuse laxatives, have profound vomiting or diarrhea, undergo nasogastric suction, or have hyperaldosteronism. Hypokalemia occurs because renal K+ excretion exceeds dietary intake of K+. Vomiting, nasogastric suction, diuretics, and diarrhea can all decrease ECF volume, which in turn stimulates secretion of aldosterone (see Chapter 35). Because aldosterone stimulates excretion of K+ by the kidneys, its action contributes to development of hypokalemia. Hypokalemic nephropathy is a chronic condition in patients with total body K+ depletion and is characterized by volume depletion and hyperaldosteronism. It is frequently a progressive form of chronic kidney disease that may lead to end-stage kidney disease. Chronic hyperkalemia ([K+] > 5.0 mEq/L) occurs most frequently in individuals with reduced urine flow, low plasma aldosterone levels, and renal disease in which the glomerular filtration rate (GFR)

falls below 20% of normal. In these individuals, hyperkalemia occurs because renal K+ excretion is lower than dietary K+ intake. Less common causes of hyperkalemia occur in people with deficiencies in insulin or aldosterone secretion or in people with metabolic acidosis caused by inorganic acids.

Na+ Delivery and Tubular Fluid Flow (K+ Sensing by Renal Epithelial Cells) K+ secretion is induced by increased Na+ delivery and flow of tubular fluid to the ASDN. This effect begins in the early portion of the DT, where Na+ transport is driven by the thiazide-sensitive Na+/Cl− cotransporter. Increased plasma [K+] is detected by the K+ sensors Kir 4.1/5.1 channels located in the basolateral membrane of the early segment of the DT (Fig. 36.6A). Sensing of increased plasma [K+] by Kir 4.1/5.1 initiates a signaling cascade that dephosphorylates and inhibits the Na+/Cl− cotransporter. Inhibition of the Na+/Cl− cotransporter results in greater Na+ delivery and tubular fluid flow to the ASDN and leads to increased renal K+ excretion. Decreased intake and decreased plasma [K+] activates the Na+/Cl− cotransporter in the early DT and limit K+ secretion by reducing Na+ delivery and flow to the ASDN (Fig. 36.6B).

FIG. 36.6 Mechanisms of epithelial K+ sensing. A, Increased plasma [K+] is detected by Kir4.1/5.1 channels in the early portion of the DT that inactivate NCC (Na+/Cl− cotransporter) leading to increased Na+ delivery to the aldosterone-sensitive distal nephron (ASDN) increasing tubular fluid flow and renal excretion of Na+ and K+. B, Decreased plasma [K+] activates the WNK pathway that phosphorylates NCC (Na+/Cl− symporter) activating it to stimulate Na+ absorption and decrease Na+ delivery to the ASDN decreasing tubular fluid flow and renal excretion of Na+ and K+. ASDN, aldosterone-sensitive distal nephron; CCD, cortical collecting duct; DT, distal tubule.

A rise in the flow of tubular fluid (e.g., with diuretic treatment, ECF expansion) stimulates secretion of K+ within minutes, whereas ECF contraction caused by hemorrhage, severe vomiting, or diarrhea reduces secretion of K+ by the ASDN. Increments in tubular fluid flow are more effective in stimulating secretion of K+ as dietary K+ intake is increased. Increased flow bends the primary cilium in principal cells, which activates the polycystin (PKD)1/PKD2 Ca++-conducting channel complex. This allows more Ca++ to enter principal cells and increases intracellular [Ca++]. The increase in [Ca++] activates BK K+ channels in the

apical plasma membrane, which enhances K+ secretion from the cell into the tubule fluid. As flow increases, such as after administration of diuretics or as the result of an increase in ECF, so does the [Na+] of tubule fluid. This increase in [Na+] facilitates entry of Na+ across the apical membrane of ASDN cells, thereby decreasing the cells’ interior negative membrane potential. This depolarization of the cell membrane potential increases the electrochemical driving force that promotes secretion of K+ across the apical cell membrane into tubule fluid. In addition, increased uptake of Na+ into cells activates the Na+,K+-ATPase in the basolateral membrane, thereby increasing uptake of K+ across the basolateral membrane and consequently elevating cell [K+]. It is important to note that an increase in flow rate during a water diuresis does not have a significant effect on excretion of K+, most likely because during a water diuresis the [Na+] of tubule fluid does not increase as flow rises.

IN THE C LIN IC Since the agricultural revolution, the human diet evolved from a high K+–low Na+ to a low K+–high Na+ diet. Recommendations for the adequate K+ intake for adults generally range between 90 and 100 mEq/day (3500–4000 mg/day). In a worldwide analysis, K+ intake (estimated from urinary K+ excretion in adults) was 40%–50% lower than the recommended intake. A low-K+ diet is associated with increased risk of adverse cardiovascular effects including hypertension and hypokalemic nephropathy. A low-K+ diet increases the activity of the Na+/Cl− cotransporter in the DT. This makes sense physiologically, because increased sodium reabsorption by the Na+/Cl− cotransporter reduces Na+ delivery to downstream K+-secreting nephron segments and therefore helps to conserve K+. The effect of a low-K+ diet, which reduces Na+ excretion by the kidneys, has been linked to the pathogenesis of salt-sensitive hypertension. Conservation of K+ and Na+ by the kidneys when there is K+ deficiency may have evolved because simultaneous deficiency of dietary K+ and Na+ was probably faced by early humans. At the present time when the dietary Na+ intake is high and K+ intake is low, the response of the kidneys to retain K+ and Na+ may lead to salt-sensitive hypertension.

Aldosterone Chronically elevated (≥24 hours) plasma aldosterone levels enhance K+ secretion across principal cells in the ASDN (Fig. 36.7): (1) by increasing the amount of Na+,K+-ATPase in the basolateral membrane; (2) by increasing expression of the epithelial sodium channel (ENaC) in the apical cell membrane; (3) by elevating SGK1 (serum glucocorticoid-stimulated kinase) levels, which also increases expression of Na+ (ENaC) channels in the apical membrane and activates K+ channels; (4) by stimulating CAP1 (channelactivating protease, also called prostatin), which directly activates ENaC; and (5) by stimulating the permeability of the apical membrane to K+. Aldosterone increases the permeability of the apical membrane to K+ by increasing the number of K+ channels in the membrane. However, the cellular mechanisms involved in this response are not completely known. Increased expression of Na+,K+-ATPase facilitates uptake of K+ across the basolateral membrane into cells and thereby elevates intracellular [K+]. The increased number and activity of Na+ channels enhance entry of Na+ into the cell from tubular fluid, an effect that depolarizes the apical membrane voltage. Depolarization of the apical membrane and increased intracellular [K+] enhance the electrochemical driving force for secretion of K+ from the cell into the tubule fluid. Taken together, these actions increase uptake of K+ into the cell across the

basolateral membrane and enhance exit of K+ from the cell across the apical membrane. Secretion of aldosterone is increased by hyperkalemia and by angiotensin II (after activation of the renin-angiotensin system). Secretion of aldosterone is decreased by hypokalemia and natriuretic peptides released from the heart.

FIG. 36.7 Effects of aldosterone on secretion of K+ by principal cells in the late segment of the distal tubule and collecting duct. Numbers refer to the five effects of aldosterone discussed in the text.

Although an acute (within hours) increase in aldosterone levels enhances the activity of Na+,K+ATPase, K+ excretion does not increase immediately. The delay results from the effect of aldosterone on Na+ reabsorption and tubular flow. Aldosterone stimulated Na+ and water reabsorption decreases tubular flow that, in turn, decreases K+ secretion (as discussed in more detail later). However, chronic stimulation of Na+ reabsorption increases the ECF volume and thereby returns tubular flow to normal. These actions allow a direct stimulatory effect of aldosterone on the ASDN to enhance K+ excretion.

AVP Although AVP does not affect urinary K+ excretion, this hormone promotes secretion of K+ by the ASDN (Fig. 36.8). AVP increases the electrochemical driving force for exit of K+ across the apical membrane of principal cells by stimulating uptake of Na+ across the apical membrane of these cells. The increased Na+ uptake reduces the electrical potential difference across the apical membrane (i.e., the interior of the cell becomes less negative). Despite this effect, AVP does not change K+ secretion by these nephron segments. The reason for this relates to the effect of AVP on tubular fluid flow. AVP decreases flow of tubular fluid by stimulating water reabsorption. The decrease in tubular flow in turn reduces secretion of K+ (explained later). The inhibitory effect of decreased flow of tubular fluid offsets the stimulatory effect of AVP on the electrochemical driving force for exit of K+ across the apical membrane (see Fig. 36.8). If AVP did not increase the electrochemical gradient favoring K+ secretion, urinary K+ excretion would fall as AVP levels increased and urinary flow rates decreased. Hence K+ balance would change in response to alterations in water balance. Thus the effects of AVP on the electrochemical driving force for exit of K+

across the apical membrane and on tubule flow enable urinary K+ excretion to be maintained constant despite wide fluctuations in water excretion.

FIG. 36.8 Opposing effects of AVP and urine flow rate on secretion of K+ by the ASDN. K+ secretion is stimulated by an increase in urinary flow rate and reduced by a fall in AVP levels. In contrast, K+ secretion is reduced by a decrease in urinary flow rate and increased by a rise in AVP levels. Because the effects of flow and AVP oppose each other, net K+ secretion is not affected by water diuresis or antidiuretics. AVP, Arginine vasopressin; CCD, cortical collecting duct; DT, distal tubule.

Glucocorticoids Glucocorticoids increase urinary K+ excretion. This effect is mediated in part by increasing GFR, which enhances the urinary flow rate, a potent stimulus of K+ excretion, and by stimulation of SGK1 activity (see earlier). As discussed, the rate of urinary K+ excretion is frequently determined by simultaneous changes in hormone levels, acid-base balance, or the flow rate of tubule fluid (Table 36.1). The powerful effect of flow often enhances or opposes the response of the ASDN to hormones and changes in acid-base balance. This interaction can be beneficial in the case of hyperkalemia, in which the increase in flow enhances excretion of K+ and thereby restores K+ homeostasis. However, this interaction can also be detrimental, as in the case of metabolic alkalosis, in which changes in flow and acid-base status alter K+ homeostasis.

Table 36.1 Effects of Hormones and Other Factors on Renal K+ Homeostasis and Effects on Plasma [K+] Condition

K+ Secretion by ASDN Tubular Fluid Flow Renal K+ Excretion Change in Plasma [K+]

Hyperkalemia

Increase

Increase

Increase

Decrease

Aldosterone









Acute

Increase

Decrease

No change

Decrease

Chronic

Increase

No change

Increase

Decrease

Glucocorticoids

Increase

Increase

Increase

Decrease

AVP

Increase

Decrease

No change

Decrease

Acidosis









Acute

Decrease

No change

Decrease

Increase

Chronic

Decrease

Large increase

Increase

Decrease

Alkalosis

Increase

Increase

Large increase

Decrease

ASDN, Aldosterone-sensitive distal nephron. Modified from Field MJ et al. In: Narins R, ed. Textbook of Nephrology: Clinical Disorders of Fluid and Electrolyte Metabolism. 5th ed. New York: McGraw-Hill; 1994.

The Acid-Base Status Both acute (within minutes to hours) and chronic (within days) acid-base disturbances have complex effects on K+ handling by the ASDN and renal K+ excretion. The effects of metabolic acidosis on renal K+ excretion are time dependent. As illustrated in Fig. 36.9, an acute acidemia (i.e., plasma pH below normal) reduces K+ secretion via two mechanisms: (1) inhibition of Na+,K+-ATPase that reduces the intracellular [K+] and the electrochemical driving force for K+ exit across the apical membrane; and (2) by decreasing permeability of the apical membrane to K+. Acute alkalemia (i.e., plasma pH above normal) has the opposite effects and increases K+ secretion.

FIG. 36.9 Acute versus chronic effect of metabolic acidosis on excretion of K+. See text for details. ECV, Effective circulating volume.

Chronic acidemia promotes renal K+ excretion leading to negative K+ balance (see Fig. 36.9). This occurs because chronic metabolic acidosis decreases reabsorption of water and solutes (e.g., NaCl) by inhibiting Na+,K+-ATPase in the proximal tubule. Hence the flow of tubular fluid is augmented along the ASDN. Inhibition of water and NaCl reabsorption by the proximal tubule also decreases ECF volume and thereby stimulates secretion of aldosterone. In addition, chronic acidosis caused by inorganic acids increases plasma [K+], which stimulates secretion of aldosterone. The rise in tubular fluid flow, plasma [K+], and aldosterone levels offsets the effects of acidosis on cell [K+] and apical membrane permeability, and K+ secretion rises. Thus metabolic acidosis may either inhibit or stimulate excretion of K+, depending on the duration of the disturbance. As noted, acute metabolic alkalosis stimulates excretion of K+. Chronic metabolic alkalosis, especially in association with ECF contraction, significantly increases renal K+ excretion because of the associated increase in aldosterone levels. The directional effects of acidemia and alkalemia on K+ excretion are similar in respiratory acid-base disturbances as in metabolic disturbances, but the effects of respiratory disorders on K+ excretion tend to be smaller than metabolic acid-base disturbances. Acute respiratory alkalosis, induced by hyperventilation, is associated with α-adrenergic stimulation that increases plasma [K+] by inhibiting

intracellular K+ uptake. Acute respiratory alkalosis is a frequent acid-base disturbance in clinical settings including chest pain, anxiety, drugs, hypoxemia, and infection. Chronic respiratory alkalosis usually increases renal K+ excretion and lowers plasma [K+].

AT THE C ELLU LAR LEVEL The cellular mechanisms whereby the dietary K+ content and acid-base status regulate secretion of K+ by the early segment of the ASDN have recently been elucidated. Elevated K+ intake increases secretion of K+ by several mechanisms. Hyperkalemia increases the activity of the ROMK channel in the apical plasma membrane of principal cells. Moreover, hyperkalemia inhibits reabsorption of NaCl and water by the proximal tubule, thereby increasing the ASDN flow rate, a potent stimulus to secretion of K+ (Fig. 36.6). Hyperkalemia also increases aldosterone levels, which increases K+ secretion by three mechanisms. First, aldosterone increases the number of K+ channels in the apical plasma membrane. Second, aldosterone stimulates uptake of K+ across the basolateral membrane by increasing the number of Na+,K+-ATPase pumps, thereby enhancing the electrochemical gradient driving secretion of K+ across the apical membrane. Third, aldosterone increases movement of Na+ across the apical membrane, which depolarizes the apical plasma membrane voltage and thus increases the electrochemical gradient, promoting secretion of K+. A low-K+ diet dramatically reduces secretion of K+ by the ASDN by increasing the activity of a tyrosine kinase, which causes ROMK channels to be endocytosed from the apical plasma membrane, thereby reducing K+ secretion. Metabolic acidosis with acidemia decreases secretion of K+ by inhibiting the activity of ROMK channels, whereas metabolic alkalosis with alkalemia stimulates secretion of K+ by enhancing ROMK channel activity.

Overview of Calcium and Inorganic Phosphate Homeostasis Ca++ and inorganic phosphate (Pi)a are multivalent ions that subserve many complex and vital functions. Ca++ is a cofactor in enzymatic reactions and a second messenger in many signaling pathways critical for the homeostasis. Pi is essential for metabolic processes, including formation of adenosine triphosphate (ATP), and it is an important component of nucleotides, nucleosides, and phospholipids. Phosphorylation of proteins is an important mechanism of cellular signaling, and Pi is an essential buffer in cells, plasma, and urine. Ca++ and Pi are critical elements of the extracellular matrix, cartilage, teeth, and bone. The kidneys regulate total body Ca++ and Pi by excreting the amount of Ca++ and Pi that is absorbed by the GI tract (normal bone remodeling results in no net addition of Ca++ and Pi to, or Ca++ and Pi release from, bone). If plasma concentrations of Ca++ and Pi decline substantially, intestinal absorption, bone resorption (i.e., loss of Ca++ and Pi from bone), and renal tubular reabsorption increase and return plasma concentrations of Ca++ and Pi to normal levels. During growth and pregnancy, intestinal absorption exceeds urinary excretion, and these ions accumulate in newly formed fetal tissue and bone. In contrast, bone disease (e.g., osteoporosis) or a decline in lean body mass increases urinary Ca++ and Pi loss without a change in intestinal absorption. These conditions produce a net loss of Ca++ and Pi from the body. Finally, during chronic renal failure, Pi accumulates in the body because the intestinal absorption of Pi exceeds the urinary excretion leading to accumulation of Pi in the body and bone remodeling (see the In

The Clinic box discussion of end-stage kidney disease). This brief introduction reveals that kidneys, in conjunction with the GI tract and bone, play a major role in maintaining plasma Ca++ and Pi levels as well as Ca++ and Pi homeostasis (see Chapter 40). Accordingly, this section of the chapter discusses Ca++ and Pi handling by the kidneys, with an emphasis on the hormones and factors that regulate urinary excretion.

Calcium Cellular processes in which Ca++ plays an important role include bone formation, cell division and growth, hemostasis, hormone-response coupling, and electrical stimulus-response coupling (e.g., muscle contraction, neurotransmitter release). Nearly 99% of Ca++ is stored in bone and teeth, approximately 1% is found in ICF, and 0.1% in ECF. The total [Ca++] in plasma is 10 mg/dL (2.5 mM or 5 mEq/L), and its concentration is normally maintained within very narrow limits. Approximately 50% of the Ca++ in plasma is ionized (i.e., free), 40% is bound to plasma proteins (mainly albumin), and 10% is complexed to several anions, including PO43−, HCO3−, citrate, and SO42− (Fig. 36.10). The pH of plasma influences this distribution (Fig. 36.11). The total measured plasma [Ca++] does not reflect the physiologically relevant ionized [Ca++]. Acidemia increases the ionized [Ca++] at the expense of Ca++ bound to proteins, whereas alkalemia decreases the ionized [Ca++] by increasing the Ca++ bound to proteins. Individuals with alkalemia are susceptible to tetany (tonic muscular spasms), whereas individuals with acidemia are less susceptible to tetany, even when total plasma Ca++ levels are reduced. The increase in [H+] in patients with metabolic acidosis causes more H+ to bind to plasma proteins, PO43−, HCO3−, citrate, and SO42−, thereby displacing Ca++. This displacement increases the plasma ionized [Ca++]. In alkalemia the [H+] of plasma decreases. Some H+ ions dissociate from plasma proteins, PO43−, HCO3−, citrate, and SO42− in exchange for Ca++, thereby decreasing the ionized [Ca++]. The plasma albumin concentration also affects [Ca++]. Hypoalbuminemia decreases the total [Ca++] and may not accurately reflect the ionized [Ca++], whereas hyperalbuminemia has the opposite effect on total [Ca++]. It is widely accepted in clinical practice to assume that the total [Ca++] falls by 0.8 mg/dL (0.2 mmol/L) for every 1 g/dL (10 g/L) fall in the serum albumin concentration.

FIG. 36.10 Distribution of Ca++ in plasma. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

FIG. 36.11 Effect of pH on plasma [Ca++]. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

A low ionized [Ca++] increases the excitability of nerve and muscle cells and can lead to hypocalcemic tetany. Tetany associated with hypocalcemia occurs because hypocalcemia causes the threshold potential to shift to more negative values (i.e., closer to the resting membrane voltage) (Fig. 36.12). An elevated ionized [Ca++] may decrease neuromuscular excitability or produce cardiac arrhythmias, lethargy, disorientation, and even death. This effect of hypercalcemia occurs because an elevated ionized [Ca++] causes the threshold potential to shift to less negative values (i.e., farther from the resting membrane voltage). The [Ca++] is regulated within a very narrow range, primarily by parathyroid hormone (PTH) and the active metabolite of vitamin D calcitriol (1,25-dihydroxyvitamin D3).

FIG. 36.12 Effect of Ca++ on nerve and muscle excitability. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

Intracellular Ca++ is sequestered in the endoplasmic reticulum and mitochondria, or it is bound to proteins. Thus the intracellular ionized [Ca++] is very low (≈100 nM). The large concentration gradient for [Ca++] across cell membranes is maintained by a Ca++-ATPase pump (PMCa1b) in all cells and by a 3Na+/Ca++ exchanger (NCX1) in some cells. Overview of Calcium Homeostasis Ca++ homeostasis depends on: (1) the Ca++ absorption from the GI tract, (2) the distribution of Ca++ between the bone and ECF, and (3) the regulation of Ca++ excretion by the kidneys. The total body Ca++ content is determined by the amounts of Ca++ absorbed from the GI tract and excreted by the kidneys (Fig. 36.13). The GI tract absorbs Ca++ through an active carrier-mediated transport mechanism that is stimulated by calcitriol produced in the proximal tubule of the kidneys. Net Ca++ absorption from the GI tract is approximately 200 mg/day, but it can increase to 600 mg/day when calcitriol levels rise. Daily Ca++ excretion by the kidneys equals the amount absorbed by the GI tract (200 mg/day), and it changes in parallel with intestinal absorption. Thus Ca++ balance is maintained because the amount of Ca++ ingested in an average diet (1000 mg/day) equals the amount lost in feces (800 mg/day) plus the amount excreted in urine (200 mg/day).

FIG. 36.13 Overview of Ca++ homeostasis. PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

The control of Ca++ distribution between the bone and ECF is mediated by PTH and calcitriol (see Fig. 36.13). PTH is secreted by the parathyroid glands in response to decreased plasma [Ca++] (i.e., hypocalcemia). PTH increases plasma [Ca++] by: (1) stimulating bone resorption, (2) increasing Ca++ reabsorption by the DT of the kidneys, and (3) stimulating the production of calcitriol, which in turn increases Ca++ absorption by the GI tract. Production of calcitriol in the kidneys is stimulated by hypocalcemia and hypophosphatemia. Calcitriol increases plasma [Ca++], primarily by stimulating Ca++ absorption from the GI tract. It also enhances renal Ca++ reabsorption by increasing expression of the Ca++ binding and transporting proteins in the kidneys (details discussed later). Plasma Ca++ is an agonist of the calcium-sensing receptor (CaSR), expressed on the surface of cells involved in Ca++ homeostasis: PTH-secreting parathyroid cells, calcitonin-secreting thyroid cells, calcitriol-producing proximal tubular cells, and TAL cells (discusser later). CaSR, activated by increased ionized [Ca++], inhibits PTH release by the parathyroid gland and calcitriol production by the proximal tubule. The net effects of CaSR activation are decreased renal Ca++ reabsorption, decreased plasma [Ca++] and blunted PTH-mediated phosphaturic effect (less renal Pi excretion). CaSR plays a major role in the steady-state plasma [Ca++] by responding immediately to small changes plasma [Ca++]. Calcitonin is secreted by thyroid C cells (parafollicular cells), and its secretion is stimulated by hypercalcemia. Calcitonin decreases plasma [Ca++], mainly by stimulating bone formation (i.e., deposition of Ca++ in bone). Although it plays an important role in Ca++ homeostasis in lower vertebrates, calcitonin plays only a minor role in Ca++ homeostasis in humans, so it will not be discussed further.

IN THE C LIN IC Conditions that lower PTH levels (i.e., hypoparathyroidism after parathyroidectomy for an adenoma) reduce plasma [Ca++], which can cause hypocalcemic tetany (intermittent muscular contractions). In severe cases, hypocalcemic tetany can cause death by asphyxiation. Hypercalcemia can cause lethal cardiac arrhythmias and decreased neuromuscular excitability. Clinically hypercalcemia is most commonly caused by primary hyperparathyroidism and malignancy. Primary hyperparathyroidism

results from overproduction of PTH by a benign tumor of the parathyroid glands. In contrast, malignant tumors such as carcinomas secrete a PTH-like hormone maned parathyroid hormone–related peptide (PTHrP). Increased levels of PTH and PTHrP lead to hypercalcemia and hypercalciuria (increased urinary calcium excretion). Calcium Transport Along the Nephron The Ca++ available for glomerular filtration consists of the ionized fraction and the Ca++ complexed with anions. Thus about 60% of the Ca++ in plasma is available for glomerular filtration. Normally 99% of filtered Ca++ is reabsorbed by the nephron (Fig. 36.14). The proximal tubule reabsorbs about 50% to 60% of the filtered Ca++. Another 15% is reabsorbed in the loop of Henle (mainly the cortical portion of the TAL), about 10% to 15% is reabsorbed by the DT, and less than 1% is reabsorbed by the collecting duct. About 1% (200 mg/day) is excreted in urine. This fraction is equal to the net amount absorbed daily by the GI tract.

FIG. 36.14 Overview of Ca++ transport along the nephron. Percentages refer to amount of filtered Ca++ reabsorbed by each segment. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

Ca++ reabsorption by the proximal tubule occurs primarily via the paracellular pathway. This passive paracellular reabsorption of Ca++ is driven by the lumen-positive transepithelial voltage across the second half of the proximal tubule and by a favorable concentration gradient of Ca++, both of which are established by transcellular Na+ and water reabsorption in the first half of the proximal tubule (see Chapter 34). Ca++ reabsorption by the loop of Henle also occurs primarily via the paracellular pathway. Like the proximal tubule, Ca++ and Na+ reabsorption in the TAL parallel each other. These processes are parallel because Ca++ is reabsorbed passively via the paracellular route secondary to Na+ reabsorption that generates a lumen-positive voltage. Loop diuretics inhibit Na+ reabsorption by the TAL of the loop of Henle, and in so doing reduce the magnitude of the lumen-positive voltage (see Chapter 34). This action

in turn inhibits reabsorption of Ca++ via the paracellular pathway. Thus loop diuretics can be used to increase renal Ca++ excretion in patients with hypercalcemia.

AT THE C ELLU LAR LEVEL Mutations in the tight junction protein claudin-16 (CLDN16) reduce the permeability of the paracellular pathway to Ca++ and Mg++ and thereby reduce the diffusive reabsorptive movement of Ca++ and Mg++ across tight junctions in the TAL of Henle’s loop. Familial hypomagnesemic hypercalciuria is caused by mutations in claudin-16, which is a component of the tight junctions in TAL cells. This disorder is characterized by enhanced excretion of Ca++ and Mg++ due to a fall in passive reabsorption of these ions across the paracellular pathway in the TAL. Affected individuals have high levels of Ca++ in their urine, which may lead to kidney stone formation (nephrolithiasis). In the DT where the voltage in the tubule lumen is electrically negative with respect to blood, reabsorption of Ca++ is entirely active because Ca++ is reabsorbed against its electrochemical gradient (Fig. 36.15). Thus Ca++ reabsorption by the DT is exclusively transcellular. Ca++ enters the cell across the apical membrane through Ca++-permeable ion channels (TRPV5). Inside the cell, Ca++ binds to calbindin-D28K. The calbindin-Ca++ complex carries Ca++ across the cell and delivers Ca++ to the basolateral membrane, where it is extruded from the cell primarily by the 3Na+/1Ca++ antiporter (NCX1); however, plasma membrane Ca++-ATPase isoform 1b (PMCA1b) may also contribute. Urinary Na+ and Ca++ excretion usually change in parallel. However, excretion of these ions does not always change in parallel, because reabsorption of Ca++ and Na+ by the DT is independent and differentially regulated. For example, thiazide diuretics inhibit Na+ reabsorption by the DT and stimulate Ca++ reabsorption by this segment. Accordingly, the net effects of thiazide diuretics are to increase urinary Na+ excretion and reduce urinary Ca++ excretion. Because thiazide diuretics reduce urinary Ca++ excretion, they are often used to reduce urinary Ca++ excretion in individuals who produce Ca++-containing kidney stones.

FIG. 36.15 Cellular mechanism of Ca++ reabsorption by the distal tubule. Ca++ is reabsorbed exclusively by a cellular pathway. Ca++ enters the cell across the apical membrane via a Ca++-permeable ion channel (TRPV5). Inside cells, Ca++ binds to calbindin (calbindin-D28K), and the Ca++-calbindin complex diffuses across the cell to deliver Ca++ to the basolateral membrane. Ca++ is transported across the basolateral membrane primarily by a 3 (or 4) Na+/Ca++ antiporter (NCX1) and also by a Ca++-H+-ATPase (PMCa1b). Claudin 8 (CLDN8) is a tight junction protein that is impermeable to Ca++ and thereby prevents the back diffusion of Ca++ across the tight junction into the tubule lumen, which is electrically negative compared to the blood side of the cell. CB, Calbindin-D28K.

Regulation of Urinary Calcium Excretion Several hormones and factors influence urinary Ca++ excretion. PTH exerts the most powerful control (Table 36.2). PTH stimulates Ca++ reabsorption by the kidneys (i.e., inhibits renal Ca++ excretion). Although PTH inhibits reabsorption of Na+ and water, and therefore Ca++ reabsorption by the proximal tubule, it stimulates Ca++ rea​bsorption by the TAL of the loop of Henle and the DT. Thus the net effect of PTH is to enhance renal Ca++ reabsorption.

Table 36.2 Summary of Hormones, Factors, and Diuretics Affecting Ca++ Reabsorption

Nephron Location

Factor/Hormone

Proximal Tubule

TAL

Distal Tubule

PTH (PTHrP)a

Decrease

Increase

Increase

Calcitriol





Increase

Volume expansion

Decrease

No change

Decrease

Hypercalcemia

Decrease

Decrease (via CaSR)

Decrease (via PTH)

Hypocalcemia

Increase

Increase



Hyperphosphatemia





Increase (via PTH)

Hypophosphatemia

Decrease



Decrease (via PTH)

Acidemia





Decrease

Alkalemia





Increase

Loop diuretics



Decrease



Thiazide diuretics





Increase

aPTH inhibits Ca++ reabsorption by the proximal tubule but stimulates reabsorption by the TAL and distal tubule. Overall the net

effect is to increase Ca++ reabsorption and thereby reduce urinary Ca++ excretion. CaSR, calcium-sensing receptor; PTH, parathyroid hormone; PTHrP, parathyroid hormone–related peptide; TAL, thick ascending limb. Modified from Mount DB, Yu A. Transport of inorganic solutes: sodium, chloride, potassium, magnesium, calcium and phosphate. In: Brenner BM, ed. Brenner and Rector’s The Kidney. 8th ed. Philadelphia: Saunders; 2008.

Changes in plasma [Ca++] also regulate urinary Ca++ excretion, with hypercalcemia increasing excretion and hypocalcemia decreasing excretion. Hypercalcemia increases urinary Ca++ excretion by: (1) reducing proximal tubule Ca++ reabsorption (reduced paracellular reabsorption due to increased interstitial fluid [Ca++]); (2) inhibiting Ca++ reabsorption by the TAL of the loop of Henle via activation of the CaSR located in the basolateral membrane of these cells (Na+ reabsorption is decreased, thereby reducing the magnitude of the lumen-positive); and (3) suppressing Ca++ reabsorption by the DT by reducing PTH levels. As a result, urinary Ca++ excretion increases. Hypocalcemia has the opposite effect on urinary Ca++ excretion, primarily by increasing Ca++ reabsorption by the proximal tubule and TAL. Calcitriol enhances Ca++ reabsorption by the DT, but it is less effective than PTH. Several factors affect renal Ca++ excretion. Increased plasma [Pi] (e.g., caused by increased dietary Pi load or reduced kidney function) inhibits renal Ca++ excretion by reducing plasma ionized [Ca++] with subsequent stimulation of PTH secretion. A decline in plasma [Pi] (e.g., caused by dietary Pi depletion) has the opposite effect (Note: with normal kidney function, changes in dietary Pi intake over a sevenfold range have no effect on plasma [Pi]). The CaSR expressed in the TAL directly increases renal Ca++ excretion in response to elevated plasma ionized [Ca++] (discussed earlier). By contrast, a fall in plasma [Ca++] leads to an increase in Ca++ absorption by the TAL and a corresponding decrease in urinary Ca++ excretion. The direct effect of plasma [Ca++] on CaSR in the TAL acts in parallel with PTH, which

regulates Ca++ absorption by the DT and controls urinary Ca++ excretion to maintain its homeostasis. Changes in ECF volume alter Ca++ excretion mainly by affecting Na+ and water reabsorption in the proximal tubule. ECF contraction increases Na+ and water reabsorption by the proximal tubule and thereby enhances Ca++ reabsorption. Accordingly, urinary Ca++ excretion declines. ECF expansion has the opposite effect. Acidemia increases Ca++ excretion, whereas alkalemia decreases excretion. Regulation of Ca++ reabsorption by pH occurs primarily in the DT. Alkalosis stimulates the apical membrane Ca++ channel (TRPV5), thereby increasing Ca++ reabsorption. By contrast, acidosis inhibits the same channel, thereby reducing Ca++ reabsorption. As noted earlier, loop diuretics inhibit Ca++ reabsorption by the TAL, and thiazide diuretics stimulate Ca++ reabsorption by the DT.

IN THE C LIN IC Mutations in the gene coding for CaSR cause disorders in the Ca++ homeostasis. Familial hypocalciuric hypercalcemia (FHH) is a haploinsufficient state caused by an inactivating mutation in the CaSR gene. The hypercalcemia is caused by altered Ca++-regulated PTH secretion (i.e., the set point for Ca++-regulated PTH secretion is shifted) such that PTH levels are elevated at any level of plasma [Ca++] and are not suppressed by hypercalcemia. Enhanced Ca++ reabsorption in the TAL and DT owing to elevated PTH levels and defective CaSR regulation of Ca++ transport in the kidneys lead to hypocalciuria. Autosomal dominant hypoparathyroidism is caused by an activating mutation in the CaSR gene. Activation of CaSR changes the set point for Ca++-regulated PTH secretion such that PTH levels are decreased at any level of plasma [Ca++]. Decreased PTH levels and defective CaSRregulated Ca++ transport in the kidneys lead to hypercalciuria. CaSR activates the thiazide-sensitive Na+/Cl− cotransporter in the early segment of the DT via the WNK (with no K = lysine) kinase signaling pathway (see Chapter 34). Activation of the Na+/Cl− cotransporter reduces Ca++ reabsorption leading to hypercalciuria. Inactivation of the cotransporter increases Ca++ reabsorption and hypercalciuria resolves. Thus activation of CaSR increases activity of the Na+/Cl− cotransporter leading to increased NaCl reabsorption, exacerbation of renal Ca++ excretion, and hypercalcuria.

Phosphate Pi is an important component of many essential cellular components, including DNA, RNA, ATP, nucleotides, nucleosides, phospholipids and intermediates of metabolic pathways. Like Ca++ it is a major constituent of bone. Its concentration in plasma is an important determinant of bone formation and resorption. In addition, urinary Pi is an important buffer (i.e., it is one of many titratable acids) involved in the maintenance of acid-base balance (see Chapter 37). Approximately 85% of Pi is located in bone and teeth, 14% is located in the ICF, and 1% in the ECF. Normal plasma [Pi] is 3 to 4 mg/dL (1–1.5 mM). Plasma Pi is ionized (45%), complexed (30%), or bound to protein (25%). Pi deficiency causes muscle weakness, rhabdomyolysis, and reduced bone mineralization resulting in rickets (in children) and osteomalacia (in adults). Overview of Phosphate Homeostasis Pi homeostasis depends on: (1) the Pi absorption from the GI tract, (2) the distribution of Pi between bone

and ECF, and (3) the regulation of Pi excretion by the kidneys (see Fig. 36.16).

FIG. 36.16 Overview of Pi homeostasis. PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

The total body Pi level is determined by the amounts of Pi absorbed from the GI tract and excreted by the kidneys. Pi absorption from the GI tract occurs via active and passive mechanisms; Pi absorption increases as dietary Pi rises, and it is stimulated by calcitriol. Despite variations in Pi intake between 800 and 1500 mg/day, in adults at the steady state the kidneys maintain total body Pi balance constant by excreting an amount of Pi in the urine equal to the amount absorbed by the GI tract (normal bone remodeling results in no net Pi addition or release from bone). By contrast, during growth, Pi is accumulated in the body. Renal Pi excretion is the primary mechanism by which the body regulates Pi balance and homeostasis. Plasma [Pi] is controlled by PTH, calcitriol, and FGF-23. PTH releases Pi from bone (see Fig. 36.16). Renal Pi excretion is increased by PTH and inhibited by calcitriol. Maintenance of plasma [Pi] is essential for optimal Ca++-Pi complex formation required for bone mineralization without deposition of Ca++-Pi in vascular and other soft tissues. A rise in plasma [Pi] directly stimulates PTH synthesis and release and decreases the ionized [Ca++], which stimulates PTH release by its interaction with CaSR. PTH enhances urinary Pi excretion by inhibiting Pi reabsorption in the proximal tubule. Hyperphosphatemia also decreases calcitriol production by the proximal tubule, which leads to a reduction in Pi absorption from the GI tract. Both the increase in PTH and the decrease in calcitriol reduce plasma [Pi]. Phosphate Transport Along the Nephron Fig. 36.17 summarizes Pi transport by the various portions of the nephron. The proximal tubule reabsorbs 80% of the Pi filtered by the glomerulus; the loop of Henle, DT, and CCD reabsorb negligible amounts of

Pi. Therefore approximately 20% of the Pi filtered across the glomerular capillaries is excreted in urine.

FIG. 36.17 Pi transport along the nephron. Pi is reabsorbed primarily by the proximal tubule. Percentages refer to the amount of the filtered Pi reabsorbed by each nephron segment. Approximately 20% of the filtered Pi is excreted. CCD, Cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

Pi reabsorption by the proximal tubule occurs by a transcellular route (Fig. 36.18). Pi uptake across the apical membrane of the proximal tubule occurs via two Na+/Pi cotransporters (IIa and IIc). Na+/Pi IIa transports 3Na+ with one divalent Pi (HPO42−), and carries positive charge into the cell. Na+/Pi IIc transports 2Na+ with one monovalent Pi (H2PO4−) and is electrically neutral. Pi exits across the basolateral membrane by a Pi-inorganic anion antiporter that has not been characterized.

FIG. 36.18 Cellular mechanisms of Pi reabsorption by the proximal tubule. The apical transport pathway contains two Na+/Pi symporters, one that transports three Na+ for each Pi(IIa) and one that transports two Na+ for each Pi(IIc). Pi leaves the cell across the basolateral membrane by an unknown mechanism. ATP, adenosine triphosphate.

IN THE C LIN IC In patients with end-stage kidney disease, the kidneys cannot excrete Pi. Because of continued Pi absorption by the GI tract, Pi accumulates in the body and plasma [Pi] rises. The excess Pi complexes with Ca++ and reduces the ionized plasma [Ca++]. Pi accumulation also decreases production of calcitriol. This response reduces Ca++ absorption by the GI tract, an effect that further reduces plasma [Ca++]. This reduction in plasma [Ca++] increases PTH secretion and Ca++ release from bone. These actions result in renal osteodystrophy (i.e., increased bone resorption with replacement by fibrous tissue, which renders bone more susceptible to fracture). Chronic hyperparathyroidism (i.e., elevated PTH levels due the elevated plasma Pi) during end-stage kidney disease can lead to metastatic calcifications in which Ca++ and Pi precipitate in arteries, soft tissues, and viscera. Deposition of Ca++ and Pi in the heart may cause myocardial failure. Prevention and treatment of hyperparathyroidism and Pi retention include a low-Pi diet and administration of a “phosphate binder” (i.e., an agent that forms insoluble Pi salts and thereby renders Pi unavailable for absorption from the GI tract) in the diet. Supplemental calcitriol is also prescribed to suppress PTH release. Fibroblast growth factor 23 (FGF-23) increases renal Pi excretion and thereby contributes to regulation of plasma [Pi] (see Fig. 36.16). FGF-23 is secreted by osteocytes and osteoblasts and inhibits Pi reabsorption and calcitriol production by the proximal tubule. Secretion of FGF-23 is stimulated by

sustained hyperphosphatemia, PTH, and calcitriol. Activating mutations in the FGF23 gene cause hypophosphatemia, low plasma calcitriol, and rickets/osteomalacia, whereas inactivating mutations cause hyperphosphatemia, high calcitriol levels, and calcifications in the soft tissue. Regulation of Urinary Phosphate Excretion Several hormones and factors regulate urinary Pi excretion (Table 36.3 and Fig. 36.19). Increased plasma [Pi] reduces [Ca++] and therefore increases PTH levels, which increases renal Pi excretion. PTH inhibits Pi reabsorption by the proximal tubule and thereby increases Pi excretion. PTH reduces Pi reabsorption by stimulating endocytic removal of Na+/Pi cotransporters from the brush border membrane in proximal tubular cells. Increased plasma [Pi] also increases FGF-23, which inhibits Pi reabsorption and calcitriol production by the proximal tubule. Elevated plasma [Pi] directly suppresses calcitriol production, which decreases intestinal Pi reabsorption. Dietary Pi intake also regulates Pi excretion by mechanisms unrelated to changes in PTH levels. Pi loading increases excretion, whereas Pi depletion decreases it. Changes in dietary Pi intake modulate Pi transport by altering the transport rate and the number of Na+/Pi IIa and IIc cotransporters in the apical membrane of the proximal tubule. Table 36.3 Summary of Hormones and Factors Affecting Pi Reabsorption by Proximal Tubule Factor/Hormone

Proximal Tubule Reabsorption

PTH

Decrease

FGF-23

Decrease

Hyperphosphatemia

Decrease

Hypophosphatemia

Increase

Metabolic acidosis: chronic

Decrease

Metabolic alkalosis: chronic

Increase

ECF expansion

Decrease

Growth hormone

Increase

Glucocorticoids

Decrease

FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone.

FIG. 36.19 Response to elevated plasma [Pi]. FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone. Dashed lines indicate negative feedback. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

ECF volume affects Pi excretion. ECF expansion enhances Pi excretion by: (1) increasing GFR and thus the filtered amount of Pi; (2) decreasing the Na+/Pi-coupled reabsorption, which reduces ECF volume; and (3) reducing plasma [Ca++], thereby increasing PTH, which inhibits Pi reabsorption in the proximal tubule. Acid-base balance also influences Pi excretion. Chronic acidemia increases Pi excretion, and chronic alkalemia decreases it. These effects of acid-base status, like the effect of PTH, are mediated by altered expression of the Na+/Pi cotransporters in the apical membrane. Metabolic acidosis increases the secretion of glucocorticoids, which inhibit Pi reabsorption by the proximal tubule and increase renal Pi excretion. This inhibition, together with the direct effect of acidosis on Pi reabsorption by the proximal tubule, enables the DT and collecting duct to secrete more H+ as titratable acid and to generate more HCO3− because Pi is an important urinary buffer. Growth hormone decreases Pi excretion.

IN THE C LIN IC Klotho is highly expressed in the early DT of the kidney. Klotho knockout mice have a phenotype that resembles chronic kidney disease (CKD), including soft tissue calcification, hyperphosphatemia, and elevated plasma FGF-23. Klotho exists as a membrane-bound and a soluble protein. The membranebound form is a coreceptor for FGF-23, thus Klotho promotes Pi excretion by the kidneys and reduces serum levels of calcitriol. Soluble circulating Klotho affects ion transport, Wnt signaling, and FGF-23dependent PTH synthesis, and inhibits the renin-angiotensin axis. Klotho may be a biomarker for CKD, and its deficiency may contribute to development of CKD. Moreover, experimental data also suggest that Klotho therapy may slow the progression of CKD.

Integrative Review of Parathyroid Hormone and Calcitriol on Ca++ and Pi Homeostasis As summarized in Fig. 36.20, PTH regulates Ca++ and Pi homeostasis. Hypocalcemia is the major stimulus of PTH secretion. PTH causes bone resorption, increases urinary Pi excretion, decreases urinary Ca++ excretion, and stimulates production of calcitriol, which stimulates intestinal Ca++ and Pi absorption. Because changes in Pi handling in bone, the GI tract, and kidneys tend to balance out, PTH increases plasma [Ca++] while having little effect on plasma [Pi]. Overall, a rise in PTH levels in response to hypocalcemia returns plasma [Ca++] to normal. Hypercalcemia has the opposite effect.

FIG. 36.20 Response to decreased plasma [Ca++]. Dashed lines indicate negative feedback. FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

Calcitriol plays an important role in Ca++ and Pi homeostasis (Fig. 36.21). The net effect of calcitriol is to increase plasma [Ca++] and [Pi]. Thus the major stimuli of calcitriol production are hypocalcemia and hypophosphatemia. The primary action of calcitriol is to stimulate Ca++ and Pi absorption from the GI tract. To a lesser degree, calcitriol acts with PTH to decrease renal Ca++ excretion. Calcitriol may enhance the bone resorptive effect of PTH to release Ca++ and Pi from bone during a Ca++ deficient diet.

FIG. 36.21 Vitamin D metabolism and effects on Ca++ and Pi homeostasis. Hypocalcemia (via PTH) and hypophosphatemia are the major stimuli of the metabolism of calcifediol to calcitriol in the kidneys. The net effect of calcitriol is to increase plasma [Ca++] and [Pi]. PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

IN THE C LIN IC In the absence of glucocorticoids (e.g., in Addison’s disease), excretion of Pi is depressed, as is the ability of the kidneys to excrete titratable acid and to generate new HCO3−. Growth hormone increases reabsorption of Pi by the proximal tubule. As a result, children are in positive Pi balance and have a higher plasma [Pi] than adults, and the elevated [Pi] is important for bone formation and growth.

Key Concepts 1. K+ homeostasis is maintained by the kidneys, which adjust K+ excretion to match dietary K+ intake, and by insulin, epinephrine, aldosterone, AVP, and glucocorticoids, which regulate the distribution of K+ between the ICF and ECF and the renal K+ excretion. Other conditions, such as cell lysis, exercise, changes in acid-base balance and plasma osmolality, affect K+ homeostasis and plasma [K+]. 2. Excretion of K+ by the kidneys is determined by the rate and direction of K+ transport by the DT

and CCD. Secretion of K+ by these tubular segments is regulated by plasma [K+], aldosterone, and AVP. Changes in tubular fluid flow and acid-base balance alter K+ excretion by the kidneys. In K+-depleted states, K+ secretion is inhibited and its reabsorption increased by the DT and CCD. 3. The kidneys, in conjunction with the GI tract and bone, regulate plasma [Ca++] and [Pi]. 4. Plasma [Ca++] is regulated by PTH and calcitriol. Calcitonin is not a major regulatory hormone in humans. Ca++ excretion by the kidneys is regulated by PTH, plasma [Ca++], and calcitriol and is altered by changes in acid-base status, ECF volume, and plasma Pi. 5. Ca++ reabsorption is stimulated by PTH and calcitriol acting in the TAL and DT, and by elevated plasma [Ca++]. 6. Plasma [Pi] is regulated by PTH, FGF-23, and calcitriol. Pi excretion is regulated by PTH, FGF23, dietary Pi, and growth hormone and is altered by acid-base dysregulation, ECF expansion, and glucocorticoids. a

At physiological pH, inorganic phosphate exists as HPO4− and H2PO4− (pK = 6.8). For simplicity, we collectively refer to these ion species as Pi.

C H AP T E R 3 7

Role of the Kidneys in the Regulation of AcidBase Balance LEARNING OBJECTIVES Upon completion of this chapter the student should be able to answer the following questions: 1. How does HCO3− operate as a buffer, and why is it an important buffer of the extracellular fluid? 2. How does metabolism of food produce acid and alkali, and what effect does the composition of the diet have on systemic acid-base balance? 3. What is the difference between volatile and nonvolatile acids, and what is net endogenous acid production (NEAP)? 4. How do the kidneys and lungs contribute to systemic acid-base balance, and what is renal net acid excretion (RNAE)? 5. Why are urinary buffers necessary for excretion of acid by the kidneys? 6. What are the mechanisms for H+ transport in the various segments of the nephron, and how are these mechanisms regulated? 7. How do the various segments of the nephron contribute to the process of reabsorbing the filtered HCO3−? 8. How do the kidneys produce new HCO3−? 9. How is ammonium produced by the kidneys, and how does its excretion contribute to renal acid excretion? 10. What are the major mechanisms by which the body defends itself against changes in acid-base balance? 11. What are the differences between simple metabolic and respiratory acid-base disorders, and how are they differentiated by arterial blood gas measurements?

The concentration of H+ in body fluids is low compared with that of other ions. For example, Na+ is present at a concentration some 3 million times greater than that of H+ ([Na+] = 140 mEq/L; [H+] = 40 nEq/L). Because of the low [H+] of the body fluids, it is commonly expressed as the negative logarithm, or pH. Virtually all cellular, tissue, and organ processes are sensitive to pH. Indeed, life cannot exist outside of a range of extracellular fluid (ECF) pH from 6.8 to 7.8 (160–16 nEq/L of H+). Normally the pH of ECF is maintained between 7.35 and 7.45. The pH of intracellular fluid (ICF) is slightly lower (7.1–7.2) but is

also tightly regulated. Each day, acid and alkali are ingested in the diet. Also, cellular metabolism produces a number of substances that have an impact on the pH of body fluids. Without appropriate mechanisms to deal with this daily acid and alkali load and thereby maintain acid-base balance, many processes necessary for life could not occur. This chapter reviews the maintenance of whole-body acid-base balance. Although the emphasis is on the role of the kidneys in this process, the roles of the lungs and liver are also considered. In addition the impact of diet and cellular metabolism on acid-base balance is presented. Finally, disorders of acid-base balance are considered, primarily to illustrate the physiological processes involved. Throughout this chapter, acid is defined as any substance that adds H+ to body fluids, whereas alkali is defined as a substance that removes H+ from body fluids.

The HCO3− Buffer System Bicarbonate (HCO3−) is an important buffer of the ECF. With a normal plasma [HCO3−] of 23 to 25 mEq/L and a volume of 14 L (for a 70-kg individual) the ECF can potentially buffer 350 mEq of H+. The HCO3− buffer system differs from the other buffer systems of the body (e.g., phosphate) because it is regulated by both the lungs and kidneys. This is best appreciated by considering the following reaction:

Equation 37.1 As indicated the first reaction (hydration/dehydration of CO2) is the rate-limiting step. This normally slow reaction is greatly accelerated in the presence of carbonic anhydrase.a The second reaction, ionization of H2CO3 to H+ and HCO3−, is virtually instantaneous. The Henderson-Hasselbalch equation is used to quantitate how changes in CO2 and HCO3− affect pH:

Equation 37.2

or

Equation 37.3

In these equations the amount of CO2 is determined from the partial pressure of CO2 (Pco2) and its solubility (α) in solution. For plasma at 37°C, α has a value of 0.03. Also, pK′ is the negative logarithm of the overall dissociation constant for the reaction in Eq. 37.1 and has a value for plasma at 37°C of 6.1. Alternatively, the relationship between HCO3− and CO2 on the [H+] can be determined as follows:

Equation 37.4

Inspection of Eqs. 37.3 and 37.4 shows that pH and [H+] vary when either [HCO3−] or Pco2 is altered. Disturbances of acid-base balance that result from a change in [HCO3−] are termed metabolic acid-base disorders, whereas those resulting from a change in Pco2 are termed respiratory acid-base disorders. These disorders are considered in more detail in a subsequent section. The kidneys are primarily responsible for regulating the [HCO3−] of ECF, whereas the lungs control the Pco2.

Overview of Acid-Base Balance The diet of humans contains many constituents that are either acid or alkali. In addition, cellular metabolism produces acid and alkali. Finally, alkali is normally lost each day in feces. As described later, although diet dependent, the net effect of these processes is the addition of acid to body fluids. For acid-base balance to be maintained, acid must be excreted from the body at a rate equivalent to its addition. If acid addition exceeds excretion, acidosis results. Conversely, if acid excretion exceeds addition, alkalosis results. As summarized in Fig. 37.1, the major constituents of the diet are carbohydrates and fats. When tissue perfusion is adequate, O2 is available to tissues, and insulin is present at normal levels, carbohydrates and fats are metabolized to CO2 and H2O. On a daily basis, 15 to 20 moles of CO2 are generated through this process. Normally this large quantity of CO2 is effectively eliminated from the body by the lungs. Therefore, this metabolically derived CO2 has no impact on acid-base balance. CO2 is usually termed volatile acid because it has the potential to generate H+ after hydration with H2O (see Eq. 37.1). Acid not derived directly from hydration of CO2 is termed nonvolatile acid (e.g., lactic acid).

FIG. 37.1 Overview of acid-base balance. The lungs and kidneys work together to maintain acid-base balance. The lungs excrete CO2 (volatile acid), and the kidneys excrete acid (renal net acid excretion [RNAE]) equal to net endogenous acid production (NEAP), which reflects dietary intake, cellular metabolism, and loss of acid and alkali (e.g., HCO3− loss in feces) from the body. See text for details. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

The cellular metabolism of other dietary constituents also has an impact on acid-base balance. For example, cysteine and methionine, sulfur-containing amino acids, yield sulfuric acid when metabolized, whereas hydrochloric acid results from metabolism of lysine, arginine, and histidine. A portion of this nonvolatile acid load is offset by production of HCO3− through metabolism of the amino acids aspartate and glutamate. On average the metabolism of dietary amino acids yields net nonvolatile acid production. Metabolism of certain organic anions (e.g., citrate) results in production of HCO3−, which offsets nonvolatile acid production to some degree. Overall, in individuals ingesting a meat-containing diet, acid production exceeds HCO3− production. In contrast, a vegetarian diet produces less nonvolatile acid. In addition to the metabolically derived acids and alkalis, the foods ingested contain acid and alkali. For example, the presence of phosphate (H2PO4−) in ingested food increases the dietary acid load. Finally, during digestion, some HCO3− is normally lost in feces. This loss is equivalent to the addition of nonvolatile acid to the body. In an individual ingesting a meat-containing diet, dietary intake, cellular metabolism, and fecal HCO3− loss result in addition of approximately 0.7 to 1.0 mEq/kg body weight of nonvolatile acid to the body each day (50–100 mEq/day for most adults). This acid, referred to as net endogenous acid production (NEAP), results in an equivalent loss of HCO3− from the body that must be replaced.

IN THE C LIN IC When insulin levels are normal, carbohydrates and fats are completely metabolized to CO2 + H2O. However, if insulin levels are abnormally low (e.g., diabetes mellitus), cellular metabolism leads to production of several organic ketoacids (e.g., β-hydroxybutyric acid and acetoacetic acid from fatty acids).

In the absence of adequate levels of O2 (hypoxia), anaerobic metabolism by cells can also lead to production of organic acids (e.g., lactic acid) rather than CO2 + H2O. This frequently occurs in normal individuals during vigorous exercise. Poor tissue perfusion, such as occurs with reduced cardiac output, can also lead to anaerobic metabolism by cells and thus to acidosis. In these conditions the organic acids accumulate and the pH of body fluids decreases (acidosis). Treatment (e.g., administration of insulin in the case of diabetes) or improved delivery of adequate levels of O2 to tissues (e.g., in the case of poor tissue perfusion) results in the metabolism of these organic acids to CO2 + H2O, which consumes H+ and thereby helps correct the acid-base disorder. Nonvolatile acids do not circulate throughout the body but are immediately neutralized by the HCO3− in ECF.

Equation 37.5

Equation 37.6 This neutralization process yields the Na+ salts of the strong acids and removes HCO3− from the ECF. Thus HCO3− minimizes the effect of these strong acids on the pH of ECF. As noted previously, ECF contains approximately 350 mEq of HCO3−. If this HCO3− were not replenished, the daily production of nonvolatile acids (≈70 mEq/day) would deplete the ECF of HCO3− within 5 days. To maintain acid-base balance the kidneys must replenish the HCO3− lost by neutralization of the nonvolatile acids, a process termed renal net acid excretion (RNAE).

Net Acid Excretion by the Kidneys Under steady-state conditions, NEAP must equal RNAE to maintain acid-base balance. Although NEAP varies from individual to individual and from day to day in anyone individual, it is not regulated. Instead, the kidneys regulate RNAE to match NEAP and in so doing replenish the HCO3− (new HCO3−) lost by neutralization of nonvolatile acids. In addition, the kidneys must prevent the loss of HCO3− in urine. This latter task is quantitatively more important because the filtered load of HCO3− is approximately 4320 mEq/day (24 mEq/L × 180 L/day = 4320 mEq/day), compared with only 50 to 100 mEq/day needed to balance NEAP. Both reabsorption of filtered HCO3− and excretion of acid are accomplished via H+ secretion by the nephrons. Thus, in a single day the nephrons must secrete approximately 4390 mEq of H+ into the tubular fluid. Most of the secreted H+ serves to reabsorb the filtered load of HCO3−. Only 50 to 100 mEq of H+,

an amount equivalent to NEAP, is excreted in urine. As a result of this acid excretion, urine is normally acidic. The kidneys cannot excrete urine more acidic than pH 4.0 to 4.5. Even at a pH of 4.0, only 0.1 mEq/L of H+ can be excreted. Therefore, to excrete sufficient acid, the kidneys excrete H+ with urinary buffers such as phosphate (Pi).b Other constituents of urine can also serve as buffers (e.g., creatinine), although their role is less important than Pi. Collectively the various urinary buffers are termed titratable acid. This term is derived from the method by which these buffers are quantitated in the laboratory. Typically, alkali (OH−) is added to a urine sample to titrate its pH to that of plasma (i.e., 7.4). The amount of alkali added is equal to the H+ titrated by these urine buffers and is termed titratable acid. Excretion of H+ as a titratable acid is insufficient to balance NEAP. An additional and important mechanism by which the kidneys contribute to maintenance of acid-base balance is through synthesis and excretion of ammonium (NH4+). The mechanisms involved in this process are discussed in more detail later in this chapter. With regard to renal regulation of acid-base balance, each NH4+ excreted in urine results in the return of one HCO3− to the systemic circulation, which replenishes the HCO3− lost during neutralization of the nonvolatile acids. Thus, production and excretion of NH4+, like excretion of titratable acid, is equivalent to excretion of acid by the kidneys. In brief the kidneys contribute to acid-base homeostasis by reabsorbing the filtered load of HCO3− and excreting an amount of acid equivalent to NEAP. This process can be quantitated as follows:

Equation 37.7

where (

) and (

) are the rates of excretion (mEq/day) of NH4+ and titratable acid

(TA), and ( ) is the amount of HCO3− lost in urine (equivalent to adding H+ to the body).c Again, maintenance of acid-base balance means that net acid excretion must equal nonvolatile acid production. Under most conditions, very little HCO3− is excreted in urine. Thus net acid excretion essentially reflects titratable acid and NH4+ excretion. Quantitatively, titratable acid accounts for approximately one-third and NH4+ for two-thirds of RNAE.

HCO3− Reabsorption Along the Nephron As indicated by Eq. 37.7, net acid excretion is maximized when little or no HCO3− is excreted in urine. Indeed, under most circumstances, very little HCO3− appears in urine. Because HCO3− is freely filtered at the glomerulus, approximately 4320 mEq/day is delivered to the nephrons and is then reabsorbed. Fig. 37.2 summarizes the contribution of each nephron segment to reabsorption of filtered HCO3−.

FIG. 37.2 Segmental reabsorption of HCO3−. The fraction of the filtered load of HCO3− reabsorbed by the various segments of the nephron is shown. Normally the entire filtered load of HCO3− is reabsorbed and little or no HCO3− appears in the urine. CCD, Cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

The proximal tubule reabsorbs the largest portion of the filtered load of HCO3−. Fig. 37.3 summarizes the primary transport processes involved. H+ secretion across the apical membrane of the cell occurs by both a Na+/H+ antiporter and H+-ATPase (V-type). The Na+/H+ antiporter (NHE3) is the predominant pathway for H+ secretion (accounts for ≈ two-thirds of HCO3− reabsorption) and uses the lumen-to-cell [Na+] gradient to drive this process (i.e., secondary active secretion of H+). Within the cell, H+ and HCO3− are produced in a reaction catalyzed by carbonic anhydrase (CA-II). The H+ is secreted into the tubular fluid, whereas the HCO3− exits the cell across the basolateral membrane and returns to the peritubular blood. HCO3− movement out of the cell across the basolateral membrane is coupled to other ions. The majority of HCO3− exits via a symporter that couples the efflux of Na+ with HCO3− (sodium bicarbonate symporter, NBC1). Some HCO3− exits the cell by other transporters, but they are not as important as the Na+/HCO3− symporter. As noted in Fig. 37.3, carbonic anhydrase (CA-IV) is also present in the brush border and basolateral membrane of the cell. The brush border enzyme catalyzes dehydration of H2CO3 in the luminal fluid, whereas the enzyme localized to basolateral membrane facilitates HCO3− exit from the cell. The movement of CO2 into and out of the cell occurs via AQP1, which is localized to both the luminal and basolateral membranes.

FIG. 37.3 Cellular mechanism for reabsorption of filtered HCO3− by cells of the proximal tubule. Only the primary H+ and HCO3− transporters are shown. ATP, Adenosine triphosphate; CA, carbonic anhydrase.

The cellular mechanism for HCO3− reabsorption by the thick ascending limb (TAL) of the loop of Henle is very similar to that in the proximal tubule. H+ is secreted by a Na+/H+ antiporter and H+-ATPase. Like in the proximal tubule, the Na+/H+ antiporter (NHE3) is the predominant pathway for H+ secretion. HCO3− exit from the cell involves both a Na+/HCO3− symporter (NBC1) and a Cl−/HCO3− antiporter (anion exchanger, AE-2). Some HCO3− may also exit the cell through Cl− channels present in the basolateral membrane. The distal tubuled and collecting duct reabsorb the small amount of HCO3− that escapes reabsorption by the proximal tubule and loop of Henle. Fig. 37.4 shows the cellular mechanism of H+/HCO3− transport by the intercalated cells located within these segments (see also Chapter 34).

FIG. 37.4 Cellular mechanisms for reabsorption and secretion of HCO3− by intercalated cells of the distal tubule and collecting duct. Only the primary H+ and HCO3− transporters are shown. ATP, Adenosine triphosphate; CA, carbonic anhydrase; HKA, H+-K+-ATPase.

One type of intercalated cell secretes H+ (reabsorbs HCO3−) and is called the A- or α-intercalated

cell. Within this cell, H+ and HCO3− are produced by hydration of CO2; this reaction is catalyzed by carbonic anhydrase (CA-II). H+ is secreted into the tubular fluid via two mechanisms. The first involves an apical membrane H+-ATPase (V-type). The second couples secretion of H+ with reabsorption of K+ via an H+-K+-ATPase similar to those found in the stomach and colon (HKα1 and HKα2). HCO3− exits the cell across the basolateral membrane in exchange for Cl− (via a Cl−/HCO3− antiporter, AE-1) and enters the peritubular capillary blood. A second population of intercalated cells secretes HCO3− rather than H+ into the tubular fluid (also called B- or β-intercalated cells).e In these cells the H+-ATPase (V-type) is located in the basolateral membrane and the Cl−/HCO3− antiporter is located in the apical membrane (see Fig. 37.4). However, the apical membrane Cl−/HCO3− antiporter is different from the one found in the basolateral membrane of the H+-secreting intercalated cells and has been identified as pendrin. The activity of the HCO3−-secreting intercalated cell is increased during metabolic alkalosis, when the kidneys must excrete excess HCO3−. However, under most conditions (e.g., ingestion of a meat-containing diet) H+ secretion predominates in these segments.f The apical membrane of collecting duct cells is not very permeable to H+, and thus the pH of tubular fluid can become quite acidic. Indeed, the most acidic tubular fluid along the nephron (pH = 4.0–4.5) is produced there. In comparison the permeability of the proximal tubule to H+ and HCO3− is much higher, and the tubular fluid pH falls to only 6.5 in this segment. As explained later the ability of the collecting duct to lower the pH of the tubular fluid is critically important for excretion of urinary titratable acids and NH4+.

Regulation of H+ Secretion A number of factors influence secretion of H+ and thus reabsorption of filtered HCO3− by the cells of the nephron. From a physiological perspective the primary factor that regulates H+ secretion by the nephron is a change in systemic acid-base balance. Thus acidosis stimulates RNAE, whereas RNAE is reduced during alkalosis.

AT THE C ELLU LAR LEVEL Cells of the kidney express receptors that monitor acid-base status and therefore play a critical role in regulating H+ and HCO3− transporters along the nephron (Fig. 37.5). For example, a G protein–coupled H+ receptor (GPCR–GPR4) has been localized to the collecting duct. Activation of this receptor by an increase in ECF [H+] stimulates H+ secretion. Also in the collecting duct, HCO3−-secreting intercalated cells (B- or β-ICs) express a basolateral insulin-related receptor (IRR) that is a tyrosine kinase. It is activated by an increase in ECF [HCO3−] and stimulates HCO3− secretion by the cell. A soluble adenylyl cyclase (sAC) regulated by intracellular HCO3− appears to also play a role in regulating collecting duct H+ secretion. In the proximal tubule, basolateral membrane receptor tyrosine kinases (ErbB1 and ErbB2) sense changes in ECF Pco2. Activation of these receptors by an increase in Pco2 results in generation of angiotensin II, which, acting from the lumen via AT-1A receptors, stimulates H+ secretion/HCO3− reabsorption. Also in the proximal tubule, the nonreceptor tyrosine kinase (Pyk2)

senses intracellular [H+]. When it is activated by an increase in intracellular [H+], H+ secretion/HCO3− reabsorption is stimulated. Finally, the gating of several ion channels (e.g., the renal outer medullary K+ channel [ROMK]) is affected by changes in either ECF or ICF pH. These too have the potential to serve as cellular acid-base sensors.

FIG. 37.5 Examples of cellular H+ and HCO3− sensors. ATP, Adenosine triphosphate; cAMP, cyclic adenosine monophosphate; GPCR, G protein–coupled receptor; IRR, insulin receptor–related receptor; Pyk2, nonreceptor tyrosine kinase; sAC, soluble adenylyl cyclase. (Adapted from: Levin LR, Buck J. Annu Rev Physiol; 2015;77:347.)

The response of the kidneys to changes in acid-base balance includes both immediate changes in the activity and/or number of transporters in the membrane and longer-term changes in the synthesis of transporters. For example, with metabolic acidosis, H+ secretion is stimulated by multiple mechanisms, depending on the particular nephron segment. First, the decrease in intracellular pH that occurs with acidosis will create a more favorable cell-to-tubular fluid H+ gradient and thereby make secretion of H+ across the apical membrane more energetically favorable. Second, the decrease in pH may lead to allosteric changes in transport proteins, thereby altering their kinetics. Lastly, transporters may be shuttled to the membrane from intracellular vesicles. With long-term acidosis the abundance of transporters increases, either by increased transcription of appropriate transporter genes or by increased translation of transporter mRNA.

AT THE C ELLU LAR LEVEL In the proximal tubule, metabolic acidosis increases the transport kinetics of the Na+/H+ antiporter (NHE3) and increases apical membrane expression of the Na+/H+ antiporter, H+-ATPase, and the basolateral Na+/3HCO3− symporter (NBCe1). In the collecting duct, acidosis leads to exocytic insertion of H+-ATPase into the apical membrane of intercalated cells. With long-term acidosis the

abundance of key acid-base transporters is increased in the proximal tubule (NHE3 and NBCe1) and in collecting duct intercalated cells (H+-ATPase and AE1). Lastly, acidosis decreases expression of the Cl−/HCO3− antiporter pendrin in HCO3−-secreting intercalated cells. Although some of the effects just described may be attributable directly to acidosis, many of these changes in cellular H+ transport are mediated by hormones or other factors. Three known mediators of the renal response to acidosis are endothelin, cortisol, and angiotensin II. Endothelin (ET-1) is produced by endothelial and proximal tubule cells. With acidosis, ET-1 secretion is enhanced. In the proximal tubule, ET-1 stimulates phosphorylation and subsequent insertion of the Na+/H+ antiporter into the apical membrane, and insertion of the Na+/3HCO3− symporter into the basolateral membrane. ET-1 may mediate the response to acidosis in other nephron segments as well. Acidosis also stimulates secretion of the glucocorticoid hormone cortisol by the adrenal cortex. Cortisol increases the abundance of the Na+/H+ antiporter and Na+/3HCO3− symporter in the proximal tubule. Angiotensin II is produced in proximal tubule cells in response to acidosis. It is secreted into the tubular fluid, where it binds to the angiotensin I receptor and thereby stimulates H+ secretion/HCO3− reabsorption by the proximal tubule. Both cortisol and angiotensin II also stimulate production and secretion of NH4+ by the proximal tubule, which as described later is an important component of the kidney’s response to acidosis. Acidosis also stimulates secretion of parathyroid hormone (PTH). PTH inhibits phosphate (Pi) reabsorption by the proximal tubule (see Chapter 36). In so doing, more Pi is delivered to the distal nephron, where it serves as a urinary buffer and thus increases the capacity of the kidneys to excrete titratable acid. The response of the kidneys to alkalosis is less well characterized. RNAE is decreased because of increased urinary HCO3− excretion and because excretion of titratable acid and NH4+ is reduced. The factors that regulate this response are not well characterized. Other factors not necessarily related to maintaining acid-base balance can influence secretion of H+ by the cells of the nephron. Because a significant H+ transporter in the nephron is the Na+/H+ antiporter, factors that alter Na+ reabsorption can secondarily affect H+ secretion. For example, with volume contraction (negative Na+ balance), Na+ reabsorption by the nephron is increased (see Chapter 35), including reabsorption of Na+ via the Na+/H+ antiporter. As a result, H+ secretion is enhanced. This occurs by several mechanisms. One mechanism involves the renin-angiotensin-aldosterone system, which is activated by volume contraction. As noted earlier, angiotensin II acts on the proximal tubule to stimulate the apical membrane Na+/H+ antiporter as well as the basolateral Na+/3HCO3− symporter. To a lesser degree, angiotensin II stimulates H+ secretion in the TAL of Henle’s loop and the early portion of the distal tubule, a process also mediated by the Na+/H+ antiporter. Aldosterone’s primary action on the distal tubule and collecting duct is to stimulate Na+ reabsorption by principal cells (see Chapter 34). However, it also stimulates intercalated cells in these segments to secrete H+. This effect is both indirect and direct. By stimulating Na+ reabsorption by principal cells, aldosterone hyperpolarizes the transepithelial voltage (i.e., the lumen becomes more electrically negative). This change in transepithelial voltage then facilitates secretion of H+ by intercalated cells. In addition to this indirect effect, aldosterone (and angiotensin II) act directly on intercalated cells to stimulate H+ secretion via H+-ATPase and H+,K+ATPase.

Another mechanism by which ECF volume (ECFV) contraction enhances H+ secretion (HCO3− reabsorption) is through changes in peritubular capillary Starling forces. As described in Chapters 34 and 35, ECFV contraction alters the peritubular capillary Starling forces such that overall proximal tubule reabsorption is enhanced. With this enhanced reabsorption, more of the filtered load of HCO3− is reabsorbed. Potassium balance influences secretion of H+ by the proximal tubule. Hypokalemia stimulates and hyperkalemia inhibits H+ secretion. It is thought that K+-induced changes in intracellular pH are responsible at least in part for this effect. Hypokalemia acidifies the cells as intracellular K+ is exchanged for H+, whereas hyperkalemia alkalinizing cells as intracellular H+ is exchanged for K+. Hypokalemia also stimulates H+ secretion by the collecting duct. This occurs as a result of increased expression of the H+,K+-ATPase in intercalated cells.

Formation of New HCO3− As discussed previously, reabsorption of the filtered load of HCO3− is important for maximizing RNAE. However, HCO3− reabsorption alone does not replenish the HCO3− lost during neutralization of the nonvolatile acids produced during metabolism. To maintain acid-base balance, the kidneys must replace this lost HCO3− with new HCO3−. Generation of new HCO3− occurs by excretion of titratable acid and by synthesis and excretion of NH4+. Production of new HCO3− as a result of titratable acid excretion is depicted in Fig. 37.6. Because of HCO3− reabsorption by the proximal tubule and loop of Henle, fluid reaching the distal tubule and collecting duct normally contains little HCO3−. Thus, when H+ is secreted it will combine with nonHCO3− buffers (primarily Pi) and be excreted as titratable acid. Because the H+ was produced inside the cell from hydration of CO2, a HCO3− is also produced. This HCO3− is returned to the ECF as new HCO3−. As noted, Pi excretion increases with acidosis. However, even with increased Pi available for titratable acid formation, this response is insufficient to generate the required amount of new HCO3−. The remainder of new HCO3− generation occurs as a result of NH4+ generation and excretion.

FIG. 37.6 General scheme for excretion of H+ with non-HCO3− urinary buffers (titratable acid). The primary urinary buffer is phosphate (HPO42−). A H+-secreting intercalated cell is shown. For simplicity, only the H+-ATPase is depicted. H+ secretion by the H+,K+-ATPase also titrates luminal buffers. ATP, Adenosine triphosphate; CA, carbonic anhydrase.

NH4+ is produced by the kidneys, and its synthesis and subsequent excretion adds HCO3− to ECF. Importantly, this process is regulated in response to the acid-base requirements of the body. NH4+ is produced in the kidneys via metabolism of glutamine. Essentially the kidneys metabolize glutamine, excrete NH4+, and add HCO3− to the body. However, formation of new HCO3− via this process depends on the kidneys’ ability to excrete NH4+ in urine. If NH4+ is not excreted in urine but instead enters the systemic circulation, it is converted into urea by the liver. This conversion process generates H+, which is then buffered by HCO3−. Thus, production of urea from renal-generated NH4+ consumes HCO3− and negates formation of HCO3− through synthesis and excretion of NH4+ by the kidneys. However, normally the kidneys excrete NH4+ in urine and thereby produce new HCO3−. The process by which the kidneys excrete NH4+ is complex. Fig. 37.7 illustrates the essential features of this process. NH4+ is produced from glutamine in the cells of the proximal tubule, a process termed ammoniagenesis. Each glutamine molecule produces two molecules of NH4+ and the divalent anion 2oxoglutarate2−. Metabolism of this anion ultimately provides two molecules of HCO3−. HCO3− exits the cell across the basolateral membrane and enters the peritubular blood as new HCO3−. NH4+ exits the cell across the apical membrane and enters the tubular fluid. The primary mechanism for NH4+ secretion into the tubular fluid involves the Na+/H+ antiporter, with NH4+ substituting for H+. In addition, some NH3 can diffuse out of the cell into the tubular fluid, where it is protonated to NH4+.

FIG. 37.7 Production, transport, and excretion of NH4+ by the nephron. Glutamine is metabolized to NH4+ and HCO3− in the proximal tubule. The NH4+ is secreted into the lumen, and the HCO3− enters the blood. The secreted NH4+ is reabsorbed in Henle’s loop, primarily by the thick ascending limb, and accumulates in the medullary interstitium. NH3 is secreted by the collecting duct via rhesus glycoproteins, and H+ secretion traps NH4+ in the lumen. For each molecule of NH4+ excreted in the urine, a molecule of “new” HCO3− is added back to the ECF. CA, Carbonic anhydrase.

A significant portion of the NH4+ secreted by the proximal tubule is reabsorbed by the loop of Henle. The TAL is the primary site of this NH4+ reabsorption, with NH4+ substituting for K+ on the 1Na+/1K+/2Cl − symporter. In addition, the lumen-positive transepithelial voltage in this segment drives paracellular reabsorption of NH4+. The NH4+ reabsorbed by the TAL of the loop of Henle accumulates in the medullary interstitium. From there it is then secreted into the tubular fluid by the collecting duct. The cells of the collecting duct express two NH3 membrane transporters known as Rhesus (Rh) glycoproteins (RhBG and RhCG).g RhBG is present in the basolateral membrane of H+-secreting intercalated cells and principal cells, and RhCG is present in both the apical and basolateral membranes of these cells. As depicted in Fig. 37.7, NH3 is transported across the collecting duct, a process traditionally termed nonionic diffusion. The secreted NH3 is protonated in the tubule lumen as a result of intercalated H+ secretion. Because the apical membrane has a low permeability to NH4+ it is effectively trapped in the tubular lumen, a process traditionally termed diffusion trapping. H+ secretion by the collecting duct is critical for excretion of NH4+. If collecting duct H+ secretion is inhibited, the NH4+ reabsorbed by the TAL of Henle’s loop will not be excreted in the urine. Instead, NH4+ will be returned to the systemic circulation, whereas described previously, it will be converted to urea by the liver and consume HCO3− in the process. New HCO3− is therefore produced during the metabolism of glutamine by cells of the proximal tubule. However, the overall process is not complete until the NH4+ is excreted (i.e., production of urea from NH4+ by the liver is prevented). In the net, one

new HCO3− is returned to the systemic circulation for each NH4+ excreted in the urine. Accordingly, NH4+ excretion in urine can be used as a marker of glutamine metabolism in the proximal tubule.

IN THE C LIN IC Assessing NH4+ excretion by the kidneys is done indirectly because assays of urine NH4+ are not routinely available. Consider, for example, the situation of metabolic acidosis, wherein the appropriate renal response is to increase net acid excretion. Accordingly, little or no HCO3− will appear in urine, urine will be acidic, and NH4+ excretion will be increased. To assess this, and especially the amount of NH4+ excreted, the “urinary net charge” or “urine anion gap” can be calculated by measuring urinary concentrations of Na+, K+, and Cl−:

The concept of urine anion gap during a metabolic acidosis assumes that the major cations in urine are Na+, K+, and NH4+ and that the major anion is Cl− (with urine pH < 6.5, virtually no HCO3− is present). The principle of electroneutrality denotes that the sum of the cations and anions in the urine should be equal. As a result, the urine anion gap will yield a negative value when NH4+, an unmeasured cation, is being excreted. Note, that an anion gap does not actually exist—the number of cations and anions is equal. The calculated anion gap simply reflects the parameters that are measured, and NH4+ is not routinely measured. Indeed, the absence of a urine anion gap or the existence of a positive value indicates a renal defect in NH4+ production and excretion. An important feature of the renal NH4+ system is that it is regulated by systemic acid-base balance. As already described, cortisol levels increase with acidosis, as does angiotensin II secretion into the lumen of the proximal tubule. Both cortisol and angiotensin II stimulate ammoniagenesis (i.e., NH4+ production from glutamine). During systemic acidosis, the enzymes in the proximal tubule cell responsible for metabolism of glutamine are stimulated. This involves synthesis of new enzyme and requires several days for complete adaptation. With increased levels of these enzymes, NH4+ production is increased, allowing enhanced production of new HCO3−. Conversely, glutamine metabolism is reduced with alkalosis. Acidosis also increases the abundance of RhBG and RhCG in the collecting duct, and thus, the ability to secrete NH4+ is also enhanced.

Response to Acid-Base Disorders The pH of the ECF is maintained within a very narrow range (7.35–7.45).h Inspection of Eq. 37.3 shows that the pH of ECF varies when either [HCO3−] or Pco2 is altered. As already noted, disturbances of acidbase balance that result from a change in [HCO3−] of ECF are termed metabolic acid-base disorders,

whereas those resulting from a change in Pco2 are termed respiratory acid-base disorders. The kidneys are primarily responsible for regulating [HCO3−], whereas the lungs regulate Pco2. When an acid-base disturbance develops, the body uses several mechanisms to defend against the change in ECF pH. These defense mechanisms do not correct the acid-base disturbance but merely minimize the change in pH imposed by the disturbance. Restoration of the blood pH to its normal value requires correction of the underlying process or processes that produced the acid-base disorder. The body has three general mechanisms to defend against changes in body fluid pH produced by acid-base disturbances: (1) extracellular and intracellular buffering, (2) adjustments in blood Pco2 via alterations in the ventilatory rate of the lungs, and (3) adjustments in RNAE.

Extracellular and Intracellular Buffers The first line of defense against acid-base disorders is extracellular and intracellular buffering. The response of the extracellular buffers is virtually instantaneous, whereas the response to intracellular buffering is slower and can take several minutes. Metabolic disorders that result from addition of nonvolatile acid or alkali to body fluids are buffered in both the extracellular and intracellular compartments. The HCO3− buffer system is the principal ECF buffer. When nonvolatile acid is added to body fluids (or alkali is lost from the body), HCO3− is consumed during the process of neutralizing the acid load, and the [HCO3−] of ECF is reduced. Conversely, when nonvolatile alkali is added to body fluids (or acid is lost from the body), H+ is consumed, causing more HCO3− to be produced from the dissociation of H2CO3. Consequently, [HCO3−] increases. Although the HCO3− buffer system is the principal ECF buffer, Pi and plasma proteins provide additional extracellular buffering. The combined action of the buffering processes for HCO3−, Pi, and plasma protein accounts for approximately 50% of the buffering of a nonvolatile acid load and 70% of a nonvolatile alkali load. The remainder of the buffering under these two conditions occurs intracellularly. Intracellular buffering involves movement of H+ into cells (during buffering of nonvolatile acid) or movement of H+ out of cells (during buffering of nonvolatile alkali). H+ is titrated inside the cell by HCO3−, Pi, and the histidine groups on proteins. Bone represents an additional source of extracellular buffering. However, with acidosis, buffering by bone results in its demineralization. When respiratory acid-base disorders occur, the pH of body fluid changes as a result of alterations in Pco2. Virtually all buffering in respiratory acid-base disorders occurs intracellularly. When Pco2 rises (respiratory acidosis), CO2 moves into the cell, where it combines with H2O to form H2CO3. H2CO3 then dissociates to H+ and HCO3−. Some of the H+ is buffered by cellular proteins, and HCO3− exits the cell and raises the ECF [HCO3−]. This process is reversed when Pco2 is reduced (respiratory alkalosis). Under this condition the hydration reaction (H2O + CO2 H2CO3) is shifted to the left by the decrease in Pco2. As a result the dissociation reaction (H2CO3 O H+ + HCO3−) also shifts to the left, thereby reducing the ECF [HCO3−].

Respiratory Compensation The lungs are the second line of defense against acid-base disorders. As indicated by the HendersonHasselbalch equation (see Eq. 37.2), changes in Pco2 alter the blood pH: a rise decreases the pH, and a reduction increases the pH. The ventilatory rate determines the Pco2. Increased ventilation decreases Pco2, whereas decreased ventilation increases it (Fig. 37.8). The blood Pco2 and pH are important regulators of the ventilatory rate. Chemoreceptors located in the brainstem (ventral surface of the medulla) and periphery (carotid and aortic bodies) sense changes in Pco2 and [H+] and alter the ventilatory rate appropriately. Thus, when metabolic acidosis occurs, a rise in the [H+] (decrease in pH) stimulates the ventilatory rate. Conversely, during metabolic alkalosis, a decreased [H+] (increase in pH) reduces the ventilatory rate. With maximal hyperventilation, the Pco2 can be reduced to approximately 10 mm Hg. Because hypoxia, a potent stimulator of ventilation, also develops with hypoventilation, the degree to which the Pco2 can be increased is limited. In an otherwise normal individual, hypoventilation cannot raise the Pco2 above 60 mm Hg. The respiratory response to metabolic acid-base disturbances may be initiated within minutes but may require several hours to complete.

FIG. 37.8 Effect of ventilatory rate on alveolar Pco2 and thus arterial blood Pco2.

Renal Compensation The third and final line of defense against acid-base disorders involves the kidneys. In response to an alteration in plasma pH and Pco2, the kidneys make appropriate adjustments in the excretion of RNAE. The renal response may require several days to reach completion because it takes hours to days to increase the synthesis and activity of the proximal tubule enzymes involved in NH4+ production. In the case of acidosis (increased [H+] or Pco2), the secretion of H+ by the nephron is stimulated and the entire filtered load of HCO3− is reabsorbed. Titratable acid excretion is increased and production and excretion of NH4+ is also stimulated, and thus RNAE is increased (Fig. 37.9). The new HCO3− generated during the

process of net acid excretion is added to the body, and the plasma [HCO3−] increases.

FIG. 37.9 Response of the nephron to acidosis. CCD, Cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; Pi, phosphate; PT, proximal tubule; PTH, parathyroid hormone; Rhbg & Rhcg, rhesus glycoproteins; RNAE, renal net acid excretion; TA, titratable acid; TAL, thick ascending limb; V̇, urine flow rate.

When metabolic alkalosis exists (decreased [H+]), the filtered load of HCO3− is increased (plasma [HCO3−] is elevated). With respiratory alkalosis (decreased Pco2), plasma [HCO3−] is decreased and thus the filtered load is decreased. In both conditions, secretion of H+ by the nephron is inhibited. As a result, HCO3− excretion is increased. At the same time, excretion of both titratable acid and NH4+ is decreased. Thus, RNAE is decreased and HCO3− appears in urine. Also, some HCO3− is secreted into urine by the HCO3−-secreting intercalated cells of the distal tubule and collecting duct. With enhanced excretion of HCO3−, plasma [HCO3−] decreases.

IN THE C LIN IC Loss of gastric contents from the body (e.g., vomiting, nasogastric suctioning) produces metabolic alkalosis secondary to the loss of HCl. If the loss of gastric fluid is significant, ECFV contraction occurs. Under this condition the kidneys cannot excrete sufficient quantities of HCO3− to compensate for the metabolic alkalosis. The inability of the kidneys to excrete HCO3− is a result of the need to reduce Na+ excretion to correct the ECFV contraction. As described previously (see Chapter 35 for details), the response of the kidneys to volume contraction is to reduce the glomerular filtration rate, which reduces the filtered load of HCO3−, and to increase Na+ reabsorption along the nephron. Because a large amount of Na+ reabsorption occurs via the Na+/H+ antiporter, this results in an increase in H+ secretion (HCO3− reabsorption) by the proximal tubule. In this setting the entire filtered

load of HCO3− is reabsorbed and new HCO3− generation may even be enhanced. The latter response occurs because aldosterone, the levels of which are elevated in volume contraction, stimulates not only distal Na+ reabsorption but also H+ secretion by intercalated cells. This stimulation of H+ secretion generates new HCO3− by enhancing titratable acid and NH4+ excretion. Thus, in individuals who lose gastric contents, metabolic alkalosis and paradoxically acidic urine characteristically occur. Correction of the alkalosis occurs only when euvolemia is reestablished.

Simple Acid-Base Disorders Table 37.1 summarizes the primary alterations and the subsequent compensatory or defense mechanisms of the various simple acid-base disorders. In all acid-base disorders the compensatory response does not correct the underlying disorder but simply reduces the magnitude of the change in pH. Correction of the acid-base disorder requires treatment of its cause. Table 37.1 Characteristics of Simple Acid-Base Disorders Disorder

Plasma pH

Primary Alteration

Defense Mechanisms

Metabolic acidosis

↓

↓ECF [HCO3−]

ICF and ECF buffers Hyperventilation (↓Pco2) ↑RNAE

Metabolic alkalosis

↑

↑ECF [HCO3−]

ICF and ECF buffers Hypoventilation (↑Pco2) ↓RNAE

Respiratory acidosis

↓

↑Pco2

ICF buffers ↑RNAE

Respiratory alkalosis

↑

↓Pco2

ICF buffers ↓RNAE

ECF, Extracellular fluid; ICF, intracellular fluid; RNAE, renal net acid excretion

Types of Acid-Base Disorders Metabolic Acidosis Metabolic acidosis is characterized by a decreased ECF [HCO3−] and pH. It can develop via the addition of nonvolatile acid to the body (e.g., diabetic ketoacidosis), the loss of nonvolatile base (e.g., HCO3− loss caused by diarrhea), or failure of the kidneys to excrete titratable acid and NH4+ (e.g., renal failure). As previously described, the buffering of H+ occurs in both the ECF and ICF compartments. When the pH falls, the respiratory centers are stimulated and the ventilatory rate is increased (respiratory compensation). Finally, in metabolic acidosis, RNAE is increased. This occurs via elimination of all HCO3− from

urine (enhanced reabsorption of filtered HCO3−) and via increased titratable acid and NH4+ excretion (enhanced production of new HCO3−). If the process that initiated the acid-base disturbance is corrected, the enhanced RNAE will ultimately return the pH and [HCO3−] to normal. After correction of the pH, the ventilatory rate also returns to normal.

IN THE C LIN IC When nonvolatile acid is added to body fluids, as in diabetic ketoacidosis, the [H+] increases (pH decreases), and the [HCO3−] decreases. In addition, the concentration of the anion associated with the nonvolatile acid increases. This change in anion concentration provides a convenient way of analyzing the cause of a metabolic acidosis by calculating what is termed the anion gap. The anion gap represents the difference between the concentration of the major ECF cation (Na+) and the major ECF anions (Cl− and HCO3−):

Under normal conditions the anion gap ranges from 8 to 16 mEq/L. It is important to recognize that an anion gap does not actually exist. All cations are balanced by anions. The gap simply reflects the parameters that are measured. In reality:

If the anion of the nonvolatile acid is Cl−, the anion gap will be normal (i.e., the decrease in [HCO3−] is matched by an increase in [Cl−]). Metabolic acidosis associated with diarrhea or renal tubular acidosis (i.e., defect in renal H+ secretion) has a normal anion gap. In contrast, if the anion of the nonvolatile acid is not Cl− (e.g., lactate, β-hydroxybutyrate), the anion gap will increase (i.e., the decrease in [HCO3−] is not matched by an increase in [Cl−] but rather by an increase in the concentration of the unmeasured anion). The anion gap is increased in metabolic acidosis–associated ketoacidosis (e.g., diabetes mellitus) with renal failure, lactic acidosis, or ingestion of toxins or certain drugs (e.g., large quantities of aspirin). Thus, calculation of the anion gap is a useful way of identifying the etiology of metabolic acidosis in the clinical setting. Albumin is a negatively charged macromolecule, and it makes a considerable contribution to “unmeasured anions.” As a result, the anion gap must be adjusted in patients who have an abnormal serum [albumin]. For each 1 g/dL change in serum [albumin], the anion gap needs to be adjusted in the same direction by 2.5 mEq/L.

Metabolic Alkalosis Metabolic alkalosis is characterized by an increased ECF [HCO3−] and pH. It can occur via the addition of nonvolatile base to the body (e.g., ingestion of antacids), as a result of volume contraction (e.g., hemorrhage), or more commonly from loss of nonvolatile acid (e.g., loss of gastric HCl because of prolonged vomiting). Buffering occurs predominantly in the ECF and to a lesser degree in the ICF. The increase in the pH inhibits the respiratory centers, which reduces the ventilatory rate, thus the Pco2 is elevated (respiratory compensation). The renal compensatory response to metabolic alkalosis is to increase excretion of HCO3− by reducing its reabsorption along the nephron. Normally this occurs quite rapidly (minutes to hours) and effectively. Enhanced renal excretion of HCO3− eventually returns the pH and [HCO3−] to normal, provided the underlying cause of the initial acid-base disturbance is corrected. When the pH is corrected, the ventilatory rate also returns to normal. Respiratory Acidosis Respiratory acidosis is characterized by an elevated Pco2 and reduced ECF pH. It results from decreased gas exchange across the alveoli as a result of either inadequate ventilation (e.g., drug-induced depression of the respiratory centers) or impaired gas diffusion (e.g., pulmonary edema, such as occurs in cardiovascular and lung disease). In contrast to the metabolic disorders, buffering during respiratory acidosis occurs almost entirely in the ICF compartment. The increase in Pco2 and the decrease in pH stimulate both HCO3− reabsorption by the nephron and titratable acid and NH4+ excretion (renal compensation). Together these responses increase RNAE and generate new HCO3−. The renal compensatory response takes several days to occur. Consequently, respiratory acid-base disorders are commonly divided into acute and chronic phases. In the acute phase the time for the renal compensatory response is not sufficient, and the body relies on ICF buffering to minimize the change in pH. In the chronic phase, renal compensation occurs. Correction of the underlying disorder returns the Pco2 to normal and RNAE decreases to its initial level. Respiratory Alkalosis Respiratory alkalosis is characterized by a reduced Pco2 and an increased ECF pH. It results from increased gas exchange in the lungs, usually caused by increased ventilation from stimulation of the respiratory centers (e.g., via drugs or disorders of the central nervous system). Hyperventilation also occurs at high altitude and as a result of anxiety, pain, or fear. Buffering is primarily in the ICF compartment. As with respiratory acidosis, respiratory alkalosis has both acute and chronic phases reflecting the time required for renal compensation to occur. The acute phase of respiratory alkalosis reflects intracellular buffering, whereas the chronic phase reflects renal compensation. With renal compensation, the elevated pH and reduced Pco2 inhibit HCO3− reabsorption by the nephron and reduce titratable acid and NH4+ excretion. As a result of these effects, RNAE is reduced. Correction of the underlying disorder returns the Pco2 to normal, and renal excretion of acid then increases to its initial level.

Analysis of Acid-Base Disorders

Analysis of an acid-base disorder is directed at identifying the underlying cause so appropriate therapy can be initiated. The patient’s medical history and associated physical findings often provide valuable clues about the nature and origin of an acid-base disorder. In addition, analysis of an arterial blood sample is frequently required. Such an analysis is straightforward if approached systematically. For example, consider the following data: The acid-base disorder represented by these values or any other set of values can be determined using the following three-step approach: 1. Examination of the pH. When pH is considered first, the underlying disorder can be classified as either an acidosis or an alkalosis. The defense mechanisms of the body cannot correct an acidbase disorder by themselves. Thus, even if the defense mechanisms are completely operative, the change in pH indicates the acid-base disorder. In the example provided, the pH of 7.35 indicates acidosis. 2. Determination of metabolic versus respiratory disorder. Simple acid-base disorders are either metabolic or respiratory. To determine which disorder is present, the clinician must next examine the ECF [HCO3−] and Pco2. As previously discussed, acidosis could be the result of a decrease in [HCO3−] (metabolic) or an increase in Pco2 (respiratory). Alternatively, alkalosis could be the result of an increase in ECF [HCO3−] (metabolic) or a decrease in Pco2 (respiratory). For the example provided, the ECF [HCO3−] is reduced from normal (normal = 24 mEq/L), as is the Pco2 (normal = 40 mm Hg). The disorder must therefore be metabolic acidosis; it cannot be a respiratory acidosis because the Pco2 is reduced. 3. Analysis of a compensatory response. Metabolic disorders result in compensatory changes in ventilation and thus in Pco2, whereas respiratory disorders result in compensatory changes in RNAE and thus in ECF [HCO3−] (Table 37.2). In an appropriately compensated metabolic acidosis, the Pco2 is decreased, whereas it is elevated in compensated metabolic alkalosis. With respiratory acidosis, compensation results in an elevation of the [HCO3−]. Conversely, ECF [HCO3−] is reduced in response to respiratory alkalosis. In this example, the Pco2 is reduced from normal, and the magnitude of this reduction (10 mm Hg decrease in Pco2 for an 8 mEq/L decrease in ECF [HCO3−]) is as expected (see Table 37.2). Therefore the acid-base disorder is a simple metabolic acidosis with appropriate respiratory compensation.

Table 37.2 Simple Acid-Base Disorders With Compensation Primary Disturbance

HCO3−

Pco 2

Compensation

Metabolic acidosis 7.4 Primary ↑

Compensatory ↑

0.6–0.75 mm Hg ↑Pco2 for every 1 mEq/L ↑[HCO3−]

Respiratory acidosis

7.4 Compensatory ↓

Primary ↓

Acute: 1–2 mEq/L ↓[HCO3−] for every 10 mm Hg ↓Pco2 Chronic: 3–4 mEq/L ↓[HCO3−] for every 10 mm Hg ↓Pco2

pH

A mixed acid-base disorder reflects the presence of two or more underlying causes for the acid-base disturbance. For example, consider the following data: When the three-step approach is followed, it is evident that the disturbance is an acidosis that has both a metabolic component (ECF [HCO3−] < 24 mEq/L) and a respiratory component (Pco2 > 40 mm Hg). Thus this disorder is mixed. Mixed acid-base disorders can occur, for example, in an individual who has a history of a chronic pulmonary disease such as emphysema (i.e., chronic respiratory acidosis) and who develops an acute gastrointestinal illness with diarrhea. Because diarrhea fluid contains HCO3−, its loss from the body results in the development of metabolic acidosis. A mixed acid-base disorder is also indicated when a patient has abnormal Pco2 and ECF [HCO3−] values but the pH is normal. Such a condition can develop in a patient who has ingested a large quantity of aspirin. Salicylic acid (active ingredient in aspirin) produces metabolic acidosis and at the same time stimulates the respiratory centers, causing hyperventilation and respiratory alkalosis. Thus, the patient has a reduced ECF [HCO3−] and a reduced Pco2. (note: The Pco2 is lower than would occur with normal respiratory compensation of a metabolic acidosis.)

Key Concepts 1. The kidneys maintain acid-base balance through excretion of an amount of acid equal to the amount of nonvolatile acid produced by metabolism and the quantity ingested in the diet (termed renal net acid excretion [RNAE]). The kidneys also prevent loss of HCO3− in urine by

reabsorbing virtually all the HCO3− filtered at the glomeruli. Both reabsorption of filtered HCO3− and excretion of acid are accomplished by secretion of H+ by the nephrons. Acid is excreted by the kidneys in the form of titratable acid (primarily as Pi) and NH4+. Both titratable acid and NH4+ excretion result in generation of new HCO3−, which replenishes the ECF HCO3− lost during neutralization of nonvolatile acids. 2. The body uses three lines of defense to minimize the impact of acid-base disorders on body fluid pH: (1) ECF and ICF buffering, (2) respiratory compensation, and (3) renal compensation. 3. Metabolic acid-base disorders result from primary alterations in ECF [HCO3−], which in turn results from addition of acid to or loss of alkali from the body. In response to metabolic acidosis, pulmonary ventilation is increased, which decreases Pco2. The pulmonary response to metabolic acid-base disorders occurs in a matter of minutes. RNAE is also increased, but this takes several days. An increase in ECF [HCO3−] causes alkalosis. This rapidly (minutes to hours) decreases pulmonary ventilation, which elevates Pco2 as a compensatory response. RNAE is also decreased, but this takes several days. 4. Respiratory acid-base disorders result from primary alterations in Pco2. Elevation of Pco2 produces acidosis, and the kidneys respond with an increase in RNAE. Conversely, a reduction of Pco2 produces alkalosis, and RNAE is reduced. The kidneys respond to respiratory acid-base disorders over several hours to days. a

Carbonic anhydrase (CA) actually catalyzes the following reaction:

b

The titration reaction is: HPO42− + H+ ↔ H2PO4−. This reaction has a pK of approximately 6.8.

c

This equation ignores the small amount of free H+ excreted in urine. As already noted, urine with a pH = 4.0 contains only 0.1 mEq/L of H+. d

Here and in the remainder of the chapter we focus on the function of intercalated cells. The early portion of the distal tubule, which does not contain intercalated cells, also reabsorbs HCO3−. The cellular mechanism appears to involve an apical membrane Na+/H+ antiporter (NHE2) and a basolateral Cl − /HCO3− antiporter (AE2). e

A third group of intercalated cells shares features of both H+-secreting and HCO3−-secreting intercalated cells. The precise function of this cell type in acid-base transport is not fully understood. f

Traditionally it was believed that intercalated cells were only involved in acid-base transport. There is now good evidence that NaCl reabsorption is also carried out by intercalated cells (B type). Reabsorption of NaCl occurs by the tandem operation of an apical membrane Cl−/HCO3− antiporter (pendrin) and an apical membrane Na+/HCO3−/2Cl− antiporter (NDCBE). This mechanism of NaCl reabsorption is inhibited by thiazide diuretics. g

Both RhBG and RhCG transport NH3. There is some evidence that RhBG may also transport some NH4+.

h

For simplicity of presentation in this chapter, the value of 7.40 for body fluid pH is used as normal, even though the normal range is from 7.35 to 7.45. Similarly, the normal range for Pco2 is 35 to 45 mm Hg.

However, a Pco2 of 40 mm Hg is used as the normal value. Finally, a value of 24 mEq/L is considered a normal ECF [HCO3−], even though the normal range is 22 to 28 mEq/L.

S E CT I ON 8

Endocrine Physiology Bruce White, John R. Harrison, and Julianne M. Hall

Chapter 38 Introduction to the Endocrine System Chapter 39 Hormonal Regulation of Energy Metabolism Chapter 40 Hormonal Regulation of Calcium and Phosphate Metabolism Chapter 41 The Hypothalamus and Pituitary Gland Chapter 42 The Thyroid Gland Chapter 43 The Adrenal Gland Chapter 44 The Male and Female Reproductive Systems

C H AP T E R 3 8

Introduction to the Endocrine System LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. Name the major endocrine glands and their hormonal product or products. 2. Map out and differentiate a simple endocrine negative feedback loop and one involving the hypothalamus, anterior pituitary and peripheral endocrine gland, and list the major endocrine glands under each type of feedback loop. 3. Define a releasing hormone and a tropic hormone. 4. Explain the chemical nature and the characteristics of protein/peptide hormones, catecholamine hormones, steroid hormones, and iodothyronines (thyroid hormones). Include such characteristics as site of regulation (synthesis or secretion), circulating form of hormone, subcellular localization of hormone receptor, and metabolic clearance. 5. Integrate the concept of peripheral conversion with the function/action of a secreted hormone. 6. Integrate the intracellular steps associated with a hormone response in a target cell.

The ability of cells to communicate with each other is an underpinning of human biology. As discussed in Chapter 3, cell-to-cell communication occurs at various levels of complexity and distance. Endocrine signaling involves (1) the regulated secretion of an extracellular signaling molecule, called a hormone, into the extracellular fluid; (2) diffusion of the hormone into the vasculature and its circulation throughout the body; and (3) diffusion of the hormone out of the vascular compartment into the extracellular space and binding to a specific receptor within cells of a target organ. Because of the spread of hormones throughout the body, one hormone often regulates the activity of several target organs. Conversely, cells frequently express receptors for multiple hormones. The endocrine system is a collection of glands whose function is to regulate multiple organs within the body to (1) meet the growth and reproductive needs of the organism and (2) respond to fluctuations within the internal environment, including various types of stress. The endocrine system is composed of three subsets of organs: 1. Glands that are solely dedicated to an endocrine function, involving the synthesis and secretion of bioactive hormones. These include (Fig. 38.1): Parathyroid glands Pituitary gland Thyroid gland Adrenal glands

2. Gonads, which have a major endocrine function, as well as gametogenic function. Gonads (testes or ovaries) (Table 38.1). A transitory organ, the placenta, also performs a major endocrine function.

3. Isolated endocrine cells or endocrine cell clusters within organs whose primary function is not endocrine (see Table 38.1). These include cells within the heart that produce atrial natriuretic peptide, liver cells that produce insulin-like growth factor type 1 (IGF-1), cells within the kidney that produce erythropoietin, cell clusters within the pancreas that produce insulin and glucagon, and numerous cell types within the gastrointestinal tract that produce gastrointestinal hormones. There also exist collections of cell bodies (called nuclei) within the hypothalamus that secrete peptides, called neurohormones, into capillaries associated with the pituitary gland.

FIG. 38.1 Glands of the endocrine system.

Table 38.1 Hormones and Their Sites of Production in Nonpregnant Adults Gland

Hormone

Hormones Synthesized and Secreted by Dedicated Endocrine Glands Pituitary gland

Growth hormone (GH) Prolactin Adrenocorticotropic hormone (ACTH) Thyroid-stimulating hormone (TSH) Follicle-stimulating hormone (FSH) Luteinizing hormone (LH)

Thyroid gland

Thyroxine Triiodothyronine Calcitonin

Parathyroid glands Islets of Langerhans (endocrine tissues of the

Parathyroid hormone (PTH) Insulin

pancreas)

Glucagon Somatostatin

Adrenal gland

Epinephrine Norepinephrine Cortisol Aldosterone Dehydroepiandrosterone sulfate (DHEAS)

Ovaries

Estradiol-17β Progesterone Inhibin

Testes

Testosterone Antimüllerian hormone (AMH) Inhibin

Hormones Synthesized in Organs With a Primary Function Other Than Endocrine Brain (hypothalamus)

Antidiuretic hormone (ADH; vasopressin) Oxytocin Corticotropin-releasing hormone (CRH) Thyrotropin-releasing hormone (TRH) Gonadotropin-releasing hormone (GnRH) Growth hormone–releasing hormone (GHRH) Somatostatin Dopamine

Brain (pineal gland)

Melatonin

Heart

Atrial natriuretic peptide (ANP)

Kidneys

Erythropoietin

Adipose tissue

Leptin Adiponectin

Stomach

Gastrin Somatostatin Ghrelin

Intestines

Secretin Cholecystokinin Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-2 (GLP-2) Glucose-dependent insulinotropic peptide (gastrin inhibitory peptide [GIP]) Motilin

Liver

Insulin-like growth factor type 1 (IGF-1)

Hormones Produced to a Significant Degree by Peripheral Conversion Lungs

Angiotensin II

Kidney

1,25-Dihydroxyvitamin D (vitamin D)

Adipose, mammary glands, other organs

Estradiol-17β

Liver, sebaceous gland, other organs

Testosterone

Genital skin, prostate, other organs

5-Dihydrotestosterone (DHT)

Many organs

Triiodothyronine

A fourth subset of the endocrine system is represented by numerous cell types that express intracellular enzymes, ectoenzymes, or secreted enzymes that modify inactive precursors or less active hormones into highly active hormones (see Table 38.1). An example is the generation of angiotensin II from the inactive polypeptide angiotensinogen by two subsequent proteolytic cleavages (see Chapter 43). Another example is activation of vitamin D by two subsequent hydroxylation reactions in the liver and kidneys to produce the highly bioactive hormone 1,25-dihydroxyvitamin D (vitamin D).

Configuration of Feedback Loops Within the Endocrine System The predominant mode of a closed feedback loop among endocrine glands is negative feedback. In a negative feedback loop, a hormone acts on one or more target organs to induce a change (either a decrease or an increase) in circulating levels of a specific component, and the change in this component then inhibits secretion of the hormone. Negative feedback loops confer stability by keeping a physiological parameter (e.g., blood glucose level) within a normal range. There are also a few examples of positive feedback in endocrine regulation. A closed positive feedback loop, in which a hormone increases levels of a specific component and this component stimulates further secretion of the hormone, confers instability. Under the control of positive feedback loops, something has got to give; for example, positive feedback loops control processes that lead to rupture of a follicle through the ovarian wall or expulsion of a fetus from the uterus. There are two basic configurations of negative feedback loops within the endocrine system: a physiological response–driven feedback loop (referred to simply as a response-driven feedback loop) and an endocrine axis–driven feedback loop (Fig. 38.2). The response-driven feedback loop is observed in endocrine glands that control blood glucose levels (pancreatic islet cells), blood Ca++ and Pi levels (parathyroid glands, kidneys), blood osmolarity and volume (hypothalamus/posterior pituitary gland), and blood Na+, K+, and H+ levels (zona glomerulosa of the adrenal cortex and atrial cells). In the responsedriven configuration, secretion of a hormone is stimulated or inhibited by a change in the level of a specific extracellular parameter (e.g., an increase in blood glucose level stimulates insulin secretion). Alterations in hormone levels lead to changes in the physiological characteristics of target organs (e.g., decreased hepatic gluconeogenesis, increased uptake of glucose by muscle) that directly regulate the parameter (in this case, blood glucose level) in question. The change in the parameter (decreased blood glucose level) then inhibits further secretion of the hormone (i.e., insulin secretion drops as blood glucose level falls). This type of feedback also involves the response of endocrine cells to changes in the contents of lumens of other organ systems, especially the lumen of the gastrointestinal tract.

FIG. 38.2 Physiological response–driven and endocrine axis–driven negative feedback loops.

Much of the endocrine system is organized into endocrine axes; each axis consists of the hypothalamus, the pituitary gland, and the peripheral endocrine glands (see Fig. 38.2). Thus, the endocrine axis–driven feedback loop involves a three-tiered configuration. The first tier is represented by hypothalamic neuroendocrine neurons that secrete releasing hormones. Releasing hormones stimulate (or, in a few cases, inhibit) the production and secretion of tropic hormones from the pituitary gland (second tier). Tropic hormones stimulate the production and secretion of hormones from peripheral endocrine glands (third tier). The peripherally produced hormones—namely, thyroid hormone, cortisol, sex steroids, and IGF-1—typically have pleiotropic actions (e.g., multiple phenotypic effects) on numerous cell types. Additionally, these peripherally produced hormones exert the primary feedback loop that inhibits the release of pituitary tropic hormones and hypothalamic releasing hormones. In contrast to response-driven feedback, the physiological responses to the peripherally produced hormone play only a minor role in regulation of feedback within endocrine axis–driven feedback loops. From a clinical perspective, endocrine diseases are described as primary, secondary, or tertiary diseases (e.g., secondary hyperthyroidism, tertiary hypogonadism). Primary disease is a lesion in the peripheral endocrine gland; secondary disease is a lesion in the anterior pituitary gland; and tertiary disease is a lesion in the hypothalamus. An important aspect of the endocrine axes is the ability of descending and ascending neuronal signals to modulate release of the hypothalamic releasing hormones and thereby control the activity of the axis. A major neuronal input to releasing hormone–secreting neurons comes from another region of the hypothalamus called the suprachiasmatic nucleus (SCN). SCN neurons impose a daily rhythm, called a circadian rhythm, on the secretion of hypothalamic releasing hormones and the endocrine axes that they control (Fig. 38.3). SCN neurons represent an intrinsic circadian clock, as evidenced by the fact that they demonstrate a spontaneous peak of electrical activity at the same time every 24 to 25 hours. The 24- to

25-hour cycle can be “entrained” by the normal environmental light-dark cycle through specialized neural input from the retina (Fig. 38.4). Under constant light or dark, however, the SCN clock becomes “free running” and slightly drifts away from a 24-hour cycle each day.

FIG. 38.3 A circadian pacemaker directs numerous endocrine and body functions, each with its own daily schedule. The nighttime rise in plasma melatonin may mediate certain other circadian patterns. ACTH, Adrenocorticotropic hormone. (Data from Schwartz WJ. Adv Intern Med. 1994;38:81.)

FIG. 38.4 Origin of circadian rhythms in endocrine gland secretion, metabolic processes, and behavioral activity. (Modified from Turek FW. Recent Prog Horm Res. 1994;49:43.)

The pineal gland forms a neuroendocrine link between the SCN and various physiological processes that require circadian control. This tiny gland, close to the hypothalamus, synthesizes the hormone melatonin from the neurotransmitter serotonin. The rate-limiting enzyme for melatonin synthesis is Nacetyltransferase. The amount and activity of this enzyme in the pineal gland vary markedly in a cyclic manner, which accounts for the cycling of melatonin secretion and its plasma levels. Synthesis of melatonin is inhibited by light and markedly stimulated by darkness (Fig. 38.4). Thus, melatonin may transmit the information that nighttime has arrived, and body functions are regulated accordingly. Melatonin feedback to the SCN at dawn or dusk may also help evoke day-night entrainment of the SCN 24- to 25-hour clock. Melatonin has numerous other actions, including induction of sleep. Another important input to hypothalamic neurons and the pituitary gland is stress, either as systemic stress (e.g., hemorrhage, inflammation) or as processive stress (e.g., fear, anxiety). Major medical or surgical stress overrides the circadian clock and causes a pattern of persistent and exaggerated hormone release and metabolism that mobilizes endogenous fuels, such as glucose and free fatty acids, and augments their delivery to critical organs. Growth and reproductive processes, in contrast, are suppressed. In addition, cytokines released during inflammatory or immune responses, or both, directly regulate the release of hypothalamic releasing hormones and pituitary hormones.

Chemical Nature of Hormones Hormones are classified biochemically as proteins/peptides, catecholamines, steroid hormones, or iodothyronines. The chemical nature of a hormone determines (1) how it is synthesized, stored, and released; (2) how it is transported in blood; (3) its biological half-life and mode of clearance; and (4) its cellular mechanism of action.

Proteins/Peptides Protein and peptide hormones can be grouped into structurally related molecules that are encoded by gene families. Protein/peptide hormones obtain their specificity from their primary amino acid sequence and from post-translational modifications, especially glycosylation. Because protein/peptide hormones are destined for secretion outside the cell, their synthesis and processing are differently from those of proteins destined to remain within the cell or to be continuously added to the membrane (Fig. 38.5). These hormones are synthesized on the polyribosome as larger preprohormones or prehormones. The nascent peptides have at their N-terminus a group of 15 to 30 amino acids called the signal peptide. The signal peptide interacts with a ribonucleoprotein particle, which ultimately directs the growing peptide chain through a pore in the membrane of the endoplasmic reticulum located on the cisternal (i.e., inner) surface of the endoplasmic reticular membrane. Removal of the signal peptide by a signal peptidase generates a hormone or prohormone, which is then transported from the cisternae of the endoplasmic reticulum to the Golgi apparatus, where it is packaged into a membranebound secretory vesicle that is subsequently released into the cytoplasm. The carbohydrate moiety of glycoproteins is added in the Golgi apparatus.

FIG. 38.5 Schematic representation of peptide hormone synthesis. In the nucleus, the primary gene transcript, a messenger RNA precursor molecule, undergoes excision of introns, splicing of exons, capping of the 5′ end, and addition of polyadenylation (poly-A) at the 3′ end. The resultant mature messenger RNA enters the cytoplasm, where it directs the synthesis of a preprohormone peptide sequence on ribosomes. In this process, the N-terminus signal is removed, and the resultant prohormone is transferred vectorially into the endoplasmic reticulum. The prohormone undergoes further processing and packaging in the Golgi apparatus. After final cleavage of the prohormone within the granules, they contain the hormone and copeptides ready for secretion by exocytosis. NH2, Amidogen.

Most hormones are produced as prohormones. Prohormones harbor the peptide sequence of the active hormone within their primary sequence. However, prohormones are inactive or less active and require the

action of endopeptidases to trim away the neighboring inactive sequences. Protein/peptide hormones are stored in the gland as membrane-bound secretory vesicles and are released by exocytosis through the regulated secretory pathway. Thus, these hormones are not continually secreted. Rather, they are secreted in response to a stimulus through a mechanism of stimulussecretion coupling. Regulated exocytosis requires energy, Ca++, an intact cytoskeleton (microtubules, microfilaments), and the presence of coat proteins that specifically deliver secretory vesicles to the cell membrane. The ultrastructure of protein hormone–producing cells is characterized by abundant rough endoplasmic reticulum and Golgi membranes and the presence of secretory vesicles (Fig. 38.6).

FIG. 38.6 Ultrastructure of a protein hormone–producing cell. Note the presence of secretory vesicles and rough endoplasmic reticulum in the protein hormone–secreting cell. (From Kierszenbaum AL. Histology and Cell Biology: An Introduction to Pathology. 2nd ed. Philadelphia: Mosby; 2007.)

Protein/peptide hormones are soluble in body fluids and, with the notable exceptions of IGFs and growth hormone, circulate in blood predominantly in an unbound form and therefore have short biological half-lives. Protein/peptide hormones are removed from blood by multiple processes, including: (1) degradation by ectoenzymes, (2) excretion by the kidney, and/or (3) endocytosis and lysosomal degradation of hormone-receptor complexes (see the section “Cellular Responses to Hormones”). Proteins/peptides are readily digested in the gastrointestinal tract if administered orally. Hence, they must be administered by injection or, in the case of small peptides, through a mucous membrane (sublingually or intranasally). Because proteins/peptides do not cross cell membranes readily, they signal through membrane receptors (see Chapter 3).

Catecholamines Catecholamines are synthesized by the adrenal medulla and neurons and include norepinephrine, epinephrine, and dopamine (Fig. 38.7). The primary hormonal products of the adrenal medulla are epinephrine and, to a lesser extent, norepinephrine. Catecholamines obtain their specificity through

enzymatic modifications of the amino acid tyrosine. Catecholamines are stored in secretory vesicles that are part of the regulated secretory pathway. They are co-packaged with adenosine triphosphate, Ca++, and proteins called chromogranins. Chromogranins play a role in the biogenesis of secretory vesicles and in the organization of components within the vesicles. Catecholamines are soluble in blood and circulate either unbound or loosely bound to albumin. They are similar to protein/peptide hormones in that they do not cross cell membranes readily and hence produce their actions through cell membrane receptors. Catecholamines have short biological half-lives (1–2 minutes).

FIG. 38.7 Chemical structures of catecholamines.

AT THE C ELLU LAR LEVEL Bioactive hormones are generated from prohormones through proteolytic cleavage of the prohormone by prohormone (also called proprotein) convertases. These enzymes are expressed in a cell-specific manner. For example, insulin-producing cells (beta cells) of the pancreatic islets express both PC1 and PC2. Insulin is produced as preproinsulin, cleaved to proinsulin in the endoplasmic reticulum, and packaged in secretory vesicles as proinsulin. While in the secretory vesicle, a portion of the center of the single chain (connecting peptide) is cleaved sequentially by PC1 and PC2. The mature secretory vesicle contains and secretes equimolar amounts of insulin and connecting peptide. Sometimes prohormones contain the sequence of multiple hormones. For example, the protein proopiomelanocortin (POMC) contains the amino acid sequences of adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormones (MSHs). Pituitary cells express only PC1 and release only ACTH as a bioactive peptide. In contrast, certain neuronal cell types and keratinocytes express both PC1 and PC2 and can produce MSHs. There are also prohormones, called polyproteins, that contain multiple copies of the same bioactive peptide. For example, the sequence for thyrotropinreleasing hormone is reiterated six times within the prepro–thyrotropin-releasing hormone sequence. Rare mutations in PC1 have been identified in humans and are associated with extreme obesity in childhood, defects in glucose homeostasis, low glucocorticoid levels, loss of menstrual cycles and hypogonadism, and problems in gastrointestinal function.

Steroid Hormones Steroid hormones are made by the adrenal cortex, ovaries, testes, and placenta. Steroid hormones from these glands belong to five categories: progestins, mineralocorticoids, glucocorticoids, androgens, and estrogens. Progestins, mineralocorticoids, and glucocorticoids are 21-carbon steroids, whereas androgens are 19-carbon steroids and estrogens are 18-carbon steroids (Table 38.2). Steroid hormones also include the active metabolite of vitamin D (see Chapter 40), which is a secosteroid (i.e., one of the rings has an open conformation). Table 38.2 Steroid Hormones Family

Number of Carbons

Specific Hormone

Progestin

21

Progesterone

Glucocorticoid

21

Cortisol Corticosterone

Primary Site of Synthesis Ovary Placenta Adrenal cortex

Primary Receptor Progesterone receptor Glucocorticoid receptor

Mineralocorticoid 21

Aldosterone Adrenal cortex 11Deoxycorticosterone

Mineralocorticoid receptor

Androgen

19

Testosterone Dihydrotestosterone

Androgen receptor

Estrogen

18

Estradiol-17β Estriol

Testis Ovary Placenta

Estrogen receptor

Steroid hormones are synthesized by a series of enzymatic modifications of cholesterol, which has a core of four carbon ring structures (Fig. 38.8). The enzymatic modifications of cholesterol are of three general types: hydroxylation, dehydrogenation/reduction, and lyase reactions. The purpose of these modifications is to produce a cholesterol derivative that is sufficiently unique to be recognized by a specific receptor. Thus, progestins bind to the progesterone receptor, mineralocorticoids bind to the mineralocorticoid receptor, glucocorticoids bind to the glucocorticoid receptor, androgens bind to the androgen receptor, estrogens bind to the estrogen receptor, and the active vitamin D metabolite binds to the vitamin D receptor. The complexity of steroid hormone action is increased by the expression of multiple forms of each receptor. In addition, there is some degree of nonspecificity between steroid hormones and the receptors to which they bind. For example, glucocorticoids bind to the mineralocorticoid receptor with high affinity, and progestins, glucocorticoids, and androgens can all interact with the progesterone, glucocorticoid, and androgen receptors to some degree. As discussed later, steroid hormones are hydrophobic and diffuse through cell membranes easily. Accordingly, classic steroid hormone receptors are localized intracellularly and act by regulating gene expression. There is mounting evidence of the presence of plasma membrane and juxtamembrane steroid hormone receptors that mediate rapid, nongenomic actions of steroid hormones.

FIG. 38.8 A, Structure of cholesterol, the precursor of steroid hormones. B, Structure of steroid hormones.

Steroidogenic cell types are defined as cells that can convert cholesterol to pregnenolone, which is the first reaction common to all steroidogenic pathways. Steroidogenic cells have some capacity for cholesterol synthesis but often obtain cholesterol from cholesterol-rich lipoproteins (low-density lipoproteins and high-density lipoproteins). Pregnenolone is then further modified by several enzymatic reactions. Because of their hydrophobic nature, steroid hormones and precursors can leave the steroidogenic cell easily and thus are not stored. Therefore, steroidogenesis is regulated at the level of uptake, storage, and mobilization of cholesterol and at the level of steroidogenic enzyme gene expression and activity. Steroids are not regulated at the level of secretion of the preformed hormone. A clinical implication of this mode of secretion is that high levels of steroid hormone precursors are easily released into blood when a steroidogenic enzyme within a given pathway is inactive or absent. The ultrastructure of steroidogenic cells is distinct from protein- and catecholamine-secreting cells. Steroidogenic enzymes

reside within the inner mitochondrial membrane or the membrane of the smooth endoplasmic reticulum. Thus, steroidogenic cells typically contain extensive mitochondria and smooth endoplasmic reticulum (Fig. 38.9). These cells also contain lipid droplets, which represent a store of cholesterol esters.

FIG. 38.9 Ultrastructure of a steroidogenic cell. Note the abundance of lipid droplets, smooth endoplasmic reticulum, and mitochondria with tubular cristae. (From Kierszenbaum AL. Histology and Cell Biology: An Introduction to Pathology. 2nd ed. Philadelphia: Mosby; 2007.)

An important feature of steroidogenesis is that steroid hormones often undergo further modifications (apart from those involved in deactivation and excretion) after their release from the original steroidogenic cell. For example, estrogen synthesis by the ovary and placenta requires at least two cell types to complete the conversion of cholesterol to estrogen. This means that one cell secretes a precursor and a second cell converts the precursor to estrogen. There is also considerable peripheral conversion of active steroid hormones. For example, the testes secrete little estrogen. However, adipose, muscle, and other tissues express the enzyme for converting testosterone (a potent androgen) to estradiol-17β (a potent estrogen). Thus, the overall production of a specific steroid hormone is equivalent to the sum of (1) the secretion of this specific steroid hormone from a steroidogenic cell type and (2) peripheral conversion of other steroids to this specific steroid hormone (Fig. 38.10). Peripheral conversion can produce (1) a more active but similar class of hormone (e.g., conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D); (2) a less active hormone that can be reversibly activated by another tissue (e.g., conversion of cortisol to cortisone in the kidneys, followed by conversion of cortisone to cortisol in abdominal adipose tissue); or (3) a different class of hormone (e.g., conversion of testosterone to estrogen). Peripheral conversion of steroids plays an important role in several endocrine disorders (see Chapters 43 and 44).

FIG. 38.10 Peripheral conversion of steroid hormones.

Because of their nonpolar nature, steroid hormones are not readily soluble in blood. Therefore, steroid hormones circulate bound to transport proteins, including albumin, but also the specific transport proteins sex hormone–binding globulin and corticosteroid-binding globulin (see the section “Transport of Hormones in the Circulation”). Excretion of hormones from the body typically involves inactivating modifications, followed by glucuronide or sulfate conjugation in the liver, which is often coupled to biliary excretion. These modifications also increase the water solubility of the steroid and decrease its affinity for transport proteins, thereby allowing the inactivated steroid hormone to be excreted by the kidneys. Steroid compounds are absorbed fairly readily in the gastrointestinal tract and may therefore be administered orally.

Iodothyronines Thyroid hormones are iodothyronines (Fig. 38.11) that are made by the coupling of iodinated tyrosine residues through an ether linkage. Their specificity is determined by the thyronine structure, as well as by where the thyronine is iodinated. Thyroid hormones cross cell membranes by transport systems. They are stored extracellularly in the thyroid as an integral part of the glycoprotein molecule thyroglobulin. Thyroid hormones are sparingly soluble in blood and aqueous fluids and are transported in blood as bound (>99%) to serum-binding proteins. A major transport protein is thyroid hormone–binding globulin. Thyroid hormones have long half-lives (7 days for thyroxine; 18 hours for triiodothyronine). Thyroid hormones are similar to steroid hormones in that the thyroid hormone receptor is intracellular and acts as a transcription factor. In fact, the thyroid hormone receptor belongs to the same gene family that includes steroid hormone receptors and vitamin D receptor. Thyroid hormones can be administered orally; the amount absorbed intact is sufficient for this to be an effective mode of therapy.

FIG. 38.11 Structure of thyroid hormones, which are iodothyronines.

Transport of Hormones in the Circulation A significant fraction of steroid and thyroid hormones is transported in blood that is bound to plasma proteins that are produced in a regulated manner by the liver. Protein and polypeptide hormones are generally transported free in blood. The concentrations of bound hormone, free hormone, and plasma transport protein are in equilibrium. If free hormone levels drop, hormone will be released from the transport proteins. This relationship may be expressed as follows:

Equation 38.1 where [H] = concentration of free hormone, [P] = concentration of plasma transport protein, [HP] = concentration of bound hormone, and K = the dissociation constant. Free hormone is the biologically active form for action on the target organ, feedback control, and clearance by cellular uptake and metabolism. As a consequence, when hormonal status is evaluated, sometimes free hormone levels must be determined in addition to total hormone levels. This is particularly important because hormone transport proteins themselves are regulated by altered endocrine and disease states. Protein binding serves several purposes. It prolongs the circulating half-life of the hormone. Many hormones cross cell membranes readily and would either enter cells or be excreted by the kidneys if they were not protein bound. The bound hormone represents a reservoir of hormone and, as such, can serve to buffer acute changes in hormone secretion. Some hormones, such as steroids, are sparingly soluble in blood, and protein binding facilitates their transport.

Cellular Responses to Hormones Hormones are also referred to as ligands, in the context of ligand-receptor binding, and as agonists, in that their binding to the receptor is transduced into a cellular response. Receptor antagonists typically bind to a receptor and lock it in an inactive state, in which the receptor is unable to induce a cellular response. Loss or inactivation of a receptor results in hormonal resistance. Constitutive activation of a

receptor leads to unregulated, hormone-independent activation of cellular processes. Hormones regulate essentially every major aspect of cellular function in every organ system. Hormones control the growth of cells, ultimately determining their size and competency for cell division. Hormones regulate the differentiation of cells and their ability to survive or to undergo programmed cell death. They influence cellular metabolism, the ionic composition of body fluids, and cell membrane potential. Hormones orchestrate several complex cytoskeleton-associated events, including cell shape, migration, division, exocytosis, recycling/endocytosis, and cell-cell and cell-matrix adhesion. Hormones regulate the expression and function of cytosolic and membrane proteins, and a specific hormone may determine the level of its own receptor or the receptors for other hormones. Although hormones can exert coordinated, pleiotropic control on multiple aspects of cell function, any given hormone does not regulate every function in every cell type. Rather, a single hormone controls a subset of cellular functions in only the cell types that express receptors for that hormone. Thus, selective receptor expression determines which cells respond to a given hormone. Moreover, the differentiated state of a cell determines how it responds to a hormone. Thus, the specificity of hormonal responses resides in the structure of the hormone itself, the receptor for the hormone, and the cell type in which the receptor is expressed. Serum hormone concentrations are typically extremely low (10−11–10−9 mol/L). Therefore, a receptor must have high affinity, as well as specificity, for its cognate hormone. How does hormone-receptor binding get transduced into a cellular response? Hormone binding to a receptor induces conformational changes in the receptor. These changes are collectively referred to as a signal. The signal is transduced into the activation of one or more intracellular messengers. Messenger molecules then bind to effector proteins, which in turn modify specific cellular functions. The combination of hormone-receptor binding (signal), activation of messengers (transduction), and regulation of one or more effector proteins is referred to as a signal transduction pathway (also called simply a signaling pathway), and the final outcome is referred to as the cellular response. Signaling pathways are usually characterized by the following properties: 1. Multiple, hierarchical steps in which “downstream” effector proteins are dependent on and driven by “upstream” receptors, transducers, and effector proteins. This means that loss or inactivation of one or more components within the pathway leads to general resistance to the hormone, whereas constitutive activation or overexpression of components can drive a pathway in an unregulated manner. 2. Amplification of the initial hormone-receptor binding. Amplification can be so great that maximal response to a hormone is achieved when the hormone binds to a small percentage of receptors. 3. Activation of multiple pathways, or at least regulation of multiple cell functions, from one hormone-receptor binding event. For example, binding of insulin to its receptor activates three separate signaling pathways. Even in fairly simple pathways (e.g., glucagon activation of adenylate cyclase), divergent downstream events allow the regulation of multiple functions (e.g., post-translational activation of glycogen phosphorylase and increased phosphoenolpyruvate carboxykinase gene transcription). 4. Antagonism by constitutive and regulated negative feedback reactions. This means that a signal is dampened or terminated (or both) by opposing reactions and that loss or gain of function of opposing components can cause hormone-independent activation of a specific pathway or hormone resistance. As discussed in Chapter 3, hormones signal to cells through membrane or intracellular receptors.

Membrane receptors have rapid effects on cellular processes (e.g., enzyme activity, cytoskeletal arrangement) that are independent of the synthesis of new protein. Membrane receptors can also rapidly regulate gene expression through either mobile kinases (e.g., cyclic adenosine monophosphate–dependent protein kinase [PKA], mitogen-activated protein kinases [MAPKs]) or mobile transcription factors (e.g., signal transducer and activator of transcription proteins [STATs], Mothers against decapentaplegic homologs [SMAD1]). Steroid hormones have slower, longer term effects that involve chromatin remodeling and changes in gene expression. Increasing evidence indicates that steroid hormones have rapid, nongenomic effects as well, but these pathways are still being elucidated. The presence of a functional receptor is an absolute requirement for hormone action, and loss of a receptor produces essentially the same symptoms as loss of hormone. In addition to the receptor, there are fairly complex pathways involving numerous intracellular messengers and effector proteins. Accordingly, endocrine diseases can arise from abnormal expression or abnormal activity, or both, of any of these signal transduction pathway components. Finally, hormonal signals can be terminated in several ways, including hormone/receptor internalization, phosphorylation/dephosphorylation, proteasomal destruction of receptor, and generation of feedback inhibitors.

IN THE C LIN IC Endocrine diseases can be broadly categorized as hyperfunction or hypofunction of a specific hormonal pathway. Hypofunction can be caused by lack of active hormone or by hormone resistance as a result of inactivation of hormone receptors or postreceptor defects. Testicular feminization syndrome is a dramatic form of hormone resistance in which the androgen receptor is mutated and cannot be activated by androgens. In patients in whom the diagnosis is not made before puberty, the testis becomes hyperstimulated because of abrogation of the negative feedback between the testis and the pituitary gland. The increased androgen levels have no direct biological effect as a result of the receptor defect. However, the androgens are peripherally converted to estrogens. Thus affected individuals are genetically male (i.e., 46,XY) but have a strongly feminized external phenotype, a female sexual identity, and usually a sexual preference for men (i.e., heterosexual in relation to sexual identity). Treatment involves removal of the hyperstimulated testes (which reside in the abdomen and pose a risk for cancer), estrogen replacement therapy, and counseling for the patient and, if one exists, the partner/spouse to address infertility and social/psychological distress.

Key Concepts 1. Endocrine signaling involves (1) regulated secretion of an extracellular signaling molecule, called a hormone, into the extracellular fluid; (2) diffusion of the hormone into the vasculature and circulation throughout the body; and (3) diffusion of the hormone out of the vascular compartment into the extracellular space and binding to a specific receptor within cells of a target organ. 2. The endocrine system is composed of the endocrine tissue of the pancreas, the parathyroid glands, the pituitary gland, the thyroid gland, the adrenal glands, and the gonads (testes or ovaries). 3. Negative feedback represents an important control mechanism that confers stability on endocrine systems. Hormonal rhythms are imposed on negative feedback loops. 4. Protein/peptide hormones are produced on ribosomes and stored in endocrine cells in membranebound secretory granules. They typically do not cross cell membranes readily and act through cell

membrane–associated receptors. 5. Catecholamines are synthesized in the cytosol and secretory granules and do not readily cross cell membranes. They act through cell membrane–associated receptors. 6. Steroid hormones are not stored in tissues and generally cross cell membranes relatively readily. They act through intracellular receptors. 7. Thyroid hormones are synthesized in follicular cells and stored in follicular colloid as thyroglobulin. They cross cell membranes and associate with nuclear receptors. 8. Some hormones act through membrane receptors, and their responses are mediated by rapid intracellular signaling pathways. 9. Other hormones bind to nuclear receptors and act by directly regulating gene transcription.

C H AP T E R 3 9

Hormonal Regulation of Energy Metabolism LEARNING OBJECTIVES Upon completion of this chapter, the student should be able to answer the following questions: 1. Explain the different requirements for and utilization by different cells of fuels during the digestive phase as opposed to the interdigestive and fasting phases. 2. Integrate the structure, synthesis, and secretion of insulin with circulating fuel levels, especially glucose. 3. Utilize the different signaling pathways regulated by insulin to link insulin to its cellular effects at the molecular level. 4. Integrate the structure, synthesis, and secretion of glucagon with the levels of circulating fuels, insulin, and catecholamines. 5. Map out and integrate the actions of insulin on the utilization and storage of glucose, free fatty acids (FFAs), and amino acids (AAs) by hepatocytes, skeletal muscle, and adipocytes during the digestive phase. 6. Map out and integrate the actions of counterregulatory hormones (glucagon, catecholamines) on the utilization of glucose, the sparing of glucose, and the utilization of FFAs and AAs by hepatocytes, skeletal muscle, and adipocytes during the interdigestive and fasting phases. 7. Integrate the changes in fuel utilization and hormonal signaling in hepatocytes during the interdigestive and fasting phases that allow for and promote hepatic glucose production and ketogenesis. 8. Compare signaling pathways that have orexigenic and anorexigenic actions via the hypothalamus. 9. Link several pathologies related to metabolism, especially those caused by the absolute or relative absence of insulin and by obesity.

Continual Energy Supply and Demand: The Challenge There are an estimated 40 trillion cells in the human body, not including the approximately 40 trillion nonhuman cells that comprise the human microbiome. All these cells must continually perform work to stay alive. This work includes maintenance of cellular composition and structural integrity, along with the integrated synthesis and breakdown (i.e., turnover) of macromolecules and organelles. This work also involves the functions of cells that contribute to the human body as a whole (e.g., contraction of the muscle fibers of the diaphragm). Additional work is required of cells when the human body is engaged in a variety of activities, including (but not limited to) manual labor, exercise, and outdoor play; body

growth spurt and maturation of the reproductive systems at puberty; pregnancy and breast-feeding; combating an infection or cancer; and the healing of damaged tissues/organs (e.g., healing from surgery). On average, the resting metabolic rate of a relaxed, awake, stationary, healthy adult human accounts for about 70% of their total energy expenditure each day (Fig. 39.1).

FIG. 39.1 Overview of energy metabolism. AAs, amino acids; FFAs, free fatty acids.

To perform this work, cells need fuels, along with the capability to convert fuels into potential chemical energy in the form of adenosine triphosphate (ATP). Cells then convert the energy within ATP into chemical and mechanical work (see Fig. 39.1). This means that the need for ATP is immediate and unending, and consequently all living cells must continually synthesize ATP. In fact, humans produce about the equivalent of their body weight in ATP daily. This places a demand on the body to continually supply fuel in some form to all cells. All fuel originates from the diet, but humans do not eat in a nonstop manner all day long. Thus, the constant cellular demand for fuels to make ATP and perform work is paired with an intermittent ingestion of fuels. Diet-derived fuels are oxidized for ATP, but in order to maintain ATP production when not eating for a while (e.g., during sleep), some fuels are stored for future use. In trying to make sense of energy metabolism, it is important to organize one’s thinking around the following: 1. Fuels (Fig. 39.1). Our diet includes both monomeric and polymeric forms (the latter are converted into monomeric forms during digestion and absorption) of the following: (1) monosaccharides, including glucose, fructose, and galactose; (2) long-chain free fatty acids (referred to in this chapter as simply FFAs); and (3) amino acids (AAs). The fourth general type of fuel is ketone bodies (KBs), which are largely absent in the diet. Instead, KBs are produced by hepatocytes via ketogenesis (reaction 14 in Fig. 39.3B), using FFAs and ketogenic AAs, both of which become abundant during the Fasting Phase. The diet also includes other fuels such as

ethanol. 2. Metabolic Phases. Metabolic Phases refer to the hourly and daily differences in fuel usage and energy metabolism, which are dictated largely by the abundance or scarcity of certain fuels and orchestrated by phase-specific hormones. In general, there are three metabolic phases (Fig. 39.2): (1) the Digestive or Absorptive Phase, which occurs during the 2 to 3 hours it takes to digest a meal; (2) the Interdigestive or Postabsorptive Phase, which normally occurs between meals; and (3) the Fasting Phase, which most commonly occurs between the last snack before bedtime and breakfast. (In fact, physicians refer to a blood value as “fasting,” e.g., “fasting blood glucose,” if the patient abstains from eating after midnight and has blood drawn about 8 AM; prolonged fasting and starvation are more extreme forms of fasting.) Physical exertion, which imposes a heightened energy demand, is another type of metabolic phase that occurs with some frequency and regularity for some individuals. This chapter primarily compares how metabolism differs between the Digestive Phase and the Fasting Phase, and how different hormones orchestrate these metabolic differences. 3. Metabolic actions of hepatocytes, adipocytes, and skeletal myocytes. All cells are involved in energy metabolism, but these three cell types have a profound impact on whole-body metabolism. During the Digestive Phase, hepatocytes, skeletal myocytes, and adipocytes function largely independently of each other. In contrast, the actions of these three cell types become highly integrated during the Fasting Phase in order to maintain adequate blood glucose levels while providing alternative energy substrates for each cell type. Key features with respect to metabolism of these three cell types are listed in Table 39.1. 4. Blood glucose levels. Cells with no or very few mitochondria (e.g., erythrocytes, lens cells of the eye) are absolutely dependent on glucose for energy. Additionally, the central nervous system (CNS) can only use glucose for ATP production under normal conditions. Thus, in the Interdigestive and Fasting Phases, maintenance of blood glucose above a certain minimal threshold is absolutely necessary to avoid CNS-related symptoms, beginning with those caused by a hypoglycemia-activated autonomic response (e.g., nausea, sweating, cardiac arrhythmias). If blood glucose continues to fall, progression to symptoms caused by neuroglycopenia (e.g., cognitive dysfunction, loss of coordinated motor function, and ultimately even coma and death) can occur. This means that whole-body metabolism during the Interdigestive and Fasting Phases must meet the challenge of maintaining blood glucose above 60 mg/dL (see Fig. 39.2, green arrow). Conversely, blood glucose levels must be maintained below an upper threshold (see Fig. 39.2, red arrow). This is because glucose is a fairly reactive molecule. High blood glucose leads to high intracellular glucose in many cells, which, in turn, becomes nonenzymatically covalently linked to proteins and other molecules, thereby disrupting their configuration, half-life, and function (see In The Clinic—Glucotoxicity Within Microvasculature).

5. Insulin and the counterregulatory hormones. Metabolism during the Digestive Phase is orchestrated almost entirely by insulin. During the Fasting Phase, insulin drops to low levels, and this alone allows for some of the metabolic adaptions during the Fasting Phase. In addition, glucagon and catecholamines (epinephrine, norepinephrine) stimulate metabolic pathways that integrate the body’s response to an absence of ingested and absorbed fuels. These hormones are referred to as counterregulatory hormones based on their opposition to insulin. Growth hormone (see Chapter 41) and cortisol (see Chapter 43) also contribute somewhat to Fasting-Phase metabolism. Table 39.1

Fate of Fuels During Digestive and Fasting Phases

Hepatocytes

Adipocytes

Skeletal Myocytes

Glucose, fed phase

• Utilization for ATP • Storage as glycogen • DNL

• Increased uptake by GLUT4 • Utilization for ATP • Utilization for G3P

• Increased uptake by GLUT4 (largest impact on glucose tolerance) • Utilization for ATP • Storage as glycogen

Glucose, fasting phase

• Breakdown of glycogen and release of glucose into blood • New synthesis of glucose from small precursors and release into blood • Use of alternative fuel for ATP

• Decreased uptake by GLUT4 • Use of alternative fuels

• Decreased uptake by GLUT4 • Use of alternative fuels • Breakdown of glycogen and use of glucose intracellularly (no export), especially during exercise

FFA/TG, fed phase

• Make FFAs from glucose by DNL • Esterify FFAs into intrahepatic TG • Uptake of chylomicron remnants

• Lipolysis of chylomicrons and uptake of FFAs • Esterification of FFAs into storage TG • Inhibition of lipolysis of stored TG

• Minimal involvement

FFA/TG, fasting phase

• Utilization for ATP • Utilization to produce KBs • Assembly of TG into VLDL • Secretion of VLDL

• Release of FFAs from TG stores • Utilization for ATP

• Utilization for ATP

AAs, fed phase

• Utilization for multiple anabolic pathways

• Utilization for multiple anabolic pathways

• Utilization for multiple anabolic pathways

AAs, fasting phase

• Utilization for gluconeogenesis • Utilization for ketogenesis

• Proteolysis and release of AAs

• Proteolysis and release of AAs

KBs, fed phase

• Should be absent

• Should be absent

• Should be absent

KBs, fasting phase

• Synthesis from FFAs and some AAs • Cannot be utilized for ATP

• Utilization for ATP

• Utilization for ATP

AA, Amino acid; ATP, adenosine triphosphate; DNL, de novo lipogenesis; FFA, free fatty acid; G3P, glycerol-3-phosphate; KB, ketone body; TG, triglyceride; VLDL, very low-density lipoprotein.

FIG. 39.2 Blood glucose levels during the three metabolic phases: D, Digestive Phase; F, Fasting Phase; I, Interdigestive Phase. B, breakfast; D, dinner; L, lunch. Red arrow indicates the upper limit for normal fasting glucose; green arrow indicates the lower limit for normal fasting glucose.

FIG. 39.3 A, Overview of glucose utilization during the Digestive Phase. GLUT transporters: G1, GLUT1; G2, GLUT2; G3, GLUT3; G4-m, functional GLUT4 localized to cell membrane. Cell types: ADIPO, adipocyte; CNS, central nervous system neurons and glia; HEPATO, hepatocyte; RBC, red blood cell; SKM, skeletal myocyte. Metabolites: G6P, glucose-6-phosphate; GLY, glycogen; G’OL3P, glycerol-3phosphate; TG, triglyceride. Metabolic reactions/pathways: 1, hexokinase/glucokinase; 2, glycolysis; 3, lactate dehydrogenase; 4, TCA cycle ± pyruvate dehydrogenase; 5, oxidative phosphorylation; 6, glycogen synthesis; 7, de novo lipogenesis; 8, esterification of FFAs to G’OL3P to form TG. B, Overview of energy metabolism during the Fasting Phase. GLUT transporters: see legend for Fig. 39.3A, plus: G4-I, inactive GLUT4 with intracellular localization. Cell types: see legend for Fig. 39.3A. Metabolites: see legend for Fig. 39.3A, plus: AAs, amino acids; FFA, free fatty acid; G’OL, glycerol; KBs, ketone bodies; LACT, lactate; PROT, protein. Metabolic reactions/pathways: see legend for Fig. 39.3A, plus: 9, glycogenolysis; 10, gluconeogenesis; 11, G-6-phosphatase; 12, lipolysis; 13, proteolysis; 14, ketogenesis; 15, ketolysis; 16, βoxidation.

Integrated Overview of Energy Metabolism The basic objectives of the Digestive Phase (see Table 39.1) include: 1. Glucose utilization, in order to prevent prolonged periods of high blood glucose (Fig. 39.2; red arrow). 2. Synthesis and storage of fuel polymers (glycogen, triglycerides, proteins) that can be accessed for fuels during the Fasting Phase. 3. Overall anabolism to maintain the molecular integrity of cells. The metabolic pathways that fulfill these objectives are driven by insulin, which is the main hormone of the Digestive Phase.

Digestive Phase During the Digestive Phase, absorbed fuels are partitioned and used for different purposes. Glucose is the primary fuel used for energy (i.e., ATP production) during the Digestive Phase (refer to Fig. 39.3A for

enzymatic pathways). Glucose is considered a universal fuel in that most cells can perform the following: 1. Import glucose via bidirectional facilitative GLUT transporters (G1, G2, G3, and G4). 2. “Trap” and “activate” imported glucose by converting glucose into glucose-6-phosphate (G6P) through the activity of one or more hexokinases (Pathway 1). G6P cannot pass through GLUT transporters (“trapping”) and is now a substrate for several enzymatic pathways (“activation”). 3. Metabolize G6P to pyruvate via the glycolytic pathway, which yields a small amount of ATP without requiring mitochondria or O2 (Pathway 2). Cells without mitochondria ferment pyruvate to lactate (Pathway 3) and export lactate to the ECF. In contrast, most cells import pyruvate into mitochondria, convert it to acetyl CoA by pyruvate dehydrogenase, and then condense acetyl CoA with oxaloacetate to form citrate. Citrate is cycled through the tricarboxylic acid (TCA) cycle back to oxaloacetate (Pathway 4). This metabolism of pyruvate through the TCA cycle releases CO2 as waste and generates guanosine triphosphate (GTP) along with flavin adenine dinucleotide hydride (FADH2) and nicotine adenine dinucleotide hydride (NADH). FADH2 and NADH are used by the electron transport system and oxidative phosphorylation to ultimately generate relatively large amounts of ATP through a process that is absolutely dependent on O2 (Pathway 5).

IN THE C LIN IC Glucotoxicity Within Microvasculature The endothelium of the microvasculature of the kidney and retina, as well as the endothelium of the vasa nervosum of the autonomic nervous system, is particularly sensitive to hyperglycemia. Chronically high blood glucose results in pathologically high intracellular levels of glucose in these endothelial cells, resulting in altered protein and lipid structure, oxidative stress, and altered signaling pathways. These insults, collectively referred to as glucotoxicity, cause pathological changes in intracellular and membrane components as well as in secreted molecules that either signal and/or make up the extracellular matrix. Indeed, glucotoxicity is the root cause of the nephropathy, retinopathy, and peripheral neuropathy that occur in poorly controlled diabetes mellitus. Therefore, whole-body metabolism during all metabolic phases must meet the challenge of minimizing the magnitude and duration of the rise in blood glucose associated with ingestion of a meal and must maintain blood glucose below a safe maximal threshold of 100 mg/dL during all other times. Fasting blood glucose between 100 and 124 mg/dL is indicative of impaired glucose tolerance, and values at 125 mg/dL and above are evidence of diabetes mellitus. Glucose is consumed by erythrocytes and the brain continually throughout all metabolic phases. In contrast, hepatocytes, skeletal myocytes, and adipocytes primarily use glucose during the Digestive Phase. Insulin stimulates glycolysis and entry of pyruvate (end product of glycolysis) into the TCA cycle and oxidative phosphorylation for ATP production in hepatocytes, skeletal myocytes, and adipocytes (see Table 39.1). Hepatocytes express the GLUT2 isoform of the glucose transporter, which is not regulated by insulin for its insertion into the cell membrane. In contrast, skeletal myocytes and adipocytes express the GLUT4 isoform. Newly synthesized GLUT4 exists in an intracellular inactive state with GLUT4 storage vesicles (G4-i in Fig. 39.3B). Insulin induces translocation and insertion of these GLUT4-rich vesicles into the

cell membrane, where GLUT4 can function as an active glucose transporter (G4-m in Fig. 39.3A). After its phosphorylation to G6P by glucokinase, hepatocytes convert some of the imported glucose into the storage form, glycogen, during the Digestive Phase (Fig. 39.3A, Pathway 6). Similarly, skeletal muscle converts some of the G6P from imported glucose into glycogen. Hepatocytes can only store a finite amount of glucose as glycogen. Hepatocytes also convert excess glucose into FFAs through the process of de novo lipogenesis (DNL; Pathway 7). These FFAs are typically esterified to glycerol-3-phosphate (G3P) to form triglyceride (TG; Pathway 8), which accumulates as intrahepatic TG during the Digestive Phase. As discussed later for insulin signaling, an excessive accumulation of intrahepatic TG (i.e., fatty liver, hepatic steatosis) can result in insulin resistance. During the Digestive Phase, AAs are used in multiple anabolic pathways to regenerate degraded molecules, including other AAs, proteins, nucleotides and nucleic acids, glutathione, and complex lipids. FFAs represent the most efficient fuel type in terms of ATP molecules made per carbon of fuel. However, utilization of FFAs competes effectively with glucose utilization in the mitochondria. High FFA levels during the Digestive Phase would promote a greater magnitude and duration of the glucose surge, thereby contributing to hyperglycemia. Thus, most of the FFAs in an average meal are prevented from entering the circulation by their reesterification into TG and packaging into chylomicrons within the intestinal enterocyte. Chylomicrons are secreted, enter lymphatic vessels and then the blood, and supply adipocytes with FFAs to be stored as TG for use during the Fasting Phase (discussed in more detail later).

Fasting Phase The basic objectives of the Fasting Phase include: 1. Glucose production that maintains blood glucose levels above the lower normal limit (Fig. 39.2; green arrow). Glucose production is achieved through glycogenolysis and gluconeogenesis in hepatocytes and kidney (see Table 39.1). 2. Glucose sparing, which involves a general decrease in the uptake of glucose by cells, especially by skeletal muscle, and the utilization of FFAs, AAs, and KBs (instead of glucose) for ATP production by most cells. This also helps to maintain adequate blood glucose levels during the Fasting Phase (Fig. 39.2; green arrow). 3. Overall catabolism, with the breakdown of polymers into alternate forms of fuel. The metabolic pathways that achieve these objectives are driven by glucagon (liver, adipose tissue), and catecholamines and intracellular metabolic signals (e.g., increased Ca2+, increased AMP/ATP ratio). Note also that decreased anabolism reduces cellular ATP needs. Hepatic glucose production is based on two metabolic pathways (refer to Fig. 39.3B). The first is the rapid catabolic process of glycogenolysis (Pathway 9). Hepatocytes express the enzyme glucose-6phosphatase (G6Pase) (Pathway 11), allowing them to convert G6P back to glucose, which can then exit the cell through a bidirectional GLUT2 transporter. Release of glucose derived from glycogenolysis is relatively short lived because the liver glycogen supply becomes exhausted by about 8 hours. The second metabolic contribution to hepatic glucose production during the Fasting Phase is the gradual pathway of gluconeogenesis (Pathway 10). The onset of gluconeogenesis during fasting is slower than glycogenolysis, but gluconeogenesis continues essentially nonstop throughout a Fasting Phase (Fig. 39.4). Gluconeogenesis requires 3-carbon precursors, especially lactate, “gluconeogenic” AAs, and glycerol. How are these precursors supplied during the Fasting Phase? Lactate is continually produced by

erythrocytes. Lactate is also produced by glycolytic skeletal muscle fibers during exercise (exercise tends to occur more frequently during the Interdigestive and Fasting Phases as opposed to “on a full stomach”), although much of this lactate is utilized by aerobic skeletal muscle and cardiac muscle during exercise. But additionally, the overall anabolism of the Digestive Phase switches over to a general catabolism during the Fasting Phase (see Fig. 39.3B). TGs within adipocytes undergo lipolysis to FFAs and glycerol (Pathway 12), and there is a general net proteolysis with the release of AAs during the Fasting state, especially from muscle (Pathway 13). The glycerol and gluconeogenic AAs are released from cells and circulate to the liver, where they are subsequently used for gluconeogenesis (Pathway 10). Thus, gluconeogenesis requires an integration of catabolic pathways in adipocytes and skeletal myocytes with anabolic gluconeogenesis in hepatocytes. Gluconeogenesis eventually supplants glycogenolysis and can continue as long as precursors flow into the liver.

FIG. 39.4 Relative contributions of the three sources of blood glucose relative to meals and time of day. The inset box stresses replacement of glycogenolysis with gluconeogenesis during the fasting phase (i.e., sleep). (Adapted from Baynes JW, Dominiczak JH [eds]. Medical Biochemistry. 3rd ed. Philadelphia: Mosby/Elsevier; 2009.)

Glucose sparing represents the other general process that contributes to maintenance of adequate blood glucose levels during the Fasting Phase. Glucose sparing means the switching of fuel utilization from glucose to a nongluconeogenic fuel in most cell types, but especially in skeletal muscle, which represents the potentially largest single consumer of glucose. First, the uptake of glucose by skeletal muscle and adipocytes is greatly reduced because the GLUT4 transporter isoform exists in intracellular vesicles and this in an inactive state (G4-i in Fig. 39.3B) during the Fasting Phase. Thus, alternative fuels need to be delivered to skeletal muscle and adipocytes. The nongluconeogenic fuels (i.e., cannot be used for gluconeogenesis by the liver) are FFAs and KBs. FFAs are primarily released from adipocytes (Pathway 12) but are also released after packaging of intrahepatic TGs into very low-density lipoproteins (VLDLs) by hepatocytes (discussed later). FFAs are then converted via multiple rounds of β-oxidation (Pathway 16) to acetyl CoAs. KBs are produced via ketogenesis (Pathway 14) in hepatocytes from acetyl CoA, which in turn originates primarily from FFAs and ketogenic AAs, both of which become abundant during the Fasting Phase. KBs are converted back to acetyl CoA via ketolysis (Pathway 15) in nonhepatic cell types. Thus, glucose sparing depends on catabolic adipocyte metabolism, which results in lipolysis of stored TGs and release of FFAs. FFAs are imported by hepatocytes, which use FFAs to produce acetyl CoA. Protein degradation in skeletal muscle and other tissues also makes certain AAs available for ketogenesis. High levels of intramitochondrial

acetyl CoA in the hepatocyte not only provides ample carbons for ATP synthesis but serves to: (1) inhibit conversion of pyruvate to acetyl CoA, (2) promote conversion of pyruvate to oxaloacetate for gluconeogenesis, and (3) promote synthesis of KBs (see Fig. 39.3B). After several days of fasting, the CNS can start using KBs for energy, thereby further sparing glucose for erythrocytes. Many other cell types with mitochondria use KBs along with FFAs for ATP production, especially skeletal muscle. Note however that hepatocytes only carry out ketogenesis, but not ketolysis, as this would form a futile cycle. The hormones that drive glycogenolysis, gluconeogenesis, lipogenesis, and hepatic ketogenesis as well as VLDL production by the liver during the Fasting Phase are glucagon and catecholamines. In the presence of low glucose, insulin levels fall, and that removes the inhibition by insulin of the secretion of another pancreatic hormone, glucagon. Thus, diminished blood glucose causes a rise in the circulating glucagon-to-insulin ratio. Hepatocytes are the primary target organ of glucagon, which directly drives glycogenolysis (Pathway 9), gluconeogenesis (Pathway 10), ketogenesis (Pathway 14), and FFA oxidation (Pathway 16). Hepatocytes also express β2- and α1-adrenergic receptors so that norepinephrine from sympathetic innervation and epinephrine from the adrenal medulla (see Chapter 43) can reinforce the actions of glucagon. Adipocytes also express the glucagon receptor, as well as the β2- and β3-adrenergic receptors that respond to catecholamines in response to hypoglycemia, exertion, or certain stresses. Skeletal muscle is not a target of glucagon but does respond to catecholamines stimulation through β2adrenergic receptors. Skeletal muscle is very responsive to intracellular signals, such as Ca++, which increases during physical exertion/movement, and to an increase in the adenosine monophosphate (AMP):ATP ratio, which activates AMP kinase. Finally, it is important to understand that the pathways upregulated during the Fasting Phase are opposed by insulin-dependent pathways that are most active during the Digestive Phase (discussed later). Thus, attenuation of insulin signaling also contributes to the ability of hepatocytes, skeletal myocytes, and adipocytes to display an integrated response to the metabolic challenges of the Fasting Phase.

Pancreatic Hormones Involved in Metabolic Homeostasis During Different Metabolic Phases The islets of Langerhans constitute the endocrine pancreas (Fig. 39.5A). Approximately 1 million islets making up about 1% to 2% of the pancreatic mass are spread throughout the exocrine pancreas (see Chapter 27). The islets are composed of several cell types, each producing a different hormone. Beta cells make up about three-fourths of the cells of the islets and produce the hormone insulin (see Fig. 39.5B). Alpha cells account for about 10% of islet cells and secrete glucagon (see Fig. 39.5C). Other endocrine cell types reside within islets, but their respective hormone products are of marginal or unclear importance and thus will not be discussed further.

FIG. 39.5 The islets of Langerhans (endocrine pancreas) from rat. A, Pancreas histology showing exocrine acini where digestive enzymes are produced to be delivered to the duodenum via the pancreatic duct, and an endocrine islet where insulin and glucagon are produced and delivered to the circulation upon uptake by a rich capillary bed. B, Staining of endocrine islet for insulin within beta cells; these are the most numerous cell type and are primarily located centrally within the islet. C, Staining of endocrine islet for glucagon with alpha cells; these are much less numerous than beta cells and are primarily located along the periphery of the islet.

Blood flow to the islets is somewhat autonomous from blood flow to the surrounding exocrine pancreatic tissue. Blood flow through the islets passes from beta cells, which predominate in the center of the islet, to alpha and delta cells, which predominate in the periphery (see Fig. 38.5B-C). Consequently, the first cells affected by circulating insulin are the alpha cells, in which insulin inhibits glucagon secretion.

Insulin Insulin is the primary anabolic hormone that dominates regulation of metabolism during the Digestive Phase. Insulin is a protein hormone that belongs to the gene family that includes insulin-like growth factors I and II (IGF-I, IGF-II) and relaxin. Insulin is synthesized as preproinsulin, which is converted to proinsulin as the hormone enters the endoplasmic reticulum. Proinsulin is packaged in the Golgi apparatus into membrane-bound secretory granules. Proinsulin contains the AA sequence of insulin plus the C (connecting) peptide. The proteases that cleave proinsulin (proprotein convertases) are packaged with proinsulin within secretory vesicles. Proteolytic processing clips out the C peptide and generates the mature hormone, which consists of two chains, an α chain and a β chain, connected by two disulfide bridges (Fig. 39.6). A third disulfide bridge is contained within the α chain. Insulin is stored within secretory granules as zinc-bound crystals. Upon stimulation, the granule’s contents are released to the outside of the cell by exocytosis. Equimolar amounts of mature insulin and C peptide are released, along with small amounts of proinsulin. C peptide has no known biological activity but is useful in assessing endogenous insulin production. C peptide is more stable in blood than insulin (making it easier to assay)

and helps distinguish endogenous insulin production from injected insulin, insofar as the latter has been purified from C peptide.

FIG. 39.6 Proinsulin is processed by prohormone convertases into a mature insulin molecule with two peptide strands linked by H-bonds and a C peptide. Both are secreted in equimolar ratios. (From White BA, Porterfield SP [eds]. Endocrine and Reproductive Physiology. 4th ed. Philadelphia: Mosby/Elsevier; 2013.)

Insulin has a short half-life of about 5 minutes and is cleared rapidly from the circulation. It is degraded by insulin-degrading enzyme (IDE; also called insulinase) in the liver, kidney, and other tissues. Because insulin is secreted into the hepatic portal vein, it is exposed to liver IDE before it enters the peripheral circulation. About half the insulin is degraded before leaving the liver. Thus, peripheral tissues are exposed to significantly less serum insulin concentrations than the liver. Recombinant human insulin and insulin analogs with different characteristics of speed of onset and duration of action and peak activity are now available. Serum insulin levels normally begin to rise within 10 minutes after ingestion of food and reach a peak in 30 to 45 minutes. The higher serum insulin level rapidly lowers blood glucose to baseline values. Glucose is the primary stimulus of insulin secretion (“steps” in glucose-stimulated insulin secretion (GSIS) described in the discussion that follows refer to Fig. 39.7). Entry of glucose into beta cells is facilitated by the GLUT2 transporter (Step 1). Once glucose enters the beta cell, it is phosphorylated to G6P by the low-affinity hexokinase glucokinase (Step 2). Glucokinase is referred to as the “glucose sensor” of the beta cell because the rate of glucose entry is correlated with the rate of glucose phosphorylation, which in turn is directly related to insulin secretion. Metabolism of G6P through glycolysis, the TCA cycle, and oxidative phosphorylation by beta cells increases the intracellular ATP:ADP ratio (Step 3) and closes an ATP-sensitive K+ channel (Step 4). This results in depolarization of the beta cell membrane (Step 5), which opens voltage-gated Ca++ channels (Step 6). Increased intracellular [Ca++] activates microtubule-mediated exocytosis of insulin/proinsulin-containing secretory granules (Step 7).

FIG. 39.7 Glucose is the primary stimulus of insulin secretion and is enhanced by sulfonylurea drugs as well as GLP-1 analogs/DPP-4 inhibitors. See text for explanation of numbered steps in glucose-stimulated insulin secretion (GSIS).

Ingested glucose has a greater effect on insulin secretion than injected glucose. This phenomenon, called the incretin effect, is due to stimulation by glucose of incretin hormones from the gastrointestinal tract. One clinically relevant incretin hormone is glucagon-like peptide 1 (GLP-1), which is released by L cells of the ileum in response to glucose in the ileal lumen (Fig. 39.7). As a hormone, GLP-1 enters the circulation and ultimately binds to the Gs-coupled GLP1 receptor (GLP1R) on beta cells. This GLP1R/Gs/adenylyl cyclase/protein kinase A (PKA) signaling pathway amplifies the intracellular effects of Ca++ on insulin secretion. GLP-1 is rapidly degraded in the circulation by dipeptidyl peptidase 4 (DPP-4).

IN THE C LIN IC Oral and Injectable Hypoglycemic Drugs The ATP-sensitive K+ channel is an octameric protein complex that contains four ATP-binding subunits called SUR subunits. These subunits are bound by sulfonylurea drugs, which also close the K+ channel and are widely used as oral hypoglycemics to treat hyperglycemia in patients with partially impaired beta cell function (see Fig. 39.7). Hypoglycemia is a significant side effect of sulfonylurea drugs if used in excess or incorrectly in combination with other drugs, owing to inappropriately high release of insulin. Both DPP-4–resistant analogs of GLP-1 and inhibitors of DPP-4 are currently approved for treatment of patients with type 2 DM with some beta cell function. Importantly these drugs are permissive to the actions of glucose on the beta cell and thus only weakly increase insulin secretion in the absence of glucose. Thus GLP-1 analogs induce hypoglycemia much less frequently than

sulfonylurea drugs. Several AAs and vagal (parasympathetic) cholinergic innervation via muscarinic receptor 3 (MR3) also stimulate insulin through increasing intracellular [Ca++] (Fig. 39.8). Insulin secretion is primarily dampened by sympathetic autonomic regulation through α2-adrenergic receptors. Binding of norepinephrine or epinephrine to relatively abundant α2-adrenergic receptors decreases cyclic adenosine monophosphate (cAMP), thereby dampening insulin secretion (see Fig. 39.8). Adrenergic inhibition of insulin serves to protect against hypoglycemia, especially during exercise. Beta cells also express Gs-coupled β2-adrenergic receptors at a low level that normally play a minor role in promoting insulin secretion (Fig. 39.8).

FIG. 39.8 Secondary regulators of insulin secretion. See text for explanation of abbreviations.

IN THE C LIN IC MODY and Beta Cell Transcription Factors Insulin gene expression and islet cell biogenesis are dependent on several transcription factors specific to the pancreas, liver, and kidney. These transcription factors include hepatocyte nuclear factor 4α (HNF-4α), HNF-1α, insulin promoter factor 1 (IPF-1), HNF-1β, and neurogenic differentiation 1/beta cell E-box trans-activator 2 (NeuroD1/β2). A heterozygous null mutation of one of these factors results in progressively inadequate production of insulin and maturity-onset diabetes of the

young (MODY) before the age of 25. MODY is characterized by nonketotic hyperglycemia, often asymptomatic, that begins in childhood or adolescence. In addition to the five transcription factors, mutations in glucokinase also give rise to MODY. Insulin Receptor The insulin receptor (InsR) is a member of the receptor tyrosine kinase (RTK) gene family (see Chapter 3). Most of the actions of insulin on metabolism involve activation of the protein kinase Akt, which in turn has pleiotropic actions on cell metabolism. The InsR is expressed on the cell membrane as a homodimer, with each monomer containing a tyrosine kinase domain on the cytosolic side (see Fig. 39.9A). Binding of insulin to the receptor induces crossphosphorylation of the subunits. These phosphotyrosine residues are then bound by the insulin receptor substrate (IRS) proteins (i.e., IRS proteins are “recruited” to the InsR). The IRS proteins themselves are phosphorylated by the InsR on specific tyrosines, which then recruits phosphoinositide-3-kinase (PI3K) to the IRS protein bound to the InsR (see Fig. 39.9B). PI3K converts phosphoinositol-4,5-bisphosphate (PIP2) to phosphoinositol-3,4,5-trisphosphate (PIP3). PIP3 is an informational lipid that recruits proteins to the membrane. In this pathway, PIP3 recruits Akt protein kinase to the cell membrane where it becomes activated. This pleiotropic Akt protein kinase signaling pathway orchestrates the numerous metabolic actions of insulin in hepatocytes, skeletal muscle, and adipocytes, including (see Fig. 39.9C): 1. Translocation of the GLUT4 glucose transporter to the cell membrane, thereby allowing import of glucose into skeletal myocytes and adipocytes. 2. Activation of multiple protein phosphatases, which in turn regulate the activity of multiple metabolic enzymes in all insulin target cells. 3. Activation of the protein complex mechanistic target of rapamycin complex 1 (mTORC1), which promotes protein synthesis and may inhibit proteasomal-mediated protein degradation in insulin target cells. 4. Activation of the transcription factor sterol response element–binding protein 1 (SREBP1). SREBP1 is especially important for insulin effects on the liver, where it orchestrates glycolysis and de novo lipogenesis (DNL) for production of phospholipids, FAs, and TGs from excessive ingested glucose and fructose. InsR/Akt signaling stimulates SREBP1 directly as well as indirectly through activation of mTORC1, which also activates SREBP1. SREBP1 also induces the enzyme that catalyzes the first reaction in the oxidative arm of the pentose phosphate pathway (PPP). This reaction generates the coenzyme NADPH, which is required co-factor for the DNL pathway.

5. Inactivation of the transcription factor FOXO1. Akt-mediated phosphorylation of FOXO1 promotes nuclear exclusion of FOXO1. In the absence of insulin/Akt signaling, FOXO1 induces expression of genes encoding gluconeogenic enzymes and proteins involved in hepatic VLDL assembly and export.

FIG. 39.9 A, Structure of dimerized insulin receptor in cell membrane. B, Simplified diagram of the Akt kinase and MAPK pathways downstream of the InsR. C, Summarized actions of insulin/InsR-activated Akt kinase.

All these actions of Akt will be discussed in more detail later. The InsR also promotes proliferation/renewal of some target cells through the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway (see Fig. 39.9B). The MAPK pathway also participates in some metabolic regulation.

Glucagon

Glucagon is the primary counterregulatory hormone that increases blood glucose levels, primarily through its effects on liver glucose output. Glucagon also enhances intramitochondrial fatty acid oxidation and ketogenesis in hepatocytes. Glucagon is a member of the secretin gene family. The precursor preproglucagon harbors the AA sequences for glucagon, GLP-1, and GLP-2 (Fig. 39.10). Preproglucagon is proteolytically cleaved in the alpha cell in a cell-specific manner to produce the peptide glucagon. Glucagon circulates in an unbound form and has a short half-life of about 6 minutes. The predominant site of glucagon degradation is the liver, which degrades as much as 80% of circulating glucagon in one pass. Because glucagon enters the hepatic portal vein and is carried to the liver before reaching the systemic circulation, a large portion of the hormone never reaches the systemic circulation. The liver is the primary target organ of glucagon, with lesser effects on adipocytes. Skeletal muscle does not express the glucagon receptor.

FIG. 39.10 Divergent proteolytic cleavage patterns of the proglucagon molecule. GLP, Glucagon-like peptide; GLUC, glucagon; GRPP, glucagon-related polypeptide.

The glucagon receptor is a Gs-linked G protein–coupled receptor that increases adenylyl cyclase activity and thus cAMP levels. Glucagon exerts many rapid actions through PKA signaling. Glucagon also exerts some transcription effects through phosphorylation and activation of transcription factors such as CREB (cAMP response element–binding) protein. The insulin-glucagon ratio determines the net effect of metabolic pathways on blood glucose. A major stimulus for secretion of glucagon is a decline in blood glucose. Insulin inhibits glucagon secretion, so low blood glucose has an indirect effect on glucagon secretion through removal of inhibition by insulin (Fig. 39.11). Some recent evidence also indicates that low glucose has a direct effect on alpha cells to increase glucagon secretion.

FIG. 39.11 Integrated regulation of blood glucose by insulin and the counterregulatory factors glucagon and catecholamines (norepinephrine, epinephrine).

Circulating catecholamines, which inhibit secretion of insulin from β cells via α2-adrenergic receptors, stimulate secretion of glucagon from α cells via β2-adrenergic receptors (see Fig. 39.11). Serum AAs also promote secretion of glucagon. This means a protein meal will increase postprandial levels of both insulin and glucagon (which protects against hypoglycemia), whereas a carbohydrate meal stimulates only insulin.

Catecholamines: Epinephrine and Norepinephrine The other major counterregulatory factors are the catecholamines epinephrine and norepinephrine. Epinephrine is the primary product of the adrenal medulla (see Chapter 43), whereas norepinephrine is released from postganglionic sympathetic nerve endings (see Chapter 11). Catecholamines are released in response to decreased glucose concentrations, various forms of stress, and exercise. Decreased glucose levels (i.e., hypoglycemia) are primarily sensed by neurons in the CNS, which initiate an integrated sympathetic response through the hypothalamus. The direct metabolic actions of catecholamines are mediated primarily by α1-, β2-, and β3-adrenergic receptors located on muscle, adipose, and liver tissue (see later). Like the glucagon receptor, β-adrenergic receptors (β2 and β3) increase intracellular cAMP.

Hormonal Regulation of Specific Metabolic Reactions and Pathways This section discusses the main pathways in hepatocytes, skeletal myocytes, and adipocytes that

contribute to integrated metabolism. For even more detailed description, the student is referred to biochemistry textbooks.

Hepatocyte Metabolism: Digestive vs. Fasting Phases Some of the key metabolic steps regulated by insulin and glucagon (and catecholamines) in the liver are as follows (refer to Fig. 39.12 for numbered pathways; “D” denotes Digestive Phase, “F” denotes Fasting Phase): 1. Trapping vs. releasing intracellular glucose. Although glucose enters hepatocytes through insulin-independent GLUT2 transporters, insulin increases hepatic retention and utilization of glucose by increasing expression of glucokinase (Pathway 1D). Insulin increases glucokinase gene expression through increased expression and activation of the transcription factor sterol regulatory element–binding protein 1C (SREBP1C), which acts as a “master switch” in the fed state to coordinately increase levels of several enzymes involved in glucose utilization and de novo lipogenesis (DNL; see Fig. 39.9C). Hepatocytes also express the enzyme gluco-6phosphatase (G6Pase; Pathway 1F), which converts G6P back to glucose, which can then exit the hepatocyte via the GLUT2 transporter. Insulin prevents the futile cycle of glucose phosphorylation-dephosphorylation by repressing gene expression of the enzyme G6Pase. The transcription factor FOXO1 stimulates gene expression of G6Pase. Insulin-activated Akt kinase phosphorylates and inactivates FOXO1 (Fig. 39.9C). During the Fasting Phase, FOXO1 is active and promotes G6Pase expression, whereas SREBP1C is inactive and does not stimulate glucokinase expression. The reciprocal regulation of SREBP1C and FOXO1 is thus regulated primarily by the presence or absence of insulin. 2. Glycogen synthesis vs. breakdown. Insulin indirectly increases glycogen synthesis through increased expression of glucokinase because high levels of G6P allosterically increase glycogen synthase activity. Through stimulation of specific protein phosphatases, insulin promotes dephosphorylation and thereby activation of glycogen synthase (Fig. 39.12, Pathway 2D). Insulin also prevents the futile cycle of glycogen synthesis to glycogenolysis through phosphatasemediated inhibition of glycogen phosphorylase (Pathway 2F). Glucagon-activated PKA phosphorylates phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase and glycogenolysis during the Fasting Phase (Pathway 2F). 3. Increasing glycolysis. A. Activating phosphofructokinase 1 (PFK1) and inhibiting fructose-2,6-bisphosphatase. Insulin increases the activity of PFK1, which phosphorylates fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (Pathway 3D). This reaction is referred to as the “commitment” reaction for glycolysis. Insulin also inhibits the reverse reaction, as catalyzed by the gluconeogenic enzyme fructose-1,6-bisphosphatase (Pathway 3F). Insulin regulates these two enzymes through an indirect two-step mechanism that is diagramed in Fig. 39.13. This mechanism involves the bifunctional enzyme called PKFBP, that catalyzes two opposite reactions: 1, a phosphofructokinase-2 (PFK2) and a fructose-2,6-bisphosphatase (F-2,6BPase). Insulin/Akt-activated protein phosphatases promote dephosphorylation of PKFBP, thereby activating the kinase function (PKF2) and lessening the phosphatase function (F2,6BPase). This phosphorylates F6P to fructose-2,6-bisphosphate (F-2,6-bisP). F-2,6-bisP, in turn, binds to and allosterically activates PFK1, thereby driving glycolysis. F-2,6-bisP

also competitively inhibits fructose-1,6-bisphosphatase (F-1,6BPase), thereby blocking the futile cycle of F6P to fructose-1,6-bisphosphate to F6P (Fig. 39.13). B. Activating pyruvate kinase (PK). PK catalyzes the irreversible conversion of phosphoenolpyruvate (PEP) to pyruvate (see Pathway 5D in Fig. 39.12). Again, insulin/Akt kinase activation of a protein phosphatase dephosphorylates PK, which activates the enzyme. Insulin also increases PK gene expression through SREBP1C. Finally, fructose-1,6bisphosphate (product of Pathway 3D) allosterically activates PK. In contrast, glucagon and catecholamines promote PK phosphorylation, thereby inhibiting this last step in glycolysis, during the Fasting Phase. 4. Activating pyruvate dehydrogenase (PDH) complex. PDH complex converts pyruvate to acetyl CoA, which can then enter the TCA cycle upon condensation with oxaloacetate (OA) to form citrate (Fig. 39.12, Pathway 6D). Insulin increases PDH complex activity through Akt kinase activation of PDH complex phosphatase, which in turn dephosphorylates and activates PDH complex 5. Increasing synthesis of intrahepatic TG. During the Digestive Phase, some acetyl CoA is transferred from the mitochondria to the cytosol in the form of citrate, which is then converted back to acetyl CoA and oxaloacetate by the cytosolic enzyme ATP-citrate lyase (Pathway 8D). Insulin increases ATP-citrate lyase gene expression through transcription factor SREBP1C. Once in the cytoplasm, acetyl CoA can enter fatty acid synthesis. The first step involves conversion of acetyl CoA to malonyl CoA by the enzyme acetyl-CoA carboxylase (Pathway 9D). Insulin stimulates acetyl-CoA carboxylase gene expression through the transcription factor SREBP1C. Insulin also promotes dephosphorylation of acetyl-CoA carboxylase, which activates the enzyme. Malonyl CoA is converted to the 16-carbon fatty acid palmitoyl CoA by repetitive additions of acetyl groups (contributed by malonyl CoA) by the fatty acid synthase (FASN) complex (Pathway 10D). FASN gene expression is enhanced by insulin through the transcription factor SREBP1C. Insulin also stimulates glycerol phosphate–fatty acyl transferases that esterify FFAs to G3P to form intrahepatic TG (Pathway 11D). Palmitate synthesis requires the coenzyme NADPH. A major source of NADPH is the pentose phosphate pathway (PPP; see Fig. 39.12). The first reaction converts G6P to 6-phosphogluconolactone by the enzyme glucose-6-phosphate dehydrogenase (G6PD; Step 4D). Insulin increases G6PD gene expression through the transcription factor SREBP1C. By activating steps that lead to generation of malonyl CoA, insulin indirectly inhibits oxidation of FFAs. Malonyl CoA inhibits the activity of CPT-I, which transports FFAs from the cytosol into the mitochondria (Pathway 12D). As a result, FFAs that are synthesized by DNL cannot be transported into mitochondria, where they would undergo β-oxidation (Pathway 13F). Thus, increased malonyl CoA prevents the futile cycle of FFA synthesis to FFA oxidation. FFAs are converted to TGs by the liver (Pathway 11D) and are either stored in the liver or transported to adipose tissue and muscle in the form of VLDL (see later). Insulin acutely promotes degradation of the VLDL apoprotein apoB-100. This keeps the liver from secreting VLDL during a meal when the blood is rich with chylomicrons from the GI tract. Thus, the lipid made in response to insulin during a meal is released as VLDL during the Interdigestive and Fasting Phases and provides an important source of energy to skeletal and cardiac muscle.

6. Activation vs. inhibition of the gluconeogenic enzymes pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). Pyruvate can also be converted to OA by PC (Pathway 6F). However, this reaction is indirectly inhibited by insulin in several ways. First, insulin activates PDH as just discussed, thereby diverting pyruvate away from the PC reaction. Additionally, PC is allosterically activated by high levels of intramitochondrial acetyl CoA. Insulin keeps intramitochondrial levels of acetyl CoA low by activation of cytosolic DNL, which promotes removal of acetyl CoA via citrate from the mitochondria. Another key mechanism is to prevent β-oxidation of FFAs within the mitochondria, which generates high levels of acetyl CoA. By stimulating DNL, insulin also increases levels of cytosolic malonyl CoA, which inhibits

transport of FFAs into the mitochondria (Pathway 12D). Also, inhibitory actions of insulin on glucagon secretion and on lipolysis of TG within adipocytes prevent release of FFAs by adipose tissue and their import into hepatocytes. In contrast, during the Fasting Phase, low insulin coupled with high glucagon and/or catecholamines stimulate release of FFAs from adipocytes (see later), which increases the flow of FFAs into hepatocytes. Glucagon also phosphorylates and activates the enzyme malonyl decarboxylase, which converts malonyl CoA back to acetyl CoA (Pathway 9F). Enhanced malonyl CoA decarboxylase, along with generally low DNL due to low insulin, reduces malonyl CoA levels and thus removes the inhibition on the CPT1 transporter. This allows FFAs to enter the mitochondria and undergo β-oxidation (Pathway 13F), generating high levels of intramitochondrial acetyl CoA, thereby activating PC (Pathway 6F) and also allosterically inhibiting PDH (Pathway 6D). The enzymes involved in β-oxidation are activated by PKA signaling. Glucagon also activates the transcription factor PPARα, which further induces expression of enzymes involved in β-oxidation. Fibrate drugs activate PPARα, promoting oxidation of intrahepatic TG and ameliorating insulin resistance. Cytosolic pyruvate can be generated by way of a multistep process that involves PC (Pathway 6F), conversion of oxaloacetate (OA) to malate (MAL), transfer of MAL out of the mitochondria, conversion to back to OA, and then to cytosolic pyruvate. Pyruvate can then enter the gluconeogenic pathway after conversion to phosphoenolpyruvate (PEP) via the action of phosphoenolpyruvate carboxykinase (PEPCK; Pathway 7F). Insulin represses gene expression of the gluconeogenic enzyme PEPCK. PEPCK is primarily regulated at the level of transcription, which partly explains the slow onset of gluconeogenesis during the Fasting Phase. Similar to its actions on G6Pase, FOXO1 stimulates transcription of PEPCK during the Fasting Phase, and insulin/Akt kinase signaling inactivates FOXO1 during the Digestive Phase. Glucagon and catecholamines also increase PEPCK gene expression through PKA-CREB signaling during the Fasting Phase. So, pyruvate can generate glucose in hepatocytes in a pathway that involves four irreversible and highly regulated reactions: PC (Reaction 6F), PEPCK (Reaction 7F), F-1,6-BPtase (Reaction 3F), and G6Ptase Reaction 1F).

FIG. 39.12 Metabolic pathways in hepatocytes during Digestive (“D” numbers) and Fasting (“F” numbers) Phases. Reactions/pathways: 1D, glucokinase; 1F; G6Pase; 2D, glycogen synthesis; 2F, glycogenolysis; 3D, phosphofructokinase 1; 3F, fructose-1,6-bisphosphatase; 4D, glucose-6-phosphate dehydrogenase; 5D, pyruvate kinase; 6D, pyruvate dehydrogenase; 6F, pyruvate carboxylase; 7F, phosphoenolpyruvate carboxykinase; 8D, ATP-citrate lyase; 9D, acetyl CoA carboxylase; 9F, malonyl CoA decarboxylase; 10D, fatty acid synthase; 11D, esterification and formation of TG; 12D, inhibition by malonyl CoA (Mal CoA) of fatty acyl CoA transporter, carnitine/palmitoyl transporter 1 (CPT1) on outer mitochondrial membrane; 13F, movement of fatty acyl CoA into mitochondrion through CPT1 (and CPT2) and beta-oxidation to acetyl CoA.

FIG. 39.13 Insulin and counterregulatory hormone regulation of phosphofructokinase 1 (PFK1; reaction 3D in Fig. 39.12) and fructose-1,6-bisphosphatase (F1,6BPase; reaction 3F in Fig. 39.12) through changing the activity of the bifunctional enzyme phosphofructokinase 2/fructose-2,6-bisphosphatase (PKFBP) and thus the levels of the allosteric regulatory metabolite fructose-2,6-bisphosphate (F-2,6-bisP). F6P, Fructose-6-phosphate.

Skeletal Muscle and Adipose Tissue Metabolism: Digestive vs. Fasting Phases 1. Skeletal muscle (Fig. 39.14). Glucose tolerance refers to the ability of an individual to minimize the increase in blood glucose concentration after a meal during the Digestive Phase. A primary way by which insulin promotes glucose tolerance is activation of glucose transporters in skeletal muscle. Insulin stimulates translocation of preexisting intracellular GLUT4 transportercontaining vesicles to the cell membrane (Pathway 1D). Skeletal muscle expresses a high affinity isoform of hexokinase that effectively converts glucose to glucose-6-P (Pathway 4D). Note that muscle does not express glucose-6-phosphatase, and thus cannot contribute directly to blood glucose. Insulin also promotes storage of glucose in muscle as glycogen (Pathway 2D) and promotes oxidation of glucose through glycolysis, PDH, TCA, and OxPhos (Pathway 3D/F). During the Fasting Phase, low insulin results in a low number of GLUT4 transporters at the membrane (reaction 1F), so these cells consume less glucose (glucose sparing). Skeletal muscle fibers break down stored glycogen for use within that fiber (Pathways 2F and 3D). Skeletal muscle fibers with mitochondria augment the use of FFAs from adipocytes and KBs from hepatocytes (Pathways 3F). Skeletal myocytes do not express glucagon receptors. Uptake of FFAs and KBs and their oxidation for ATP is largely upregulated by intracellular Ca++ levels and a high AMP:ATP ratio, as well as adrenergic stimulation.

2. Adipocytes→ glucose (Fig. 39.15A). Insulin also stimulates GLUT4-dependent uptake of glucose. Adipocytes also expresses a hexokinase that effectively converts glucose to glucose-6-P (Pathway 5D), and, like muscle, does not express glucose-6-phosphatase. Insulin stimulates subsequent glycolysis in adipose tissue (Pathways 1D and 2D). Adipose tissue uses glycolysis for energy needs but also for generating G3P (Pathway 3D), which is required for esterification of FFAs into TGs (Pathway 4D). During the Fasting Phase, insulin is low, so GLUT4 movement to the cell membrane is blocked (Pathway 1F).

3. Adipocytes → FFAs and TG (see Fig. 39.15B). Insulin stimulates expression of lipoprotein lipase (LPL) within adipocytes and its migration to the apical side of endothelia in adipose capillaries (Pathway 1D). This action of insulin allows LPL to extract FFAs from chylomicrons within adipose tissue capillary beds (Pathway 2D). The chylomicron remnants (CRs; discussed later) are removed by the liver. Insulin also stimulates activation of imported FFAs by their conversion to fatty acyl CoAs (Pathway 3D). As discussed earlier, insulin stimulates glycolysis in adipocytes, which generates the G3P required for reesterification of FFAs with glycerol-3-P into TGs (Pathway 4D). TG droplets in adipocytes are coated by perilipins (PL). Insulin directly inhibits hormone-sensitive lipase (HSL; Pathway 5D), thereby promoting storage of FFAs as opposed to their release. During the Fasting Phase, glucagon and catecholamines phosphorylate and activate HSL (Pathway 5F), thereby promoting release of FFAs and glycerol from stored TG (Pathway 6F). In the absence of insulin these two products of lipolysis are exported into the blood.

FIG. 39.14 Metabolism in skeletal muscle during digestive (“D” reactions) vs. fasting (“F” reactions) phases. Reactions/pathways: 1D, translocation of GLUT4 transporter to cell membrane; 1F, loss of translocation of GLUT4 transporter to cell membrane; 4D, conversion of glucose to G-6-P; 2D, glycogen synthesis; 2F, glycogenolysis; 3D, glycolysis and lactate dehydrogenase, or pyruvate dehydrogenase/TCA cycle/oxidative phosphorylation (OxPhos), depending on muscle fiber type; 3F, β-oxidation of FFAs or ketolysis followed by the TCA cycle and OxPhos.

FIG. 39.15 A, Glucose metabolism in an adipocyte during Digestive (“D” pathways) and Fasting (“F” pathways) phases. Reactions/pathways: 1D and 1F, insertion or lack thereof of GLUT4 transporters into cell membrane (see Fig. 39.14 legend); 5D, conversion of glucose to G-6-P; 2D, glycolysis, pyruvate dehydrogenase/TCA cycle/OxPhos; 3D, glycerol-3-phosphate dehydrogenase; 4D, esterification of FFAs to G3P to form triglyceride. B, Lipid metabolism in adipocyte during Digestive (“D” pathways) and Fasting (“F” pathways) phases. Reactions/pathways: 1D, synthesis of lipoprotein lipase (LPL) and LPL secretion into subcapillary space, binding to GPI-anchored protein (thick red line), and migration to luminal side of capillary endothelial cell; 2D, lipolysis of chylomicron TG and releasing free FFA (after digestion, chylomicron remnant (CR) is cleared from circulation by liver); 3D, activation of imported FFAs by transfer

to acetyl CoA to form fatty acyl CoA; 4D, esterification of fatty acyl CoAs to G3P to form TG); 5D, dephosphorylation and inactivation of hormone-sensitive lipase (HSL), thereby promoting storage of TG; 5F, phosphorylation and activation of HSL, which contributes to complete lipolysis of TG; 6F, final step in TG lipase by monoglyceride lipase releases FFA and glycerol.

Protein Metabolism in All Hormone Target Cells: Digestive vs. Fasting Phases Insulin promotes protein synthesis in muscle and adipose tissue by stimulating AA uptake and mRNA translation. Insulin also inhibits proteolysis. Although the liver uses AAs for ATP synthesis, insulin also promotes synthesis of proteins during the Digestive Phase and attenuates the activity of urea cycle enzymes in the liver. Glucagon and catecholamines activate proteasomal degradation of proteins and release of AAs during the Fasting Phase.

Metabolic Roles of Lipoproteins: Digestive vs. Fasting Phases This section provides an overview of lipoprotein metabolism, as depicted in Fig. 39.16. For more details, please consult a biochemistry textbook.

FIG. 39.16 Role of lipoproteins in energy metabolism. A, Digestive phase. B, Fasting phase.

FFAs circulate in the blood primarily bound to albumin. However, TG, free cholesterol, cholesterol esters, phospholipids, and some lipid-soluble vitamins, all of which are hydrophobic and would partition

into the membranes of endothelial cells instead of circulating, are transported through blood within lipid aggregations (i.e., a mix of the above) bound by specific apoproteins. These lipid-protein complexes are referred to as lipoproteins. The TG-rich lipoproteins are chylomicrons and VLDL and primarily function to deliver FFAs (as TG) to skeletal and cardiac muscle for energy and to adipocytes for storage. The cholesterol-rich lipoproteins include low-density lipoprotein (LDL) and high-density lipoprotein (HDL), which deliver cholesterol to proliferating cells, steroidogenic cells, and bile-producing hepatocytes. HDL also removes excess cholesterol (i.e., from macrophage-engulfed dead cells) from the periphery. There are also “remnants” of lipoproteins that have their lipid cargo partially digested and then cleared from the circulation by the liver. Digestive Phase: Chylomicrons and Chylomicron Remnants (Fig. 39.16A) TGs in a meal are enzymatically digested to FFAs and 2-monoglycerides within the lumen of the intestine. Intestinal enterocytes import both of these lipids and reesterify them to form TGs. TGs, along with fatsoluble vitamins, cholesterol, cholesterol esters, and phospholipids, are complexed with the protein ApoB48 to form chylomicrons. Chylomicrons are secreted, move into lymphatics, and then ultimately enter the circulation. While in the blood, other apoproteins such as ApoE and ApoC2 are transferred to the chylomicrons from HDL particles (one function of HDL is to provide a circulating reservoir of various apoproteins). This converts nascent chylomicrons into mature chylomicrons. When chylomicrons enter the capillaries of adipose tissue during the Digestive Phase, they are partially digested by lipoprotein lipase (LPL). LPL is synthesized by adipocytes and secreted into the subendothelial space. LPL then binds to an endothelial membrane GPI-anchored protein, which transports LPL to the luminal (apical) surface of the capillary endothelial cell. Once in this position, LPL molecules come into contact with chylomicrons. ApoC2 within chylomicrons is an activator of LPL dimerization and activity. FFAs are released from chylomicrons by LPL-mediated lipolysis of TG. (See earlier discussion and Fig. 39.15B for an explanation of the processing of FFAs to stored TG within adipocytes.) LPL is also expressed in cardiac and skeletal muscle. Cardiac muscle preferentially uses FFA for energy and obtains most FFAs from lipoprotein particles (Fig. 39.16). Thus, cardiac muscle also extracts FFA from chylomicrons during the Digestive Phase. The activity of LPL in cardiomyocytes is highly regulated by local factors such as the local concentration of FFAs within the coronary capillary beds. LPL activity in skeletal muscle is relatively low during the Digestive Phase. After lipolytic digestion within adipose and cardiac muscle capillary beds, chylomicrons are converted to smaller, denser chylomicron remnants (CRs) that now have reduced TG content. CR particles are able to penetrate the tunica intima of blood vessels at sites with endothelial dysfunction and thus are atherogenic. Because they still have ApoE protein associated with them, they can bind to one of several membrane receptors that recognize ApoE. Bound CRs are then endocytosed by hepatocytes (see Fig. 39.16). Remaining FFAs that are released after endocytosis of CRs are reesterified into intrahepatic TG. Fasting Phase: VLDL, IDL, and LDL (Fig. 39.16B) The source of circulating TG during the Fasting Phase is primarily the liver (see Fig. 39.16). During the Digestive Phase, intrahepatic TGs accumulate from DNL and from endocytosed CRs. Intrahepatic TG, along with other lipids including cholesterol and cholesterol esters, is exported by hepatocytes as VLDL. VLDL particles are assembled as lipids complexed to the ApoB100 protein. Expression of ApoB100, along with other components involved in VLDL assembly, is stimulated by transcription factor FOXO1. FOXO1, in turn, is inhibited by the insulin signaling pathway. This means that hepatic VLDL production is minimal during the time when blood is rich in chylomicrons. During the Fasting Phase, insulin levels are

low, so FOXO1 activity is high, and VLDL assembly and secretion resumes. Once VLDL particles enter the circulation, they accept other apoproteins (e.g., ApoE, ApoC2) and become mature VLDL. Adipocytes display low LPL activity during the Fasting Phase, in part owing to low insulin levels. However, cardiomyocytes and skeletal myocytes express LPL, which digests VLDL and provides FFA to these muscle cell types during the Fasting Phase. Lipolytic extraction of some FFAs from VLDL generates a remnant particle called intermediate-density lipoprotein (IDL). IDL circulates to the liver, where it is processed in one of two ways (see Fig. 39.16). About half of the IDL binds to one of several ApoErecognizing receptors on hepatocytes, undergoes receptor-mediated endocytosis, and is digested in endolysosomes. Released lipids can be reassembled into VLDL particles and returned to the circulation to provide fuel for cardiac and skeletal muscle as the Fasting Phase progresses. The other half of the IDL undergoes further digestion from the hepatocyte-specific LPL-related enzyme hepatic lipase (HL). HL extracts most of the remaining TG in the IDL, forming the final remnant of VLDL, namely LDL. LDL is TG poor but cholesterol rich. It should be noted that both mature chylomicrons and VLDL can receive additional cholesterol from HDL while in the circulation through the action of cholesterol ester transport protein (CETP), so cholesterol content of the remnant particles (ChyR, IDL, and LDL) can vary. In any case the LDL particle is a small, dense, cholesterol-rich particle that is potentially very atherogenic in the face of endothelial damage. LDL particles are safely imported into cells through the LDL receptor. It should be noted that in the conversion of IDL to LDL, the ApoE protein disassociates from the particle. This means that only ApoB100 receptors can remove LDL from the blood. In contrast to the multiple ApoE receptors, only one receptor, the LDL receptor, can recognize and bind ApoB100. Thus, loss or decrease of a functional LDL receptor has significant clinical consequences (see At the Cellular Level box). LDL receptor is expressed on proliferating cells, including some cancer cells, which need to synthesize new cell membranes. LDL receptor is also expressed on steroidogenic cells, which use cholesterol to make steroid hormones. The major site of LDL uptake is the liver, which secretes cholesterol as well as cholesterol-based bile acids, as bile into the biliary tree. Some cholesterol is excreted by the intestines. Other cholesterol by-products (e.g., steroid hormones) are excreted primarily at the kidney.

AT THE C ELLU LAR LEVEL SREBP2 was discovered as a transcription factor that resides in the membrane of the endoplasmic reticulum (ER). In the presence of high intracellular cholesterol, SREBP2 is held in the ER by a lipidsensing protein called SCAP (SREBP cleavage–activating protein). In response to depleted sterols, SCAP escorts SREBP2 to the Golgi, where SREBP is cleaved sequentially by proteases and released into the cytoplasm. SREBP2 then translocates to the nucleus and increases the transcription of genes involved in synthesis and uptake of cholesterol. A more recently discovered member of this transcription factor family is SREBP1C, which is highly expressed in adipose and liver. In contrast to SREBP2, SREBP1C stimulates genes involved in synthesis of FA and TG. Regulation of SREBP1C occurs at the transcriptional level of the SREBP1C gene, with cleavage induced by polyunsaturated fatty acids and activation by the MAPK pathway. Peroxisome proliferation activator receptors (PPARs) belong to the nuclear hormone receptor superfamily that also includes steroid hormone receptors and thyroid hormone receptors. PPARs heterodimerize with the retinoid X receptors (RXRs). Unlike steroid and thyroid hormone receptors, PPARs bind to ligands in the micromolar range (i.e., with lower affinity). PPARs bind saturated and unsaturated fatty acids as well as natural and synthetic prostanoids. PPARγ is highly expressed in adipose tissue and at a lower level in skeletal muscle and liver. Its natural ligands include several

polyunsaturated fatty acids. PPARγ regulates genes that promote fat storage. It also synergizes with SREBP1C to promote differentiation of adipocytes from preadipocytes. Tissue-specific knockout of PPARγ in mice and PPARγ-dominant negative mutations in humans give rise to lipodystrophy (i.e., lack of white adipose tissue), which leads to deposits of TG in muscle and liver (called steatosis), insulin resistance, diabetes, and hypertension. The thiazolidinediones are exogenous ligands for PPARγ. Although they promote weight gain, moderate levels of thiazolidinediones significantly improve insulin sensitivity. PPARγ also stimulates secretion of adiponectin, which promotes oxidation of lipids in muscle and fat and thereby improves insulin sensitivity. PPARα is abundantly expressed in liver and to a lesser extent in skeletal and cardiac muscle and kidney. PPARα promotes uptake and oxidation of FFAs. Thus, PPARα is an antisteatotic molecule. The fibrates are exogenous ligands of PPARα and are used to reduce TG deposits in muscle and liver, thereby improving insulin sensitivity. A third member, PPARδ, similarly promotes fatty acid oxidation in adipose and muscle tissue. PPARδ promotes development of slow-twitch oxidative muscle fibers and increases muscle stamina. PPARδ has a positive effect on lipoprotein metabolism by increasing production of ApoA apoproteins and the number of HDL particles. Another family of lipid-sensing transcription factors is the liver X receptor (LXR) family, which is composed of LXRα and LXRβ. LXRα is expressed primarily in adipose tissue, liver, intestine, and kidney, whereas LXRβ is ubiquitously expressed. LXRs are related to PPARs in that they are members of the nuclear hormone receptor family and heterodimerize with RXR. LXRs are cholesterol sensors. In high-cholesterol conditions, LXRs upregulate expression of ATP-binding cassette (ABC) proteins. In the face of excess cholesterol, LXRs also increase ABC protein expression in the gastrointestinal tract, which promotes efflux of cholesterol from enterocytes to the lumen for excretion. Mutations in these transporters (ABCG5 and ABCG8) cause sitosterolemia, characterized by excessive absorption of cholesterol and plant sterols. In the liver, LXRs promote conversion of cholesterol to bile acids for excretion or to cholesterol esters for storage. In the latter action, LXRs increase SREBP1C expression, thereby increasing the fatty acyl CoAs needed for esterification.

IN THE C LIN IC Diabetes mellitus is a disease in which insulin levels or responsiveness of tissues to insulin (or both) is insufficient to maintain normal levels of plasma glucose. Although the diagnosis of diabetes is based primarily on plasma glucose, diabetes also promotes imbalances in circulating levels of lipids and lipoproteins (i.e., dyslipidemia). Major symptoms of diabetes mellitus include hyperglycemia, polyuria, polydipsia, polyphagia, muscle wasting, electrolyte depletion, and ketoacidosis (in T1DM). With normal fasting (i.e., no caloric intake for at least 8 hours), plasma glucose levels should be below 110 mg/dL. A patient is considered to have impaired glucose control if fasting plasma glucose levels are between 110 and 126 mg/dL, and the diagnosis of diabetes is made if fasting plasma glucose exceeds 126 mg/dL on two successive days. Another approach to the diagnosis of diabetes is the oral glucose tolerance test. After overnight fasting the patient is given a bolus of glucose (usually 75 g) orally, and blood glucose levels are measured at 2 hours. A 2-hour plasma glucose concentration greater than 200 mg/dL on two consecutive days is sufficient to make the diagnosis of diabetes. The diagnosis of diabetes is also indicated if the patient has symptoms associated with diabetes and has a nonfasting plasma glucose level greater than 200 mg/dL. Diabetes mellitus is currently classified as type 1 (T1DM) or type 2 (T2DM). T2DM is by far the more common form and accounts for 90% of diagnosed cases. However, T2DM is usually a

progressive disease that remains undiagnosed in a significant percentage of patients for several years. T2DM is often associated with visceral obesity and lack of exercise—indeed, obesity-related T2DM is reaching epidemic proportions worldwide. Usually there are multiple causes for the development of T2DM in a given individual that are associated with defects in the ability of target organs to respond to insulin (i.e., insulin resistance), along with some degree of beta cell deficiency. Insulin sensitivity can be compromised at the level of the InsR or at the level of postreceptor signaling. T2DM appears to be the consequence of insulin resistance, followed by reactive hyperinsulinemia, but ultimately by relative hypoinsulinemia (i.e., inadequate release of insulin to compensate for the end-organ resistance) and beta cell failure. The underlying causes of Insulin Resistance differ among patients. Three major underlying causes of obesity-induced insulin resistance are: 1. Decreased ability of insulin to increase GLUT4-mediated uptake of glucose, especially by skeletal muscle. This function, which is specifically a part of glucometabolic regulation by insulin, may be due to excessive accumulation of TG in muscle in obese individuals. Excessive caloric intake induces hyperinsulinemia. Initially this leads to excessive glucose uptake into skeletal muscle. Just as in the liver, excessive calories in the form of glucose promote lipogenesis and, through generation of malonyl CoA, repression of fatty acyl CoA oxidation. Byproducts of fatty acid and TG synthesis (e.g., diacylglycerol, ceramide) may accumulate and stimulate signaling pathways (e.g., protein kinase C–dependent pathways) that antagonize signaling from the InsR or IRS proteins, or both. Thus, insulin resistance in the skeletal muscle of obese individuals may be due to lipotoxicity. 2. Decreased ability of insulin to repress hepatic glucose production. The liver makes glucose by glycogenolysis in the short term and by gluconeogenesis in the long term. The ability of insulin to repress key hepatic enzymes in both these pathways is attenuated in insulin-resistant individuals. Insulin resistance in the liver may also be due to lipotoxicity in obese individuals (e.g., fatty liver or hepatic steatosis). Visceral adipose tissue is likely to affect insulin signaling at the liver in several ways, in addition to the effects of lipotoxicity. For example, visceral adipose tissue releases the cytokine tumor necrosis factor (TNF)-α, which has been shown to antagonize insulin signaling pathways. Also, TG in visceral adipose tissue has a high rate of turnover (possibly because of rich sympathetic innervation), so the liver is exposed to high levels of FFAs, which further exacerbates hepatic lipotoxicity. 3. Inability of insulin to repress hormone-sensitive lipase or increase LPL in adipose tissue (or both). High HSL and low LPL are major factors in the dyslipidemia associated with insulin resistance and diabetes. Although the factors that resist the actions of insulin on HSL and LPL are not completely understood, there is evidence for increased production of paracrine diabetogenic factors in adipose tissue, such as TNF-α. The dyslipidemia is characterized as hypertriglyceridemia with large TG-rich VLDL particles produced by the liver. Because of their high TG content, large VLDLs and IDLs are digested very efficiently, thereby giving rise to small, dense LDL particles that are very atherogenic. In addition, HDL takes on excess TG in exchange for cholesterol esters, which appears to shorten the circulating half-life of HDL and ApoA proteins. Thus, there are lower levels of HDL particles, which normally play a protective role against vascular disease. T1DM is characterized by destruction of beta cells, almost always by an autoimmune mechanism.

T1DM is also termed insulin-dependent diabetes mellitus. Characteristics of T1DM are: 1. People with T1DM need exogenous insulin to maintain life and prevent ketosis; virtually no pancreatic insulin is produced. 2. There is pathological damage to the pancreatic beta cells. Insulitis with pancreatic mononuclear cell infiltration is a characteristic feature at the onset of the disorder. Cytokines may be involved in the early destruction of the pancreas. 3. People with T1DM are prone to ketoacidosis. 4. Ninety percent of cases begin in childhood, mostly between 10 and 14 years of age. This common observation led to application of the term juvenile diabetes to the disorder. This term is no longer used because T1DM can arise at any time of life, although juvenile onset is the typical pattern. 5. Islet cell autoantibodies are frequently present around the time of onset. If T1DM is induced by a virus, the autoantibodies are transient. Occasionally antibodies will persist long term, particularly if they are associated with other autoimmune disorders. About 50% of T1DM is related to problems with the major histocompatibility complex on chromosome 6. It is correlated with an increased frequency of certain human leukocyte antigen (HLA) alleles. The HLA types DR3 and DR4 are most commonly associated with diabetes.

Leptin and Energy Balance White adipose tissue (WAT) is composed of several cell types. The TG-storing cell is called the adipocyte. These cells develop from preadipocytes during gestation in humans. This process of adipocyte differentiation, which may continue throughout life, is promoted by several transcription factors. One of these factors is SREBP1C, which is activated by lipids as well as insulin and several growth factors and cytokines. Another important transcription factor in WAT is PPARγ. Activated PPARγ promotes expression of genes involved in TG storage. Thus, an increase in food consumption leads to activation of SREBP1C and PPARγ, which increase the differentiation of preadipocytes into small adipocytes and upregulation of enzymes within these cells to allow storage of excess fat.

Leptin Adipose tissue produces multiple paracrine and endocrine factors. Leptin is an adipocyte-derived protein that signals information to the hypothalamus about the degree of adiposity and nutrition, which in turn controls eating behavior and energy expenditure. Leptin-deficient mice and humans become morbidly obese. These findings originally raised hope that leptin therapy could be used to combat morbid obesity. However, administration of leptin to individuals who suffer from diet-induced obesity does not have a significant anorectic or energy-consuming effect. In fact, obese individuals already have elevated endogenous circulating levels of leptin and appear to have developed leptin resistance. Leptin has an important role in liporegulation in peripheral tissues. Leptin protects peripheral tissues (e.g., liver, skeletal muscle, cardiac muscle, beta cells) from accumulation of too much lipid by directing storage of excess caloric intake into adipose tissue. This action of leptin, though opposing the lipogenic actions of insulin, contributes significantly to maintenance of insulin sensitivity (as defined by insulindependent glucose uptake) in peripheral tissues. Leptin also acts as a signal that the body has sufficient

energy stores to allow reproduction and to enhance erythropoiesis, lymphopoiesis, and myelopoiesis. For example, in women suffering from anorexia nervosa, leptin levels are extremely low and result in low ovarian steroids, amenorrhea (lack of menstrual bleeding), anemia from low red blood cell production, and immune dysfunction. Structure, Synthesis, and Secretion Leptin, a 16-kDa protein secreted by mature adipocytes, is structurally related to cytokines. Thus, it is sometimes referred to as an adipocytokine. Circulating levels of leptin have a direct relationship with adiposity and nutritional status. Leptin output is increased by insulin, which prepares the body for correct partitioning of incoming nutrients. Leptin is inhibited by fasting and weight loss and by lipolytic signals (e.g., increased cAMP and β3-agonists). Diet-induced obesity, advanced age, and T2DM are associated with leptin resistance. Thus, mechanisms that turn off leptin signaling are potential therapeutic targets.

Energy Storage The amount of energy stored by an individual is determined by caloric intake and calories expended as energy per day. In many individuals, input and output are in balance, so weight remains relatively constant. However, the abundance of inexpensive high-fat, high-carbohydrate food, along with more sedentary lifestyles, is currently contributing to a pandemic of obesity and the pathological sequelae of obesity, including T2DM and cardiovascular disease. The preponderance of stored energy consists of fat, and individuals vary greatly in the amount and percentage of body weight that is accounted for by adipose tissue. About 25% of the variance in total body fat appears to be due to genetic factors. A genetic influence on fat mass is supported by (1) the tendency for the body mass of adopted children to correlate better with that of their biological parents than with that of their adoptive parents; (2) the greater similarity of adipose stores in identical (monozygotic) twins, whether reared together or apart, than in fraternal (dizygotic) twins; (3) the greater correlation between gains in body weight and abdominal fat in identical twins than in fraternal twins when they are fed a caloric excess; and (4) the discovery of several genes that cause obesity. In addition, the gestational environment has a profound effect on body mass of the adult. The effect of maternal diet on the weight and body composition of offspring is called fetal programming. Low birth weights correlate with increased risk for obesity, cardiovascular disease, and diabetes. These findings suggest that the efficiency of fetal metabolism has plasticity and can be altered by the in utero environment. The development of a “thrifty” metabolism would be advantageous to an individual born to a mother who received poor nutrition and into a life that meant chronic undernourishment.

Body Mass Index A measure of adiposity is the body mass index (BMI). The BMI of an individual is calculated as:

Equation 39.1

The BMI of healthy lean individuals ranges from 20 to 25. A BMI greater than 25 indicates that the individual is overweight, whereas a BMI higher than 30 indicates obesity. The condition of being overweight or obese is a risk factor for multiple pathologies, including insulin resistance, dyslipidemia, diabetes, cardiovascular disease, and hypertension. WAT tissue is divided into subcutaneous and intra-abdominal (visceral) depots. Intra-abdominal WAT refers primarily to omental and mesenteric fat and is the smaller of the two depots. These depots receive different blood supplies that are drained in a fundamentally different way in that venous return from intraabdominal fat leads into the hepatic portal system. Thus, intra-abdominally derived FFAs are mostly cleared by the liver, whereas subcutaneous fat is the primary site for providing FFAs to muscle during exercise or fasting. Regulation of intra-abdominal and subcutaneous adipose tissue also differs. Abdominal fat is highly innervated by autonomic neurons and has a greater turnover rate. Furthermore, these two depots display differences in hormone production and enzyme activity. Men tend to gain fat in the intra-abdominal depot (android [apple-shaped] adiposity), whereas women tend to gain fat in the subcutaneous depot, particularly in the thighs and buttocks (gynecoid [pear-shaped] adiposity). Clearly an excess of abdominal fat poses a greater risk factor for the pathologies mentioned earlier. Thus, another indicator of body composition is circumference of the waist (measured in inches around the narrowest point between the ribs and hips when viewed from the front after exhaling) divided by the circumference of the hips (measured at the point where the buttocks are largest when viewed from the side). This waist-hip ratio may be a better indicator of body fat than BMI, especially as it relates to risk for development of diseases. A waist-hip ratio of greater than 0.95 in men or 0.85 in women is linked to a significantly higher risk for development of diabetes and cardiovascular disease.

Central Mechanisms Involved in Energy Balance In recent years, numerous hormones and neuropeptides have been implicated in both chronic and acute regulation of appetite, satiety, and energy expenditure in humans. One simplified model involves two peptide hormones, leptin and insulin (see Fig. 39.17), already discussed. Leptin acts on at least two neuron types in the arcuate nucleus of the hypothalamus. In the first, leptin represses production of neuropeptide Y (NPY), a very potent stimulator of food-seeking behavior (energy intake) and an inhibitor of energy expenditure. Norepinephrine, another appetite stimulator, co-localizes with NPY in some of these neurons. At the same time, leptin represses production of agouti-related peptide (AGRP), an endogenous antagonist that acts on MC4R, a hypothalamic receptor for the anorexigenic peptide αmelanocyte–stimulating hormone (α-MSH), which inhibits food intake. In another type of arcuate neuron, leptin stimulates production of proopiomelanocortin (POMC) products, one of which is α-MSH, and production of cocaine-amphetamine–regulated transcript (CART), both of which inhibit food intake. Thus, leptin decreases food consumption and increases energy expenditure by simultaneously inhibiting NPY and the α-MSH antagonist AGRP and by stimulating α-MSH and CART (see Fig. 39.17). These secondorder neuropeptides are transmitted to and interact with receptors in neurons of the paraventricular hypothalamic nucleus (“satiety” neurons) and lateral hypothalamic nucleus (“hunger” neurons). In turn these hypothalamic neurons generate signals that coordinate feeding behavior and autonomic nervous system activity (especially sympathetic outflow) with diverse endocrine actions on thyroid gland function, reproduction, and growth.

FIG. 39.17 Leptin and hypothalamic centers involved in regulation of appetite. See text for explanations of abbreviations.

Another regulator of food intake and body energy stores is melanin-concentrating hormone (MCH). This neuropeptide increases food seeking and adipose tissue by antagonizing the satiety effect of α-MSH downstream from the interaction of α-MSH with its MC4R receptor. The probable importance of this molecule is demonstrated by the fact that it is the only regulator whose ablation by gene knockout actually results in leanness. To maintain overall energy homeostasis, the system must also balance specific nutrient intake and expenditure—for example, carbohydrate intake with carbohydrate oxidation. This may account for some specificity in neuropeptide and neurotransmitter responses to meals. Serotonin produces satiety after ingestion of glucose. Gastrointestinal hormones such as cholecystokinin and GLP-1 produce satiety by humoral effects, but their local production in the brain may participate in nutrient and caloric regulation. The recently discovered hormone ghrelin is an acylated peptide with potent orexigenic activity that arises in cells of the oxyntic glands in the stomach. Plasma levels of ghrelin rise in humans in the 1 to 2 hours that precede their normal meals. Plasma levels of ghrelin fall drastically to minimum values about 1 hour after eating. Ghrelin appears to stimulate food intake by reacting with its receptor in hypothalamic neurons that express NPY.

Key Concepts

1. Cells make ATP to meet their energy needs. ATP is made by glycolysis and by the TCA cycle coupled to oxidative phosphorylation. 2. Cells can oxidize carbohydrate (primarily in the form of glucose), AAs, and FFAs to make ATP. Additionally, the liver makes KBs for other tissues to oxidize for energy in times of fasting. 3. Some cell types are limited in the energy substrates they can oxidize for energy. The brain is normally exclusively dependent on glucose for energy. Thus, blood glucose must be maintained above 60 mg/dL for normal autonomic and CNS function. Conversely, inappropriately high levels of glucose (i.e., fasting glucose > 100 mg/dL) promote glucotoxicity and thereby lead to the longterm complications of diabetes. 4. The endocrine pancreas produces the hormones insulin, glucagon, somatostatin, gastrin, and pancreatic polypeptide. 5. Insulin is an anabolic hormone that is secreted in times of excess nutrient availability. It allows the body to use carbohydrates as energy sources and store nutrients. 6. Major stimuli for insulin secretion include increased serum glucose and some AAs. Activation of cholinergic (muscarinic) receptors also increases insulin secretion, whereas activation of α2adrenergic receptors inhibits insulin secretion. The gastrointestinal tract releases incretin hormones that stimulate pancreatic insulin secretion. GLP-1 is particularly potent in augmenting glucose-dependent stimulation of insulin secretion (GSIS). GLP-1 is degraded by dipeptidyl peptidase (DPP)-4. DPP-4–resistant GLP-1 analogs and inhibitors of DPP-4 are currently used to increase GSIS in patients with type 2 diabetes. 7. Insulin binds to the insulin receptor (InsR), which is linked to multiple pathways that mediate the metabolic (Akt kinase) and growth effects (MAPK) of insulin. 8. During the digestive phase, insulin acts on the liver to promote trapping of glucose as G6P. Insulin also increases glycogenesis, glycolysis, and de novo lipogenesis (DNL) in the liver. Insulin inhibits gluconeogenesis, glycogenolysis, and assembly of lipids into VLDL. 9. Insulin increases GLUT4-mediated glucose uptake in muscle and adipose tissue. 10. Insulin increases glycogenesis, glycolysis, and in the presence of caloric excess, lipogenesis in muscle. 11. Insulin increases glycolysis and generation of G3P in adipocytes. Insulin induces expression of LPL and its transport to the luminal side of capillary endothelial cells. Insulin promotes uptake and activation of FFAs and esterification of fatty acyl CoAs to G3P to form TG, and it decreases hormone-sensitive lipase activity in adipocytes. 12. Insulin increases AA uptake and protein synthesis in skeletal muscle but also essentially all insulin target cells. Insulin/Akt kinase signaling activates mTORC1 and S6K to promote synthesis of ribosomal proteins and proteins involved in mRNA translation, as well as other types of proteins. Insulin inhibits proteasomal degradation of protein. 13. Glucagon is a catabolic counterregulatory hormone. Its secretion increases during periods of food deprivation, and it acts to mobilize nutrient reserves. It also mobilizes glycogen, fat, and even protein. 14. Glucagon is released in response to decreased serum glucose (and therefore decreased insulin) and increased serum AA levels and β-adrenergic signaling. 15. Glucagon binds to the glucagon receptor, which is linked to PKA-dependent pathways. The primary target organ for glucagon is the liver. Glucagon increases liver glucose output by increasing glycogenolysis and gluconeogenesis. It increases β-oxidation of fatty acids and ketogenesis.

16. Glucagon regulates hepatic metabolism both by regulation of gene expression and through posttranslational PKA-dependent pathways. 17. The major counterregulatory factors in muscle and adipose tissue are the adrenal hormone epinephrine and the sympathetic neurotransmitter norepinephrine. These two factors act through β2- and β3-adrenergic receptors to increase cAMP levels. Epinephrine and norepinephrine enhance glycogenolysis and fatty acyl oxidation in muscle and increase hormone-sensitive lipase in adipose tissue. 18. Diabetes mellitus is classified as type 1 (T1DM) and type 2 (T2DM). T1DM is characterized by destruction of pancreatic beta cells, and exogenous insulin is required for treatment. T2DM can be due to numerous factors but is usually characterized as insulin resistance coupled to some degree of beta cell deficiency. Patients with T2DM may require exogenous insulin at some point to maintain blood glucose levels. 19. Obesity-associated T2DM is currently at epidemic proportions worldwide and is characterized by insulin resistance due to lipotoxicity, hyperinsulinemia, and inflammatory cytokines produced by adipose tissue. T2DM is often associated with obesity, insulin resistance, hypertension, and coronary artery disease. This constellation of risk factors is referred to as the metabolic syndrome. 20. Major symptoms of diabetes mellitus include hyperglycemia, polyuria, polydipsia, polyphagia, muscle wasting, electrolyte depletion, and ketoacidosis (in T1DM). 21. The long-term complications of poorly controlled diabetes are due to excess intracellular glucose (glucotoxicity), especially in the retina, kidney, and peripheral nerves. This leads to retinopathy, nephropathy, and neuropathy. 22. Adipose tissue has an endocrine function, especially in terms of energy homeostasis. Hormones produced by adipose tissue include leptin and adiponectin. Leptin acts on the hypothalamus to promote satiety.

C H AP T E R 4 0

Hormonal Regulation of Calcium and Phosphate Metabolism LEARNING OBJECTIVES Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the pool of serum calcium and phosphate, including ionized, complexed, and protein bound. Describe the normal concentration ranges of these ions and the major routes of influx and efflux. 2. Discuss the role of the parathyroid gland in the regulation of serum calcium and explain the role of the calcium-sensing receptor in the regulation of parathyroid hormone (PTH) secretion. 3. Describe the production of 1,25-dihydroxyvitamin D, including sources of vitamin D precursor, sites and key regulators of vitamin D hydroxylation, and transport of vitamin D metabolites in the blood. 4. List the target organs of PTH and describe its effects on calcium and phosphate mobilization or handling at each of these sites. 5. List the target organs and key actions of 1,25-dihydroxyvitamin D. 6. Discuss the regulation of phosphate metabolism by FGF23. 7. Predict the hormone responses that would be triggered by perturbations of serum calcium and phosphate or by vitamin D deficiency, and discuss the consequences of these compensatory hormone actions.

Calcium (Ca) and phosphate are essential to human life because they play important structural roles in hard tissues (i.e., bones and teeth) and important regulatory roles in metabolic and signaling pathways. In biological systems, inorganic phosphate (Pi) consists of a mixture of dihydrogen phosphate (H2PO4−) and hydrogen phosphate (HPO4−). The two primary sources of circulating Ca and Pi are the diet and the skeleton (Fig. 40.1). Two hormones, 1,25-dihydroxyvitamin D (also called calcitriol) and parathyroid hormone (PTH), regulate intestinal absorption of Ca and Pi and release of Ca and Pi into the circulation after bone resorption. The primary processes for removal of Ca and Pi from blood are renal excretion and bone mineralization (see Fig. 40.1). 1,25-Dihydroxyvitamin D and PTH regulate both processes. Fibroblast Growth Factor-23 (FGF23) regulates serum Pi by inhibiting its renal reabsorption.

FIG. 40.1 Daily Ca++ and Pi flux.

Crucial Roles of Calcium and Phosphate in Cellular Physiology Ca is an essential dietary element. In addition to obtaining Ca from the diet, humans contain a vast store (i.e., >1 kg) of Ca in bone mineral, which can be called upon to maintain normal circulating levels of Ca in times of dietary restriction and during the increased demands of pregnancy and nursing. Circulating Ca exists in three forms (Table 40.1): free ionized Ca++, protein-bound Ca, and Ca complexed with anions (e.g., phosphates, HCO3−, citrate). The ionized form represents about 50% of circulating Ca. Since it is critical to so many cellular functions, [Ca++] in both the extracellular and intracellular compartments is tightly controlled. Circulating Ca++ is under direct hormonal control and normally maintained within a relatively narrow range. Either too little calcium (hypocalcemia; total serum calcium < 8.7 mg/dL [2.2 mM]) or too much Ca (hypercalcemia; total serum Ca > 10.4 mg/dL [2.6 mM]) in blood can lead to a broad range of pathophysiological changes, including neuromuscular dysfunction, central nervous system dysfunction, renal insufficiency, calcification of soft tissue, and skeletal pathology. Table 40.1 Forms of Ca and Pi in Plasma Ion

mg/dL

Ionized

Protein Bound

Complexed

Ca

8.5–10.2

50%

45%

5%

Pi

3–4.5

84%

10%

6%

Ca++ is bound (i.e., complexed) to various anions in plasma, including HCO3−, citrate, and SO42−. Pi is complexed to various cations, including Na+ and K+. From Koeppen BM, Stanton BA. Renal Physiology. 4th ed. Philadelphia: Mosby; 2007.

Pi is also an essential dietary element, and it is stored in large quantities in mineral. Most circulating Pi is in the free ionized form, but some Pi (