Pathophysiology of Peptic Ulcer: Proceedings of the Second World 9780773593855

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PATHOPHYS I OLOGYOFPEPTI CULCER

Edi t o r

S t a nl e yC. S k o r y na

M. D. , M. S C. , PH. D. BI OL. ) , F. A. C. S . Di r e c t o ra ndAs s o c i a t ePr o f e s s o r Ga s t r o I nt e s t i na l Re s e a r c hLa b o r a t o r y , De pa r t me nto f Ex pe r i me nt a l S ur g e r y Mc Gi l l Uni v e r s i t y , Mo nt r e a l , Ca na da

Fo r e wo r d

He nr yL. Bo c k us

M. D. , F. A. C. P. Eme r i t usPr o f e s s o ro f Me di c i ne Gr a dua t eS c ho o l o f Me di c i ne Uni v e r s i t yo f Pe nns y l v a ni a Phi l a de l phi a , Pa . , U. S . A.

PATHOPHYSIOLOGY OF PEPTIC ULCER

Stanley C. Skoryna M.D., M.SC., PH.D. BIOL.), F.A.C.S.

Director and Associate Professor Gastro-Intestinal Research Laboratory, Department of Experimental Surgery McGill University, Montreal, Canada

Editor

Henry L. Bockus M.D., F.A.C.P.

Foreword

MONTREAL

Emeritus Professor of Medicine Graduate School of Medicine University of Pennsylvania Philadelphia, Pa., U.S.A.

McGILL UNIVERSITY PRESS

MCMLXIII

COPYRIGHT CANADA

1963

McGill University Press

ALL RIGHTS RESERVED, PRINTED IN CANADA

Foreword

PEPTIC ULCER, because of its frequency and wide distribution throughout the world, continues to be the subject of numerous investigations, both experimental and clinicopathological. In this respect it occupies a place secondary only to carcinoma in the field of gastroenterology. Because of great differences in the epidemiologic and clinical aspects of peptic ulcer disease in many parts of the world, as the president of the Organisation Mondiale de Gastroenterologie, I assigned to peptic ulcer a place of eminence in the deliberations of the First World Congress of Gastroenterology in Washington in 1958. A panel made up of essayists from all corners of the globe discussed geographic and environmental variants in behavior patterns of the disease and pointed out a great need for further investigation. It is indeed fortunate that during the Second World Congress of Gastroenterology in Munich 1962, a review of the basic pathologic and physiologic characteristics of ulcer disease has been undertaken as a logical sequence of the proceed-

Henry L. Bockus

ings of the previous Congress. In spite of great advances in our knowledge in many areas concerned with the etiology of peptic ulcer during recent years, the basic mechanism responsible for the development of chronic ulcer is not clear. Much investigative work has been devoted to gastric secretory behaviour patterns in peptic ulcer, and rightly so. Unfortunately, the solution has not been forthcoming as a result of these studies. Now the scope of investigation is widening. New experimental approaches and new techniques are appearing. Many of these facets are explored and discussed in the Skoryna panel. Further leads are supplied which bring us nearer to an answer to the question: "How does chronic peptic ulcer develop in man?". It is possible that these studies may well contribute toward the eventual discovery of the basic mechanism responsible for this disease. Doctor Skoryna hails from the research laboratory at McGill University, where his former chief, the late Professor B. P. Babkin, contributed so much to our knowledge of gastrointestinal physiology. v

I know of no other symposium concerned with ulcer pathogenesis which has covered such a wide scope and with such an illustrious group of scientists coming from nineteen countries and five continents. Doctor Skoryna is to be congratulated for having brought together workers in various fields of peptic ulcer research, who are well-known experts in the

vi

subjects discussed. Obviously this is only possible when organized for presentation at a world gathering such as the recent Munich meeting. The medical profession is indebted to the Editor for making available in this monograph highly important contributions on pathologic physiology of peptic ulcer.

A Message from the Congress President

THE WORLD-WIDE clinical importance of peptic ulcer made it imperative for the organizers of the Second World Congress of Gastroenterology to select this theme for a panel discussion. Therefore, as the president of the Second World Congress of Gastroenterology I most cordially welcome the efforts and ingenuity of Professor Skoryna in organizing a symposium on the pathologic physiology of peptic ulcer. Dr. Skoryna and a group of internationally known workers in this field of research have met the challenge to present the actual experimental data of gastric physiology and ulcer etiology with great success. The topics included in the presentation were gastric secretory and motor mechanisms, the methods of experimental ulcer production in animals as well as studies on the effects of pharma-

Norbert Henning

cological agents and local and systemic factors in ulcer formation. The lively discussion of the participants in this symposium and the exchange of scientific experience were additional proof of the high value of this international meeting to research workers. The material presented shows that investigation of etiology and pathogenesis of peptic ulcer must continue. The contributors to this monograph should be credited for expressing clearly the results of their studies and reasons for differences of opinion. I would like to wish this publication many interested readers in a world-wide dissemination of data on pathophysiologic factors of peptic ulcer and to hope it will stimulate research work in this important field. vii

Preface

Stanley C. Skoryna

THE FIELD of peptic ulcer research has Secondly, we tried to cover the field of expanded considerably during the past peptic ulcer research as completely as decade. New methods of study have been possible, giving it a historical perspective, employed and old views reconsidered. As as well as including the most recent my chief at McGill University, Professor studies. Finally, the Symposium has offerDonald Webster, once said, we have been ed a possibility of bringing together reliving on Pavlovian principles of gastric searchers from various parts of the world physiology and these need a re-evaluation for the common good. in light of modern methods. The new There is one more point which is close fields of gastrointestinal chemistry, phy- to my heart, and which I wish to mention sics and biology, which have so tremen- here. This concerns the co-operation of dously enlarged, might be considered the various disciplines in gastroenterological most important single factor in the de- research. This important system, diseases velopment of methods which allow more of which form perhaps the core of the precise identification of pathophysiologic practice of internal medicine and surgery, processes involved in development of has recently been joined by investigators from such fields as chemistry, biophysics, peptic ulcer in man. When I was invited by Professor Nor- hematology and microbiology. In medical bert Henning to organize a symposium research, which is basically interdisciplinon pathophysiology of peptic ulcer at the ary in nature, this seems to be not only a Congress, I realized the difficulties in- desirable but a necessary way to achieve volved in evaluation of this large and a maximum of understanding of the comever-changing field. We have attempted plex problems involved. to reach three objectives in this work. In The help I have received in the organthe first place, we obtained contributions izational work for the Symposium and the from authors who have carried out editing of this book is too extensive to original investigations of the subject. be detailed. I consider myself fortunate

ix

to have been able to work with such a distinguished group. I wish to mention particularly professor Norbert Henning and the Officers of the World Congress of Gastroenterology in Munich, who were most helpful in the preparatory work for the Symposium. To professors R. E. Davies, Eivind Myhre, Andre Lambling, J. N. Hunt and P. Raghavan I am indebted for chairing of the respective sessions at the Congress. Dr. Deirdre Edward is thanked sincerely for editorial assistance and my secretaries, Miss Unni Mürer and Mrs. Anna Maria Eccles, for carrying out the large volume of correspondence and the preparation of the manuscripts. Dean Lloyd G. Stevenson, Honorary Librarian of the Medical Library at McGill University, has served me with constant advice and encouragement during preparation of this monograph. The facilities of this large library proved invaluable for checking the numerous references, a task which was carried out by

x

Mrs. M. Benjamin and Miss Lucille Lavigeur. Finally, I wish to thank Miss Elspeth McGreevy for editing of the manuscript, Mr. Robert Reid for the design and supervision of production of the book and Mr. Robin Farr, Director of the McGill University Press, for making this publication possible. The results of this work are presented to the critical reader not without realization of our shortcomings. It may not be inappropriate to quote from the introduction by the American army surgeon William Beaumont, when he described in X 8 33 his findings in the case of the Canadian voyageur, Alexis St. Martin: "I submit a body of facts which cannot be invalidated. My opinions may be doubted, denied, or approved, according as they conflict or agree with the opinions of each individual who may read them; but their worth will be best determined by the foundation on which they rest — the incontrovertible facts."

CONTRIBUTORS ADESOLA, AKIN O. M.Ch., F.R.C.S., Lecturer in Surgery and Head, Department of Surgery, Lagos University Teaching Hospital, Lagos, Nigeria. ARABEHETY, JULIAN T. M.D., Miles-Ames Research Fellow, Harvard Medical School, Boston, Mass., U.S.A., and Universidad de Buenos Aires Buenos Aires, Argentina BALG, JOSEPH M.D., Professor and Chairman of the First Department of Pathological Anatomy and Experimental Cancer Research, Medical University, Budapest, Hungary. BERG, GERHARD W. M.D., (Dr. med.),

Privatdozent, Medizinische Universitäts Klinik, University of Erlangen, Erlangen, Germany. BOCKUS, HENRY L. M.D., F.A.C.P., Emeritus Professor of Medicine, Graduate School of Medicine, University of Pennsylvania, Philadelphia, Pa., U.S.A. BONFILS, SERGE M.D., Directeur de Recherches å l'Institut National d'Hygiene, Medecin de l'Höpital Bichat, Paris, France. BRODIE, DAVID A. Ph.D., Senior Investigator in Gastroenterology, Merck Institute for Therapeutic Research, West Point, Pa., U.S.A. BURGEN, ARNOLD S. V. M.A., M.D., M.R.C.P.,

Professor of Pharmacology, Department of Pharmacology, Cambridge University, Cambridge, England. xi

CARD, WILFRED INGRAM M.D., F.R.C.P.E., F.R.C.P.(Lond.), Reader in Medicine and Physician in Charge, Gastro-Intestinal Unit, Western General Hospital, Edinburgh, Scotland. CHAPMAN, NILES D. M.D., Instructor in Surgery, Department of Surgery, University of Washington School of Medicine, Seattle, Wash., U.S.A. CHEN, JEANNE K. B.A., Research Associate, The Gastrointestinal Research Laboratory, George Washington University and Veterans Administration Hospital, Washington, D.C., U.S.A. CLARKE, STEWART D. M.Ch., F.R.C.S., Lecturer in Surgery, Department of Surgery, Royal Infirmary, Sheffield, England. CSERNAY, L. M.D., Research Fellow, First Department of Medicine, University of Szeged, Szeged, Hungary. DAVIES, ROBERT E.

M.A., M.Sc., Ph.D., DSc., Professor of Biochemistry, Chairman of the Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pa., U.S.A.

DOLCINI, HORACIO M.D., Research Fellow, Harvard Medical School, Boston, U.S.A., Universidad de Buenos Aires and Stapler Clinic, Buenos Aires, Argentina. ERSPAMER, VITTORIO M.D., Professor and Chairman of the Department of Pharmacology, University of Parma Medical School, Parma, Italy. xii

FORTE, JOHN G. PhD.,

Instructor of Physiology, Department of Physiology, University of Pennsylvania, Philadelphia, Pa., U.S.A. GRAY, SEYMOUR J. M.D., Ph.D.,

Associate Clinical Professor of Medicine, Department of Medicine, Harvard Medical School, Boston, Mass., U.S.A. GREGORY, RODERIC A. D.Sc., Ph.D., M.Sc., M.R.C.S., L.R.C.P.,

Professor and Chairman of the Department of Physiology, University of Liverpool, Liverpool, England. GUPTA, S. K. M.B., B.S.(Cal.), D.C.P.(Lond.),

Lecturer in Pathology, Department of Pathology, Nilratan Sircar Medical College, Calcutta, India. HALLENBECK, GEORGE A. M.D.,

Professor of Surgery and Physiology, Section of Surgical Research, Mayo Clinic and University of Minnesota, Rochester, Minn., U.S.A. HARRISON, R. C. M.D., M.S., F.A.C.S., F.R.C.S.(Can.),

Professor of Surgery and Director of Surgical Research, Department of Surgery, University of Alberta, Edmonton, Canada. HEINKEL, KLAUS M.D., (Dr. med.),

Privatdozent, Medizinische Universitiits Klinik, University of Erlangen, Erlangen, Germany. HELLEMANS, J. M.D.,

Aspirant, National Fonds voor Wetenschappelyk Onderzoek, University of Louvain Medical School, Louvain, Belgium.

HENNING, NORBERT

M.D., Professor of Medicine and Director, Medizinische Universitäts Klinik, University of Erlangen, Erlangen, Germany.

HIRSCHOWITZ, BASIL I. B.Sc., M.B., B.Ch., M.D., M.R.C.P.(Lond.), M.R.C.P.E., Associate Professor of Medicine, Director, Division of Gastroenterology, University of Alabama College of Medicine, Birmingham, Alabama, U.S.A. HUNT, JACK NAYLOR M.D., DSc., PhD., Professor, Department of Physiology, Guy's Hospital Medical School, University of London, London; England. ISHIHARA, KEIZO M.D., Professor and Head of the Department of Surgery, Gunma University, Maebashi, Japan. JOHNSON, F. R. M.D., B.A.O., Reader in Anatomy, London Hospital Medical College, University of London, London, England. KAHN, DAVID S. M.D., Pathologist in Chief, St. Mary's Memorial Hospital, Assistant Professor, Department of Pathology, and Consulting Pathologist, Gastro-Intestinal Research Laboratory, McGill University, Montreal, Canada. KAY, ANDREW WATT M.D., Ch.M., F.R.C.S.(Engl. & Ed.), F.R.C.PS.G. Professor of Surgery, Department of Surgery, The Royal Infirmary and University of Sheffield, Sheffield, England. KYLE, JAMES M.Ch., F.R.CS.(Engl. & I.), Consulting Surgeon and Clinical Lecturer in Surgery, Royal Infirmary, Aberdeen, Scotland. xiv

LAMBERT, RENE E. M.D., Professeur agrege Charge de Recherches au Centre National de la Recherche Scientifique, University of Lyon, Lyon, France. LAMBLING, ANDRE M.D., Professeur å la Faculte de Medecine Directeur du Centre de Gastroenterologie de 1'Höpital Bichat, Paris, France. MALLIK, BASU K. C. M.D., PhD., Professor and Director of the Department of Pathology, Nilratan Sircar Medical College, Calcutta, India. McMINN, R. M. H. M.D., Ph.D., Reader in Anatomy, King's College, University of London, London, England. MYHRE, EIVIND

Dr. Med., Prosektor, Institutt for Generell og Eksperimentell Patologi, Rikshospitalet, University of Oslo, Oslo, Norway.

NYHUS, LLOYD M. M.D., F.A.C.S., Associate Professor of Surgery, Department of Surgery, University of Washington Medical School, Seattle, Wash., U.S.A. PALMER, EDDY D. B.A., M.D., F.A.C.P., Colonel, M.C., Chief, Department of Gastroenterology, Brooke Army Hospital, Fort Sam Houston, Texas, U.S.A. PHILLIPS, MELVILLE J. M.D., C.M., F.R.C.P.(C), Pathologist, Department of Pathology, Toronto General Hospital, University of Toronto, Toronto, Canada. xv

PROHASKA, GERTRUDE N. M.D., M.Sc., Research Associate, Gastro-Intestinal Research Laboratory, McGill University, Montreal, Canada. RAGHAVAN, P. M.D., Professor and Director of the Department of Medicine, Seth Gordhandas Sunderdas Medical College, Bombay, India. RETZER, OSKAR

M.D., Teaching Fellow in Surgery, Department of Surgery, University of Alberta, Edmonton, Canada.

ROTH, JAMES L. A. M.D., Ph.D., Professor of Clinical Gastroenterology and Director, Division of Gastroenterology, Graduate School of Medicine, University of Pennsylvania, Philadelphia, Pa., U.S.A. SHERMAN, JACQUES L. Jr. B.S., M.D., F.A.C.P., Lt. Colonel, M.C., Chief, Research Division, U.S. Army Medical Service, Research and Development Command, Washington, D.C., U.S.A. SIRCUS, WILFRED M.D., Ph.D., F.R.C.P.E., M.R.C.P.(Lond.), Physician, Gastro-Intestinal Unit, and Lecturer in Medicine, Western General Hospital, Edinburgh, Scotland. SKORYNA, STANLEY C.

M.D., M.Sc., Ph.D., F.A.C.S. Director and Associate Professor, Gastro-Intestinal Research Laboratory, McGill University, Montreal, Canada.

SMITH, ADAM N. M.D., F.R.C.S.(Ed.), Reader in Surgery, Department of Clinical Surgery, University of Edinburgh, Edinburgh, Scotland. xvi

STAUFFER, MAURICE H. M.D., Consultant in Gastroenterology and Assistant Professor of Medicine, Mayo Clinic and University of Minnesota, Rochester, Minn., U.S.A. SUN, DAVID C. H. M.D., D.Sc., (Med.), Director, Gastro-Intestinal Research Laboratory, and Associate Clinical Professor, Department of Medicine, The George Washington University and Veterans Administration Hospital, Washington, D.C., U.S.A. UVNÄS, BÖRJE Professor of Pharmacology and Chairman, Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden. VALDES-DAPENA, ANTONIO M.D., Assistant Professor of Pathology, Graduate School of Medicine, University of Pennsylvania, Philadelphia, Pa., U.S.A. VANDENBROUCKE, J. M.D., A.H.O., Professor of Medicine and Chairman of the Department of Medicine, University of Louvain Medical School, Louvain, Belgium VANTRAPPEN, G. M.D., A.H.O., Associate Professor of Medicine, Department of Medicine, University of Louvain Medical School, Louvain, Belgium. VARRO, VINCE M.D., Docens, First Department of Medicine, University of Szeged, Szeged, Hungary. VERBEKE, S. M.D., Aspirant, National Fonds voor Wetenschappelyk Onderzoek, Department of Medicine, University of Louvain Medical School, Louvain, Belgium. xvii

WALDRON, EDWARD D.

BSc., PhD.,

Research Associate in Biochemistry, Gastro-Intestinal Research Laboratory, McGill University, Montreal, Canada. WARD, JOHN T. M.B., M.Ch.,

Tutor, Department of Surgery, Queen's University, Belfast, Northern Ireland. WATT, JAMES M.B., Ch.B., M.D.,

Lecturer in Pathology, Department of Pathology, University of Liverpool, Liverpool, England. WEBSTER, DONALD R. O.B.E., M.D., C.M., M.Sc., Ph.D., F.A.C.S., F.R.C.S.(Can.), Hon. F.R.C.S.(Engl.),

Professor of Experimental Surgery and Head, Department of Experimental Surgery, McGill University, Montreal, Canada. WELBOURN, RICHARD B. iM.A., M.D., F.R.C.S.,

Professor of Surgery, Postgraduate Medical School, University of London, London, England. WOODWARD, E. R. M.D.,

Professor and Head, Department of Surgery, University of Florida Medical School, Gainesville, Fla., U.S.A. ZAIDMAN, ISODORO M.D.,

Research Fellow, Harvard Medical School, Boston, Mass., U.S.A., and Universidad Central de Venezuela, Caracas, Venezuela.

xviii

CONTENTS

PART I. GASTRIC PHYSIOLOGY PAGE

3. Secretion of Acid by the Stomach R. E. DAVIES AND J. FORTE

23. The Physiology of Pepsinogen BASIL I. HIRSCHOWITZ

59. Secretion of Gastric Mucin K. HEINKEL AND G. BERG

73. The Biochemistry and Degradation of the Mucus of the Upper Gastrointestinal Tract DEIRDRE WALDRON EDWARD

87. Gastrin Release BÖRJE UVNÄS

lot. Recent Advances in Preparation of Gastrin R. A. GREGORY

109. The Osmotic Activity of Gastric Secretion A. S. V. BURGEN

I15. Gastric Motility G. VANTRAPPEN, T. VANDENBROUCKE, S. VERBEKE AND J. HELLEMANS

PART II. EXPERIMENTAL ULCER PRODUCTION AND METHODOLOGY 135. The Mann-Williamson Ulcer MAURICE H. STAUFFER AND GEORGE A. HALLENBECK

141. Experimental Ulcer Production in the Pylorus-Ligated Rat DAVID C. H. SUN AND JEANNE K. CHEN

153. Psychological Factors and Psychopharmacological Actions in the Restraint-Induced Gastric Ulcer SERGE BONFILS AND ANDR] LAMBLING

xix

PAGE 173.

Experimental Chronic Gastric Ulcer in the Rat DAVID S. KAHN AND M. J. PHILLIPS

183.

Experimental Methods of Collection of Gastric Secretion R. C. HARRISON AND 0. RETZER

193.

The Microcirculation of Gastric Mucose: Effects of Neural, Hormonal and Pharmacological Factors SEYMOUR J. GRAY, J. T. ARABEHETY, H. DOLCINI AND I. 'ZAIDDIAN

PART III. EFFECT OF PHARMACOLOGICAL COMPOUNDS 213.

Experimental Histamine Ulceration JAMES WATT

233.

The Effect of Corticotrophin and Cortisone on the Healing of Gastric Ulcer EIVIND MYHRE

245.

Mechanisms of Salicylate Gastrointestinal Erosion and Hemorrhage JAMES L. A. ROTH AND ANTONIO VALDES-DAPENA

253. Experimental Studies of the Effects of Reserpine on Gastric Secretion RENT; LAMBERT 273.

Experimental Caffeine Ulceration JAMES L. A. ROTH AND ANTONIO VALDES-DAPENA

z

i. Experimental Production of Peptic Ulcer by Administration of Cincophen V. VARRO AND L. CSERNAY

291.

Experimental Studies on the Ulcerogenic Effect of Phenylbutazone V. VARRO AND L. CSERNAY

301.

An Experimental Study of Gastric Ulcers Produced by Pilocarpine K. C. BASU MIALLIK AND S. K. GUPTA

311. Experimental Production of Gastric Ulceration by Gastrotoxin and the Compound 48/8o ADAM N. SMITH XX

PART IV. LOCAL FACTORS IN PEPTIC ULCER FORMATION PAGE 325.

Gastric Secretion in Experimental Animals E. R. WOODWARD

333• The Nature of Basal Hypersecretion in Man with Duodenal Uker J. N. HUNT, A. W. KAY, W. I. CARD AND W. SIRCUS

339.

Hypoxia of Vascular Origin in the Development of Gastroduodenal Ulcer Disease JACQUES L. SHERMAN, JR. ANI) EDDY I). PALMER

353•

The Gastric Antrum and the Regulation of Acid Secretion LLOYD M. NYHUS AND NILES D. CHAPMAN

369.

The Cytology of Mucosal Regeneration in Experimental Gastric Ulcer R. M. H. MC MINN AND F. R. JOHNSON

PART V. SYSTEMIC FACTORS IN PEPTIC ULCER FORMATION 389.

Role of the Central Nervous System in Peptic Ulcer Development JOSEPH BALG

403. Neurogenic Factors in Experimental Peptic Ulceration DAVID A. BRODIE

413.

Effects of Corticotrophin Release Produced by Pitressin and Pitocin on Gastric Secretion KEIZO ISHIHARA

423.

5-Hydroxytryptamine (Enteramine, Serotonin) and Gastrointestinal Tract VITTORIO ERSPAMER

445. The Relationship of the Major Endocrine Glands to Experimental Peptic Ulceration

JAMES KYLE, STEWART D. CLARKE, JOHN T. WARD, A. 0. ADESOLA AND RICHARD B. WELBOURN

xxi

PAGE

457. Effects of Insulin and Glycemic Factors an Gastric Secretion D. R. WEBSTER AND G. N. PROHASKA

465. Role of Nutritional Factors in Peptic Ulcer P. RAGHAVAN

PART VI. GENERAL PROBLEMS 481. Stochastic Processes in The Causation of Peptic Ulcer STANLEY C. SKORYNA

PART I GASTRIC PHYSIOLOGY

Secretion of Acid by the Stomach*

Robert E. Davies John G. Forte**

Origin of Gastric Acid THE problem of the origin and mechanism

proposes a precursor at some level even of gastric acid secretion has intrigued bio- though it might be at some microbounlogists for very many years. As far back dary within the cell. as 1803, John R. Young (i ), a medical student at the University of Pennsylvania, reported his experiments on the digestive Fine structure of acid secreting cells process in his doctoral dissertation. His The tubules which occur in the stomexperiments can now be understood as ach are lined by various cell types. Among proving that the stomach makes hydro- these in the mammal are the parietal cells chloric acid, but he interpreted them which have intracellular canaliculi or wrongly in concluding that the stomach lumina extending into the cell from the secreted phosphoric acid. It remained for apical or secretory surface. Lining the William Prout to show that the acid in walls of the canaliculi are numerous exthe stomach was in fact "free, or at least tensions, or microvilli (4). Fingerlike prounsaturated muriatic acid" (in 1824) (2). jections or villi are usually associated with The next problem was to find out where an absorptive function as in the kidney it is formed. Claude Bernard (3) believed or intestine, though the parietal cells are that the stomach produced some precur- thought to be secretory cells. Proof that parietal cells are the source sor which only turned to hydrochloric acid in the lumen. The major difficulty of acid secretion in mammals was with such a theory is that analyses of originally gathered by a process of elimgastric secretions show no evidence for ination through the physiological and the presence of a precursor in the gastric histological examinations of Langley (S). contents. However, the problem of a pre- All of the other cell types in the tissue had cursor to hydrochloric acid production clear functions; e.g. the mucous neck cells is more subtle in that any theory propos- obviously secreted mucus; muscle cells ing the transport of the hydrogen and the were concerned with contractions; chief chloride ions (or the hydrochloric acid cells were the source of pepsinogen; etc., molecule) by a carrier substance, also thus leaving the parietal cells to secrete 'Supported by a grant from the National Institute of Health. "From the Department of Animal Biology, School of Veterinary Medicine; Department of Biochemistry and Physiology, School of Medicine, University of Pennsylvania, Philadelphia, U.S.A.

3

Silk thread

HC0 Cl A Open"

acid. In the frog stomach one cell type apparently can secrete acid and some pepsinogen, although in amphibians most of the pepsinogen is secreted by the esophagus (5,6,7). The acid-secreting cells of the stomach are called oxyntic or parietal cells. The oxyntic cells are remarkably full of mitochondria, indicating a very active oxidative metabolism, a fact which has been borne out by biochemical evidence (8,9,1o). Electron microscopists have shown that numerous microvesicles (zo-zoomµ) are randomly distributed throughout the whole of the cytoplasm in the resting stomach (11,12,13) and Sedar has shown that these vesicles apparently migrate to the apical secretory surface of parietal cells in the in vivo dog under vagal stimulation (14). He has demonstrated similar effects in the in vivo and in vitro frog after histamine stimulation (15,16) These microvesicles might be filled with HC1 and deposit their contents at the secretory surface on stimulation; they may be concerned with water movement across the gastric mucosa; they may be reserves for membrane synthesis that might occur when the cells are actively secreting, etc. The function of these microvesicles certainly requires further investigation. It is interesting to note that electron-microscopic studies of the "chloride secretory cell" of the gills of teleost fishes show structures remarkably similar to those in the oxyntic cell (17,18,19). Of particular interest is the presence of numerous dense microvesicles which might pick up electrolytes at the mitochondria and discharge their contents at the plasma membrane (19). Bradford and Davies (zo) used pH indicator dyes to determine whether the parietal cell secretion was acid. They recorded acidities corresponding to a pH less than 1.4 in the canaliculi of the polecat parietal cell. These studies show that 4

Thin rubber balloon

B "Tied" Glass tube CO2+ H2O 1+HCO; Mucosax insideout

H+ CI-

"Inside-out"

H2O

OH-

+CO2HCO3

FIG. r. Diagrammatic representation of the over-all reactions in A "open," B "tied," and C "inside-out" frog gastric mucosa.

the acid first appears in the intracellular canaliculi of the oxyntic cell. The source of hydrogen ions In 1859 Brücke hypothesized that if a free acid was liberated in the gastric tubules, an alkaline fluid of corresponding strength would at the same time pass into the blood and lymph systems (21). Several authors ( 22,23,24,25) working with in vivo and in vitro preparations had demonstrated, qualitatively, that alkali was released into the blood or serosal chamber when the stomach was made to secrete acid. In 1948 Davies (9), working with isolated frog gastric mucosa, showed that an exactly equivalent amount of alkali was formed for each mole of acid produced. As indicated diagrammatically in Fig. 1, in mucosae that were tied in bags so as to secrete acid inwards, it was found by direct titration that the amount of bicarbonate liberated into the bathing medium was equivalent to the acid produced and the quantity of gas (carbon dioxide) absorbed during the experiment. When the mucosae were tied inside out there was a transfer of bicarbonate from outside to the lumen. No net uptake of gas occurred because the acid produced

DAVIES & FORTE

by the inverted mucosa liberated just as much carbon dioxide from the external bicarbonate as was absorbed to make the internal bicarbonate. Open sheets of mucosae did not change the pH of the incubating solution, although there was an increase in oxygen consumption when histamine was added, this proving that exactly equivalent amounts of acid and alkali were formed and able to mix. Teorell (26), who also worked on isolated frog gastric mucosa, confirmed this point by direct titration. Actively acid-secreting stomachs require external supplies of carbon dioxide and have negative respiratory quotients. The early work on the relationship between the rate of acid secretion and the availability of external supplies of carbon dioxide was carried out by Delrue (24), and Gray, Adkison and Zelle (25). Davies (g) and Davies and Longmuir (27, 28) using tied bags of isolated frog gastric mucosa, found that large amounts of hydrochloric acid could be secreted as long as the rate of acid secretion remained below the rate at which the mucosa itself produced carbon dioxide by metabolic processes. Since for every mole of acid secreted there was an uptake of a mole of carbon dioxide, external supplies of carbon dioxide were needed when the rate of acid secretion rose above the rate of oxygen uptake, or of metabolic carbon dioxide output. In the absence of external supplies of carbon dioxide, mucosa with high rates of acid secretion were ulcerated or perforated. This damage was interpreted as being due to a disturbance of the acid-base balance within the cell so that unneutralized alkali accumulated and disorganized the oxyntic cells. This damage never occurred if adequate external supplies of carbon dioxide were available to the mucosa. Davenport and Fisher (29) found carbonic anhydrase in the stomach, and

Davenport (3o) showed that large amounts of the enzyme occurred in oxyntic cells. Carbonic anhydrase catalyzes the hydration and dehydration of carbon dioxide to form carbonic acid. Davenport (30,31) suggested that this enzyme played a role in the acid secreting mechanism, a hypothesis which was strengthened when he found that thiocyanate, which inhibits carbonic anhydrase, also inhibits acid secretion. However, Feldberg, Keilin and Mann (32) found in cats that when acid secretion was largely inhibited by thiocyanate, there was only a to per cent reduction in carbonic anhydrase activity. They also found that concentrations of sulfonamide that reduced the activity- of carbonic anhydrase more than 8o per cent had little or no effect on acid secretion. Davies and Roughton (3 3) offered an explanation of the results of the work involving carbonic anhydrase from calculations based on rate constants of the hydration of carbon dioxide. They concluded that in mammalian cells the uncatalyzed rate of carbon dioxide uptake was too slow to neutralize alkali within the actively secreting cell by a factor which depends on the extent to which the pH of the cell is maintained alkaline to the equilibrium value. They further showed that the amount of carbonic anhydrase in the cells was in apparent excess of the amount necessary for catalysis even at high secretory rates. Their calculations showed that even o.3 per cent of the carbonic anhydrase activity in the oxyntic cells would catalyze the reaction by the sufficient amount of forty-fold. Davenport and Jensen (34) used several sulfonamides, which are powerful inhibitors of carbonic anhydrase, on in vitro mouse stomachs. In two-hour experiments they found significant depressions of acid secretion (23 per cent) with 1.4 X le M thiophene-2-sulfonamide. Davies and Edelman (34) subjected isolated frog 5

I / SECRETION OF ACID BY THE STOMACH

gastric mucosae to several sulfonamides. These substances caused only slight inhibition of acid secretion during the first half hour after addition, but after subsequent half-hour intervals, acid secretion was apparently completely inhibited. However, the back diffusion of acid already secreted prevented a precise estimate of the inhibition. The oxygen uptake of non-secreting gastric mucosa and several other tissues was unaffected

by these inhibitors, but that of acidsecreting gastric mucosa was reduced by the amount expected if the oxyntic cells had been damaged. The finding of Janowitz, Colcher and Hollander (36) that acetazolamide inhibited acid secretion in in vivo dogs by some 85 per cent is further evidence involving carbonic anhydase in the overall process of acid secretion.

The Mechanism of Acid Secretion Energetics of the secretory process Any secretory process is limited by the over-all thermodynamics of the system. In '943, Gray (37) applied the following relationship to the parietal secretion: A F = nRTlog. G

G where the minimum free energy needed (A F) is proportional to the log of the concentration gradient (C, and G are concentrations of the substance in the plasma and in gastric juice respectively), n is the number of gram ions secreted, R the gas content, and T the absolute temperature. Gray calculated that there were required 9,65o calories gram mole to effect a secretion of 159 mN acid. This value is fixed, and is a minimum for any theory to describe the immediate energetics of the acid-secreting machinery. Gray neglected to include the effect of potential difference across the mucosa; however, the help given to the hydrogen ion of hydrochloric acid would be just equal to the hindrance of the negatively charged chloride ion, so the figure remains correct for the over-all production of hydrochloric acid although the individual contributions he gave for hydrogen ions and chloride ions are wrong. Acid secretion is abolished in the ab6

sence of oxidative metabolism (2 4,38,39, 40,41). This has been established both by depriving the tissue of its oxygen supply, and by the use of various inhibitors which have their influence at some point in the respiratory cycle. Davenport has suggested that energy for acid secretion can also come from glycolysis provided there is concomitant oxygen consumption (42 ,43) . Either the contributory effect from glycolytic metabolism becomes greater as the partial pressure of oxygen in the system decreases, or the efficiency of the utilization of the energy derived from oxidative metabolism is increased. Both azide and 2,4-dinitrophenol increase the respiration but completely abolish acid secretion and the potential difference across the mucosa (44). A linkage between acid secretion and "energy-rich phosphate" metabolism is suggested by this latter observation. Red ox pump hypothesis Lund first proposed that an oxidationreduction mechanism was responsible for the origin of bio-electric currents (45,46). He discussed the significance for electrical phenomena that in some animal tissues, during respiration, four electrons react with each oxygen molecule in the presence of water. Lundegardh (47,48)

DAVIES & FORTE

presented evidence for the conclusion that anions were actively transported across root hairs of plants by an oxidationreduction mechanism such as the ferrousferric system of the cytochromes. In 1948 three groups of workers (Crane and Davies (49), Roberston and Wilkins (5o), and Conway and Brady (51) ) suggested that, for the formation of gastric acid, oxygen might react with substrate hydrogen atoms via the cytochrome system to form hydrogen and hydroxyl ions instead of water. Crane and Davies pointed out that for glucose as substrate, and allowing for water uptake in the citric acid cycle, the over-all reaction would be as follows: C0HLO0 + 602+ 18H20 —) 6CO2 + 24H+ + 240HIf the transport of any monovalent ion were dependent upon this process alone, in which oxygen was the sole electron acceptor, a quantitative upper limit of 4 would exist between the number of ions transported and the amount of oxygen consumed. The above mechanism would correspond to Mechanism I, proposed by Davies and Ogston (52), to account for acid production by the stomach, and is the basis of the Redox Pump (S3). If the source of hydrogen ions were from the dissociation of water driven by metabolic energy in a process independent of, or in addition to, the above reaction, the stoichiometric efficiency need only be imposed by the over-all thermodynamic limit of the system. This latter hypothesis was included in Mechanism II of Davies and Ogston.

acid secretion and then after acid secretion induced by histamine. The onset of acid secretion was accompanied by an increase in respiration. The experimental design permitted the calculation of the ratio of the change in acid secretion (AQHC1) to the change in oxygen consumption (AQOf). The results of thirtythree experiments showed that two-thirds of the ratios were above 4.o and values as high as 11.5 were obtained. The basic assumption of these calculations was that the increase in respiration induced by histamine was responsible for the whole of acid secretion; all other respiration was due to the non-acid secreting elements of the gastric mucosa. However, the authors pointed out that some of the resting respiration could have been channeled into secretory work upon histamine stimulation. When they averaged all the values after histamine stimulation, they reported a value of 0.9 for the ratio of mean total QHC1/mean total QO,. Teorell (54) found the over-all ratio of hydrogen ions secreted to oxygen molecules consumed to be 2.2 (probable variation 1.1 to 4.4) for the frog gastric mucosa, when calculated from the temperature coefficients of hydrogen ion secretion and oxygen consumption. Since this ratio included all of the oxygen consumption, this value is clearly a minimum one. In 1952 Davenport (55) reported the results of experiments to determine the efficiency of acid secretion of tied bags of frog gastric mucosa. His experimental design differed from that of Crane and Davies in that he measured and plotted Investigations on the stoichiometric the acid secretion (,umoles./mg. dry efficiency of HCI secretion wt./hr.) against the oxygen consumption Crane and Davies (10,49) investigated (same units) in a non-stimulated series of the stoichiometric efficiency of the rela- mucosae and in a series which were stimution between HC1 secretion and oxygen lated with histamine. A regression line consumption. They measured the QO: was calculated for all of the plotted data of tied bags of frog gastric mucosa before and from this Davenport assumed a) that 7

I / SECRETION OF ACID BY THE STOMACH

the intercept on the abscissa (oxygen consumption) represented the resting oxygen consumption, and b) that the slope of the line for the entire group of data was a measure of the ratio of acid produced for a given amount of oxygen consumed. His calculated ratios for both unstimulated and histamine-stimulated mucosae were below 3.o, but the slope of the line joining the means of both of his samples (this would correspond to the AQHC1/AQO, upon histamine stimulation) was 5.23 which Davenport considered as not being significant. Davenport and Chavre (56) repeated these experiments using mouse gastric mucosa and got similar results. The interpretations of these experiments were critically reviewed by Davies (S7), who pointed out that the values do not fall on a straight line as assumed by Davenport. On the basis of the curve which best fits the data, there is a large segment of the population which gives a slope corresponding to an average ratio of 4.6. Davies further pointed out that any experiment designed to test the maximum efficiency should maintain the mucosa under optimal conditions (CO! must be present to obtain maximum rates of acid secretion), and should include simultaneous measurements on the same tissue. The calculation of the ratio from average values of acid secretion and respiration will only yield the average efficiency for that population. When evaluating the applicability of a theory which imposes a rigid stoichiometric limit, the demonstration of a number of experimentally valid ratios above the theoretical limit would make the mechanism untenable. Conway (58) reviewed both Davies' and Davenport's position, but it is clear that no agreement existed as to the stoichiometric efficiency of gastric acid production. 8

Electrical activity of gastric mucosa Before proceeding further in this consideration of respiration directly associated with acid secretion and the ultimate source of hydrogen ions, it is useful to outline some of the electrical characteristics of the gastric mucosa and its relation to acid secretion. In 1834 Donne (S9) observed that a potential difference was maintained across the stomach wall, the secretory side being negative to the nutrient side in an external circuit. A possible relationship between this potential and the secretion of acid was studied by numerous investigators throughout the nineteenth century (see review by Biedermann (6o), 1895). Later developments on the relation between hydrochloric acid secretion and the electrical activity of the gastric mucosa came from the work of Rehm et al. (39,61,62,63) using an in situ dog stomach preparation, and from experiments of Davies and his colleagues (9,38, 64,65,66) using the isolated frog gastric mucosa. Many theories of gastric acid secretion have been concerned with the source of the electromotive force of the stomach which is about —3omv across the isolated frog gastric mucosa and about —7omv across the dog stomach wall (secretory side negative to the nutrient side in an external circuit). Changes in the secretory state of the tissue and changes in the metabolism will alter the magnitude of the potential difference. The stomach is capable of doing electrical work by passing a current through an external circuit that connects the secretory to the nutrient side. Passage of electric current from an external source increases, or decreases, the rate of secretion of acid by a secreting mucosa according to whether the applied current enhances or opposes the natural membrane-potential difference.

DAVIES & FORTE

In order to assess the role that a mem- could be maintained at zero electrobrane-potential plays in a secretory pro- chemical potential difference. Here an cess, one has to measure several para- equilibrium exists so that any net transmeters, most important being the elec- port of a substance across the membrane trical characteristics of the membrane can only be achieved by an "active pro(potential, current, and resistance) and cess," that is, a process requiring metathe composition of the solutions on both bolism for its function. sides of the membrane. Ussing (67,68) The normal secreting stomach must briefly defined active transport as a trans- transport chloride uphill (endergonicalfer which cannot be accounted for by ly) against both a chemical and an elec"physical" forces. The physical forces trical gradient (37,72,73). Hogben was he cites are diffusional forces, electric able to show, via radioactive chloride fields, and solvent drag. With these flux measurements, that a net transport of physical forces eliminated, one could this ion occurred in the short-circuited measure directly the active transport of gastric mucosa. However, the measured any substance. (However, it should be short-circuit was less than the net flux of noted that some of these physical forces chloride. This difference was found to be depend on biochemical metabolism for equal to the hydrochloric acid transport their maintenance and are in a sense to the secretory side which does not pro"active") . duce a current since it is a movement in Ussing applied this concept to the frog the same direction of two equally but skin. The natural membrane-potential oppositely charged ions. across the frog skin was "short-circuited" by passing an external current through Chloride ion secretion the skin. Net passive diffusion from one Forte, Nauss, Winters and Davies (74) side to the other was eliminated by bath- used the Ussing short-circuit technique ing the frog skin on both sides with to measure chloride ion transport by identical solutions. With the two solu- bullfrog gastric mucosa, while oxygen tions thus at zero electrochemical poten- consumption was being measured by an tial difference, Ussing and Zerahn (69) oxygen electrode in each chamber of the demonstrated that a net transport of assembly. This approach of correlating sodium, which was dependent on meta- metabolism with HC1 secretion afforded bolism, occurred across the frog skin, and an opportunity to make simultaneous that this ionic movement could be exactly measurements on the mucosa of respiraaccounted for in the short-circuit current. tion, chloride ion transport and hydrogen In 1951 Hogben (7o,71), working in ion production, and at the same time, Ussing's laboratory, used the short-cir- maintain favorable conditions for acid secuiting technique on isolated frog gastric cretion (i.e. 5 per cent CO dissolved in mucosa. His method consisted of mount- the bathing solutions). They compared ing the mucosa between two halves of a the ratio of the short-circuit current in chamber in which both sides could be uequiv./cm'./hr. to the total oxygen conbathed by identical solutions—hence, the sumption in umoles./cm 2/hr. Only two abolition of any chemical gradient, such of the fifty reported values were above 4.0 as the large ionic gradients that exist (range 0.9 to 5.4 with a mean of 2.83). across the in vivo secreting stomach. Thus only rarely was the ratio found to When an external current was passed he higher than the electrochemical equithrough the mucosa, the two solutions valent of oxygen. 9

AO

OF CASES

30

10P PER CODS 22 MUCOSAE

NUMBER

20

10

Since the total amount of actively transported chloride consisted of hydrochloric acid as well as chloride ions that were responsible for the short-circuit current, the real ratio for chloride ion movement must include both of these. Forte et al. further compared the sum of the shortcircuit current and the HC1 in pequiv/ cm'/hr to the oxygen consumed, and found many values significantly above 4.0. (It had been discovered by the authors that after some months of use, the Plexiglass chambers began to absorb oxygen from the bathing solution, resulting in an under-estimation of the true values by some 5-10 per cent (discussed by Forte (75)). Glass chambers were used in place of the Plexiglass and the experiments were repeated. Fig 2 shows a frequency distribution of ratios of the chloride current plus hydrochloric acid production to the oxygen consumption of 102 periods on twenty-two mucosae. Since more than one third of the values were above 4.0, it is obvious that any theory of chloride transport must provide that more than four chloride ions could be transported per oxygen molecule consumed. Villegas and Durbin (76) independently ion out measurements of hydrogen on secretion, short-circuit current production, and oxygen consumption (oxygen electrodes) on mucosae mounted in a Plexiglass chamber. In their communication they only give the average values for these measurements before and after histamine stimulation. The average ratio of the short-circuit current to the oxygen consumption was 2.6, not significantly different from the average (2.83) obtained by Forte et al. If the acid secretion and the short-circuit current were summed (thus giving total chloride transport), and the value compared to the oxygen consumption, a ratio of 3.2 is obtained, but the standard errors reported 10

1.0

1.0

30

4

0

3.0

FIG. 2. Frequency distribution of the ratios of chloride current (ttequiv/cm`/hr) plus hydrochloric acid secretion (Itequiv/cn?/hr) over oxygen consumption (pmoles/cm'/br) from isolated, short-circuited gastric nnrcosae in glass chambers (22° -2g C ).

for each of the average values indicate that at least 5 per cent of the population would be expected to be 4.0 or greater. Hydrogen ion secretion In sixty-two experiments we have found that the ratios of H' secreted to total oxygen molecules consumed by the whole mucosa under optimal conditions varied between 0.5 and 3.o with a mean of 1.89. Although this value was considerably below 4.0, the measurements of respiration were made on the entire mucosa including all the cells and processes not concerned with hydrogen ion production; e.g. the work necessary to transport chloride ions as an electric current would not be available for hydrogen ion transport. The approach taken by Crane and Davies (10,49) to measure the change in oxygen consumption correlated with changes in acid secretion was an effort to eliminate non-secretory respiration from the measurements. We have used various methods to enhance or inhibit acid secretion, and to measure accompanying changes in oxygen consumption. In four experiments where histamine was added to mucosae that were already secreting, there was an average increase in acid production of o.8z ,ttequiv./cm'/hr. while oxygen consumption increased by

Q0

DAVIES & FORTE

0.34 ,umoles./cm.'/hr., giving a ratio of 2.41. However, this might be an underestimate since histamine has been shown to stimulate secretions other than pure hydrochloric acid (77,78). An interesting point here is that the Rana catesbiana that we used for these experiments were already secreting acid at relatively high rates, and hence the changes brought about by histamine were not very great (20-37 per cent) with a resulting efficiency ratio of less than 4.o. The Rana teznporaria used by Crane and Davies (10,49) were not secreting any acid before histamine stimulation, thus the changes in H. production were large compared to the increase in respiration. A similar effect can be seen in the data of Davenport (S5) as discussed by Davies (S7) with mucosae having a different efficiency depending upon the secretory rate. The concomitant changes in acid secretion and oxygen consumption were measured when thiocyanate was introduced into the solutions bathing the mucosa, and when the thiocyanate ion was washed out and acid production resumed. The average value for the inhibitory change in hydrogen ion secretion when I omM NaSCN was added to five secreting mucosae was 3.7 ,uequiv./cm °/hr. (S.E. ' 0.S3), while the average decrease in oxygen consumption was 0.5 umoles./ cm.'/'hr. (± 0.69) giving a ratio for iH`/ DO, of 7.4. Since the effect of thiocyanate is readily reversible, the above mucosae were washed and the changes in H.secretion (2.4 pequiv./cm.'/hr.±o.41) and oxygen consumption (0.4 pmoles./ cm.'/hr. 0.13) give a value of 6.o for the ratio of AH'/AO_. When an electric current opposing the natural membrane-potential was passed through the mucosa, there was a drop in the rate of acid secretion with a concomitant fall in oxygen consumption

(except in one experiment where hydrogen ion secretion was inhibited with no measurable effect on the respiration of the tissue). This effect is readily reversible, and the average ratio for the AH'/DO_ of 14.4 was obtained from experiments on three mucosae where measurements were made before, during, and after current flow. These results indicate that the real ratio of oxygen directly associated with acid production can apparently be greater than 4.o. It must be pointed out that in these experiments the thermodynamic limit based on the large concentration of hydrogen ions across the intact secreting stomach does not hold. There is only a small H. gradient (8o to 1), the pH of the secretory solution being maintained at 5.5 by the pH-stat method. We have run a series of experiments on five different mucosae where the pH of the secretory solution was varied so that large gradients of H.ions could be maintained across the mucosa. Oxygen consumption, short-circuit current, and hydrogen ion secretion were measured throughout the experiments. The pH of the secretory solution was lowered from either 7.0 or 5.5 down to 3.o or 2.5 and elevated again at least two times during any run. For every mucosa the ratio of hydrogen ion secretion to the oxygen consumed was less (about 15 per cent) at the lower pH value where the gradient of hydrogen ions across the mucosa was between Io,000-Ioo,000 fold. Thus these experiments afford a better estimation of the stoichiometric relationship between hydrochloric acid production and associated metabolism. Clearly, a simple redox hypothesis, with molecular oxygen as the only final acceptor of electrons, cannot be the only operating mechanism to account for the movement of hydrogen and chloride ions across the gastric mucosa. II

I / SECRETION OF ACID BY THE STOMACH

Proposed mechanisms to account for the high values of the observed stoichiometric efficiencies The simple Redox Pump hypothesis has been shown to be inadequate to explain secretion of hydrochloric acid by gastric mucosa, for sodium transport by frog skin (79,80), for sodium transport by toad bladder (81), and for sodium transport in the kidney (82). Davies and Ogston (52) proposed their Mechanism II in order to explain ratios greater than 4.0 for the secretion of acid by the stomach, and for ion transport in other systems. This mechanism employs high-energy phosphate compounds, and could yie.d ratios up to 12.o when coupled in parallel with the redox mechanism. In 1957, Davies (83) presented a scheme in which all of the energy comes from the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The power comes from the breakdown of the phosphate compound rather than an electron (redox) pump, and the mechanism can transport a great number of ions for each ATP molecule against small electrochemical gradients. It would be unlimited by stoichiochemical considerations applying to previous hypotheses, and it, or some similar scheme, must be invoked since it has been shown in several transport systems that apparently more than 12 ions can be moved with the utilization of one molecule of oxygen, or two ions per ATP broken down (see Prague Symposium (84)). Such an analysis could account for the high AHCI/DO_ values obtained when an electric current inhibits acid production. (However, Hokin (84) has pointed out that the active transport of sodium in frog skin could be accounted for by a process involving phosphatidic acid with a ratio of two sodium ions per ATP if the basal respiration is utilized). I2

Specificity of the chloride transporting mechanism The transport of hydrogen ions by the oxyntic cells is a very specific mechanism. No other cation can be transported in its place. However, chloride ion transport, the source of the mucosal potential, might well be termed anion transport in that many other anions will substitute for chloride to some degree. As far back as 1887, Kiilz (85) observed that other halogens injected intravenously could replace some of the chloride in the gastric juice. Davenport and Fisher (86) injected bromide into the plasma of dogs, and showed that the concentration of bromide in the gastric juice was always greater than in the plasma. In 1 943, Davenport (87) found that the concentration of iodide in the gastric juice was dependent upon the iodide concentration in the plasma, but was not dependent upon the rate of secretion. However, Mason and Bloch (88) found that the accumulation ratio (amount of substance in gastric juice/amount of substance in plasma) for iodide was markedly influenced by the rate of acid secretion. Using radioactive P", Schiff et al. (89) found that the radioisotope could be one to seventy times more concentrated in basal gastric juice than in plasma. Lagergreen (90), using a Heidenhain pouch dog, observed that as the flow of acid gastric juice increased, the iodide accumulation ratio decreased. Independently, Gabrieli (91) showed that the proportion of iodide, bromide, and thiocyanate in the gastric juice decreased with increasing rates of secretion as induced by histamine. Both of these authors concluded that these anions replace chloride, but only the neutral chloride of the gastric juice. Friedman et al. (92) noted that iodide secretion was more related to mucous output than to "parietal secretion"

DAVIES & FORTE

in dogs with pouches in various areas of sed the potential and diminished acid the stomach. secretion. These findings by Kitahara Heinz, Öbrink and Ulfendahl (93) could not be duplicated in our laborastudied the accumulation ratios of bro- tory. mide and chloride when bromide was inWhen they replaced some of the crementally added to the plasma. The chloride in the bathing solution with observed differences in the accumulation other anions, Hogben and Green (99) ratios were ascribed to the fact that the found that the flux of bromide across relative output of bromide was constant- the gastric mucosa in both directions ly 1.55 times that of chloride. The au- was approximately the same as the fluxes thors claimed that their results lent sup- of chloride, whereas for iodide the nuport to the concept that halogens do not trient to secretory flux was 35 per cent, pass the mucosal membrane by a simple and the secretory to nutrient iodide flux diffusion process, but rather in some was 45 per cent of the respective chlorcombined state. (It is of interest that ide fluxes. Consistent with Hogben and Hogben (94) demonstrated that the net Green, we have found that when all of transport of chloride ions was greater the chloride was removed from the than the total conductance of the tissue bathing solution, bromide Ringers (see when put into similar units. Since a par- Table I.) did not bring about any major ticle which traverses the membrane in changes in acid production or in the the ionized state must contribute to the electrical properties of the mucosa. Iodielectrical conductance, Hogben conclud- de Ringers caused a slight change in the ed that at least some chloride was trans- transmucosal potential (14 per cent deported in a bound state and hence this crease), but the acid secretion was inhibited by 62 per cent. was evidence for a carrier.) It is interesting that acid secretion in Nitrate was found in gastric secretions of dogs who had up to 7o per cent of the nitrate Ringers was only about one tenth total body chloride replaced by nitrate. of the amount observed in chloride RingThe electrical behavior of the gastric ers, yet the short-circuit current, transmucosa in the presence of various anions mucosal potential difference, and mucosal has been extensively studied. Mond (96) resistance were maintained at approxifound no significant changes in the po- mately the control values. This would tential difference across a frog stomach indicate that the mechanism in the gastric whether the lumen was perfused with mucosa which generates the anion cursodium chloride, sodium sulfate or so- rent does not distinguish between chlordium nitrate. Similarly, Crane and Davies ide and nitrate, and is in agreement with (97) observed that isosmotic sodium ni- an unpub.ished observation by Hogben trate (and various other ionic solutions) (loo). In sulfate Ringers, the mucosa behaved on the secretory side of an isolated frog stomach did not appreciably affect the quite differently than in chloride Ringpotential or resistance of the mucosa. ers, in that the potential and the current Kitahara (98) reported that sodium ni- were reversed. Heinz and Durbin (1 o i ) trate on the secretory side of the mucosa demonstrated that mucosae bathed in caused the potential to increase along sulfate Ringers would respond to histawith an increase in hydrogen ion secre- mine stimulation by secreting acid, the tion, whereas nitrate on the nutrient side, maximum rates being some two-thirds as as well as on the secretory side, depres- great as that produced in chloride Ring£3

I / SECRETION OF ACID BY THE STOMACH

ers. They also reported a short-circuit flux in one direction or the other (I03). current in sulfate Ringers which was op- (Recently, Hogben (104) has claimed posite in direction to the current flow in that there is a small net transport of sulnormal Ringers, and which was directly fate of the order of 0.15 uequiv./cm=./hr. proportional to the rate of hydrogen ion from the nutrient to the secretory sursecretion. Recent experiments by us, and face.) Since in these experiments acid independently by Rehm and his col- secretion can continue at rates up to 1.3 leagues (Ioz), are in complete agreement uequiv./cm-./hr., it woud be interesting with these results. Various effects were to know what anion accompanies the H. produced when other anions were sub- The relatively low permeability of the stituted for chloride in the Ringers. mucosa to sulfate may account for its inTable I. gives a summary of the results of ability to enter the anion transport bathing bullfrog gastric mucosae in Ring- mechanism. ers solution where the chloride ion was It can be concluded that many anions replaced by the indicated anion. When can be transported across the mucosa by thiocyanate completely replaced chlor- substitution in the chloride transport ide, acid production was abolished al- mechanism. When this occurs, even in though the transmucosal potential differ- the absence of chloride in the bathing ence and short-circuit current were not solution, the electrical behavior of the inhibited by more than 5o per cent of mucosa varies only slightly from that obthat observed in chloride Ringers. served in the presence of normal Ringers. Experiments in this laboratory indi- However, certain anions cannot substicated that sulfate moves at a rate of o.3 to tute for chloride, and the potential dif0.4 pequiv./cm2./hr. across both sides of ference falls to zero. the gastric mucosa with no consistent net

TABLE I. Acid secretion and transmucosal potential difference of gastric mucosae when chloride was replaced by various anions (3-5 washes during approximately one hour) Chloride corNelely replaced by

No. of experiments

Average % change in acid secretion

Range of transmucosal P.D.* —25, —27mv

Bromide

(2)

T

2%

Iodide

(I)

1

63%

—25mv

Sulfate

(10)

70%

—2 to +19mv

Nitrate

(8)

j 84%

—25 to —36mv

Thiosulfate

(3)

1

—4 to —27mv

Thiocyanate

(3)

j 100%

—13 to —23mv

Nitrate

(1)

1100%

Omv

*Range of P.D. in chloride Ringers —28 to —45mv. 14

94%

DAVIES & FORTE

Factors Affecting Variations in Gastric Acidity Primary acid secretion Many factors operate in the control of gastric secretion: the central nervous system; local neural responses; hormonal influences; metabolic factors; etc. All of these will moderate to some degree the secretion of acid by the stomach. However, the basic question still remains: in what form is gastric acid elaborated? In seeking the answer to this question, the analysis of "gastric" contents should be made solely on secretions originating in the stomach, and this should not be contaminated with saliva or intestinal secretions. The gastric pouch (both innervated and denervated) has been a useful device for the study of gastric secretion in vivo. Likewise, the in situ stomach flap has been a useful preparation (61,62, 63). Most mammalian stomachs are too thick, and cannot be isolated from their blood supply and still remain active for any length of time, since the oxygen cannot diffuse to areas deep within the respiring tissue. Amphibian stomachs can be dissected so that the outer muscle coat is removed, leaving the gastric mucosa intact. Such a preparation is thin enough to be properly oxygenated, and has been useful for many types of experiments. Even after isolating the system so that one obtains only secretions originating from the gastric glands, "pure gastric juice" consists of a mixture from at least three different secretory processes. Thus, the study of acid secretion as it originates from the gastric glands is complicated by all of these factors. This complication is echoed in the controversies that have existed on the subject since the turn of the century, when Pavlov and Rosemann put forth their respective views. Pavlov (I 05) believed that the secretion of the acid-producing cells was of a constant composition, and that this could be alter-

ed by dilution with other secretions. The view held by Rosemann (io6) was that oxyntic cells could secrete acid at any concentration, a conclusion which was based largely upon the fact that the chloride concentration showed very little fluctuation in experiments with any given animal, although there were marked variations in the IT content. The points of difference that arise from these views center around the composition of the acid as it is secreted by the oxyntic cells. Does pure primary oxyntic cell secretion have a constant acidity, or can it be secreted at any concentration depending upon the stimulation? More recently, Teorell (107) put forward a theory that hydrogen ions that have been secreted can exchange for the sodium ions of the blood. Teorell's viewpoint was supported by his experiments showing that hydrogen ions diffused out of a cat's stomach at a rate directly proportional to the concentration of hydrogen ions (Io8). He proposed a model (109) which would account for the high concentration of hydrogen ions in the stomach as a result of rapid rates of secretion and the low concentration of hydrogen ions (high Na concentration) at slow rates of secretion. Terner (II o), using the isolated gastric mucosa of a frog, confirmed that hydrochloric acid could diffuse out of the stomach passively, and that this diffusion was linearly related to the hydrogen-ion concentration of the gastric contents. In an effort to determine what the primary concentration of hydrogen ions was, Teorell devised an experiment where the secretory surface was bathed by an isosmotic glycine buffer so as to prevent a large gradient of hydrogen ions developing across the mucosa (III ). From the change in volume of the secretory solution and the increase in acidity, he could 15

I / SECRETION OF ACID BY THE STOMACH

calculate what he proposed was the pri- chloric acid was forced through a colmary acidity of gastric acid secretion. lodion membrane into a protein solution An extension of the technique by Linde, than the corresponding fluid volume. This Teorell and Obrink (11 z) found that the effect increased with decreasing rates of primary acidity was related to the secre- flow through the membrane. He sugtion rate. For cats at high secretory rates, gested a mechanism for acid secretion the primary acidity was found to be whereby the primary hydrogen ion secreconstant and isotonic (approximately 169 tion was diluted and buffered by the mN), whereas at low rates of secretion glycine, resulting in a concentration the primary acidity was found to in- gradient of hydrogen ions. He reasoned crease (highest value obtained was 35o that at very low secretory rates the diffumN). sion of hydrogen ions was sufficiently Conway (113) pointed out that the faster than the flux of water to result in glycine buffer used by Linde et al. may the observed, apparent high acidities. have been the cause of the high values of Heinz further presented some calculaprimary acidity. That is, when the gly- tions which showed that glycine could cine molecule could buffer the hydrogen diffuse to the depths of the gastric pits ion at the site of secretion, i.e. at low even at maximum volume outputs by the secretory rates, the osmotic effect of the stomach. Heinz and Obrink claimed hydrogen ion would be lost, and thus, (116) that this latter point invalidated the only half of the normal water volume explanation offered by Conway that the would be drawn into the lumen. He con- number of osmotically active particles cluded that the value obtained by Linde was reduced at low secretory rates. Howet al. at low secretory rates (35o mN) ever, Heinz's calculations only included was exactly the concentration one would the depth of the pits, which is only a part expect if all of the hydrogen ions were of the entire gastric gland. The gastric buffered at the site of secretion, and tubule extends two to three times deeper hence the data support the view that than the gastric pit, and it is from the oxyntic cells lining the tubule that the secretion is isosmotic. Dayson (114) has proposed that the acid emanates. Also to be considered are results of Teorell, and Linde et al. might the intracellular canaliculi of the parietal be explained on the basis that the work cells which would present a finite distance (secretion gradient of hydrogen ions) for the diffusion of glycine buffer, and might be less in the presence of a buffer. it may well be that this membrane (at the The slower the rate of secretion, the wall of the canaliculus) is the site of most more effectively the acid-secreting rapid osmotic equilibration. Thus, the mechanism could build up the maximal explanation by Conway, that the glycine concentration gradient, hence the greater can diffuse to the effective site of secrethe hydrogen-ion concentration in the tion at low rates of secretion, and there cause a reduction in the osmotically active secretory solution. Heinz (11 S) experimentally verified on particles resulting in a more concencats the values of high acid concentration trated acid secretion, remains plausible at low secretory rates found by Linde et for this system. Hirschowitz (11 7) has recently preal. He showed that water absorption from the stomach could not account for the in- sented a theory on gastric secretion which creasing primary acidity observed. In a consists of having the peptic cells deep model system he found that more hydro- in the gastric glands, secreting chloride 16

DAVIES & FORTE

followed by sodium and potassium to maintain electroneutrality. In the region of the parietal cells he proposes an exchange of Na` in the tubule for H' from the parietal cells—this phenomenon being the determinant of the acidity of gastric juice. (It is to be noted that amphibians do not have the different cell types required by this theory, and any mechanism of acid secretion should apply to these species as well as to the higher forms.) In one of his studies, Hirschowitz plots the "gastric sodium clearance" (V . Og . Nan) against the total acid plus Op sodium secreted V(Nag + H). Where V is volume of gastric juice, On is osmolarity of gastric juice, On is osmolarity of plasma, Na, and Nag are sodium concentrations of plasma and gastric juice respectively, and H is hydrogen-ion concentration in gastric juice. He reasoned that since the correlation of the line was highly significant (correlation coefficient, r = + .9987) with a slope of regression of about 1.0, a hydrogen for sodium ion exchange on a 1 : I basis occurred. Hirschowitz claimed that he could measure the extent of Na-H exchange by the formulation H`/(H` + Nat) (the value being 1.00 if exchange were complete, and zero in a completely anacid stomach). If the H`/(H` + Na') ratio could be varied with no effect on the chloride, one could test the degree of exchange of sodium for hydrogen. In a separate group of studies, Hirschowitz found that acetazolamide decreased the value of the H`/(H` + Na') ratio, while the concentration of chloride in the gastric juice remained constant. However, such a result might have been observed with any inhibitor of gastric secretion. A more crucial observation would have included the volume output, and if this value had remained constant while the H`/(H' + Na') ratio decreased then the suggestion

for a sodium-hydrogen exchange would be more likely. It should also be pointed out that Hogben (I oo) has presented evidence that acetazolamide will inhibit chloride transport in isolated frog gastric mucosa. According to the theory of Hirschowitz, if the basal secretion consisted of chloride accompanied by sodium and potassium with a cation exchange mechanism (sodium-hydrogen) accounting for the increase in acidity, then one would expect that at very high rates of flow of gastric juice the hydrogen-ion concentration would fall because it would not be able to be presented to the sites of exchange. Another point would be that when chloride was replaced with anions which fit into the anion transport mechanism of the gastric mucosa (e.g. bromide, iodide, nitrate, thiocyanate, etc.), there should be no effect on the rates of acid secretion. That is, if the anion can be secreted at sufficient rates (as evidenced from electrical measurements of potential difference, short-circuit current, etc.) accompanied by sodium and potassium to maintain electroneutrality, then one should obtain copious secretions of the acid or sodium salt of the anion, dependent upon whether there was stipulated an additional adverse effect on the sodium-hydrogen exchange mechanism. In no instance does the data presented by Hirschowitz invalidate or distinguish either a two-component secretion mechanism or an exchange mechanism where primary hydrogen secretion exchanges for sodium. It can be concluded that there has been no substantial evidence that the basal or primary parietal secretion can be anything less than isosmotic. The high values obtained by Teorell, Linde et al. (112) and Heinz (115) at low secretory rates can be explained on the basis of the technique employed by these workers. 17

I / SECRETION OF ACID BY THE STOMACH

The final composition of gastric secretion

and would fit most of the experimental data; however, complete verification on the mechanism of acidity changes must await further evidence.

There are two possible methods by which the collected gastric juice attains its composition: a) by dilution with a nonacid, nonparietal solution, the view Potassium content of gastric secretion originally formulated by Pavlov and expanded by Hollander; b) that the acid The source of potassium ions in the can disappear by diffusion in exchange gastric juice has remained an enigma, comfor other cations, the hypothesis advanced plicated by the massive amounts of data by Teorell. The literature on this subject and copious flow of theories as to its is voluminous and has been reviewed by origin. Much of the literature has been Conway (58), Heinz and Obrink (1 16), reviewed by Conway (113), Heinz and and Jåmes (118). Lifson, Varco and Obrink (116) and James (118). The most Visscher (119) have shown that gastric recent review of the problem by Holsamples from. Pavlov pouch dogs show a lander (12o) has been very illuminating. variation in osmolarity dependent upon He points out why some of the older data the rates of acid secretion. At high acidi- have been misleading, and presents rather ties (140-160 mN) and at zero acidity, convincing evidence that the potassium the samples were either isosmotic with content of the gastric juice is not related plasma or slightly hypertonic. However, to the acidity, the volume-rate of secreat intermediate acidities most of their tion, or the sodium content. Colcher and samples were hypo-osmolar with respect Hollander have shown very clearly in to the plasma of the dog. Proponents of Heidenhain pouch dogs that the potasboth the two-component hypothesis and sium is related to the stimulus for secreof the diffusion hypothesis have claimed tion. That is, stimuli such as histamine that these results are in complete agree- will cause the potassium concentration in ment with their respective theories and the gastric juice to rise and then fall. The both have derived formal proofs in favor fall occurs even with sustaining doses of of their views. the drug. However, the increase in potasMost authors have agreed that the sium concentration can be provoked again sodium content of the gastric juice varies with another injection of histamine (the inversely with the acidity although the second injection must be larger than the argument persists as to the precise means first if sustaining doses were used). These by which the final "mixture" is achieved. effects were demonstrated as well in the On the basis of the experimental evidence human (121,122). alone, one finds it difficult to accept one The general conclusion by Hollander theory to the complete exclusion of the was that the primary parietal secretion other. Diffusion of acid out of the was one of pure hydrochloric acid, the stomach has been definitely shown to sodium coming from extraparietal secreoccur (io8,i 1o), while non-parietal com- tion (and/or exchange of hydrogen with ponents of secretion most certainly exist. sodium), while the potassium comes from There are, however, several observations a passive efflux as a result of the stimulus. in the literature which are difficult to He presented and discussed five possiinterpret on the basis of either of these bilities as to the source of the potassium theories (i18). A more accurate theory efflux. Notable amongst these is a) the would be intermediate between the two possibility that potassium is actively trans18

DAVIES & FORTE

ported into the stomach, and b) the possibility that potassium is naturally released (passively) when tissues, such as the gastric mucosa, are under the influence of histamine. Hollander dismissed the former as being obscured by the "melange of findings", while he introduced a host of references citing evidence for the release of potassium by many tissues, induced by various stimuli. As to the mechanism of potassium release, one can still not truly distinguish whether this process be active or passive, from the existing data. The membrane might simply become leaky to potassium with the proper stimulus (potassium efflux from muscle and nerve upon stimulation). Alternatively the microvesicles (see above), seen in electron micrographs, which apparently migrate to the secretory surface after histamine,

might contain potassium (the major intracellular cation), and the stimu.us causes them to deposit their contents at the secretory surface. Once deposited, the potassium efflux into the gastric juice would decline, yet hydrogen ion secretion might continue with proper reinforcement of the stimulus. Only a larger stimulus could effect an additional migration of microvesicles with a concomitant potassium release. Regardless of the mechanism of potassium release, it appears that the composition of pure parietal secretion is pure hydrochloric acid. Sodium gains entry into the gastric juice via other secretions and by a direct exchange for hydrogen ions. Potassium is apparently an artifact of the stimulus and is transiently released at that time.

Summary The structure of the oxyntic cells within the gastric trrucosa has been discussed with special reference to the morphological link to hydrochloric acid production. The site of acid secretion appears to be the pericanalicular zone around the intracellular canaliculi of the oxyntic cells. For every mole of hydrogen ions secreted by the stomach, there is an uptake of an equal amount of carbon dioxide with an output of bicarbonate ions to the blood or nutrient side. In actively acidsecreting stomachs, inadequate supplies of carbon dioxide result in tissue damage in the form of ulceration of the nrucosa, probably because of an accumulation of hydroxyl ions within the cell. Carbonic anhydrase is important in the over-all process of acid production. The literature concerning the stoichiometric efficiency of hydrochloric acid

production has been reviewed. Evidence is presented which shows that a simple redox hypothesis of active transport with oxygen as the sole electron acceptor cannot be the only operating mechanism for the active transport of chloride across the gastric mucosa. When the ratio is measured for the change in the rate of hydrogen ion production (AQHCI) to the change in the rate of oxygen consumption (AQO_) for periods when the mucosa is inhibited by sodium thiocyanate or by electric currents opposing the natural membrane-potential, or when these inhibitions are reversed, values well above 4.0 are obtained. If the "resting respiration" cannot be channeled into secretory work, these values would also invalidate a simple redox mechanism for hydrogen-ion production. Many anions can substitute for chloride in the active chloride transport system of 19

I / SECRETION OF ACID BY THE STOMACH

the gastric mucosa. Secretion of acid is largely independent of the mucosal potential which results when some of these anions completely replace chloride in the bathing solution. Some anions apparently cannot substitute for chloride, yet the secretion of acid continues. The primary oxyntic cell secretion is most probably isosmotic with the blood and contains only hydrogen and chloride ions. Dilution with other secretions and a secondary back diffusion of hydrogen ions (in exchange for sodium and potas-

sium ions of the blood) can occur, whilst transient increases in the concentration of potassium in the gastric juice can follow the stinmlation of acid secretion by histamine. ACKNOWLEDGEMENT

Figure I is reproduced from the Mechanism of Hydrochloric Acid Production by the Stomach by R. E. Davies, unpublished Ph.D. Thesis, University of Sheffield, 1948.

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chem. J., 42: 609-621, Appendix 618-621, 1948. 34. DAVENPORT, H. W. and JENSEN, V. Gastroenterology, 1r: 22 7-2 39, 1 948. 35. DAVIES, R. E. and EDELMAN, J. Biochem. J., SO: 1 90-1 94, 1951. 36. JANOWITZ, H. D. COLCHER, H. and HOLLANDER, F. Amer. J. Physiol., 171: 325-330, 1952.

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37. GRAY, J. S. Gastroenterology, I: 390-400, 1 943. 38. CRANE, E. E., DAVIES, It. E. and LONGMUIR, N. M. Biochem. J., 4o: xxxvi-xxxvii, 1946. i9. REHM, W. S. Amer. J. Physiol., 147: 69-77,

1946. 40. DAVENPORT, H. W. Fed. Proc. 6: 94, 1947. 41. DAVENPORT, H. W. Gastroenterology, 9: 293-3o2, 1 947. 42. DAVENPORT, H. W. and CHAVRE, V. J. Gastro-

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68. ussING, H. Metabolic Aspects of Transport Across Cell Membranes, Murphy, Q. R., ed., 39-56. Madison; University of Wisconsin Press, 1957. 69. USSING, H. H. and ZERAHN, K. Acta Physiol. Scand., 23: 110-127, 1951. 70. HOGBEN, C. A. M. Proc. Nat. Acad. Sei., U.S.A., 37: 393-395, 1951. 71. HOGBEN, C. A. M. Proc. Nat. Acad. Sei., U.S.A., 38: 13-18, 1952. 72. GRAY, J. S. Fed. Proc., 1: 255-260, 1942. 73. DENNIS, NV. H., BORNSTEIN, A. M., WHITE, T. D. and REHM, W. s. Abstract, Amer. J. Physiol., 183: 6o8, 1 955. 74. FORTE, J. G., NAUSS, A., WINTERS, R. W. and DAVIES, R. E. Science, 132: 1491-1492, 1960.

75. FORTE, J. G. Ion Transport by Frog Gastric Mucosa, Ph.D. Thesis, University of Pennsylvania, 1961. 76. VILLEGAS, L. and DURBIN, R. Biochim. Biophys. Acta, 44: 612-613, 1960. 77. VILL ARREAL R. Proc. Soc. Exp. Biol. Med., 83: 817-819, 1953. 78. REHM, W. S. Abstracts: Fifth Annual Meeting of the Biophysical Society, St. Louis, 1961. 79. ZERAHN, K. Acta. Physiol. Scand., 36: 300318, 1956. 80. LEAF, A. and RENSHAW, A. Biochem. J., 65: 82-90, 1957. 81. LEAF, A. PAGE, L. B. and ANDERSON, J. J. Biol. Chem., 234: 1625-1629, 1959. 82. DAVIES, R. E. Metabolic Aspects of Transport across Cell Membranes, Murphy, Q. R., ed. 246, Madison; University of Wisconsin Press, 1 957. 83. DAVIES, R. E. Metabolic Aspects of Transport across Cell Membranes, Murphy, Q. R., ed. 244-250, Madison, University of Wisconsin Press, 1957. 84. HOKIN, A. Symposium on Membrane Transport an Metabolism; Kleinzeller, A. and Kotyk, A., ed. 3o5-34o, New York, Academic Press, 1962. 85. KüLZ, E. Z. Biol., .23: 460-474, 1887. 86. DAVENPORT, II. W. and FISHER, R. B. Amer. J. Physiol., 131: 165-175, 1940. 87. DAVENPORT, H. W. Gastroenterology, 1: 1055-1061, 1943. 88. MASON, E. E. and BLOCH, H. S. Proc. Soc. Exp. Biol. Med., 73: 488-491, 1950. 89. SCHIFF, L., STEVENS, C. D., MOLLE, W. E., STEINBERG, H. and KUMPE, C. W. J. Nat.

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94. HOGBEN, C. A. M. Amer. J. Physiol., do: 641-649, 1955• 95. HIATT, E. P. Amer. J. Physiol., 129: 597609, 1940. 96. MOND, R. Arch., f.d. ges. Physiol., 215: 468480, 1927. 97. CRANE, E. E. and DAVIES, R. E. Trans. Faraday Society, 46: 598-610, 1950. 98. KITAHARA, S. Kumamoto Med. J., 12: 121132, 1 959. 99. HOGBEN, C. A. M. and GREEN, N. D. Abstract. Fed. Proc., 17: 72, 1958. 100. HOGBEN, C. A. M. Electrolytes in Biological Systems, Shanes, A. M., ed. 176-204, Wash., Amer. Physiol. Society, 1955. . 101. HEINZ, E. and DURBIN, R. Biochem. Biophys. Acta. 31: 246-2 47, 1959. 102. DAVIS, T. L., CHANDLER, C. and REHM, W. S. The Physiologist, 4: 28, (Abstract), 1961. 103. NEVILLE, M. Unpublished Observations, 1960. 104. HOGGEN, C. A. M. Abstract, Fed. Proc. 20: 1;9, 1961. 105. PAVLOV, I. P. Die Arbeit der Verdauunsderüsen Wiesbaden, Bergmann, 1898. 106. ROSEMANN, R. Arch. f.d. ges. Physiol., 118: 467-524, 1907• 107. TEORELL, T. Skandinav. Arch f. Physiol., 66: 22 5-317, 1933.

22

108. TEORELL, T. J. Gen. Physiol., 23: 263-274, 1 939. 109. TEORELL, T. Gastroenterology, 9: 425-443, 1947• 110. TERNER, T. Biochem. J., 45: 150-158, 1949. III. TEORELL, T. J. Physiol., (Lond.) 308-315, 1940. 112. LINDE, S., TEORELL, T. and OBRINK, K. J. Acta Physiol. Scand. 14: 220-232, 1947. 113. CONWAY, E. J. Biochemistry of Gastric Acid Secretion. Springfield, Thomas, 1 953. 114. DAVSON, H. A Textbook of General Physiology, Boston, Little, Brown, 1959. 115. HEINZ, E. Biochim. Biophys. Acta., 6: 434444, 1951. 116. HEINZ, E. and OBRINK, K. J. Physiol. Rev., 34: 643-673, 1954. 117. HIRSCHOWITZ, B. I. Amer. J. Dig. Dis., 6: 199-228, 1961. 118. JAMES, A. H. The Physiology of Gastric Digestion. London, Arnold, 1957. 119. LIFSON, N., VARCO, R. L. and VISSCHER, M. B. Gastroenterology, 1: 784-802, 1943. 120. HOLLANDER, F. Gastroenterology, 40: 477490, 1961. Ill. BERNSTEIN, R. E. J. Lab. Clin. Med., 4o: 707717, 1952. 122. WERTHER, J. L., PARKER, J. G. and HOLLANDER, F. Gastroenterology, 38: 368-373, 1960.

The Physiology of Pepsinogen*

WHILE it had been long known that the gastric juices owed their power to digest to a ferment (r ), it was not until 1836 that Schwann discovered pepsin (2) and a half century later that Langley and his students (3,4) published their classical descriptions of pepsinogen and the peptic cell, all of it just as valid as now. In recent years, a great deal of interest has revived in the secretion and the pathophysiologic significance of pepsin and its precursor pepsinogen. This interest has centered mainly on the diagnostic usefulness of measurement of pepsinogen in urine and blood, reminiscent of the numerous similar studies at the turn of

Basil I. Hirschowitz**

the century (1,5). Almost as a by-product of this revival, interesting advances have been made in the physiology of pepsinogen — the nature of the gastric enzymes, the factors effecting secretion and the physiologic significance of changes in blood and urine pepsinogen. Independently in the last twenty-five years important advances have been recorded in the chemistry of pepsin, pepsinogen and the nature of proteolysis, which is still obscure. This chapter will attempt a cohesive look at these many aspects of the subject, emphasizing more the physiology than its application to natural or experimental peptic ulcer.

Chemistry of Pepsinogen Pepsinogen is the precursor and inactive form of the proteolytic enzyme pepsin. It is in this form that it exists in the peptic cell, and is found in the blood and other body fluids and in the urine where it is known as uropepsin (6). Since pepsinogen has no measurable proteolytic or other catalytic activity, it has to be converted to pepsin for assay.

Pepsinogen is a protein with a molecular weight of 42,50o which has been crystallized from gastric mucosa (7). It is relatively resistant to denaturation, showing only a partially reversible denaturation at temperatures over 70°C, and it is not destroyed even by boiling in salt-free solutions. It is reversibly denaturated at pH over 9.o and irreversibly

*Supported by a grant from the U.S. Public Health (uspits, c-498o, 2A-5286). "From the Division of Gastroenterology, College of Medicine, University of Alabama, Birmingham, U.S.A. 23

I / THE PHYSIOLOGY OF PEPSINOGEN

denaturated at pH over 12.o. These and other properties distinguish it from pepsin which is irreversibly denaturated at pH over 7.o and at temperatures over 7o°C (8, p. 8o). Conversion of pepsinogen to pepsin occurs in solutions more acid than pH 6.o, by an autocatalytic process. At pH 6.o conversion is extremely slow with the formation of a pepsin-inhibitor complex which is dissociated at pH < 5.4 to give pepsin and an inhibitor which is the terminal part of the original pepsinogen molecule (9) and has been crystallized by Herriot (Io), having a molecular weight of about 3,10o (9). The inhibitor is digested rapidly by pepsin at pH 3.54.0 (8, p. 90), and plays no further part in the subsequent proteolysis by pepsin. At pH z.o the conversion of pepsinogen to pepsin is very rapid and complete. Pepsin catalyzes this conversion and this action is interchangeable between species, e.g. chicken and swine pepsin will each activate pepsinogen from the other (1I). Pepsin was first crystallized from bovine gastric juice by Northrop in 1933 (12). It was found to be a protein of molecular weight 35,50o and a molecular volume of about 8o,000 (13), suggesting that the molecule is either non-spherical or hydrated. Crystalline pepsin has been found to contain at least two proteins, identified by their differing solubilities, the more soluble having twice the specific activity per mg protein than the less soluble one (14), but they appear indistinguishable by ultracentrifugation or by electrophoresis. Loss of activity in weak alkali is exactly equal to the rate of formation of denaturated protein (8, p. 55). In acid solutions a gradual loss of activity occurs, which is appreciable below pH 1.o, due to autodigestion of pepsin (15). Autodigestion of pepsin in a dialysis system at pH 4.o-5.6 has been reported to 24

produce proteolytically active dialyzable fragments of low molecular weight (16, 17,18). These studies, if correct, suggest that pepsin as described is not a pure single protein or that the active proteolytic moiety can be separated from a skeleton. The amino acid composition of pepsinogen, pepsin and the pepsin inhibitor produced in the conversion have been described by Van Vunakis and Herriott (9,19) and others (20,21). By the dinitro fluorobenzene method the N-terminal amino acids and second amino acids have been determined as follows: SECOND AMINO N-TERMINAL ACID PEPSINOGEN

PEPSIN

isoleucine/ leucine isoleucine Glycine (19, 2o) (-Asp-Asp-

leucine

His-Glu-) (z I )

PEPSIN INHIBITOR

leucine

Glutamic acid

Principles of Peptic Assay Pepsin acts on a wide variety of substrates (Table I) and at varying pH indicating its relative non-specificity, which one might expect from a digestive enzyme. The choice of substrates has led to the use of a variety of assay methods and even a variety of units within each method, so that much of the reported material cannot be compared. Our main concern in the application of these techniques to the choice of a method is that it shall be sensitive, reproducible, as specific as possible, relatively rapid and simple, applicable to biological fluids (gastric juice or mucosal extracts, blood and urine), and have some solid basis in the kinetics of hydrolytic enzymes (22). In order of speed, the changes in a protein solution resulting from peptic ac-

H IRSCHOWITZ

TABLE I. An outline of methods used for measuring proteolytic enzyme activity Substrate

Basis of Method

Quantity Measured

Technique

Protein

Change in physical properties of Substrate

Viscosity Optical rotation Volume Conductivity Clot formation Loss of solid substrate Turbidity Loss of protein N2 Tyrosine and Tryptophan

Visometer Polarimeter Dilatometer Conductivity bridge Time measurement Mett's tube Weight of dry residue Nephelometry RNA—age, etc. UV absorption (280µ)*

Optical rotation Refractive index

Folin Ciocalteu reagent* Polarimeter Refractometer

Liberation of colored peptides or absorbed dye Liberation of bound radioactivity

Color intensity

Colorimeter

Bonds Broken

—COO —

Disappearance of Substrate Appearance of peptides

Modified Proteins

Proteins, Peptides or Amides

RISA*

— NH3+ — NH3+ — NH3+ + CO2 — NH3 — NH3 — NH2

Acetone titration Alcohol titration Formal titration Dilatometer Ninhydrin colometric Gasometric HNO2 Gasometric ninhydrin

Synthetic Peptides Amino acids produced e.g. CO2 from tyrosine Manometry with and many of the above decarboxylase methods *Methods of Choice

tivity, and which are relatively specific (mainly centered around the acid pH optimum) are: altered viscosity, loss of protein nitrogen, appearance of peptides and slowest of all, the increase in titratable amino or carboxyl groups. Changes in optical rotation, volume, conductivity, and refractive index are less specific, and are neither recommended, nor generally used in biological studies (23). The actions of pepsin (and presumably other proteolytic enzymes) probably should be divided into a) SUBSTRATE DENATURATION, occurring as the principle change at pH above the proteolytic range 1.5-3.5 and

which is probably the basis for changes in viscosity, optical rotation, volume, conductivity, and refractive index and b) SUBSTRATE PROTEOLYSIS measured by loss of substrate, broken bonds or end products with incubation of substrate in the "proteolytic range" of pH 1.5-3.5. Substrate proteolysis seems more specific, and with our present state of techniques, easier to measure and more quantitative. Pepsin is one of the few enzymes which apparently requires no cofactors (unless it be hydrogen ions), and there are no known accelerators of peptic action except prior denaturation of substrate. 25

I / THE PHYSIOLOGY OF PEPSINOGEN

Viscosity Changes While pepsin does liquefy gelatin, and viscosity can be measured accurately to within 2-3 per cent, the presence of other enzymes in gastric juice and mucosa which also liquefy gelatin and at rates far greater than pepsin, e.g. gelatinase (24), parapepsin I and II (25,26), make this method unsuitable for measurement of peptic activity in biologic fluids. Other substrates used are edestin and casein (8). The difficulty of preparing reproducible substrates and the high optimum pH (5.4) are further disadvantages. Clot Formation The commonly used substrates for clotting measurements are casein and milk (27,28,29,30,31), and the unit is derived from linearity of log time vs log concentration of enzyme. Inequalities in substrate batches may be eliminated by reference use of crystalline pepsin if properly standardized. Though the method is simple and has been used extensively, especially for urine pepsinogen, it is relatively non-specific, depending as it does on the power to denature the substrate rather than to digest it. Furthermore clotting is common to many proteolytic enzymes and other non-enzymic factors (3o). It is thus not entirely suitable for biological work with mixed fluids. Disappearance of Substrate The first method employed by Schwann (3 2) in the measurement of pepsin depended on the disappearance of solid or suspended substrate in the form of coagulated egg albumen. The widest application of this principle has been the Mett egg white tube method, which has little to recommend it because it lacks both sensitivity and accuracy. Other substrates 26

used in the past have included ricin, fibrin, globulin and others. Appearance of Peptides The hemoglobin method of Anson and Mirsky (3334) has for several reasons gained the widest acceptance for peptic assay, particularly in biological use. In this method, a hemoglobin solution is denatured by acidification and after the enzyme solution has been allowed to act on the hemoglobin for a specified period, the reaction is stopped by the addition of trichloracetic acid which also precipitates the undigested protein. After filtration or centrifugation, tyrosine and tryptophancontaining peptides arc measured using either the Folin Ciocalteu procedure or measuring optical density at 28o mu (8, 35), with 1-tryosine as the standard. The method fulfills the criteria mentioned above: it is reproducible, sensitive and quantitative without the involved and often contentious mathematics attached to the interpretation of milk clotting (29) or Metts tubes, reasonably specific at the appropriate pH, relatively simple and applicable to the biological fluids (36,37,38, 3940,4i). In this category many other methods are available and possible, differing in substrate (e.g. serum albumin (42), or dried plasma (43) ), in method of precipitation and in assay of reaction products, but all are the same in principle and the choice is arbitrary. Use of Modified Proteins The difficulty of making an adequate substrate with an adsorbed dye (e.g. carmine-fibrin (Ø)) has been overcome by using i" labelled serum albumen and measuring the i"` split off by standard radioactive counting equipment (45)• This method appears to have the same

HIRSCHOWITZ

advantages of the hemoglobin method except that the substrate does not keep due to decay of radioactivity, and fresh supplies have to be obtained frequently. Bonds Broken Methods in this category give more specific and significant information about the course and nature of a proteolytic reaction than the empiric methods just described, and include the use of synthetic substrates (46,47), an extensive list of which are given by Green and Neurath (48). However, these methods are tedious, the reactions are slow, requiring prolonged incubation with the concomitant problems of change in pH, bacterial overgrowth and peptic autodigestion and are not recommended for routine use. Schlamowitz and Peterson (2) have suggested that different bonds may be broken at different pH levels depending on the denaturation of the substrate by pH alteration. Comment on Currently used Methods a) SPECIFICITY. Simply because a commercial or even a crystalline preparation of pepsin performs a certain reaction, e.g. ng gelatin, clotting milk or digestliquifyi ing a protein under certain conditions, it may not be at all applicable to the measurement of pepsin(ogen) in any biological system. Thus gelatinase (24) or parapepsin I and II (9,22) will liquefy gelatin much faster than pepsin, and minor contamination with either will give false results. The same is true of milk clotting and the many other things that will clot milk (3o). The studies of substrate and pH optima of gastric juice (49,50,51,52, 53,54,55,56) and of the careful analysis of these matters in the discovery of parapepsins I and II by Ryle and Porter (25, 26) indicate some of the problems under-

lying the conclusions which may be drawn from simple (one system, one pH) analyses (46, 47, 57). For the most part, however, digestion of hemoglobin at pH 1.7-2.0 seems fairly specific for pepsin (ogen), with different pH optima for other substrates (58, 59). b) UNITAGE AND REPORTING OF RESULTS. If a reliable assay system (e.g. Hb, albumen) is used, it might be best to report this in terms of activity- of crystalline pepsin having a specific activity of 0.26 [PU]"bmgN at 2 5.5° C (8,33,34)* as measured by the release of tyrosine in a ten minute digestion. The common usage in terms of release of tyrosine would be acceptable if everybody would agree on the amount of tyrosine which represented a unit—at present mg, mEq, I x I o-' inEq, I x I o-' mEq, log units and moles of tyrosine are used. Thus I mg. of crystalline (Northrop) pepsin ([0.26 PU] Hb o", 35.5° C)* should produce the equivalent of 382.3 X le mEq or 6.92 mg. tyrosine in a ten minute digestion of i oo mg. (5 ml. 2 per cent) Hb at 35.5°. In general clinical use a unit has been defined as i x io-` mEq tyrosine released from a ten minute assay at 37° C. Contrary to a previous suggestion (5) it might be best to label this the clinical unit of peptic activity or [C PU] "b I o", 37° C. The author is still hopeful that the people who measure pepsin (ogen) for clinical purposes may speak a common language and that editors insist on a definition of units with each paper on pepsin(ogen) measurement. C) DIFFICULTIES WITH TILE ASSAY ME-

A number of papers have appeared, especially in regard to urine pepsinogen, in which it has been reported that on consecutive days the urine (sometimes the THOD.

`A pepsin of higher specific activity than the Northrop pepsin (0.36 vs 0.26 [PU] "bmgN) has been prepared by Miyamoto and Matsumara (6o). 27

I / THE PHYSIOLOGY OF PEPSINOGEN

gastric juice) may contain large quantities of pepsinogen, and the next day or next specimen, none. Any report of this nature is very suspect and implies serious errors in techniques of assay. One of the least suspected errors arises from glassware contaminated with detergents containing sodium lauryl sulfate (seen inhibitors). Related Enzymes A great deal has been written about proteolysis at pH 3.5 which is assumed to be cathepsin in gastric juice and urine (61). More recently, other proteolytic enzymes active at this pH range such as parapepsins I and II (25,26) have been identified, the latter probably the same as gastricsin (62). Other studies have shown that pH optima depend on the substrate (49,58,63) rather than on the enzymes, and several authors (50,64,65) deny the existence of a cathepsin in gastric juice, claiming that it merely represents an "extended pH range" of action of pepsin. It is not even certain whether they represent different enzymes or different forms of pepsin. These related enzymes should be kept in mind when such non-specific methods

as milk-clotting are related to standard curves with purified pepsin. For more detailed discussions of the chemistry of pepsinogen and pepsin and on the nature of proteolysis, the reader is referred to more comprehensive descriptions (7,8,9,19,22,23, 25,33,34,48). Distribution of Pepsinogen in Various Tissues Pepsinogen has been found in various body fluids in concentration roughly equal to that of the plasma, viz. sweat (1,6), cerebrospinal fluid (i), pleural fluid (1), ascitic fluid (66), but not in peripheral edema fluid in congestive heart failure (1) or in tuberculous pus, in which there appeared to be a pepsin inhibitor present in the cells (1). Pepsinogen in high concentration has been found in human semen (67) arising from the seminal vescicles, and in human leucocytes (40). It is not known whether these are made in these sites or simply concentrated. Buchs (68) has described acid active proteolytic enzymes to be present, in decreasing concentration, in the entire gastrointestinal tract. Because pepsin is denatured in neutral or alkaline medium, it is found only in the acid gastric juice.

Pepsin Inhibitors There is recurrent interest in the inhibi- though the pH in the stomach soon retion of peptic action as being potentially turns to acid values, the pepsin is not useful in the management of peptic ulcer. reactivated; other physical agents such as Inhibition of peptic action may be pressure (8), ultraviolet light (8,69), radibrought about by two means—destruction ation (7o) and heat (8) are obviously unof pepsin and true inhibition. Destruction suited to clinical use. At neutral pH pepof pepsin (irreversible denaturation) may sin gives up 5.6 moles of H*, with 4 of be accomplished almost instantly by an these arising from carboxyl groups which environmental change of pH to values are stabilized in the native protein moleover 7.0 (8), which happens for a very cule by hydrogen bonds (71), and thus short while with the ingestion of an alkali denaturation near neutral pH may be such as sodium bicarbonate, and even promoted by the binding of carboxyl 28

HIRSCHOWITZ

groups by salts of Pb, Cd, Cu and Zn (7i ) and by urea and its analogues thiourea and guanidine chloride (72), the latter promoting denaturation at pH > 3.5 (73)• Dephosphorylation does not inactivate the pepsin molecule (74), and binding of sulfhydryl groups by various metals likewise does not affect denaturation of pepsin (71). Of the antacids, bismuth carbonate (7) and sodium lauryl sulfate are apparently the only ones in vitro able to inhibit without changes in pH, though this needs further analysis, since neither is very effective in vivo (76). Among the inhibitors which appear to act without denaturing pepsin is the natural pepsin inhibitor derived from the conversion of pepsinogen to pepsin (1 o), but it is only active at pH 5.5 or over, being destroyed at lower pH by pepsin.

It thus is not useful clinically. A pepsin inhibitor found in Ascaris (77) has not been reported on again. Therapeutically, there is reason for interest in the sulfate containing mucopolysaccharides (78,79, 8o) and the sulfated esters of starch (81), and possibly in an inhibitor of milkclotting by pepsin found associated with TSH (82). Benzaldehyde is inhibitory of pepsin and is reported to be non-toxic in short term administration (83). Thus no certain inhibition of pepsin in vivo has been described, and though it makes an interesting approach, much still needs to be learned about the structure and mode of action of pepsin before a logical attack can be made on its denaturation or inhibition. The subject is well reviewed and studied from a clinical point of view by Koskinen (76).

The Peptic Cell The peptic cell possesses characteristics which are common to zymogen secreting cells of the pancreas, stomach and salivary glands (84,85,86). All have a common origin from the endoderm and similar cellular structure and ultrastructure (85), including granules of the respective proenzyme and a lamellar arrangement of the cytoplasm (85) or a-cyto membranes (87) . They are all arranged as part of tubular cell systems of which they occupy the base, and are all concerned with digestion of food and go through distinct histologically observed cycles related to secretory activity (84,86). Peptic or chief cells were first independently described as different from parietal cells by Rollet (88) and by R. Heidenhain (89), who rightly believed that these cells secreted a proenzyme and that the parietal cells secreted acid. The granules of the peptic cell, its most prominent constituent, contain and probably

comprise pepsinogen and are best stained by crystal violet (90). By electron microscopy the cytoplasm can be shown to contain both droplets (0.6-0.85 ,u) and a variable number of vacuoles (0.9,u) (85,87), containing finely granuar material, the two being inversely proportional in number, the droplets apparently representing the secretory material (85). Where they abut on the free end of the cell, the microvilli or filaments which line the surface (0.2 ,u long and o.o6 ,u wide) are absent (85). A more recent study (91) of the pepsinogen granules in man reports that they are pale and homogenous oval structures o.5 to 3 µ in diameter, those of less than 1 ,u being enclosed in a double membrane and those larger in a single membrane peculiar to the peptic cell granules. The basal basophilic filaments or ergastoplasm represent RNA (92), which on electron microscopy comprise numerous filaments or lamellae about 29

I / THE PHYSIOLOGY OF PEPSINOGEN

o.o8 µ wide and arranged in parallel rows or concentric layers between the nucleus base of the cell (85). A quantitative and qualitative relationship between RNA and pepsinogen secretion has been shown, both showing involution in pituitary or adrenal insufficiency in rats (93). Cytochemical studies have shown a number of esterases (94, 95), the functions of which are unknown. Relation of Structure to Function There seems little doubt about the localization of pepsinogen in the chief cells (3,4,94,96,97). Ultracentrifugal studies of gastric mucosa are at variance. Degkwitz and Lang (1960) (98) report a distribution of 40 per cent pepsinogen in the nuclear layers, 19 per cent mitochondral, 4 per cent microsomal layer and 33 per cent in the cytoplasmic layer, while Taylor (5o) reports about 6o per cent in the microsomal layers and 25 per cent in the mitochondral layers, values which appear more probable. It is therefore an unresolved matter as to where exactly pepsinogen is synthesized, but the weight of evidence about protein synthesis would favor RNA as the site of manufacture with transport to the granules acting in a storage capacity. Microscopically, mitochondria apparently play no part in the synthesis of pepsinogen (99). More electron microscopic and ultracentrifugal separation studies under various secretory conditions of the peptic cell will have to be done in order to resolve these inconsistencies. Secretory Cycle of the Chief Cell A well defined secretory cycle in the chief cells has been described (4,84,99, I 00,101,102 ) . In the rabbit, there is a complete cycle which can be initiated by a single dose of pilocarpine (wo). After 30

one hour, granules have disappeared from all but the base of the cell, which becomes smaller and mitochondria are scattered throughout the cell. Depletion of granules continues until between three and six hours when only a few cells in the depths of the tubules contain any granules, and the mucosal content of pepsinogen is at a minimum. After six hours, signs of replacement appear — the cells enlarge, mitochondria (?) become bigger and organize at the base of the cell and granules begin to appear at the free border. After sixteen hours, the cycle appears to be complete, with repletion of granules and the mucosal content of pepsinogen. The Golgi body goes through a cycle, being small and compact in the full cell to being large and coarse in the area of appearance of the granules in the empty cell; apparently a similar cycle occurs after feeding (99) and the granules appear in close association with the Golgi apparatus. It has been suggested that the smallest granules seen by phase-contrast are propepsinogen (99) and the larger argentophobic granules pepsinogen. Atropine accentuates the resting phase with increased granules (103,104,105 ), a decrease in visible mitochondria (104) and the Golgi body made even more compact. Also promoting excessive storage of granules are extremes of body temperature, hyperventilation (which in other studies increases pepsin secretion (1 o6) ), cerebral compression (107), and hyperglycemia (Io8). Despite the histological evidence of depletion of granules and even chemical evidence of depletion of pepsinogen content of the mucosa after stimulation, the stomach can secrete large amounts of pepsinogen for prolonged periods (103,108,1 Io). This indicates that pepsinogen can be made and secreted without having to undergo the granule phase of storage, which presumably is only used

HIRSCHOWITZ

as a ready stock to tide the cell over the period of inertia between the start of the stimulus and the time when synthesis can catch up with the demand (Fig. 1). Distribution of the Chief Cells In inframammalian vertebrates—birds, fish, reptiles and amphibians—there is only one gastric cell type combining both enzyme and acid secretion (1 1 1,1 12,1 13 ), though some specialized esophageal glands in most (112) amphibians, mainly frogs and Necturus, secrete only pepsinogen (4,84,101,111,112,114) and resemble mammalian chief cells (111). Systematic histological and biochemical studies in cats (9o) and pigs (96,97) show maximum peptic cell concentration in the middle three-quarters of the stomach, with few peptic cells in the fundus and very few in the pyloric areas. Other, unsystematic, histological observations confirm the general pattern for other mammals, though the exact areas occupied by fundus or pyloric area vary (1oz). In the dog, pepsin is secreted by pyloric mucosa, though no typical peptic cells can be

found (115). However, since the mucus neck cells, which are precursors of the peptic cells (84,102,112,116), occur in this area, it may well be that these cells secrete pepsinogen but form no granules. The embryologic development of the peptic cell varies considerably between species as to the time of first appearance of granules and measurable proteolytic activity (112,117,i 18,1 19). In the human, granules and peptic activity appear between four and eight months, with a maximum increase of 55 cm. fetal length (start of tenth month), developing roughly in parallel with pancreatic tryptic activity (120,1 2 1 ). The peptic cells embryologically and in repair of gastric mucosa are thought to differentiate from mucus neck cell (mucoid neck cell) (84,92,102,11 2,116). The turnover rate of the mucus neck cell is 155 hours (63,92), which is far in excess of growth requirements, and though there is no clear evidence of gross loss of peptic cells by extrusion or degeneration in the absence of injury, any cells that are lost are replaced from this source (116).

Synthesis of Pepsinogen Synthesis of pepsinogen is normally a continuous process (105) as shown schematically in Fig. I (5) . Thus, under basal conditions, the store of pepsinogen is almost full and remains constant because it is only fractionally depleted by secretion and simultaneously repleted by synthesis at a low level of activity. The immediate response to stimulation is a discharge of accumulated granules (initial stimulated secretion), the purpose of the stock proenzyme granules being to tide the cell over the early period of inertia in protein synthesis, which is set in motion by the acute upset of the intracellular

pepsinogen equilibrium. If the stimulus is removed, there is a return to a low level of basal secretion, and the cell enters a restorative phase, with a gradual accumulation of granules and a return of the equilibrium x —* y to basal conditions. If, however, the stimulus to secretion continues, pepsinogen secretion in man attains a plateau in 30-45 minutes (110, 12 2) and the cell approaches a new, dynamic equilibrium proceeding to the right x —(y) —> z with little zymogen storage. The rate of maximum secretion is then limited by the rate of pepsinogen synthesis, so that the concentration of pep31

PEPSINOGEN ,,,~~~

,PEPSINOGEN

. STORE PRECURSORS RNA.–: (X)A.

ATROPINE -

STIMULANTS SECRETED PEPSIN acetyl chloline (Z) histamine hyperventilation

(Synthesis)• ✓ ~ _°.(Y)` dom` .-

RNA DYNAMIC EQUILIBRIUM

OUT PUTOF PEPSIN

( Maximum rate of synthesis)

EXAGGERATED RESTING PHASE

ATROPINE no secretion) X

Y PEPSINOGEN STORAGE

e

BASAL SECRETION (resting phase)

0 co

RNA

O,

NEAR -4 EQUILIBRIUM STATIC EQUILIBRIUM

X —Y RESTORATION

S TIM ULAT ION —•

ATROPINE BASAL Y I X ~=YI X

It

CONTINUOUS

~X

Z X INITIAL STIMULUS ~ of granules)

(Y)1\

~~

Z

B.

FIG. t. Schematic representation of synthesis and secretion of pepsinogen by the chief cells. A. The chief cell is shown diagrannnatically in the basal state, with the lumina' end depicted as a door allowing zymogen granules to escape. Atropine is shown pushing the door shut while

X~z(Y)tiZ

C.

IISSOLVED PEPSINOGEN

CONTINUOUS STIMULATION

stimulants to secretion will open it. B. The various stages of activity of the chief cells are shown schematically. C. The secretion of pepsin by the chief cell has been graphically related to the state of equilibrium of pepsinogen synthesis in various stages of activity.

sinogen in gastric juice falls as the volume (coo), presumably because the pylorus reaches maximum values, but the output ligated rat secretes continuously (105). Secretion and synthesis have been always exceeds that of the initial stimulation period (i io). Inhibition of secretion shown to be well correlated in pylorus e.g. by atropine causes the accumulation ligated rats (105). Under atropine, synof pepsinogen in the mucosa (103,104, thesis, even though reduced in rate, still 105,1 23) and a significant reduction in the occurred at greater rate than secretion rate of synthesis (105). Synthesis is de- (which was greatly reduced), causing pressed without a primary reduction in both the accumulation of an excess of secretion by adrenal cortical insufficiency pepsinogen in the mucosa and the secre(i24,125) presumably due to a decrease tion of pepsinogen in very high conin RNA (93) and may be restored to centration (105), suggesting that synnormal by adequate doses of cortisone, thesis may in part be responsible for secrecorticosterone, hydrocortisone or adrenal tion which takes place under these cortex extract, (124). Synthesis is not in- conditions, presumably to avoid rupture creased in the normal rat given adreno- of cells. Similar findings related to low cortical hormones or ACTH and large volume of gastric secretion are reported doses of cortisone may actually depress after administration of amphenone to synthesis (124). Vitamin B deficiency rats (127). does not depress the mucosal content of In vitro, synthesis by mucosal expepsinogen (126). tracts and even by an acetone powder of The rate of pepsinogen synthesis in the gastric mucosa were reported by Ullman pylorus ligated rat has been found to be and Straub (128), but these observations roughly six hours for complete turnover could not be confirmed (129). No studies have been done on the inof the fasting content of the mucosa (105). This differs from the histologic corporation of labelled amino acids into observation of a single stimulus and a six- the pepsinogen molecule to determine the teen hour restorative cycle in the rabbit rate and course of synthesis, but such 32

HIRSCHOWITZ

studies together with substrate and energy requirements will have to wait on a reliable in vitro system. Now that the

amino acid composition and N-terminal amino acids of pepsinogen are known, studies of this sort should be possible.

Secretion of Pepsinogen Under most conditions the secretion of pepsinogen is part of the secretion of gastric juice. It is thus most important when considering any effect on the secretion of pepsinogen to determine whether the effect is on gastric secretion in general or on pepsinogen secretion, independent of the secretion of water, acid or other electrolytes. For example when one speaks of an intermittent secretion of pepsinogen, this means intermittent secretion of all gastric juice; suppression of pepsinogen secretion by atropine falls into the same category. To arrive at a true estimate of pepsinogen secretion, the pepsin content of gastric juice should be related to some reference measurement in gastric juice, either acid or the sum of acid and sodium (122). In addition, of course, the absolute secretion of pepsin under the influence of a test material or situation should be compared with that under suitable control conditions. Basal Secretion Gastric secretion in the unstimulated state may be continuous or intermittent depending on species differences. Continuous basal secretion occurs in man (109,130,131,132), the spider monkey (133), the rat (105,134), herbivora (118, 135) and in Necturus (136). In man, at least, pepsin may be found in appreciable quantities even in the anacid stomach under basal conditions. Gastric juice is secreted intermittently or at very low rates between feedings in birds (137), dogs (109), and cats (109,138). During hibernation in the frog, hedgehog (Io1, 11 1) and salamander (139) there is little

or no secretion but warming fairly rapidly restores secretion (101,111,140). The isolated stomach of the mouse and the frog (114,129) secrete pepsin. The maintenance of basal secretion probably depends mainly but not entirely on cholinergic mechanisms, since vagotomy greatly reduces basal secretion of all elements of gastric juice (141,142,143), whereas atropine in adequate doses reduces gastric secretion even more (105). The action of atropine, however, is not absolute proof of cholinergic mechanisms, since atropine has apparently actions other than simply blocking the action of acetylcholine (105). It is possible, and indeed likely, that gastrin (17) or gastrozymin (144), perhaps partly under vagal control, maintain basal secretion. The recovery of some basal secretion long after a vagotomy and the persistence of high rates of secretion after vagotomy in the Zollinger-Ellison syndrome suggest further that the hormonal maintenance of pepsin secretion is not entirely vagally dependent. In man basal pepsin output is 20-25 per cent that of insulin stimulated secretion (1 29,131) and about 3o per cent that of histamine stimulated secretion (122), in both normal and pathological conditions of stomach or duodenum. Basal, interdigestive, secretion of gastric juice in the dog is uninfluenced by a high protein diet (145) but may be continuous, even in the denervated pouch, at a very low rate when measured by sensitive methods ( 1 43) In part, at least, basal secretion of pepsinogen occurs independently of other gastric secretion because synthesis of 33

I / THE PHYSIOLOGY OF PEPSINOGEN

pepsinogen is continuous and an "overflow secretion" occurs when the peptic cells fill up and discharge granules to accommodate newly synthesized proenzyme (105) . This occurs when there has been marked suppression of secretion by atropine (105) or Amphenone (127). It may well be that this represents the only true basal secretion, beyond which any pepsinogen secretion is stimulated— endogenously in the basal state or exogenously when we apply external stimuli. Stimulation of Pepsinogen Secretion Most stimuli of pepsinogen secretion, in fact, stimulate all elements of gastric secretion, and to be precise about pepsinogen, stimulation independent of stimulation of other elements of gastric juice ratios pepsin:acid should be known. Since both acid and pepsin concentrations in gastric juice have a finite limit (122) although the latter is more subject to variation between subjects, animal or human, the pepsin:acid (P:H) ratio is hard to define, and few reports have attempted any such description. It is important, however, in the comparison of stimuli in the same subject to use this ratio as an assessment of the relative pepsinogogic action of each stimulus. In most species cholinergic (i.e. vagal) mechanisms are the clearest and strongest stimuli of pepsinogen secretion. They are, however, not the only stimuli and the secretion of pepsinogen is in itself no absolute indication of vagal or cholinergic activity. Cholinergic Mechanisms With the possible exception of the frog (111) and Necturus (136), pepsin secretion can be stimulated via the vagus nerves in all species studied, either by adequate (>1 ma) direct electrical excitation of the nerve trunks (103,146) or indirectly by hypoglycemia (131,147), by 34

alcohol (110,148), by sham feeding (109, 148,149) and by psychic stimuli. Vagotomy in dogs eliminates and may reverse secretory responses to these stimuli (150,151) and reduces pepsin responses to other stimuli except to pilocarpine (17). In man, vagotomy does not reduce the pepsin stimulating effect of histamine (1o6). Chemically cholinergic (local) stimulation can be accomplished either by parasympathomimetic drugs such as choline (152), acetyl choline (138,153) or its active derivatives notably methyl beta choline (mecholyl) and urecholine (138, iso,1 51,154,155), or by the inhibition of choline esterase by pilocarpine, or eserine among others (17, 100,15 5,156,157) . The action of pilocarpine after vagotomy (17 ) supports the contention that acetyl choline made locally in the gastric mucosa by choline acetylase (158), possibly related to the intrinsic nerve networks of the mucosa, may be important in maintaining secretion. However, while atropine can apparently block all cholinergic transmission(103), basal secretion cannot be entirely eliminated in the rat (105) or mouse stomach (1 59), while in the dog it does (129). In man, hypoglycemia stimulates pepsin about 10-20 per cent more strongly, relative to acid than does histamine, whereas in the dog, the P: H ratio may be from ten to sixty times greater with insulin than histamine in different dogs (148). The dog is a particularly good animal for the study of pepsin stimulation because secretion of pepsin and acid can be pretty well dissociated during histamine stimulation, and trained unanesthetized dogs do not have any appreciable basal secretion of gastric juice (160). Histamine The recognition of species differences and the employment of refined techniques in the collection and measurement of

HIRSCHOWITZ

pepsin has mainly clarified the position of histamine in relation to pepsinogen secretion. Thus histamine in various doses and in various species may stimulate, inhibit or be without effect on pepsin secretion. a) Stimulation of pepsin secretion by histamine has been confirmed repeatedly in intact man (zIo,122,131,156,161,162, 163,165,166,167), as well as after vagotomy, in the spider monkey (34), guinea pig (118), snakes and tortoise (113). The evidence for such stimulation in frogs is contradictory (111,114). Some of the confusion stems from the measurement of concentration alone, where an initial peak of concentration, preceding the maximum acid concentration, is followed by a decrease to pepsin concentration levels below those in the control period. This has been interpreted as a "washingout" (131). However, in observing the output of pepsin, there is a smooth rise from basal values to maintained output averaging three and a half times basal values during sustained histamine stimulation (110,122), and if a sustained increased output defines stimulation of secretion, then there is absolutely no doubt that histamine stimulates the secretion of pepsinogen in man. b) Lack of stimulation of pepsinogen secretion by histamine has been described in dogs (103,138,155,18), in cats (138), the groundhog (135), in birds (137), in the skate and Necturus (136). c) Inhibition of pepsinogen secretion by histamine. Of the species in which histamine stimulates pepsin secretion differently from man, the dog has been the most extensively studied, and a true species difference is now recognized, though not explained. Two types of experiments should be differentiated— those on innervated stomachs or pouches and those on denervated (Heidenhain) pouches, the latter showing poorer secretion responses to all stimuli than the

former (17). Under all conditions when histamine alone is used, the intact dog stomach secretes very small quantities of pepsinogen (1/10 to 1/6o as much as hypoglycemia (148) ), and in this sense histamine never really stimulates pepsinogen secretion, even though it can be shown that there is an increasing output which can be related to increasing doses up to about 0.1 mg./Kg. single subcutaneous dose of histamine base (148) or 0.025 mg./Kg./hour I.V. (Grossman, personal communication). This level of secretion, relative to acid secretion is still from ten to fifty times smaller than the response to insulin stimulated secretion (148). In larger doses (o.os to 1.4 mg./Kg./hour in dogs and cats) (138) there is an inverse relation between pepsin output and the dose of histamine, suggesting active suppression, similar to that seen with cholinergic stimulation in excessive dosage (118,153,169 ) . In longcontinued (three to four and half hours) stimulation with intravenously administered histamine at moderate dose levels (0.05 mg./Kg./hour), there is a continuous maintained output of pepsin at low but measurable levels (170,171). After about three hours, both concentration and output increase (171) to about twice the preceding control period. The apparent increase in secretion is due to the forcing out of pepsin from cells with excess stores of pepsinogen, as demonstrated histologically by Bowie and Vineberg (103), after histamine, and analogous to the apparent increase in pepsinogen secretion after prolonged atropinization of rats (105). This is thus a result of and a sign of suppression of secretion in the first three hours of histamine, and not in fact due to stimulation by histamine. Evidence on the suppression of pepsinogen secretion by histamine is clearly shown by a significant reduction (about 40 per cent) of insulin stimulated pepsin secretion 35

I / THE PHYSIOLOGY OF PEPSINOGEN

(but not acid) if the same dose of insulin is used in the same dogs during a histamine infusion (171). In the intact trained dog, priming with small doses of cholinergic drugs, such as mecholyl, prior to histamine administration, will apparently induce a situation similar to that obtaining in the human stomach, in which histamine will then stimulate fairly large quantities of pepsinogen secretion (129), but, on the other hand, histamine given during spontaneous, (vagal) gastric secretion will suppress secretion (129). However, a synergism between acetylcholine, mecholyl or pilocarpine and histamine (or gastrin) has been shown (1 37,1 53,1 54, 156,17 2,17 3) to cause a greater secretion of pepsinogen than could be predicted from a simple additive effect. What little secretion there is, is not affected by vagotocry during histamine administration (175). Synergism between histamine and cholinergic stimulation has also been found in the tortoise (176). The nature of the continuous secretion of pepsinogen during histamine administration may thus be due to a washing-out of pepsinogen from the peptic cells by coincident secretion of water from the peptic cell (1zz), or to a maintained basal secretion secondary to continued synthesis in the cell which forces some pepsinogen out (see above). A further clue to the possible mechanism is the demonstration that histamine, with concomitant sympathetic nerve electrical stimulation, greatly augments pepsin secretion, which can be blocked by dibenzylene (177). The difference, then, between man and dog is not clearly vagal preconditioning in the former and lack of it in the latter. Gastrin and the Antrum GASTRIN. With the methods currently used for the preparation of gastrin from pyloric antral mucosa, there is a great

36

similarity between the actions of histamine and gastrin on pepsin secretion (138,1 78,179,18o), neither apparently stimulating pepsin. Some stimulation of pepsin by gastrin has, however, been reported (144,18o). While two reports on a pancreatic Zollinger-Ellison tumor (181,182) indicate that the active secretagogue may be gastrin and does not stimulate pepsinogen secretion; studies by the author with a Zollinger-Ellison tumor, extracted by a different procedure (148), indicate that it contains a very potent stimulus, presumably gastrin, which promotes pepsin secretion to the same extent as does insulin hypoglycemia, as well as stimulating acid at least as powerfully as histamine in dogs. THE ANTRUM. While the results with crude gastrin may be at present at variance because of different methods of preparation, results of experiments designed to elucidate the role of the antrum in situ in pepsinogen secretion are also inconclusive. Though vagal stimulation has been shown capable of stimulating pepsin secretion despite cocainization, devascularization or removal of the antrum (138), and though distension of the antrum of man does not stimulate secretion of pepsin (149), experiments in which an antrum pouch in dogs was stimulated by liver extracts or by transplantation to the colon where they were stimulated by constant alkalinization, show an increase in pepsin secretion almost as great as the increase in acid secretion (119), but less than that produced by pilocarpine (183). In dogs, antrectomy has been shown to eliminate the pepsin response of a denervated pouch to vagal stimulation (151). In frogs, mechanical stimulation of the antrum stimulates both gastric and esophait) . geal secretion of pepsinogen (IOI,I Whether such stimulation is produced by gastrin or by another hormone produced

HIRSCHOWITZ

together with gastrin is not known. Of various amino acids tested for stimulation of the antrum, 3-alanine (149) and lysine (184), but not arginine, stimulated pepsin secretion. Intestinal Phase of Gastric Secretion In man, a variety of proteins introduced into the intestine by tube do not augment pepsin secretion (149), whereas in dogs both HC1 and a variety of foods may stimulate pepsin secretion (109, p. 505), especially in the innervated pouch (143), and in Necturus the only known stimulus to pepsin secretion is the introduction of food into the intestine (136). Crude preparations of secretin stimulate pepsin secretion but apparently purified preparations do not (185), and a fraction which can be removed by butanol extraction from crude secretin can stimulate pepsin secretion (185). There might then be a case made for gastrozymin as a separate hormone (144). Burstall and Schofield (I 51) have found a gastric secretagogue as a contaminant of commercial insulin, indicating the pancreas as another possible source of such a hormone. Miscellaneous Stimuli Increased intracranial pressure (107) and head injuries (186) cause excessive pepsin secretion possibly via the vagus. Caffeine, itself having an action comparable to that of histamine, is apparently additive to histamine in stimulating pepsin in man (187). Hyperventilation in man increases pepsinogen output by as much as 100 per cent with a slight decrease in acid through mechanisms at present unknown, whether performed in the basal state or during histamine infusion, in intact, vagotomized or atropinized man and may increase the

concentration to levels exceeding by up to 3o per cent maximal levels seen after insulin (106). Diamox, (106,188), Amphenone (1 2 7) and, atropine (105), produce pseudo-stimulation of pepsinogen by reducing the volume of gastric juice and a corresponding increase in concentration, but no increase in output. Komarov has described a substance extracted from canine gastric juice which stimulates pepsin secretion during histamine stimulation (189). Inhibition of Pepsin Secretion Decrease of pepsin secretion may be produced by reduced synthesis, in adrenal or pituitary insufficiency (124,125,127) when RNA is decreased (93). Inhibition of pepsin secretion while acid is being secreted may be obtained in some species, notably the dog and cat, by histamine (see above). Other decreases in output of pepsin are related to an over-all decrease in gastric secretion e.g. vagotomy (142); atropine (105,138); hypertonic glucose in birds (137) (but not in dogs (160)); enterogastrone in innervated pouches (19o), insulin in denervated pouches (150,151) and many others; these are thus not true inhibitors of pepsinogen secretion but inhibitors of all gastric secretion. There is, in fact, no separate inhibitor of pepsin secretion except histamine under certain conditions. Endocrine Influences on Pepsinogen Secretion ANTERIOR PITUITARY GLAND. The pituitary is important in maintaining the structural and functional integrity of the gastric mucosa. Hypophysectomy leads to permanent decrease (up to 8o per cent) in pepsinogen and acid secretion starting within three to seven days (93), together with involution of the chief cells which

37

I / THE PHYSIOLOGY OF PEPSINOGEN

show a decrease in cell and nuclear size, zymogen granules and RNA. The decrease in content of mucosal pepsinogen after hypophysectomy is more pronounced than after adrenalectomy (124), suggesting that factors other than those operating through the adrenal may be involved. Thus growth hormone given to rats for three to six days increases pepsinogen secretion almost five fold (191) and a single injection in cats may double the output of gastric juice. In denervated pouch dogs given anterior pituitary extract there is an initial small increase with return to control levels by the sixth day (192). Rats given anterior pituitary extract also have a small increase in pepsin secretion after seven days (193). There is no direct action of pitressin on pepsin secretion (129). ADRENAL CORTEX. While there may be considerable controversy regarding a stimulant role of the adrenal cortex, there is unanimity regarding the effects of adrenocortical deficiency which results in a moderate to marked depression of gastric secretion in man (194,195) and which may be restored to normal by replacement therapy (194). Almost all the information on the role of adrenocortical deficiency has been obtained in rats in which the principal secretion is the salt-retaining hormone corticosterone (196). Thus, interpretation extrapolated to man, who secretes mainly hydrocortisone (196), should be cautious. In the rat adrenocortical insufficiency produces moderate involution of the chief cells, less extreme than that produced by hypophysectomy (93,197), as well as a decrease in mucosal content of pepsinogen (124); in the adrenalectomized rat, these findings may be accentuated by further salt-depletion (124.12 5,1 93) by large doses (25 mg./rat/day) of cortisone (124,125) and by ACTH (124). The reduction in pepsin secretion starts within 38

a few days after adrenalectomy (125, 199), and is proportional to histologically observed cellular involution (197,198) and due to decreased synthesis of pepsinogen (125). The involution appears to be relatively nonspecific, and normal function can be restored by adrenal cortex extract (124,199), corticosterone, desoxycorticosterone, cortisone and hydrocortisone in adequate amounts (124). Evidence for a stimulant role of the adrenal cortex in gastric pepsin secretion is contradictory and not very convincing, mainly because any changes that do occur are relatively small and capable of interpretation either way. Conclusions based on measurement of pepsinogen in blood or urine regarding the adrenocortical effects on gastric secretion are generally invalid and misleading. There is no evidence that the adrenal cortex or its hormones can stimulate gastric secretion in the sense that cholinergic, histamine or even gastrin stimuli do — the so called "adrenal phase of gastric secretion" needs much more proof than has been given to support it. Though it has been reported that ACTH may stimulate gastric secretion within four to six hours (20o), no such effect could be determined in man by an eight-hour infusion of 25 mg. ACTH (where a fifteen-fold increase in urinary 17-hydroxycorticoid excretion occurred within four hours), with too mg. hydrocortisone or 25 mg. prednisolone (130), or within six hours of a single dose of ACTH (201). More prolonged administration of ACTH or cortisone has been reported to increase basal pepsin secretion in man (202,203) and the dog (204), though this could not be confirmed in man by others using ACTH, cortisone or hydrocortisone (142,205,206) in the dog or in the rat given ACTH for one to three weeks (125). Changes in the gastric mucosa of rats after ten to twenty days administration of cortisone or hydrocor-

HIRSCHOWITZ

tisone comprise an increase in the number of cells, which become smaller and show an increase in RNA and nuclear size, with no apparent change in mitochondria (198). ACTH causes an increase in mucosal content of pepsinogen within one week but a return to control levels by three weeks (125). Amphenone suppresses secretion, possibly by a local effect (1 27) with an increase in mucosal storage, its effect being the same with concomitant administration of ACTH, aldosterone, desoxycorticosterone acetate, corticosterone or cortisone (127) and resembling the effect of atropine (105), i.e. mucosal accumulation due to suppression of secretion. ADRENAL MEDULLA. In frogs small doses of epinephrine stimulate and large doses depress the secretion of pepsin (1 11,114), while in dogs adrenalin appears to block pepsin secretion less markedly than acid

secretion (207) and to inhibit vagal stimulation more than it does histamine stimulation (138). THYROID. Experimental hypothyroidism in rats produces a modest depression of volume of gastric juice after eight weeks (208) but without any significant changes in the appearance of the chief cells (197) or in the concentration of pepsin in gastric juice (193,197,208). PARATHYROIDS. Total parathyroidectomy in rats produces no significant changes in pepsin output by eight weeks (208) even if combined with thyroidectomy. GONADS. Gonadectomy alone produces little effect; but combined removal of gonads, thyroid and adrenals results in profound suppression of gastric pepsin output (93 per cent) with involution of chief cells greater than produced by hypophysectomy (197).

Clinical Studies of Gastric Pepsin Secretion Much of the clinical material reported suffers from a lack of uniformity, and in many cases inadequacy of method of sampling, stimulating, measuring and reporting pepsin secretion. Where test meals have been used and only concentrations reported, the results are generally of little value because of the wide individual variations in volume of gastric juice to which fixed volumes of test meal of varying composition have been added. This applies even more to the measurement of HCI. Sex and Age Pepsin output or concentration is not significantly different between the sexes at all ages (129,132,163,164), though males tend to run higher in output, but

not when reckoned on a body weight basis. Age. The lower values for pepsin concentration reported by Heck and Pelikan (209) in small children are probably due to the use of fixed volumes of test meal with different sized children (see above). In fact the output of pepsin in children aged three to eight years per unit time per lb. body weight is the same as in normal adults (122). Normal Variations In the adult there is a relatively stable output of pepsin during the day, with a mean hour-to-hour variation of 35 per cent (130). The day to day variations in basal output is about 5o per cent (210), the data being normally distributed about 39

I / THE PHYSIOLOGY OF PEPSINOGEN

the mean. During maintained stimulation, the variation in pepsin output from one fifteen minute period to the next is less than zo per cent (122) and from week to week to about zo per cent (129). Duodenal Ulcer By a variety of methods of stimulation, collection, and measurement, duodenal ulcer patients have been found to secrete more pepsin and acid (because of greater volume of secretion) than do normal subjects or patients with gastric ulcer alone, (122,131,132,147,158,203,211,212, 213,214, 215). However, the composition of the gastric juice of duodenal ulcer patients, especially when stimulated by histamine (122) or by insulin (141,142,21 1), is the same as in normal subjects, and the explanation of the difference in pepsin output between duodenal ulcer and controls is seen to be due to the differences in volume of secretion (122). The differences in composition of gastric juice, especially the apparently high concentrations of pepsin found in patients with duodenal ulceration and more so with pyloric stenosis reported by Vanzant et al. (215,216), is accounted for by the use of a standard volume of test meal where the juice would be more diluted in normals (low volumes) than in duodenal ulcer, where gastric juice volumes are high, and even more so in pyloric stenosis. Gastric Ulcer In those gastric ulcers, which are not secondary to duodenal ulcer, the volume and composition of gastric juice is the same as in normals (28,I22,131,142,158) in both basal and stimulated conditions, whereas in those with both gastric and duodenal ulcers, secretions resemble the findings of the duodenal ulcer group (129). 40

Gastritis In gastritis both the concentration and output of pepsin are lower than normal (122), and the mucosal content of pepsinogen always decreased in atrophic gastritis and usually so in superficial gastritis (217) . Since gastritis is not a single entity and may be of differing severity, more work needs to be done with emphasis on exact diagnosis and stage of gastritis. Gastric Cancer In gastric cancer the average concentration of pepsin is about half that of controls (218), while the output is about one quarter (122) or less (214) . The overlap between pepsin values in gastric cancer and ulcer are such that pepsin measurements are of little value in differential diagnoses (218). Pernicious Anemia Only minute quantities of pepsin (0-2 per cent of normal) are secreted by the stomach in pernicious anemia (219,220, 221), and this is not improved by treatment with liver extracts (221). Patients with juvenile pernicious anemia may have both acid and pepsin in normal amounts (222,223), and thus have only a deficiency of intrinsic factor. It has been speculated that advanced gastric atrophy may result from intrinsic factor or vitamin B. deficiency, but one should then see many more patients with pernicious anemia in an intermediate stage of acid and pepsin secretion depression. In the anacid stomach, the presence of pepsin in significant quantities distinguishes the pernicious anemia stomach from the non-pernicious anemia anacid stomach. Other Anemias In patients with iron-deficiency anemia who secrete acid in response to histamine,

H I RSCHOW ITZ

pepsin secretion is normal while those who do not secrete acid have a gross reduction (to 20 per cent of normal) in pepsin secretion (219). Other anemias (posthemorrhagic, pregnancy, malnutrition or malabsorption, hemolytic) have normal pepsin secretion (163,221). Miscellaneous Disorders CIRRHOSIS. Patients with cirrhosis (both compensated and decompensated) have decreased secretion of pepsin and acid

compared to controls, unless they have a coincident peptic ulcer, in which case secretion is in the range of ulcer without cirrhosis (66). EMPHYSEMATOUS patients do not have increased secretion of pepsin by the stomach (129). Patients with sprue have normal pepsin secretion (22 1). VITAMIN B deficiency in man has no effect on pepsin secretion (224), but pantothenic acid deficiency reduces gastric secretory responses (including pepsin) to histamine (zzs).

Pepsinogen in the Blood Like many other enzymes which are normally intracellular or secreted into the lumen of the gastrointestinal tract, pepsinogen is present in the blood in small but significant quantities which are relatively constant in any one individual, (40, 226,227,228,229,230).

Identity of the Enzyme The presence of a proteolytic enzyme active in highly acid medium has been demonstrated repeatedly (40,220,226,zz8, 229), but its identity with pepsinogen has been derived from indirect evidence. It disappears rapidly from the plasma after total gastrectomy (within two hours in some instances) (40,229,230,23 1), which indicates a direct origin of the enzyme from the stomach. Temperature and pH optimum and inactivation studies indicate that it behaves like pepsinogen (40,229). The "second proteolytic enzyme" reported to be active in this pH range (229, 231,232) was found to be an artefact and due to non-enzymic acid hydrolysis of the plasma protein of the tested sample (40), and not to a proteolytic enzyme. Its persistence, unchanged after total gastrectomy, in pernicious anemia (229,233)

and after bilateral nephrectomy performed together with total gastrectomy (231), is further indication of its artefactual nature. Passage of Pepsinogen from the Peptic Cells into the Blood Recent studies showing changes in pepsinogen in gastric venous blood, where no change was evident in peripheral blood (230,231), have placed a different complexion on the interpretation of results of studies of pepsinogen in peripheral venous blood. Studies on the simultaneous pepsinogen secretion into the stomach and on the content of gastric venous effluent under various conditions would go far to clear up many of the questions on how and why and when pepsinogen moves from the peptic cell into the blood. However, since pepsinogen is excreted in the urine, the simultaneous measurement of peripheral venous blood and urine pepsinogen should supply adequate clues to the conditions which might increase gastric venous blood pepsinogen concentrations, such as ACTH. Much the same information is obtained with other materials, such as adrenocortical hormones, 41

I / THE PHYSIOLOGY OF PEPSINOGEN

where adrenal effluent blood cannot be readily obtained. The finding of pepsinogen in higher concentration in gastric vein blood than peripheral blood (40,230,231,234), suggests that transfer to the blood takes place directly rather than through lymphatic channels. As with other enzymes, the mechanisms of this transfer are at present unknown. The two main possibilities are a) diffusion through cell/capillary contact walls or b) loss of enzyme from disintegrated cells into intercellular fluid and absorption from there into the blood. Several facts tend to support the impression that the blood pepsinogen derives from disintegrated cells. In peripheral blood, pepsinogen levels are unaffected by food or other gastric stimulants in physiologic doses (231,232,233), but are increased by procedures which damage the gastric mucosa e.g. destructive doses of histamine in dogs and guinea pigs (231, 235,236) cincophen (23o), alcohol (237), and X-irradiation (238) but there is some direct variation with changes in basal secretion of pepsin (40). Measurement of changes in concentration in the gastric vein blood, which are much more subject to controlled alteration than in peripheral blood, indicates an increase in pepsinogen three to four hours after mecholyl stimulation (231) and four hours after large doses of histamine (236), i.e. at a time when the stimulation of gastric pepsin secretion has already subsided (156,236). It is also decreased by gastric mucosal cooling (239). Distribution of Pepsinogen in the Blood The concentration of pepsinogen is the same in plasma as in serum (40,220,226, 229), none being attached to fibrinogen or to euglobulin (226), and the levels are unaffected by the amounts of heparin used for anticoagulation (226). The blood cells in man contain pepsinogen, red cells 42

in about the same concentration as plasma, white cells considerably more (40). Pepsinogen in the white cells persists after gastrectomy, even when the plasma contains no enzyme (40). In the rabbit (240), rat (40), and, in one report, in man (241) white cells were found to contain no acid-active proteolytic activity. Passage of blood through the liver does not affect the concentration in the blood (236) arising from the stomach, and the lower level in peripheral venous blood is presumably due to dilution. Factors Influencing the Level of Plasma Pepsinogen wrrHIN INDIVIDUAL VARIATION. There is little (± 10 per cent) variation of pepsinogen concentration during the day (13o), from day to day (60,220,226,229, 230,242) and from month to month (229) . AGE AND SEX. There is a moderate increase in the first three decades of life after which the levels remain unchanged (229,243) or show a slight decline in old age (232). No significant sex differences have been noted (229,232,243). Meals, whether mixed or comprised solely of protein, carbohydrate, or fat do not affect the level of plasma pepsinogen within two and a half hours of eating (229, 232), but in a dog changed from bread to meat diet there is an increase (220). HISTAMINE. In man, histamine in the usual stimulant doses has no noticeable effect on plasma pepsinogen concentrations (40,226,2 32,235) and does not increase pepsinogen in the urine, even though it stimulates pepsin secretion three to four fold (see above). In guinea pigs, massive doses of histamine (75 mg./ Kg.) caused a great increase in plasma pepsinogen within four hours (235,244), but also caused gastric mucosal ulceration, and in the dog these doses of histamine (which killed all the dogs in from two to thirty-six hours) also increased

HIRSCHOWITZ

plasma pepsinogen (231). Smaller but renal clearance of pepsinogen during adstill excessive doses of histamine (2-5 renocortical stimulation (2 2 7) . mg./Kg.) may (236,245) or may not Ephedrine in man (252) or epinephrine (231) increase plasma pepsinogen in the in rats (25o) have no significant effect, or peripheral circulation. Because of the cause a small decrease (253) in plasma doses used, it is doubtful that these studies pepsinogen, but posterior hypothalamic imply anything more than destruction of stimulation in anesthetized cats caused an the gastric mucosa. The histamine libera- increase in plasma pepsinogen in two tor 48/80 increased plasma pepsinogen in peaks, possibly related partly to symrats more than ten fold in three hours pathetic stimulation either directly or after acute injections, but chronic admi- through the adrenal medulla (254) as nistration (four days) produced a fall judged by the simultaneous measurements of blood catecholamine levels. ( 246). BLOOD SUGAR. Insulin hypoglycemia has PREGNANCY. A small increase is noted little effect on plasma pepsinogen (226, in the last trimester with return to con232), despite adequate gastric stimulation. trol levels by 140 days after delivery Hyperglycemia produced by glucagon or (2 55)• PSYCHIC STRESS. In monkeys, seventyby I.V. glucose caused a fall in plasma pepsinogen which occurred in intact as two hours of psychic stress produced a suppression of plasma pepsinogen (and well as vagotomized dogs (247). urine pepsinogen) during the avoidance RENAL FAILURE. Pepsinogen accumulates rapidly in the blood of a dog following period with a post-stress rise. These ligation of the ureters (248) and after changes were in directions opposite to bilateral nephrectomy (231), but not changes in blood and urine 17-hydroxywhen the stomach has been removed. It corticosteroids (256). In rats, subjected is increased in the plasma of patients with to immobilization stress which produces uremia (40,220,248,249), roughly in pro- gastric erosions, there is no significant portion to the accumulation of urea or correlation between plasma pepsinogen and ulceration (2 57). non-protein nitrogen (1,233) ENDOCRINE INFLUENCES. In the peripheral blood in man, neither ACTH nor the adrenocortical steroids increase Relation of Plasma Pepsinogen to pepsinogen concentration in peripheral Gastric Pepsin Secretion blood whether given for eight hours inThere is a fair correlation between travenously or intramuscularly for as plasma pepsinogen concentration and the long as three weeks (130,205,227,250). maximum concentration of gastric pepThe same is true in rats (125) or dogs sinogen during histamine stimulation (z 3z) (251). However, when the measurement or with basal pepsinogen secretion (4o, is made in gastric venous blood, ACTH 132), especially in any one individual does increase pepsinogen concentrations (4o). However, the level of pepsinogen (231,251) and cincophen which produces may bear absolutely no relation to gastric gastric ulcers in dogs only increases secretion as shown in two instances after plasma pepsinogen in dogs with intact the development of steroid induced ulcers adrenals (251). The failure to find in- where plasma levels five to ten times concreased levels in peripheral blood after trol were found at a time when there was ACTH is probably due to the dilution in almost no pepsin in the gastric juice (124, the general circulation and the increased 205). 43

I / THE PHYSIOLOGY OF

PEPSINOGEN

Plasma Pepsinogen in Disease A number of clinical studies have appeared in the past few years and for the most part agree that there is an increased average level of plasma pepsinogen in duodenal ulcer, normal levels in gastric ulcer and normal to low values in gastric cancer. Duodenal Ulcer Mean levels of plasma pepsinogen are between one and a half and two times control values in reports from various parts of the world (40,226,232,234,242, 243,249,253,258,259,260,261) with no appreciable difference between the sexes, and no change with duration of disease (232,234), the state of acitvity or bleeding (232), with therapy or with healing of the ulcer (232,234,242), or following vagotomy (242). These findings agree with those for gastric acid or pepsin secretion (122), urinary pepsinogen (see beow), and with what is known of the pathologic physiology of duodenal ulcer. Experimental Ulcer and Ulcer Prediction Two studies in army recruits indicate that ulcers occur with greater frequency among those men with the highest plasma pepsinogen values (119,262) (or vice versa), while another study among recruits (263) finds plasma pepsinogen differences between ethnic groups, but no specific relation to social factors or incidence of ulcer. In two cases observed by the author in which adrenocortical steroids produced peptic ulceration in experimental subjects (124,205), the plasma (and urine) pepsinogen was not elevated before the ulcer developed but was increased aftenvards during healing. In animals, restraint ulcer in rats (257) cincophen ulcers in adrenalectomized dogs (251), Mann-Williamson ulcers in dogs and antrum to colon transplant ulcers of 44

the Dragstedt type (23 1) are not accompanied by any changes in plasma pepsinogen. It would seem to the author a little naive to speak of gastric secretion on the basis of plasma pepsinogen measurements, and even more so of the etiology of duodenal ulcer on this basis. Gastric Ulcer Even though most reports only contain a few patients with gastric ulcer, and no attempt is made to differentiate into those which are primary and those which are secondary to duodenal ulcer disease, gastric ulcer subjects seem to have statistically normal (40,232), or slightly increased (234,242,260) levels of pepsinogen in the blood. Gastric Cancer Most patients with gastric cancer have normal or slightly depressed levels of plasma pepsinogen (232,260,261,264) in contrast to the markedly suppressed values of gastric pepsin secretion (122, 218), seen in this disease. Pernicious Anemia Very low levels of plasma pepsinogen have been reported (220,226,265), and may precede the hematologic changes (5). Gastritis Variable levels are found in gastritis, depending on the cause and stage of gastritis. In acute damage, e.g. by alcohol or X-irradiation, values are high, in atrophic quiescent gastritis levels are low. Other Diseases Hiatus hernia, esophageal stricture (with or without ulcer), and bleeding

HIRSCHOWITZ

varices are not associated with changes in plasma pepsinogen (z 3 2) . In hyperparathyroidism, with or without ulcer disease, pepsinogen is increased in the plasma (233). In children, acute infections cause an increase in pepsinogen levels, and this is probably due to the fever (z66). In leukemia, pepsinogen is low and increases after x-ray therapy (267). Clinical Value of Plasma Pepsinogen Measurements As with most other tests of gastric function, plasma pepsinogen measurements are of limited diagnostic value. The relation to basal pepsin secretion, even at best (r = +.6o, (40) ), leaves 65 per cent of the derivation from regression unexplained and it gives no indication of the gastric response to stimulation. In about one-third of the patients with duodenal ulcer, plasma pepsinogen levels

clearly above 95 per cent of normal may be found, but it is in just this sort of patient where such a confirmatory test is redundant in almost every instance. In most reports, patients with gastric cancer have normal or only slightly depressed levels of pepsinogen and the test is in this case much inferior to most other gastric secretory tests. While in group studies they may possibly sort out the duodenal ulcer subjects, changes in the individual may actually follow the clinically obvious ulcer. As an index of stress or endocrine imbalance there are other and more direct means of discovery and measurement of the response. While many physiologically important questions remain to be answered in regard to the access of pepsinogen to the blood and its excretion, its clinical usefulness is so limited that the case for its abandonment as a clinical tool should be self-evident by now.

Pepsinogen in the Urine The presence of an acid-active proteolytic enzyme in the urine, described a century ago by Brucke in 1 861 (1 72) and named uropepsin by Benderskey (6) in 189o, has been the subject of intense study over the past fifteen years, and in the past five years at least fifty papers a year with major emphasis on plasma or urine pepsinogen have been published. Pepsinogen has been found in the urine of a wide range of vertebrates (5), and may be absent during hibernation in frogs and salamanders (z68). Frouin in 1904 (269) and Fuld and Hirayama in 1912 (270,271) showed that uropepsin derived solely from the stomach, and identified it by inactivation studies. Subsequent investigators amply confirmed these findings, and it is now generally held that pepsinogen is the only enzyme in the urine which is

proteolytic at pH 1.5-2.5. While pepsinogen is found in high concentration in semen (67) and human leucocytes (4o), the virtual absence from the urine after total gastrectomy makes it unlikely that extragastric sources contribute much, if any, pepsinogen to the urine. ;Mechanisms of Excretion of Pepsinogen by the Kidney Even though pepsinogen is a fairly large protein molecule (42,500), it is fairly readily excreted by the healthy kidney. In a group of young men with an average creatinine clearance or glomerular filtration rate (GFR) of 17o L/24 hours, the pepsinogen clearance was found to average 44 ± 14 L/24 hours, and, in the individual, to be relatively 45

I / THE PHYSIOLOGY OF PEPSINOGEN

constant during the day (13o) and from day to day (2 27) and varying with the GFR. Since the pepsinogen clearance is much lower than the GFR, pepsinogen is either filtered and reabsorbed in the tubule or is only partially filtered. Several facts tend to support the belief (5) that pepsinogen is filtered and reabsorbed by the tubule, as is the case with other and even larger protein molecules (272); thus pepsinogen excretion is increased by phlorizin (139) and during the start of a forced diuresis (256). Cross circulation from a nephrectomized uremic dog with high plasma pepsinogen to a gastrectomized dog with no plasma pepsinogen shows only a short-lived large excretion of pepsinogen in the recipient dog (231), experimental evidence against the suggestion by Gregor and Schiick ( 2 73,2 74) that pepsinogen excretion is non-threshold. The increased output during ACTH administration has been shown to be due to an increased clearance of pepsinogen, correlated with the increase in 17-hydroxycorticoids and related to the increase in GFR (227). By contrast the increased output in duodenal ulcer occurs with a high plasma pepsinogen and a normal renal clearance of pepsinogen (227). Studies of glomerular filtrate would rapidly resolve the question of whether pepsinogen is partly or completely filtered. Factors Affecting Pepsinogen Excretion AGE AND sEx. Premature infants excrete less pepsinogen than full-term infants, but when expressed per unit of body weight there is no difference (275). The amount of pepsinogen excreted increases from age three to maturity (276), but when expressed as a function of body surface area there is no difference between children and adults (277), the same being true of gastric pepsin expressed per unit of 46

body weight (izz). In adult life, excretion in males age i8-6o is higher than in all other groups (276,278,279) declining in the older age groups in men (276,279), whereas in females at all ages, from 18 to 1o0 years, there is no significant difference. Others have found no significant sex difference in normal controls (39, 28o). INDIVIDUAL AND DIURNAL VARIATIONS. A coefficient of variation of io per cent or less in day to day pepsinogen excretion in the individual has been reported in a number of studies (37,227,264,281). This constancy may be maintained for weeks or months (264,281). The constancy of pepsinogen excretion is independent of normal fluctuations in urine volume (37,264,281,282). In one group of twelve fasting normal students at rest, pepsinogen secretion varied by less than 12 per cent from one two hour period to the next during a fourteen hour day, despite, in some cases as much as an eightfold change in urine output from one period to the next (13o). t1-lany reports have indicated much larger variations from day to day—such variations suggest errors in urine collection or, more likely, in pepsinogen assay, and should be cautiously accepted. While a number of authors have reported about 20 per cent lower pepsinogen excretion at night (130,283,284), others have found no significant difference in controls (2 81) and a reversal, under mental stress, in members of a university boat-race crew (284). The diurnal rhythm has been compared and related to diurnal changes in adrenocortical secretions (283). Relation between Uropepsin and Gastric Secretory Activity Because so many assumptions have been made regarding gastric secretion from information derived only from urinary

HIRSCHO%VITZ

pepsinogen measurements, it is important to point out that no correlation between gastric pepsin secretion and uropepsin in the individual has been demonstrated under a variety of conditions. Those studies which do show a positive correlation between uropepsin output and gastric pepsin output (285,286), or concentration (219), are studies of groups of patients in which both hyposecretors and hypersecretors are included, and examination of the normal population between the two extremes shows poor correlation. Others have shown no good correlation between uropepsin and gastric pepsin or acid (1 30,256,287,288). In the individual, there is little correlation between gastric pepsin secretion and uropepsin, especially under the influence of gastric stimuli which increases gastric pepsin and not uropepsin and adrenocortical steroids which have the opposite effect (130). In two subjects recovering from steroid-induced peptic ulcer, gastric secretion of pepsin was very low and uropepsin very high (124,205), and similar discrepancies under other conditions have been reported from time to time (132). This lack of correlation in the individual would appear to invalidate any discussion of gastric secretion when only uropepsin or plasma pepsinogen have been measured. Gastric Secretory Stimulants and Uropepsin Excretion Meals per se have no effect on the amount of pepsinogen excreted in the urine (37,132,203,281,289,290). Pilocarpine (256) and histamine, in physiologically stimulant doses in man (235,291, 292) and in the cat, (293) do not affect uropepsin excretion. Caffeine has no effect in cats (293) or man (289) on urinary pepsinogen. Insulin hypoglycemia which stimulates gastric pepsin secretion

has no effect on uropepsin in the usual hypoglycemic doses (294), but when used for shock therapy in schizophrenics in doses of 80-900 units inconstantly increased uropepsin in subjects who also had an eosinopenia (295), the uropepsin change presumably depending on adrenocortical stimulation. Schizophrenics have no increase after electroshock therapy (295). Gastric Secretory Depressants and Uropepsin Excretion Vagotomy or atropine which cause a marked decrease in gastric pepsin secretion, do not generally decrease urinary pepsinogen excretion (256,296,297,298). Other anticholinergic drugs, probanthine and banthine, have been reported to decrease pepsinogen excretion (299), though others have been unable to confirm this (203,297,300). Hyoscine is without effect on uropepsin. Effects of Dietary Factors A high protein diet in contrast with a low protein diet has been reported to increase urinary pepsinogen excretion in man (37) and in the dog (264), though this could not be confirmed in infants (275) and in one adult eating 150 grams protein daily for seven days (301). Changes in pepsinogen excretion in animals (268) and in infants (228) have been interpreted as showing adaptive enzyme changes to feeding of various foodstuffs, both in quantity of uropepsin and in the nature of the substrate optimum of the excreted enzyme. There are no reliable recent studies on adaptive enzyme changes with more specific attention to assay conditions. Effects of Starvation Starvation is said to reduce the output of uropepsin (302). Peczenik (139) re47

I / THE PHYSIOLOGY OF PEPSINOGEN

ported that this reduction started after Lion between pepsinogen excretion and eight days and by sixteen days uropepsin urinary 17-hydroxycorticoids and 17was reduced by 5o per cent recovering keto steroids in Cushing's syndrome, in within two hours of the first feed to con- Addison's disease (308) and after the adtrol levels. In famine edema, pepsinogen ministration of ACTH (130,308), wheredisappears from the urine (256). Starva- as there is a poor correlation in healthy tion also reduces pepsinogen excretion in persons not receiving ACTH (284,308). dogs (264), in cats (303) and in rats (1 39). In Cushing's syndrome, there is often an In rats a pre-starvation diet of protein increased excretion of pepsinogen (296) prevents a reduction in uropepsin until and in Addison's disease a decrease which just before death from starvation, where- can be restored to normal by cortisone as if pre-fed with carbohydrate, uropep- (203,282,296). In hypopituitarism, the desin decreases early in starvation (139). creased excretion of pepsinogen can be It is not known what changes occur in restored to normal by ACTH or cortithe gastric mucosa in starvation, but only sone (296). While uropepsin excretion two of twelve patients with malnutrition has been reported to be increased in had a decrease in gastric acid or pepsin in chronic aldosteronism (309), an eight the study made by Fouts et al. (304). hour infusion of 400 mcgm caused a small but definite decrease in excretion (130). Desoxycorticosterone acetate (DCA) has Endocrine Influences no effect on urinary pepsinogen, and one THE ADRENAL CORTEX. ACTH in doses might suppose that the increased excresufficient to stimulate the adrenal cortex, tion in chronic aldosteronism is due to promptly and almost invariably increases renal tubular damage. uropepsin excretion in persons with inThough the changes in pepsinogen extact adrenals and stomach (31,202,203, cretion with glucocorticoid activity are 205,227,282,290,305,306,307). Such in- consistent enough, it still requires intactcreases occur within four to six hours ness of stomach, adrenal glands and kidafter the start of an ACTH infusion neys to give a response to ACTH, and (130). As expected, hydrocortisone in- probably other mechanisms are involved creases uropepsin excretion, whether as well. It seems a little roundabout to given orally (113,203) or intravenously measure adrenocortical responses by uro(130), though the effect is not as con- pepsin excretion, when direct measuresistent as it is with ACTH. Cortisone has ment of steroids in blood or urine make an even less consistent effect than hydro- the measurement directly and more precortisone ( 203,278,282). Prednisone in- cisely, or total circulating eosinophils increases the excretion of uropepsin (13o, directly but at least more so than uropep203) while corticosterone, either orally sin. It seems to me hardly defensible to (113,203) or intravenously (13o) pro- equate "stress", "gastric secretion", "pituduces a small and variable increase. The itary-adrenal axis" and the "etiology of changes in pepsinogen excretion have duodenal ulcer", (to name but a few of shown a negative linear correlation with the extrapolations appearing in print) circulating eosinophils (130) during the with an increase in urinary pepsinogen ACTH or adrenocortical steroid adminis- excretion. tration, and are apparently related to an OTHER ENDOCRINE INFLUENCES. The increased renal clearance of pepsinogen low levels or urinary pepsinogen in (130,227). There is also a positive correla- myxedema (203) which can be increased 48

HIRSCHOWITZ

by ACTH or thyroid replacement (310) lation with urinary corticoid excretion. are not mirrored by an increase in thyro- Patients with anxiety and schizophrenia toxicosis (296). No increases have been have uropepsin levels midway between noted in pepsinogen excretion in man or normal and duodenal ulcer (321); schizorats after administration of thyreotropin, phrenics have also been reported to have gonadotropin, desoxycorticosterone normal uropepsin (176). The lack of cor(DCA), growth hormone, adrenalin, relation during stress between uropepsin adrenal and testicular androgens, proges- and corticoid excretion (256,320,322) terone or estrogen (203), norethandrolone suggests alternative pathways for the in(311) or noradrenalin, though one report crease. Some observers have found an claims an increase with adrenalin (312). increased urine volume associated with an PREGNANCY. There is an increase in uroincrease in urinary pepsinogen (256,317, pepsin in pregnancy with a peak at four 318) and it is not clear whether the supmonths (313), and a progressive increase pression of antidiuretic hormone plays a to term (302,313,314) with a return to role in these changes, though it is possibly control levels within ten days post par- involved in some way (315). Unfortuntum. In hyperemesis gravidarum, there is ately few, if any, reliable studies are availa diminished output (313) but in toxemia able on the simultaneous effect on gastric of pregnancy a significant increase (314). secretion, and therefore the significance Uropepsin is not abnormally increased in of changes in uropepsin excretion, related those pregnant women who have gastro- to mental stress, are obscure. intestinal symptoms (314). Physical Stress Psychological Influences Mental stress has been reported to increase the excretion of pepsinogen either during the stress (315,316) or after the stress (256,317,318). The study of Mason et al. (256) in monkeys showed an inverse relationship between uropepsin and 17hydroxycorticoids in the blood during and after stress. Hunter (319) found no increase in uropepsin in servicemen subjected to three days of emotional stress. In a study of six subjects under psychiatric observation for two years and in eighteen healthy men keeping diaries (316), increased urinary excretion was related to oral-receptive tendencies and occurred in situations which threatened or mobilized the individuals unconscious wish for love but not from any conscious conflict. In a one year study of a manicdepressive psychotic (32o), there was only a slight increase in excretion associated with the manic phases but no corre-

In athletes, physical exertion per se may (307,323) or may not (284) increase urinary pepsinogen excretion with a decline if exertion is continued for several days (323). Pyrogen administration produces a biphasic curve, first increasing, and then decreasing pepsinogen excretion (307). Acute stress is more likely to increase urinary pepsinogen than chronic stress (203), but most operations other than gastric operations, which cause a steep increase in pepsinogen for five days (1), generally do not increase excretion (1,278, 319), though cerebral trauma and fractures (324) and very extensive operations, e.g., pneumonectomy (325) may cause increases in pepsinogen excretion inversely proportional to the blood eosinophil count (324), and thus presumably related to adrenal glucocorticoid secretion. The stress of hemorrhage in cirrhosis of the liver or in peptic ulcer disease (296) apparently does not increase pepsinogen 49

I / THE PHYSIOLOGY OF PEPSINOGEN

excretion. The combination of fatigue and mental stress, such as in long operational air force flights causes an increase in uropepsin excretion and a fall in eosinophils (326), suggesting that the fatigue is the important element. Following myocardial infarction, uropepsin excretion increases two to five times, and rises in parallel with blood transaminase (327). In rats, immobilization which causes gastric erosions greatly increases urinary pepsinogen but not gastric pepsin secretion (325). Urinary Pepsinogen in Upper Gastrointestinal Disease GASTRIC OPERATIONS AND IRRADIATION.

There is a very steep rise, often to ten times control values from the second to fifth day after partial gastrectomy for either gastric or duodenal ulcer (203,278, 2 97,302,319). The rise is not immediate, indicating that it is not due to handling of the stomach, but starts twenty-four hours after operation, and reaches a peak on the third or fourth postoperative day. At the same time, plasma pepsinogen levels and renal clearance of pepsinogen are moderately elevated, (1) probably resulting from adrenocortical stimulation (227). The large amounts of pepsinogen released into the circulation therefore probably come from disintegrating peptic cells at the suture lines or in the remaining mucosa which had been traumatized at operation. Vagotomy alone does not affect urinary pepsinogen (296,297), and the late result of partial gastrectomy is a reduction in urinary pepsinogen (296,297), except in the group of patients who have marginal ulcers, where uropepsin is above control values (291,328). By contrast with gastric operations, other types of surgery cause a small or no increase in pepsinogen excretion (see above). 50

Between one and eight weeks after therapeutic X-irradiation of the stomach for peptic ulcer there is a five-fold increase in urinary pepsinogen while gastric secretion of acid and pepsin is much diminished (238). It thus seems likely that pepsinogen is released from degenerating cells, because when gastric secretion is recovering, urinary pepsinogen returns to normal. DUODENAL ULCER. The average excretion of pepsinogen has been found to be one and a half times normal in patients with duodenal ulceration (30,39,203,278,285, 301,310,329). As with any other gastric secretion tests, there is considerable overlap with the normal values, and consequently the measurement of uropepsin is only an occasionally helpful diagnostic test. In some studies, females with duodenal ulcer have been found to excrete normal amounts of pepsinogen (39,302), while others find that this group excretes less than males but more than controls (296). Such a sex difference has been denied (28o). The amount of pepsinogen excreted has been reported to increase with the duration of the disease (39), but this finding could not be confirmed in another study (28o). The age of the patient bears no relation to the uropepsin excretion (39,203). Some authors (280,330) have reported increased values in bleeding or otherwise active duodenal ulceration, while others could not confirm this (296). Thus uropepsin changes in duodenal ulcer are very much like those found in gastric secretion and plasma pepsinogen. BENIGN GASTRIC ULCER. Most patients with benign gastric ulceration excrete normal amounts or a slightly increased average amount of uropepsin (39,278,280, 285,301,310,331). In those reports in which the excretion of pepsinogen has been related to the site of the ulcer, those which occur beyond the angulus were

H IRSCHO W ITZ

associated with a higher output of pepsinogen (39,310), and are probably those antral ulcers which are secondary to chronic duodenal ulcer disease. Lesser curvature, i.e. "primary" gastric ulcers, all had uropepsin in the normal range (39,310).

The Anemias PERNICIOUS ANEMIA. Little or no pepsinogen is found in the urine of patients with pernicious anemia (280,285,310,3 29, 3 34, 335), and these levels are unaffected by cortisone or ACTH (282,336), by liver extract, vitamin B,: or intrinsic factor therapy (336). Absence of uropepsin helps to distinguish pernicious anemia from simple achlorhydria (337), though presumably vitamin B„ absorption studies would do it more directly.

STOMAL OR JEJUNAL ULCERATION is associated with uropepsin values which are somewhat above normal, but significantly greater than in those postgastrectomy subjects without ulcer (1,291,296). Both NON - PERNICIOUS MEGALOBLASTIC ANE the recurrent ulceration and the high uropepsin indicate inadequate gastrectomy; AIIA. In sprue with anemia, uropepsin is it is unlikely that the ulceration produces normal (1,219,335), whereas in patients with diphyllobothrium latum anemia the high uropepsin. (336) about half have normal and half CANCER OF THE STOMACH. The great depressed pepsinogen excretion. In five to interest in uropepsin around the turn of ten days after expulsion of the worm, half the century centered mainly on a simple the treated cases have an increase in peptest for cancer of the stomach (5), but sinogen. more recent studies have shown that IRON DEFICIENCY ANEMIA. In those pamany of the patients afflicted with gastric tients with iron deficiency anemia able to cancer may have normal amounts of pep- secrete free acid after histamine, both sinogen in the urine, but that a signifi- gastric and urinary pepsin are normal cant number excrete less pepsinogen (219) whereas those achlorhydric to histhan the lower limits of normal subjects tamine excrete significantly depressed (310), levels which are generally propor- amounts (219,280), with some as low as tional to the ability to secrete acid (302), in the pernicious anemia range. Those paa factor which may be important in re- tients achlorhydric to o.5 mg. histamine sectability. Fuld and Hirayama (271) re- but not to 2.0 mg. have more uropepsin ported an inhibitor of pepsin in the urine than those that remain achlorhydric even of patients with gastric cancer. This has with the larger dose of histamine (z8o). not yet been confirmed or denied. Exfoliative Dermatitis GASTRITIS. Because gastritis denotes many different things, and the diagnosis In five patients with generalized exfoliof the exact type of gastritis is still diffi- ative dermatitis, uropepsin excretion was cult, interpretation of uropepsin data as low as in pernicious anemia (1). Two among this group of disorders is not easy. of these patients studied some months laIn gastric atrophy or atrophic gastritis ter in remission excreted normal amounts pepsinogen excretion is depressed (332). of pepsinogen. A third receiving 100 mg. In superficial gastritis, uropepsin may be ACTH for five days showed neither clinidepressed, normal or elevated, especially cal improvement nor increase in uropepin alcoholic gastritis (237). Hradsky et sin. al. (333) in eighty-seven patients found no correlation between mucosal histology Miscellaneous Influences DISEASE. Normal values are found in and uropepsin excretion.

51

I / THE PHYSIOLOGY OF PEPSINOGEN

hypertension (338), tuberculosis (279), sease, (chronic cholecystitis) (340). In rheumatoid arthritis (276), adult diabetes measles, uropepsin is increased (341). (276), but uropepsin is elevated in juveDRUGS. Rauwolfia (338) and chlorpronile diabetics proportionally to the dura- mazine (312) do not influence uropeption of the disease, being more than twice sin excretion, though Rauwolfia does control values in those having diabetes stimulate gastric secretion. Salicylates inover ten years (339). In sick cirrhotics, crease uropepsin excretion (342) in noruropepsin excretion is depressed (66), and mal subjects. In frogs small doses of may return to normal with improvement. uranium nitrate, a renal tubular poison, In hepatitis, uropepsin is depressed in the increases uropepsin (139), whereas large initial stages, as it is in gall bladder di- doses suppress excretion.

Summary Pepsinogen, the precursor of the proteolytic enzyme pepsin exists in the peptic cell, and is found in the blood and other body fluids. Conversion of pepsinogen to pepsin occurs in solutions more acid than pH 6.o by an autocatalytic process. Pepsin acts on a wide variety of substrates and at varying pH, indicating its relative non-specificity. The actions of pepsin can be divided into substrate denaturation and proteolysis; pepsin is one of the few enzymes which require no cofactors (unless it be hydrogen ions) and there are no known accelerators of peptic action except prior denaturation of substrate. Inhibition of peptic action may be brought about either by destruction of pepsin or by true inhibition. The peptic cell possesses characteristics common to zymogen secreting cells. The granules of the peptic cell contain pepsinogen and are best stained by crystal violet. The embryologic development of peptic cell varies considerably between species. In the human, zymogenic granules and peptic activity appear between four and eight months. The peptic cells are thought to differentiate from mucus neck cells. The weight of evidence favors RNA as the site of synthesis of pepsinogen with transport to the granules action in a stor52

age capacity. Synthesis is normally a continuous process, well correlated with the secretion. Thus under basal conditions the store of pepsinogen is almost full and remains constant. The secretion is part of the secretion of gastric juice, the maintenance of it depending on cholinergic mechanisms. However, in part at least, the secretion of pepsinogen occurs independently, when the peptic cells fill up and discharge granules to accommodate newly cynthetized proenzyme. In most species, cholinergic mechanisms are the strongest stimuli of pepsinogen secretion. However, other stimuli such as histamine in man and intestinal phase of gastric secretion in dogs are also capable of inducing this effect. Inhibition of pepsin secretion may be 'produced by reduced synthesis, in adrenal or pituitary insufficiency, when RNA is decreased. Other decreases are related to an over-all effect on gastric secretion. Duodenal ulcer patients have been found to secrete more pepsin because of greater secretory volume; in gastric ulcer cases, the volume and composition of gastric juice is the same as in normal subjects. Pepsinogen is present in the blood in small but significant and relatively constant quantities; the concentration is the same in plasma as in serum and red blood

HIRSCHOWITZ

cells; white blood cells contain consider- found in the urine which is proteoly tic at ably more. There is little variation in pH 1.5-2.5. Urinary pepsinogen excrepepsinogen concentration during the day. tion is increased in duodenal ulcer and There is a fair correlation between plasma decreased in gastric cancer but neither and gastric pepsinogen concentration dur- finding is consistent enough to provide ing histamine stimulation or under basal great clinical usefulness. Urinary pepsinoconditions. However plasma pepsinogen gen level changes have been also reported measurements are of limited diagnostic in a number of diseases of upper gastroinvalue, and give no indication of the gas- testinal tract; pernicious anemia and other tric response to stimulation in the ma- miscellaneous influences, such as juvenile jority of cases. diabetes, hepatitis and gallbladder disease. Even though pepsinogen represents a ACKNOWLEDGEMENT fairly large protein molecule (42.500), it Table I adapted from Green, N. M., is fairly readily excreted by the healthy and Neurath, H. (Ref. 48), with permiskidney. Pepsinogen is the only enzyme sion of the publishers.

References B. 1. A Study of the Physiology of Pepsinogen in the Human. M.D. Thesis, Witwatersrand University, Johannesburg, 1953. 2. SCHLAMOWIT'L, M. and PETERSON, L. U. J. Biol. Chem., 234: 3 1 37-3 147, 1 959. 3. LANGLEY, J. N. J. Physiol. (Lond.) 3: 269291, 1881. 4. LANGLEY, J. N. and SEWALL, H. J. Physiol. (Lond.) 2: 281-301, 1879. 5. HIRSCHOWITZ, B. I. Physiol. Rev., 37: 475511, 1957• 6. BENDERSKY, J. Virchow Arch. Path. Anat. 121: 544-597, 189o. 7. HERRIOTT, R. M. J. Gen. Physiol., 21: 501540, 1938. 8. NORTHROP, J. H., KUNITZ, M. and HERRIOTT, R. M. Crystalline Enzymes. (2nd ed.) N.Y. Columbia Univ. Press, New York, 1948. 9. VAN VUNAKIS, H. and HERRIOTT, R. M. Biochim. Biophys. Acta, 22: 537-544, 1957. 10. HERRIOTT, R. M. J. Gen. Physiol., 24: 3 25338 , 1 941 . 11. HERRIOTT R. M., BARTZ, Q. R. and NORTHROP, J. H. J . Uken. Physiol., 21: 575-582, 1938. 12. NORTHROP, J. H. J. Gen. Physiol., 16: 61562 3, 1933. 13. ANSON, M. L. and NORTHROP, J. H. J. Gen. Physiol., 20: 575-588, 1939. 14. DESREUX U. and HERRIOTT, R. M. Nature, (Lond.) 144: 287-288, 1939. 15. NORTHROP, J. H. J. Gen. Physiol., 16: 33-58, 1932. ,6. FUNATSU, M. and TOKUYASU, K. Proc. Japan. Academy, 35: 1 39-143,1959. 17. FUNATSU, M. and TOKUYASU, K. J. Biochem. (Tokyo) 46: 1441-1451, 1959. 18. PERLMAN, G. E. Nature (Land.) 173: 406, 1954. 1. HIRSCHOWITZ,

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Reumatismo, 1 1: 258-266, 1956.

Secretion of Gastric Mucin

Klaus Heinkel Gerhard Berg*

DESPITE decades of research work on the position of specific mucoid substances resecretion of gastric mucin, the composi- presents a pathogenic factor in the inception of mucoid substances and their tion of inflammatory or ulcerogenic changes under pathological conditions re- changes of gastric mucosa. The present main largely unknown. Although some of paper will necessarily deal only with the the physiological functions of mucin have fragmentary knowledge available at prebeen elucidated, it is not yet possible to sent, and point out certain avenues for state whether the occurrence of abnormal future investigation. mucoid components or a change in corn-

Mucin-Secreting Cells It has been known for a long time that the gastric mucosa is covered by visible mucus. One can remove this layer only incompletely because of its close contact with the epithelium. The clinician knows that gastric secretion contains flocculent, gelatinous or fluid mucus. These substances were supposed to originate in the goblet cells. With the introduction of more refined techniques in histochemistry for the detection of mucoid substances (polysaccharides, mucopolvsaccharides and mucoproteins), the occurrence of such components in the cells can be ascertained and their site of origin inferred. We do not intend to discuss the well

known anatomical pictures seen in cadavers or tissues obtained from gastrectomies, but rather the results of recent research work, in order to obtain a better understanding of the mucin-secreting elements of gastric mucosa under normal and pathological conditions. Gastric suction biopsy is undoubtedly the method of choice for obtaining material for histological examination (1,i,3). By application of staining methods, which comprise oxidizing reactions as part of the procedure (such as the periodic acidSchiff technique), dark blue to violet stained cells, or parts of cells, can be observed in the gastric mucosa (q.). (Fig. I ).

From the Medizinische Universitets Klinik, University of Erlangen, Germany, (Director Prof. Dr. N. Henning). 59

FIG. t. Biopsy specimen of normal gastric mucosa from the fundic area stained with periodic acid-Schiff reagent. H & E x too.

FIG. 3. Mucous-producing glands in the fundic area in a case of atrophic gastritis (Section of a lymph follicle visible in the lower field). H & E x too.

FIG. 4. Appearance of surface epithelium in a case of intestinal metaplasia (gastric biopsy specimen) H & E x 800.

HEINKEL & BERG

A well-defined, intensively colored zone numbers (6). (Fig. 2). Thus it may be in the surface epithelium is always pre- inferred that other cells also secrete gassent. The cells on the bottom of the tric mucin, the cardiac and pyloric glands foveolae do not produce substances which being the main producers. With Haemareact in this way in detectable quantities. toxylin-eosin-staining, these latter cells Further treatment with colloidal iron ac- show a typical picture. The cytoplasm cording to Muller(5), produces in over 90 appears optically as an empty space, with per cent of cases a typical blue stain (I o2 the nucleus positioned at the base of the from I Io cases). Alcian blue 8-B-S seems cell. These cells are easily recognized to react only with a part of the PAS- without histochemical staining (7). The stainable substances. This stain was posi- cells which appear to be optically empty tive only if goblet cells were present in with H & E staining show intensive the gastric mucosa; slides without goblet PAS-staining, and thus contain increased cells showed minimal staining. In the sur- mucoid substances; this suggests that these face epithelium of normal and pathologi- glands have mucus-producing properties cal mucosa, we could find only traces of (Fig. 3). The accumulation of these the dye on staining with mucicarmine, glands in the normal stomach towards but the goblet cells and their secretion the cardia and pylorus demonstrates their showed the typical red color (4). functional importance. In pathological Until recently, mucicarmine has been conditions, formations of these cells apthe classical staining method for mucus. pear also in the fundic area (8). In chronic This method induced earlier investigators gastritis, the number of goblet cells found to believe that goblet cells are the main appears to be increased. This transformasite of origin of the gastric mucus. Mo- tion of the mucosa results in a change dern biopsy investigations demonstrated of function: there is a diminution of pephowever, that the normal gastric mucosa sin, cathepsin and acid secretion with a does not contain goblet cells in noticeable marked increase in production of mucoid substances. A small number of cells with a positive NUMBER OF EXAMINAT ONS PAS-reaction can also be found in normal 975 972 403 331 452 100mucosa. (Fig. 1). The biopsy material did not demonstrate higher concentrations of mucoid substances in the chief 75 and neck cells (4). Several approaches have been used in 50 studies of gastric mucoid substances and their distribution in the gastric mucosa. 25Earlier work by Zimmermann (q) indicated that the region of the glandular neck has a high regenerating activity. NI I NORMAL SUPERF CIAL A TROPIC Florey (I o) had remarkable results with GASTRITIS MUCOSA GASTRITIS the injection of Na_S'O,. He found S' in the mucus of goblet cells of all animals -% OF CASES IN WHICH GOBLET CELLS WERE FOUND ■ examined. In the surface epithelium of the FIG. 2. Occurrence of goblet cells in the fundic stomach, he could not find radioactivennscosa in cases of gastritis (3,136 examinations). labelled substances. Some of the species Histological diagnosis was made on specimens showed S35-labelled compounds in the obtained by gastric suction biopsy. 41.

6i

I / SECRETION OF GASTRIC MUCIN

basal area of the foveolae, but the mucoid neck cells were free of S'". Similar examinations using S'sO. have been performed with the same results by Cornet et al.( 1). The authors prepared autoradiograms from biopsy material obtained from human stomachs. The distribution of amino acids marked by S' was studied by Maurer (12). Using autoradiography he found the region of the foveolar neck especially photoactive, indicating that this area has a high amino acid uptake. Glass (13) et al., applying chemical analyses, suggested that the glandular mucoprotein secretion originates in the neck cells. This glandular mucoprotein is separable from other substances of the gastric secretion and also from visible gastric mucus, which is produced by the surface epithelium of the entire stomach. Summarizing the results of studies on the origin of the gastric mucus, by gastric biopsy, it can be stated that certain cells of the stomach show typical histochemical reactions (4). The surface epithelium contains a clearly demarcated layer of mucoid substances with a high content of

protein, demonstrated by standard staining methods (Haemotoxylin-eosin). It is probable that this layer is identical with the visible mucus. A few cells distributed in the gastric mucosa are oxidized and stained by PAS; one can assume that some production of mucus also occurs here. The main part of the gastric mucus seems to be produced by the cardiac and pyloric glands. In cases of chronic gastritis, one will find this type of gland in the fundic area also (14). In extensive atrophic gastritis, the mucoid glands can be found distributed all over the gastric mucosa. Because this transformation is the result of an inflammation, it must be of a metaplastic nature (8). Goblet cells appear in less than one per cent of cases with normal gastric mucosa (6). Thus they cannot be the sole site of origin of gastric mucus. On the other hand an increased number of goblet cells is present in chronic gastritis; this is a so-called "goblet cell metaplasia." The final stage of these changes appears to be "intestinal metaplasia", with a histological picture similar to the intestinal mucosa.

The Secretory Process The activity of single cell types has not been sufficiently investigated to warrant definite conclusions concerning the exact biochemical nature of their secretory product. It appears that at least three sources of mucin exist in the stomach: a) superficial mucous cells lining the gastric surface and pits; these are similar throughout the organ and secrete mucin constantly (15,22), the exact rate of secretion being unknown; b) mucous neck cells which occur in the neck portion of the gastric glands of the fundus; c) mucus-producing glands of the cardiac and pyloric regions. The mucus of the surface epithelial cells 62

and that of mucoid neck cells stain differently, for example with periodic acidSchiff reagent the surface mucous cells stain deep purple, while the mucous neck cells show paler coloration. In cases of the so-called "intestinal metaplasia" of gastric epithelium, goblet cells become more numerous and constitute another source of secretion of mucoid substances ( Fig. 1 ). Much of the original knowledge of the secretory process of mucin-producing cells is based on the work of Sir Howard Florey (16). It is now currently believed that synthesis of mucoproteins takes place in the basophilic substances of the cyto-

HEINKEL & BERG

plasm called ergastoplasm. This material is then transported toward the Golgi zone of the cell where it takes a granular configuration. These mucigen granules migrate then toward the apical portion of the cell, where they are stored for an unknown period of time. Finally, the granules are extruded at the apical pole of the cell. Factors influencing mucin secretion affect differently various types of mucinproducing cells. Neurogenic and hormo-

nal stimuli cause emptying of mucous neck cells, but do not affect the surface epithelial cells (15,17). This effect may be a direct one, but it is more likely that it is due to the compression of the mucous neck cells by contraction of the smooth musculature. Such contraction will obviously have no effect on the surface mucous cells. Irritative stimuli, such as local application of alcohol or chemical compounds affect the discharge of mucin from superficial cells.

Correlation of Biopsy Material and Chemical Analysis of Gastric Mucin It is difficult to define human gastric mucus exactly. Under the heading gastric mucus, one can include all viscous secretions of the mucosa, as well as the viscous products of enzymic activity. It has been shown that the mucous substances of the stomach are glycoproteins. Recent results of research on protein chemistry show that nearly all proteins contain some carbohydrate (18). Mucoproteins differ in the proportion of carbohydrate in the total molecule. There is no well defined boundary between glycoproteins and mucoproteins. A large spectrum of substances containing different quantities of protein and carbohydrate is to be found in gastric secretion. The proportion of these constituents is responsible for its physical characteristics. According to the above definition, viscosity is the most important property. Physical measurements will give only composite data. According to Bucher (19), freshly secreted mucus is acid to litmus. However, Hollander (zo) and many others have shown that this mucus is slightly alkaline. In the secretory cells, its pH is probably the same as that of serum. Freshly secreted gastric mucus is viscous

in the pH range of 4.5-8.0 with a maximum of about pH 5.0. At lower pHvalues, the mucus is precipitated, and gastric juice loses its viscosity. Viscosity measurements are not diagnostically important, as the data from normal and pathological conditions are not yet exactly known. However, more attention should be paid to such determinations because of the growing interest in mucoviscidosis (21). In this disease, an abnormal highlyviscous secretion of all mucus-producing glands is supposed to be the cause of alterations in different organs. Use is made of solubility differences in order to fractionate the soluble gastric mucins. Glass et al. (13,22) separated visible mucus and soluble mucus. The latter was divided into three further fractions, soluble mucin, glandular mucoprotein and soluble mucoproteoses. Glass suggests that mucoproteose is a product of the autodigestion of visible mucus. Electrophoresis is widely used in the analysis of mucoproteins of gastric juice. While there are a large number of reports about the protein components of gastric juice (23,24), only a few data are given on the carbohydrate part of the muco63

POINT OF APPLICATION

si SERUM

GASTRIC JUICE

POINT OF APPLICATION

FIG. 5. Inzmuno-electrophorograms of serum and gastric juice (33).

proteins ( 27,28,29,30,3 1 ) . Diffuse alteration of gastric mucosa, as in gastritis, causes changes in the protein content, and also in the number of fractions obtained on electrophoresis. The findings of Henning, Kinzlmeier and Demling (23) in atrophic gastritis are characteristic. Some of the protein in gastric juice originates from serum as shown by experiments with I131-marked serum albumin (25). Cases with atrophic gastritis showed a high counting rate in the juice. Exudative gastropathy showed that a considerable amount of albumin passes into the gastrointestinal tract (3 2) . Both protein and carbohydrate determinations are necessary for mucoprotein analysis. The best method for the detection of carbohydrates is the periodic acidSchiff-stain after paper electrophoresis. Deviations from the normal are less likely to be caused by disturbed function than by alterations or the morphological structure, with or without clinical symptoms (26). Proteins with both low and high amounts of carbohydrate can be demonstrated in gastric juice, if a sensitive method is used. By staining the glycoproteins after electrophoretic fractionation, six to eight peaks appeared (33,34,35). The fast migrating proteins are for the most part rich in protein and poor in carbohydrate. This fraction includes intrinsic factors and pepsin (36). According to available information, the slowly migrating fractions have no group with a specific biological function. A large number of individual mucoprotein components can be observed by immunological analysis. Götz, Scheiffarth 64

and Dübeler (37), and Fasel and Scheidegger (38) found up to nine groups of mucoproteins in the gastric juice, using immuno-electrophoresis (Fig. 5). A correlation is evident between the results of biopsy-morphological examinations of the gastric mucosa and the glycoprotein-stained electrophoretic fractions of gastric juice (26) (Fig. 6). In superficial gastritis, glycoprotein content increases, and more and better defined glycoprotein-containing fractions appear (39)• In cases with atrophic gastritis, there are also deviations from normal. A high albumin content is a prominent feature. Glycoproteins are increased but not as markedly as in superficial gastritis. These results are also found by other investigators (27,36). Variations in the results can be explained on the basis of the different methods used. Analysis of the composition of the carbohydrate moiety, in particular, gives further information on the structure of the mucins. Glass (35) determined the amount of hexose, hexosamine, fucose, sialic acid and sulfate in electrophoretic fractions. Hexose concentration ranged from S4 per cent in the fast-moving components to 67 per cent in the more slowly migrating fractions. Hexosamines range from 4.8 to 15 per cent in the same order. Fucose rose from 4.8 in the fast migrating fraction to a maximum of 22 per cent in the fraction M_. Sialic acid concentration was in inverse relation to the hexose. The fraction with the highest content of hexose (slow migrating fraction X) had the lowest concentration of sialic acid ( c per cent), and conversely the fastest migrating fraction P had the highest concentration of sialic acid (38 per cent). The presence of the acidic groups of sialic acid in proteins increases their mobility in electrophoresis (34). A simple method has been developed to assay the mucoprotein content of mucin

A

B

A

B

III

II samples from patients with superficial gastritis, atrophic gastritis, and from subjects with a normal mucosa, as evidenced by gastric biopsy (26). The method is based on the well-known reaction of galactose and mannose treated with orcinol in concentrated sulphuric acid. Mannose shows a higher light absorption than galactose at 425 mu while the reverse is true at S40 mu. The optical densities of mucin treated with orcinol —H_SO, were measured at these two wavelengths, and a quotient E 425 mu

E S40 mg was calculated. Patients with normal mucosa gave a mean quotient of 0.93 and with superficial gastritis o.75. The quotient for cases of atrophic gastritis was similar to that of normals. These results indicate that there is a change in the composition of the mucins accompanying alterations in the gastric mucosa. Glucosamine and fucose concentrations are higher in cases with superficial gastritis (39,4o). In cases of atrophy

FIG. 6. Comparison of histological findings and electrophoresis of gastric juice. A. Protein stain. B. Glycoprotein stain. I Normal mucosa. lI Superficial gastritis. Ill Atrophic gastritis.

of the parenchyma, mannose and other hexoses constitute a large portion. It is not known which type of gland is responsible for these phenomena. Further investigations on larger numbers of patients is in progress, using morphological examinations together with chromatographic analysis of gastric juice. The secretion of glycoproteins and mucoproteins is supposed to be constitutionally determined, as is evidenced by examination of the blood group factors. Individuals whose gastric juice and saliva contain these factors are called "secretors". It is too early to say whether constitutional characteristics influence the secretion of proteins with a higher proportion of carbohydrate into the stomach. In these investigations, one should not overlook the high percentage of chronic gastritis diagnosed by gastric biopsy in cli-

FIG. 7. Protein and carbohydrate content of gastric juice in cases of gastritis Normal mucosa Total Protein mg/% Total Hexose mg/% 425 mµ 540 my Quotient 425 mu 540 my

280

a 30

230.8 245.8 0.93

114 153

13

Superficial gastritis 280

30

15 391.3 13 518.3 0.75

183 282

n

Atrophic gastritis

18 270

23 298.2 19 335.3

n 20

8

129 173

12 10

0.93

65

I / SECRETION OF GASTRIC MUCIN

nically healthy persons (41). Today the basis for such analysis should be a biopsymorphologic examination of the gastric mucosa. Only after considering these

findings, can constitutional factors responsible for the mechanism of secretion be evaluated.

Mucous Barrier It was very natural to conclude that the are not able to influence the actual pH visible mucous layer in the stomach has a value of gastric juice (44,45,56). We have studied histologically the protective activity against peptic digesstainable mucoproteins of the surface epition. Hollander (42) pointed out the in- thelium and of the gastric glands in cases teresting and little known dissertation of of gastric ulcer and duodenal ulcer. In 22 Glover (43) who suggested in 1 Soo .. . per cent of eighty-three patients with "it must likewise defend the internal sur- gastric ulcer, no changes were observed; face of the stomach and intestines, from 49 per cent showed slight alterations, and the action of gastric juice...." Hollander 29 per cent exhibited severe changes. (42) supported this thesis, believing that judging only patients with gastric ulcer, the layer of mucus constitutes the first we found normal appearance in 19 per line of defence and the layer of columnar cent of these cases and pathological and cuboidal cells of the surface and changes in 3z per cent. We considered as crypts constitutes the second defence normal a zone which takes uniformly a line. Our biopsy examinations (5,80o moderate to intensive stain following cases) do not show a marked layer of treatment with the periodic acid-Schiff mucus acting as the first barrier against reagent. (Fig. 1). The mucous layer was autodigestion. Seldom is there a mucous sharply demarcated toward the gastric layer in biopsy material that has the lumen and also toward the base of the character of a second epithelial layer de- cells. We considered goblet cells in the scribed by Duchenne-Aran. The mucus surface epithelium as pathological. In a portion of the columnar epithelial cells group of patients with acute gastric ulcer, can be clearly demonstrated. In super- we found atrophic gastritis in 45 per cent, ficial and atrophic gastritis, the mucous superficial inflammation in 42 per cent, layer is very often minimal, sometimes and a normal gastric mucosa in 13 per even missing in small areas (Fig. 9). De- cent of cases. In duodenal ulcer cases, the spite this, there is no colliquative necrosis results were different, especially where as an initial stage of peptic autodigestion. acute ulceration had occurred: 51 per This is true even in cases with high peptic cent were normal, 46 per cent had slight activity in the gastric juice. Such a col- alterations and 3 per cent severe changes liquative necrosis would prevent further in the mucous layer of the surface epienzymatic attack on the tissues. thelium. The routine histological diagAcid conditions only arise on the sur- noses showed normal findings in 49 per face and not within the mucosa. It has cent, superficial inflammation in 40 per been suggested that gastric mucin exerts cent, mostly slight, and atrophic gastritis a protective effect by a buffering action, in II per cent. By comparing the frebut most authors now agree that mucus quency of histological changes in gastric has a low buffer capacity. Mucoproteins ulcer cases and those with a duodenal 66

FIG. 8. Gastric biopsy from fundic area. Superficial gastritis with a mucous layer on the surface PAS Stain x too.

FIG. 9. Superficial chronic gastritis with alterations of the surface epithelium and minimal defects of the epithelial layer. PAS Stain x i80.

FIG. to. Pseudo pyloric glands in the fundic area— atrophic gastritis diagnosed by suction biopsy. PAS Stain x too.

I / SECRETION OF GASTRIC MUCIN

ulcer, a relationship between the histological alterations and changes in the mucous substances could be observed. In cases of duodenal ulcer, the normal findings predominated; in cases with gastric ulcer, pathological alterations could be observed in routine H & E-stained sections as well as by mucoid staining (Fig. Io). These results demonstrate that in cases of ulcer, there are no typical changes in the mucoid substances responsible for the development of the lesions. We interpret the changes in mucoid substances as a re-

sult of chronic inflammation. It is not established whether the mucoid staining will give diagnostic information. According to Hollander (42), there must be several factors acting together to produce peptic ulceration. Otherwise it is difficult to understand why only single ulcers arise, although there are diffuse general changes in the epithelium and within the mucous barrier. Erosive gastritis, according to Henning and Schatzki (47), seems to be an exception. Research in this field is still in progress.

Variation in Gastric Examination by biopsy of the gastric mucosa showed variations in the mucoid substances as described above. In chronic gastritis, there were alterations in the mucin content of the epithelial cells. A change in the production of mucus would appear in goblet cells present in the mucosa. In cases of atrophic gastritis, numerous cells with positive mucoprotein staining occur in the fundic area (Fig. I I ). In the final stage, pseudopyloric glands appear. In consequence, it should be possible to detect increased mucoprotein in the gastric juice. However, the exact quantitative differentiation of the mucoproteins is not yet possible. Secretory stimulants also produce variations in the composition of gastric mucin (49). It has not been possible to show that in pathological conditions individual fractions of gastric mucin have an abnormal composition. Our examinations of the mucoproteins are valid only in respect to total gastric juice, and do not refer to single components. This problem has the greatest importance in the discussion of protective function against peptic digestion. In vivo, the composition of gastric 68

i

Mucin

MUCOID SUBSTANCES IN SURFACE EPITHELIUM:

50-

25-

NORMAL SLIGHT EXTENSIVE CHANGES MUCOSA CHANGES HISTOLOGICAL DIAGNOSIS: 50-

2 5-

NORMAL SUPERFICIAL ATROPHIC MUCOSA GASTRITIS GASTRITIS

GASTRIC ULCER (83 CASES)

DUODENAL ULCER (49 CASES)

FIG. uu. Comparison of content of mucoid substances in surface epithelium with histological diagnosis in cases of gastritis.

HEINKEL & BERG

mucin is not readily altered by external agents. We have started new experiments on the digestion of gastric mucin, utilizing modern methods, but it is too early to make conclusive statements. At the beginning of this century, Pekelharing (49) showed that pepsin digestion changed the colloidal properties of gastric mucin, making some of it soluble, the remainder being soluble in alkali only. In vitro, Mahlo and \lulu (5o) found that only one third of the mucin was digested when a Io per cent solution of mucin in duodenal juice containing trypsin was incubated at pH 8.o. Anderson and Farmer (5 1) reported that much from hog's

stomach is resistant to pepsin. Trypsin liberated approximately 7.7 per cent and erepsin 3 per cent of the nitrogen from gastric mucoproteins. Brestkin and Bykow (5z) found that mucus absorbs pepsin, while Henning and Norpoth (S3) observed that mucus combines with acid and alkali. These properties may have a function in the role of mucus as a protective barrier. Heatley (54,55) has suggested the interesting concept of a dynamically maintained mucous barrier, in which the alkali secreted from the mucosal cells meets acid from the lumen of the stomach, providing a pH gradient through the mucous layer.

Summary Despite decades of work on the secretion of gastric mucin our present knowledge of the composition of mucoid substances and of aberrations which occur under pathological conditions is only fragmentary. Gastric suction biopsy is undoubtedly the method of choice for studies of the histological changes, which can be correlated with the electrophoretic pattern of secreted gastric mucin. In superficial gastritis, glycoprotein content increases, and more and better-defined glycoprotein-containing fractions appear. In atrophic gastritis, a high albumin content is a prominent feature; glycoproteins are increased but not as markedly as in superficial gastritis. The surface epithelium contains a clearly demarcated layer of mucoid substances with a high content of protein. Normal gastric mucosa does not contain goblet cells in noticeable numbers; their number increases in chronic gastritis. Factors controlling mucin secretion affect various types of mucin-producing cells differently. Neurogenic and hormonal stimuli cause emptying of mucous

neck cells, but do not affect the surface epithelial cells (t 5,17). The effect may be a direct one, but it is more likely that it is due to the compression of the mucous neck cells by contraction of the smooth musculature. Such contraction will obviously have no effect on the surface mucous cells. Irritative stimuli, such as local application of alcohol or chemical compounds, affect the discharge of mucin front superficial cells. Glass and his co-workers have separated visible mucus and soluble mucus; the latter was divided into three further fractions. The mucoprotein content of mucin samples has been assayed by us, using a method which is based on the well-known differential color reaction of galactose and mannose with arcinol in concentrated sulfuric acid. The comparison of the ratio of optical densities at different wave lengths of mucins in cases of superficial gastritis indicates that there is a change in composition of mucins accompaying inflammatory alterations in the gastric mucosa. No specific qualitative change in this ratio was observed in cases of peptic ulcer 69

I / SECRETION OF GASTRIC MUCIN

apart from gastritis. It is difficult to explain why single ulcers arise, although there are diffuse changes in the epithelium and within the mucous barrier, unless, as suggested by Hollander, other etiological factors are also involved in the development of peptic ulcer. Heatley has suggested the interesting concept of a dynamically maintained mucous barrier in which the alkali se-

creted from the mucosal cells meet acid from the lumen of the stomach, providing a pH gradient through the mucous layer. Brestkin and Bykow observed that mucus absorbs pepsin while Henning and Norpoth found that mucus combines with acid and alkali. These findings may be of importance in determination of the role of mucus as a protective barrier.

References I. TOMENIUS, J. Gastroenterology, 1 5: 498-504, ' 1 950. 2. WOOD, I. J., DOIG, It. K., MOTTERAM, R., and HUGHES A., Lancet 1: 18-21, 1 949. 3. HENNING, N., and HEINKEI., K., Munch. Med.

Wschr. 97: 832, 1955.

4. QUINA, M., HEINKEL, K., HENNING N., LANDGRAF, J. and ELFTER, K. Munch. Med. Wschr.

104: 878-882 (1962). 5. HEINKEL, K., LANDGRAF, J., ELSTER, K. HENNING, N. and CONINX, G. Gastroenterologia (Basel) 93: 269-287, 1960. 6. LIPP, W. ed. Histochemische Methoden.

Munch Med. Wschr. 1 954.

7. HEINKEL, K., HENNING, N., BUCHAC, I., LANDGRAF, J. and ELSTER, K. Munch. Med. Wschr.

104: 837-8777 (1962). 8. ELFTER, K. Bibl. Gastroent. 5: 83, 1961. 9. ZIMMERMANN, K. W. Ergebn. Physiol. 281-307, 1925. 10. FLOREY, H. Gastroenterologia (Basel),

24: 85:

140-141, 1956. 11. CORNET, A., BESCOI: LIVERSAC, J., GUILLAM, C. and DEBUSSCHE, c. Bull. Soc. Med. Hop.

Paris, 76: 293-309, 196o. W. Coll. Ges. Physiol. Chemie Mosbach, 1959. GLASS, G. B. J. and BOYD, L. J. Gastroenterology, 12: 821-878, 1 949. HEINKEL, K. Bibl. Gastroent. 5: 101, 1961. CLASS, G. B. J. Gastroenterology, 23: 636-658, 1 953• FLOREY, H. Proc. Roy. Soc. (Lond.), Ser. B 143: 147-1 58, 1955. WOLF, S. and WOLFF, H. G. Gastroenterology to: 251-255, 1948. SÜDHOF, H., and KELLNER, H., Bibl. Paediat. 65: 1-104, 1957. BUCHER, R. Deutsch Ztschr. f. Chir., 236: 515559, 1 932. HOLLANDER, F. and LAUBER, F. U. Fed. Proc. 7: 56, 1948.

12. MAURER, 13. 14. 15. 16. 17. 18. 19. 20.

21. BOHN, H., KOCK, E., RICK, W., VON KUGELGEN, B., GRÜTZNER, A., GUMBEL, W., and JESCH, W.

Deutsch. Med. Wschr. 86: 1384-1 394, 1961. 70

22. GLASS, G. B. J. and BOYD, L. J. Gastroenterology, 20: 442-457, 1952. 23. HENNING, N., KINZLMEIER, Il. and DEMLING, L. Munch. Med. Wschr. 95: 423-426, 1953. 24. HENNING, N. Lehrbuch der Gastrokopie, Leipzig, Barth, 1934• 25. KIMISEL, K. H., HEINKEL, K., and BORNER, W. Ärztl. Wschr. II: 602-607, 1956. 26. BERG, G., HENNING, N., HEINKEL, K. and LENTZEN, W. Klin. Wschr. 38: 262-265, 1960. 27. NORPOTH, L., SURMANN T., and CLOSGES, J. Gastroenterologia, (Basel) 85: 10-19, 1956. 28. GROSSBERG, A. L., KOMAROV., S. A. and SHAY H. Amer. J. Physiol. 165: 1-9, 1951. 29. RICHMOND, V., CAPUTTO, R. and WOLF, S. Arch. Biochem. 65: 155-166, 1956. 30. KATZKA, I. Gastroenterology, 36: 593-598, 1 959• 31. GRASBECK, R. Acta Med. Scandinay. Suppl. 314, 1956. 32. HOROWITZ, M. I. and HOLLANDER, F. Ann. N.Y. Acad. Sci. 99: 67-73, 1962. 33. GLASS, G. B. J., STEPHANSON, L., and RICH, M. Gastroenterologia (Basel) 86: 384-615, 1958. 34. SCHULTZE, H. E. and HEIDE, K. Med. Grundlangenforschung. Bd. III. Gg. Thieme, Stuttgart, 1960. 35. GLASS, G. B. J., RICH, M. and STEPHANSON, L. Gastroenterology 34: 598-615, 1958. 36. GLASS, G. B. J., STEPHANSON-LIOUNIS, L., RICH, M. and MITCHELL, S. E. World Congress of

i7. 38. 39. 40. 41.

42.

Gastroenterology Proceedings, 598-615, Baltimore, Williams & Wilkins„ 1958. GOTZ, H., SCHEIFFARTH, F. and DÜBELER, T. Gastroenterologia (Basel), 98: 30, 1962. FASEL, J. and SCHEIDEGGER, J. J. Gastroenterologia, (Basel), 94: 236-250, 1960. BERG, G. Bibl. Gastroent. 5: 195, 1962. BERG, G., and PREISSER, F. Gastroent. Suppl. 97: 238-240 (1962). HEINKEL K., LANDGRAF, J. and ELSTER, K. Praktische Ergebnisse neuer klinischer Forschung, 197-204, Stuttgart, 1962. HOLLANDER, F. A.M.A. Arch. Int. Med. 93: 107-120, 1954.

HEINKEL & BERG

Dissertation Univ. of Pennsylvania, 1800, cited by 42. 44. BALTZER, F. Arch. f. Verdauungskr. 56: 3546, 1934. 45. KAPP, H. Gastroenterologia (Basel), 76: 1951 99, 1 950-51 . 46. KATSCH, G. Handbuch der Innern Medizin, Bergmann-Stachelin ed., 3rd ed., 198, Berlin Springer, 1938. 47. HENNING, N. and SCHATZKI, R. Fortschr, a.d. Geb. d. Röntgenstrahlen 48: 177-182, 1933. 48. HOLLANDER, F. Ann. N.Y. Acad. Sci. 99: 4, 43.

GLOVER, J.

49.

1962. PEKELHARING, C. A.

Hoppe-Seyler Z. Physiol.

Chem. 35: 8-30, 1902. 50. MAHLO, and MULLI Deutsch. Med. Wschr. 6o: 937-938, 1632, 1934. 51. ANDERSON, R. K. and FARMER, C. J. Proc. Soc. Exp. Biol. Med. 32: 21-23, 1934. 52. BRESTKIN, M. P. and BYKOV, K. M. J. Russe Physiol. 7: 301, 1924, cited after 15. S3. HENNING N. and NORPOTH, L. Arch. f. Verdauungskr. 55: 143- 148, 1 934• S4. HEATLEY, N. G. Gastroenterology, 37: 312, 1959. 55• HEATI.EY, N. G. Gastroenterology, 37: 31331 7, 1959.

71

The Biochemistry and Degradation of the Mucus of the Upper

Deirdre Waldron Edward**

Gastrointestinal Tract*

many years the viscous secretions of the gastrointestinal tract, ovarian cysts, umbilical cords and many other organs have attracted the attention of chemists. Pure homogenous entities are required before chemists can carry out the structural studies, which should precede studies of function and metabolism. Until recently, however, it has been impossible (with a few exceptions) to separate, characterize and classify these intractable substances. New developments in technique now permit the fractionation of these difficult materials. With the aid of electrophoresis, the ultracentrifuge and other physicochemical methods, the purity of the materials may then be established and structural studies begin. Of particular interest to gastroenterologists, are the mucins in the epithelial mucosa and the secretions of the gastrointestinal tract. The main function of these mucins is probably to provide a protective layer over the delicate, enzyme secreting tissues, lubricating ingested foodstuffs in the course of their passage towards digestion, absorption and elimination. They FOR

appear to be very stable and resistant to attack, forming water-insoluble gels and viscous fluids, whose properties are dependent on their chemical constitution and structure. The mucins from the gastrointestinal tract are, however, difficult to obtain in reasonably large quantities, particularly from human sources. Consequently few well-defined components have been isolated. With the technical experience gained in handling more readily available mucins such as those from ovarian cysts and bovine salivary glands, progress in the future should be more rapid. It is generally believed that salivary and gastric mucin are degraded by intestinal bacteria (1). It is known that no blood group activity nor mucins are present in the feces of the normal adult, whereas they are abundant in the meconium from the sterile tract of the newborn. Some evidence exists, however, that the first stages of degradation may take place in the oral cavity and in the stomach, by chemical and by enzymic processes.

The experimental work carried out by the author was supported by a grant from the Medical Research Council of Canada. (Misr 775). ••From the Gastro-Intestinal Research Laboratory and the Department of Experimental Surgery, McGill University, Montreal, Canada.

73

I / THE BIOCHEMISTRY AND DEGRADATION OF MUCUS

Classification and General Chemistry of Mucin Mucins can now be divided into two main groups: the glycoproteins, which are high molecular weight materials, containing both carbohydrate and protein, linked by stable chemical bonds, and the acidic mucopolysaccharides, which are polymers of uronic acids and amino sugars. To the latter group belong hyaluronic acid from synovial fluid and umbilical cord; chondroitin and chondroitin sulfates from connective tissue; and mucoitin sulfuric acid and heparin (mucoitin disulfate). Some of these acidic mucopolysaccharides also contain esterified sulfuric acid. In vivo, they are often found combined with the more basic proteins, to which they are joined by electrostatic dissociable bonds. The structural chemistry of the acidic polysaccharides has been much more thoroughly investigated than that of the glycoproteins. Protein can be removed from them by various physical processes, and this simplifies analysis of the polysaccharide. The glycoproteins, in which the protein and carbohydrate are covalently

bound, can for convenience be further subdivided into three broad subgroups according to their carbohydrate moiety. (Table I) The neutral fucomucins, such as the blood group substances, contain hexosamine, galactose, fucose and traces of sialic acid. The sialomucins contain principally hexosamines and sialic acid (derivatives of neuraminic acid) in equal proportions, while the glycoproteins of plasma, such as seromucoid, contain hexosamine, galactose, mannose, sialic acid and a small quantity of fucose. Both of the former types of glycoprotein are to be found in most epithelial mucosa and secretions in varying proportions. The protein moiety of glycoproteins is believed to exist as one or more polypeptide chains, to which are attached short polysaccharide side chains. The nature of the linkage between sugars and specific amino acids and the structure of the carbohydrate chains is being studied by various workers in several typical, welldefined glycoproteins, such as bovine

TABLE I. Carbohydrate Composition of Typical Glycoproteins

Fucomucins

e.g. ovarian cyst contents (a)

Hexose

Pentose

Hexosamine

Galactose

Fucose

N-Acetyl galactosamine N-Acetyl glucosamine 19.3% dry wt.

10.0%*

9.0%

Sialomucins

N-Acetyl galactosamine

e.g. ovine submax. mucin (b)

18.5%

Serum Mannose & glycoproteins galactose e.g. orosomucoid (c) 16.3%

Fucose 1.3%

a) Werner, see reference No. 1. b) Gottschalk & Simmonds, see reference No. 17. c) Winzler, R. J., see reference No. 85. *All percentages expressed on dry weight of glycoprotein.

74

N-acetyl glucosamine 11.9%

Sialic Acid Trace 1.6% N-Acetyl neuraminic acid 25.0% Variable 10.6%

EDWARD

sialomucin (z), egg albumin (3a, b, 4, 5), the blood group substances from ovarian cysts (6, a, b) and serum a,-acid glycoprotein (7). Sialomucin and fucomucins (as represented by the blood group substances) resemble each other chemically more than either resembles the serum type glycoproteins. Both types contain little or no aromatic amino acids and a high proportion of glycine, proline and the i-hydroxy amino acids, serine and threonine; neither contains appreciable

amounts of mannose. The characteristic feature of all glycoproteins so far investigated seems to be that sialic acid or fucose take terminal positions on the polysaccharide side chains. The more sialic acid present, the more highly charged and more acidic the glycoprotein. Further discussion on the mucins of the epithelial mucosa will be limited in this paper to those of the upper gastrointestinal tract, particularly the mouth and stomach.

Composition of Saliva Saliva is the mixed secretion of the parotid, sublingual and submaxillary glands. Parotid secretion has generally, in the past, been considered as serous in nature, while the other secretions are mucinous. Each of these secretions from human sources has been examined by Tiselius electrophoresis and in the ultracentrifuge (8). Many different components were observed in each case. The electrophoretic pattern obtained from parotid secretion was markedly different from serum, however, although four components could be identified with serum proteins. Immunologically, saliva has been shown to contain some serum proteins (9), but also seems to contain many which are unique to saliva. The only protein which has been unambiguously identified so far is the enzyme a-amylase. Isolation of mucins Little isolation work has been carried out on mixed whole saliva, because it loses many of its characteristic properties shortly after collection. It has been shown, however, that normal human saliva contains both of the typical epithelial types of glycoprotein, fucomucins and sialo mucins, with traces of the mannose, galactose, N-acetyl glucosamine-containing

type of glycoprotein, which is present in blood (lo). Fucomucins predominate in mixed saliva, but the submaxillary and sublingual secretions are rich in sialomucins. Sialomucin Direct extraction of submaxillary glands from cattle and sheep have afforded the first homogenous glycoprotein for structural studies. A mucin was prepared from these glands by Hammersten (it) in 1888. Blix (12), in 1936, demonstrated that the characteristic carbohydrate component of this mucin was sialic acid, which is very readily split off the parent molecule by warming it to 37° at pH 3.o. Crystalline sialic acid was obtained. Sialic acids from different species are not identical, but all are derivatives of the neuraminic acid of Klenk (13) . The N-acetyl and R, = R2 = H in neuraminic acid CHOH /\ OH CH2 CHNR, I_ \ CH-CH-CHOH-CH2OH / \ / CO OH

/

Rs

75

I / THE BIOCHEMISTRY AND DEGRADATION OF MUCUS

N-glycolyl derivatives are found in ovine maxillary mucin showed that approxiand porcine mucin while N -O diacetyl mately 5o per cent of the protein moiety derivatives are found in bovine and equine was comprised of prolinc, glycine, serine and threonine (i7). It is believed by these mucin (14). Nisizawa and Pigman (15) extracted workers that the remaining zo per cent the main viscous component from mucin disaccharide units are linked in o-glycoclots of submaxillary glands by mild sidic linkage with the hydroxyl groups of methods, and established that it is a sialo- serine and threonine. mucin containing about 32 per cent sialic Tsuiko, Hashimoto and Pigman (18) acid. These workers showed that the have isolated a sialomucin from bovine familiar mucin clot, which is obtained glands by a different method. This prowhen glandular secretions are acidified, duct has a much higher carbohydrate is due to a salt-like complex formation of content, approximately 65 per cent of the sialomucin with protein. The reaction is molecule. They also found traces of gareversible. Sialomucin is very resistant to lactose and fucose, which they were unproteolytic enzymes. After liberation of able to detach during the process of purisialic acid groups however, proteolysis fication, and which they think may be an intrinsic part of the molecule. The averreadily takes place. The first evidence of the sequence of age molecular weight of this bovine submonosaccharides in this type of epithelial maxillary mucin is about four millions mucin was given by Gottschalk and Gra- ('9). Extracts of sublingual glands have been ham (16a,b), who obtained a sialomucin in 95 per cent pure form from bovine and fractionated by Tsuiko and Pigman (zo). ovine submaxillary glands, by mild aque- No homogenous component was obous extraction, followed by precipitation tained, but most of the material belonged with methanol at -40°. The carbohydrate to the fucomucin group. moiety, consisting of a-D-acetyl or a-Dglycolyl-neuraminyl-N-acetyl galactosamine represents 42 per cent of the total Trachea, esophagus and complex. On the basis of kinetic and other nasal mucosa epithelia Werner (1) has shown by paper chrostudies, Gottschalk (2) postulates that 8o per cent of these disaccharide units are matography, that the predominating glylinked as glycoside esters to 1 and y car- coproteins of the mucous epithelia and boxyl groups of aspartyl and glutamyl the secretions of these organs are comresidues of the polypeptide chain. An posed of hexosamine, galactose and fucose, amino acid analysis made on ovine sub- similar to the gastric fucomucins.

Gastric Mucosa and Mucin Secretion Historically, the main component of gastric mucin has been considered to be mucoitin sulfuric acid (zi). The claim for the presence of this polymer of glucuronic acid, glucosamine and esterified sulfuric acid was based on the work of 76

Levene and Lopez-Suarez (22a,b) on commercial pig gastric mucin, and on canine gastric juice (23). However, Meyer, Smyth and Palmer (z 3) showed that the acidic polysaccharide comprised only a small part of the carbohydrate

EDWARD

material in the mucin, and that the main component was a neutral carbohydrateprotein complex. Wolfrom and Rice (25) confirmed the presence of glucuronic acid in the acidic polysaccharide of hog mucosa by an oxidative hydrolysis to glucosaccharic acid. Smith et al. (26a,b) isolated heparin, which is mucoitin disulfuric acid ester, and two chondroitin sulfates from commercial hog mucosa, but no mucoitin monosulfuric acid as claimed by earlier workers. Werner (i) found a main component of the acid polysaccharide, as prepared by the method of Meyer et al., to have the same mobility on paper electrophoresis as chondroitin sulfate. There seems to be no definite proof of the presence of mucoitin monosulfuric acid in gastric mucosa or secretion. Uronic acids have not so far been isolated and identified in human gastric juice. Colorimetric methods have been used to detect and estimate these substances. All these methods depend upon the absence of hexoses and sialic acids, which interfere to different extents in the colour value of the reaction. Glass (27) made suitable corrections for such interference in his analysis of gastric juice fractions, and found the amount of uronic acid present was almost negligible. The author (28) found free glucuronic acid on paper chromatograms of pyridine extracts of lyophilized, neutralized gastric secretion from Heidenhain pouches in dogs. No uronic acid was detected in pyridine extracts of lyophilized, neutralized human gastric juices. The bulk of the mucinous material obtained from gastric mucosal scrapings and from whole gastric juice, however, belongs to the neutral glycoprotein group containing glucosamine, chondrosamine, galactose, fucose and a peptide containing several amino acids (1), in composition quite similar to the purified blood group

substances, and designated fucomucins. Traces of sialic acid are found in all material from this source. The hexosamines together constitute about half of the total carbohydrate moiety. The relative amounts of galactose and fucose are variable. Human gastric juice, separated from the solid or gel-like "visible" mucin, has been fractionated by precipitation with trichloracetic acid, followed by acetone, into three fractions (29), and called "soluble mucin", "gastric mucoproteose" and "glandular mucoprotein". None of these three soluble fractions is homogenous, but they all contain widely varying proportions of each of the carbohydrate components typical of the group of fucomucins. On paper electrophoresis of pooled gastric juice in borate buffer (3o) pH 9.o, the more mobile, the more negatively charged components are found to contain a higher proportion of sialic acid, while the more nearly neutral components are characterized by their high fucose content. Richmond et al. (3 i) have fractionated soluble gastric mucin on ion exchange resins. Carbohydrate rich and protein rich fractions were prepared, but no homogenous material was obtained. The biologically highly active blood group substances comprise only a very small part of the total glycoprotein in gastric juices or saliva, but are the only well-defined, homogeneous fucomucins that have so far been prepared and studied. The blood group substances The A,B, and H group substances are known to be secreted in water-soluble form in the saliva and gastric juice of 75 per cent of persons. These people are called secretors. The Le substance is found in those individuals who do not secrete the A,B, and H substances (32,33). Blood 77

1 / THE BIOCHEMISTRY AND DEGRADATION OF MUCUS

group activity has been demonstrated by Glass (34) to occur in several fractions of gastric juice separated by continuous electrophoresis on paper curtain. Specimens of active material have been isolated from the soluble mucins of human gastric juice and saliva by many workers, but in such small quantities that detailed studies of their chemical structure have not been possible. The neutral polysaccharide isolated by Meyer et al. (24) from the mucosa of pig's stomach, by means of peptic hydrolysis, contains two components, one with high A activity and the second with H activity. The most important human sources of such substances for analysis, however, are the fluids from ovarian cysts and from meconium. Most preparations of blood group substances involve treatment of the tissue with go per cent phenol, which extracts unspecific protein and other impurities. There is no evidence that phenol brings about any modification of the specific serological character of the native mucopolysaccharides, and therefore, of the spe-

cific chemical groupings necessary for their activity. This does not eliminate the possibility that limited and undetected changes in physical and chemical properties may occur as a result of such treatment (35). However, further methods for the isolation of undergraded active materials have been investigated, and the evidence obtained seems to indicate that the materials obtained by phenol extraction are largely unchanged and exist as such in native secretions. The highly purified substances all have the same qualitative composition: L-fucose, D-galactose, D-glucosamine and Dchondrosamine with fifteen amino acids. The most important of these latter are threonine, proline and serine, comprising about 15-2o per cent of the total mucopolysaccharide molecule (36). The molecular weight ranges from 200 to 300 X 103 (37a,b,c) to 1o° (38). There are welldefined quantitative differences in the A,B, Le° and H substances in the amount of each sugar present. Specific substances have now been iso-

TABLE II A terminal structure of the antigenic polysaccharide from Type XIV pneumococcus.

Galactose 1

6 N - Acetyl

1p

4 Glucosamine

3

Galactose

1(3

6 N - Acetyl 1/3 Glucosamine 4

II Galactose

10 Glucose 4

Galactose 1(3

I

N - Acetyl I~ 4. Glucosamine 1(3 Galactose

3 Galactose

1~

6 N — A ce tyl Glucosamine

4

I Glucose

EDWARD

lated that are substantially homogeneous, and more detailed structural analyses have been carried out. The site of serological activity on the molecule has been studied in detail, although the macromolecular structure remains undetermined. The sequence of some of the sugars in the prosthetic groups has been determined for the A,B and H substance, by mild acid and enzymic hydrolysis and other chemical techniques. Disaccharides and trisaccharides are released by hydrolysis and are then tested for their ability to inhibit agglutination. 0-a-N acetyl-Dgalactosamine- (i —>3) -D-galactose liberated from Group A substance inhibited the agglutination of A cells by human anti-A serum (39). This activity indicates that the structure is closely similar to, or identical with, the dominant serologically active group in the A substance molecule.

A trisaccharide, probably O-a-N-acetyl galactosaminoyl -(1 —* 3) -0-3 galactosyl -(1 —* 4)-N-acetyl galactosamine has the same properties (40). The removal of fucose by mild acid hydrolysis from A,B and H substances produces materials which react with a pneumococcus Type XIV antibody (41). Some of the specific antigenic structure of the Type XIV capsular polysaccharide has been elucidated by Barker et al. (42). (Table II). It seems very probable that this structure or one closely similar also participates in the whole AB and H substances. Enzymes from Trichomonas foetus degrade B substance in a steplike fashion (43a,b) to yield substances with different specific activities. The course of the degradation can be illustrated as follows:

B enzyme B substance

H enzyme H active substance

mucopolysaccharide possessing type XIV pneumococcus activity -I-

galactose These results, together with other chemical evidence support the conclusion that galactose and fucose are the important determinant structures in B and H specific substances respectively. From many similar studies by Morgan, Kabat and their collaborators, it seems that the blood group substances, which are genetically controlled, are chemically closely related, and that the H substance, common to the great majority of tissues, irrespective of the ABO group, represents a less developed mucopolysaccharide than either A or B specific substances. It may be impossible to obtain a homogeneous blood group substance with a single

fucose

specificity, constant analytical composition and properties characteristic of a single group specificity (35) In the same way it is possible that the non-blood-group-active fucomucins, both soluble and insoluble are composed, not of a simple mixture of a few substances with chemically well-defined structures, but of a series of closely interrelated molecular species. The structure of their various polysaccharide end-groups, which in turn are genetically controlled, would determine their specific biological properties. Such a hypothesis would account for the many diverse biological activities of apparently homogeneous materials. 79

I / THE BIOCHEMISTRY AND DEGRADATION OF MUCUS

Serum proteins in gastric juice Anacid gastric juice from Heidenhain pouches stimulated with acetylcholine appears to contain some serum albumin detected by radioactive tracer studies with l'-labeled albumin, and by immunochemical techniques (44). Small quantities of serum albumin have been found by similar techniques in anacid human gastric juice under normal conditions (45), while there is a marked increase in albumin in some gastric diseases (46,47,48). Albumin has also been detected in the gastric secretion from explanted rat mucosa (49), in which biochemical findings are very similar to the secretions of patients with protein-loosing gastropathies. The presence of gamma globulin and other serum proteins in all these conditions has been observed. In acid gastric juice, serum proteins rapidly undergo peptic digestion. Several peptide components have been found on paper electrophoresis which may be derived from this source (50,51, 52). Three possible sources of these proteins in gastric juice may be postulated a) from the exudation of serum from superficial ulcerations, b) in the absence of lesions, as a result of transudation of the interstitial fluid into the lumen of the stomach, c) from swallowed saliva where the presence of serum proteins has been shown in normal conditions. Traces of the sugar, mannose, have been reported from time to time in isolated mucin fractions. This sugar is characteristic of the serum glycoproteins (see Table). It seems possible that its presence in the gastric mucopolysaccharide fractions indicates a contamination with serum proteins. Site of origin of gastric mucin fractions There are four sources of mucin in the stomach: the superficial epithelial cells, 8o

the mucoid neck cells in the fundal region, and the mucous cells of the cardiac and pyloric glands (S3). Whether each of these sources contributes a different type of mucin is not easy to determine. Present methods of histological staining of the mucus-bearing cells do not differentiate between the different types of neutral carbohydrate complex. Glass (54) believes that his "glandular mucoproteose" is derived from the fundal glands of the stomach, because this fraction is absent in the gastric juice of patients with pernicious anemia and in the secretions of explants of rat gastric mucosa (4955)• James (56) in his monograph suggests that it is probably secreted by the mucous cells of the neck. Werner (i) analyzed separately the mucosal scrapings of the cardia, corpus and canalis ventriculi of hog stomach. The cardia contained very little carbohydrate. Both other sections were rich in neutral carbohydrates, the corpus containing, in addition, fairly large amounts of an acidic mucopolysaccharide. Barker et al. (57) found that cancerous tissue in both the pyloric and the cardiac region of the stomach, and also duodenal ulcer tissue, contain mainly neutral glycoproteins, with a trace only of acidic polysaccharide. In addition, malignant tissue from a Ca of the stomach, and also duodenal ulcer tissue, contain mainly neutral glycoproteins, with a trace only of acidic polysaccharide. In addition, malignant tissue from a Ca of the stomach contains twice as much polysaccharide bound neuraminic acid as tissue from ulcers of the stomach and duodenum. N-acetyl neuraminic acid was identified in all ulcer material and in most Ca tissue, but N-Odiacetyl neuraminic acid was also found in a Ca of the cardium, involving i in. esophagus. The significance of these findings is not known.

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Degradation of Mucins The process of disintegration of epithelial mucins in normal conditions is of vital interest in attempting to elucidate the mechanism of pathological changes in the gastrointestinal tract, but very little is known of these processes. A certain amount of degradation of salivary and gastric mucin can take place by simple changes in pH, particularly within the acid physiological range, with breaking of acid labile bonds of the carbohydrate moiety. Thus sialic acid is liberated when submaxillary gland sialomucin is held at pH 3.0 and 37°. (Vide infra). The mucin solution rapidly loses its viscosity. Similarly fucose and galactose are slowly liberated when gastric fucomucins are treated in acid at pH 1.1 to 1.85 at 37° under conditions in which enzyme action is excluded (28). Hydrolytic enzymes may break other, more stable bonds. Proteolytic enzymes are required to attack the peptide links of the protein moiety, while mucolytic enzymes (mucinases) attack the carbohydrate links. Such enzymes may be endogenous or produced by bacteria or viruses in the gastrointestinal tract. Proteolytic enzymes Voss (58) and Ito, Hiroshe and Takeuchi (59) demonstrated proteolytic activity in human saliva. The latter workers measured the appearance of free iodine liberated from l'-labeled casein incubated with saliva at pH 7.6. Taylor (6o) and Schafer (61) noted protease activity in saliva. As a high percentage of the activity was associated with cellular debris, Taylor suggested that the protease may be derived from broken cells of buccal mucosa. The main proteolytic enzyme of the stomach, pepsin, and its associates Parapepsins I and II (6z) and Gastricsin (63), which is probably related to the others, are discussed in detail in another

paper in this book (64). Gastric mucin in its native state is not digested by pepsin (65). A gastric gelatinase (66) has also been detected. In addition to these proteinases which are active in the acid pH range, Taylor (6o) has also demonstrated proteolytic activity in human gastric juice, and in pig and calf mucosal extracts, which is optimal at pH 6.8-7.2 and at pH 7.5-7.7. Activity was detected by measuring amino acid nitrogen liberated from substrates of plasma proteins or casein. Typical glycoproteins have not been used as substrates in these experiments so their susceptibility to attack is not known. However, Glass and Boyd (67a,b) observed that gastric mucus, incubated at 37° and pH 6-7 was liquified. At the same time, tyrosine-containing split products were obtained, which had the "precipitation and solubility characteristics" of mucoproteose. They concluded that an enzyme which they called mucolysine was responsible for the degradation. Hollander (68a) and Janowitz and Hollander (68b) reached the same conclusion, and deduced that gastric mucus is under constant attack by a mucolytic enzyme. It seems possible that this enzyme is the same as that described by Taylor. Mucinases Reports of the presence of mucolytic enzymes in saliva have rested largely on the observation of the loss of viscosity of saliva when it is allowed to stand at room temperature for thirty minutes. This is particularly apparent with the more viscous saliva which is collected without the stimulus of chewing. The origin of this mucolytic enzyme (or enzymes) and also its specific action are not yet clear. Cavelli (69) states that parotid saliva contains an enzyme which splits the salivary mucin of submaxillary glands, reducing its vis81

I / THE BIOCHEMISTRY AND DEGRADATION OF MUCUS

cosity and raising the titer of reducing substances. Similar activity was noted by Simmons (7o), who further observed that the agent was non-dialysable, thermolabile and inhibited by I_ and mercury salts. Rogers (71) believed that oral bacteria were responsible for mucin breakdown. He found that the consumption of bound glucosamine and reducing sugars in saliva was inhibited by such bacteriostatic substances as thymol and toluene. However, in the presence of these inhibitors, a dialysable substance reacting like N-acetyl glucosamine in the Morgan and Elson reaction accumulated, which he attributed to enzymic activity by the cellular material of the saliva. Knox (72) showed that there is both an endogenous and an exogenous mucinase (or combination of several enzymes) in saliva. The mucinase was partially purified by precipitation and absorption methods. All preparations contained hyaluronidase and a-amylase, which is inactive against mucins. The enzyme is active only at neutral pHs, and is inactivated at pHs lower than 4.5. It depolymerized salivary mucoid which had been precipitated by weak acid with the liberation of reducing sugars. Mucolytic enzymes from cultures of oral bacteria were also capable of depolymerizing sterile, enzymefree salivary mucoid. Enzymes which attack specific glycoside bonds There are known to be several enzymes present in the oral cavity which can break specific linkages in carbohydrate molecules, among them lysozyme, (3-glucuronidase, hyaluronidase, (i-galactosidase and the starch splitting enzyme a-amylase (73). a-Mannosidase and (3-N-acetyl glucosaminidases have not been reported as definitely occurring in saliva, but Conchie et al. (74) have demonstrated that they 82

are widely spread throughout mammalian tissues, and point out that these enzymes may have a function in mucoid catalolism. Some, or perhaps most of the carbohydrate splitting enzymes present in saliva, are of bacterial origin except lysozyme, a-amylase and p-glucuronidase, which can be both exogenous and endogenous. (3-GLUCURONIDASE

(3-glucuronidase has been shown to be present in parotid saliva and in most animal tissue associated with the microsomal fragments of tissue cells (75). The enzyme is usually detected by its activity in splitting a chromogenic aglycone from a glucuronide derivative; but it can also act on oligosaccharides containing uronic acid. Tnus li-glucuronidase, together with the endohexosaminase, testicular hyaluronidase, can act alternately to cause the steplike degradation of hyaluronic acid or chondroitin (76). Hyaluronic acid has not been identified in epithelial mucus, but it has already been indicated that chondroitin is sometimes present (26). LYSOZYME

Pure crystalline lysozyme has been prepared from hen's egg white and several other sources. Its molecular weight is about 14,600; it is a very stable enzyme and has the remarkable property of retaining its activity even when heated at an acid pH at 1 oo°. Lysozymes from different species and even from different organs of the same species have slightly different chemical compositions, but all are characteristic in their ability to lyse the cell walls of certain bacteria, in particular .!licrococcus lysodeikticus. These cell walls are polysaccharide in nature; lysozyme releases N-acetyl hexosamine and a number of other, larger compon-

EDWARD

ents, including a di- and a tetrasaccharide, namely 13(1 -* 6) N-acetyl glucosamineN-acetyl muramic acid and its dimer (77). It is believed that lysozyme acts specifically on repeating units of N-acetyl glucosamine and N-acetyl muramic acid joined by a (3(i -4 4) linkage (78). Egg white lysozyme also hydrolyses chitin, indicating 3-glucosaminidase activity (79). Lysozyme is present in both the parotid and submaxillary saliva in man and in animals. It has frequently been suspected of mucinolytic activity in the stomach where it has been found to be increased at the same time and under the same conditions in which mucoproteose is increased (8o). Rat's gastric and colonic mucosa when treated with lysozyme develop erosions and hemorrhages, increasing the injurious effect of pepsin and hydrochloric acid. Insoluble gastric mucus, however, does not appear to be attacked when treated directly with the enzyme (81,82), indicating the absence or shielding of the specific groups in the mucin. The drastic effect that takes place on

lysozyme-treated mucosa suggests that the enzyme must act on the cell walls or structural tissue. NEURAMINIDASE

Another enzyme of viral and bacterial origin which may be of importance in attacking epithelial mucins in infections of the respiratory and gastrointestinal tract is neuraminidase. This enzyme has been demonstrated in influenza -(A,B,C) virus, mumps, Newcastle disease and several other viruses and in Vibrio cholera, Cl. welchii, P. pyocyaneus and in several other bacteria. Ada and French (83) have prepared it in a pure crystalline form from Vibrio cholera, and have shown that it attacks bovine and ovine submaxilIary gland sialomucins, orosomucoid, meconium, and pituitary gonadotropin, releasing neuraminic acid in each case. Neuraminidase, however, has not been found in animal tissues (84), and cannot be considered as a metabolic enzyme.

Summary Little progress has been made in the past in the study of the breakdown of the mucins of the upper gastrointestinal tract; major physical changes such as loss of viscosity and solubility were observed, but minor chemical changes were more difficult to detect. The secretions of the nmcus glands and surface epithelium are complex mixtures of carbohydrate and protein-containing materials. The recent isolation and identification of highly purified nrucopolysaccharides should enable a more methodical study of their breakdown in vitro and in vivo. Some of the initial stages in the fragmentation of the high molecular weight mucopoly saccharide secreted by the sub-

maxillary glands, sialorrucin, probably take place in the gastric lumen by the action of acid, and later by the proteolytic action of pepsin. Certain of the terminal sugar units of the f ucoimicins of the surface epithelium, such as fucose, are also hydrolysed by gastric acid. The presence of several endogenous and exogenous proteolytic enzymes in saliva has been demonstrated by various workers, and there is evidence that oral bacteria can degrade the salivary nnmcins under suitable conditions. However, there is no direct chemical evidence that lysozyme, produced endogenously, has any action on the salivary or gastric zmtcins. The action of pepsin seems to be limited 83

I / THE BIOCHEMISTRY AND DEGRADATION OF MUCUS

to the partially degraded sialonrucin, and is not known to attack the native surface epithelial nnzcin of the stomach. The presence of an endogenous proteolytic enzyme in gastric juice acting at neutral pH has been demonstrated by Taylor (6o). This enzyme may be responsible for the degradation of ?micro clots when held at pH 7.o, observed by Glass (67a,b) and by Hollander (68a,b). Such an enzyme could hydrolyze the nnlcin as it is freshly

secreted under neutral or mildly alkaline conditions on localized areas of the gastric nmcosa. It is suggested that several enzymes, both of physiological and bacterial origin, known to be present in the upper gastrointestinal tract, may act synergistically with the normal gastric acidity in the steplike degradation of fucomucins or sialonnrcins.

References I. WERNER,

1, 1953.

1. Acta Soc. Med. Upsaliensis, 58:

and MURPHY, W. H., Biochem. Biophys. Acta., 46: 81, 1961. 3. JOHANSEN, P. G., MARSHALL, R. D. and NEUBERGER, A., a) Biochem. J., 77: 239, 1960. b) Biochem. J., 78: 518, 1961. 4. CUNNINGHAM, L. w., KUENKE, B. J. and NUENKE, R. B., Biochem. Biophys. Acta 26: 660, 1957. 5. JEVONS, F. R., Nature, (Lond), 181: 1346, 1958. 6. PUSZTA[, A. and MORGAN, W. T. J., a) Biochem. J., 8o: 107, 1961. b) Biochem. J., 8i: 639, 1961. 7. EYLAR, E. H. and JEANLOZ, R. W., J. Biol. Chem., 237: 1021, 1962. 8. PATTON, J. R. and PIGMAN, w., J. Amer. Chem. Soc., 8,: 8035, 1959. 9. ELLISON, S. A. and MASHIMO, P. A., J. Dent. Res., 37: 28, 1958. 9.2. STOFFER, H. R., KRAUS, F. W., HOLMES, A. C. Proc. Soc. Exp. Biol. Med., iii: 467, 1962. 10. BERGGARD I. and WERNER, I. Acta Odontol. Scand., 16: 43, 1958. 11. HAMMERSTEN, o. Hoppe-Seyl. A., 12: 163, 1888. 12. BL1x, G. Z. Physiol. Chem., 240: 43, 1936. 13. KLENK, E., Hoppe-Seyl. A., 273: 77, 1942. 14. BLIR, G., LINDBERG, E., ODIN, L. and WERNER, I. Acta Soc. Med. Upsaliensis, 6r: 1, 1956. 1 5. NISIZAWA, K., and PIGMAN, \V., Biochem. J., 7S: 293, 1 96o. 16. GOTTSCHALK, A., and GRAHAM, E. R. B. a) Biochem. Biophys. Acta: 34: 380, 1959. GRAHAM, E. R. B. and GOTTSCHALK. b) Biochem. Biophys. Acta 38: 513, 1960. 17. GOTTSCHALK, A. and SIMMONDS, D. H., Biochem. Biophys. Acta., 42: 141, 1960. 18. TSUIKO, S., HASHIMOTO, Y., and PIGMAN, W. J. Biol. Chem., 236: 2172, 1961. 19. BETTELHEIM, F. A., Biochem. Biophys. Acta, 63: 235, 1962. 2. GOTTSCHALK, A.

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20. TSUIKO, s. and PIGMAN, w., Arch. Oral Biol., 2: 1, 1960. 21. BABKIN, D. P. Secretory Mechanisms of the

Digestive Glands 240. N.Y. Paul B. Hoeber Inc. 1950. 22. LEVENE, P. A. and LOPEZ-SUAREZ, J. a) J. Biol. Chem., 25: 11, 1916. b) J. Biol. C m., he 36: 105, 1918. 23. KOMAROV, S. M. J. Biol. Chem., 109: 177, 1 935. 24. MEYER, K., SMYTH, E. M. and PALMER, J. W. J. Biol. Chem., tip: 73, 1 937. 25. WOLFROM, M. L. and RICE, F. A. H., J. Amer. Chem. Soc., 69: 1833, 1947. 26. SMITH, H., GALLOP, R. C., HARRIS-SMITH, P. W., STANLEY, J. L. a) Biochem J., 52: 23, 1952. SMITH, H., and GALLOP, R. c., b) Biochem. J., 53: 666, 1953. 27. GLASS, G. B. JERZY, RICH, M. and STEPHANSON, L., Gastroenterology, 34: 598, 1958. 28. WALDRON EDWARD, D., Unpublished Observations. 29. GLASS, G. B. JERZY and BOYD, L. J., Gastroenterology, 12: 821, 1949. 30. GLASS, G. B. JERZY, STEPHANSON, L., and RICH, M., Gastroenterologia, 86: 384, I956. 31. RICHMOND, V., CAPUTTO, R. and WOLF, S. Arch. Biochem & Biophys., 66: 1 55, 1957. 32. GRUBB, R., Nature (Lond.) 162: 933, 1948. 33. GRUBB, R., and MORGAN, W. T. J., Brit. J. Exp. Path., 3o: 198, 1 949. 34. GLASS, G. B. JERZY, Gastroenterology, 43: 310, 1962. 35. MORGAN, W. T. J. Proc. Roy. Soc. (Lond.) Series B. 151: 308, 1959-60. 36. CARSTEN M. E. and KABAT, E. A. J. Amer. Chem. , Soc. 78: 3083, 1956. 37. KEKWICK, R. A. a) Biochem. J., 46: 438, 195o. b) Biochem J., 52: 2 59, 1952. c) Biochem J., 50: 471, 1952. 38. CASPARY, E. A. Biochem J., 57: 295, 1956. 39. COTE, R. H. and ISORGAN, W. T. J. Nature (Lond.), 178: 1171, 1956.

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and MORGAN, W. T. J. Nature (Lond.), 191: 149, 1961. 41. KABAT, E. A., BAER, S., BEZER, A. E., and KNAUB, v., J. Exp. Med., 88: 43, 1948.

40. CHEESE, I. A. F. L.

42. BARKER, S. A., HEIDELBURGER, M. STACEY, M., and TIPPER, D. J., J. Chem. Soc., 3468, 958. 43. WATKINS, W. M. a) Biochem J., 54: XXXISII,

The physiology of pepsinogen. In: Physiopathology of Peptic Ulcer, McGill University Press, 1963. 65. PLACER, A., ROUBEL, A. and SLABOCHOVA Z. Ceskoslov. gastroenterol. Vyziva., 13: 517, 1959• 66. NORTHRUP, J. H. J. Gen. Physiol., 15: 29, 64. HIRSCHOWITZ, B. I.

1953• 67. b) Biochem J., 64: 21, 1956. 44. HOROWITZ, M. I. and HOLLANDER, F. Gastroenterology, go: 785, 1961. 45. GULLBERG, R. and OLHAGEN, B. Nature (Lond.) 68. 184: 1848, 1959• 46. GLASS, G. B. and ISHIMORI, A. Amer. J. Dig. Dis., 6: 103, 1961. 47. OHARA, H. and GLASS, G. B. J. Unpublished 69. 7o. Observations (as quoted in Ref. 34.) 71. 48. ISHIMORI, A. and GLASS, G. B. J. Unpublished Observations (as quoted in Ref. 4) 49. GLASS, G. B. J. and SKORYNA, S. C. c. ProSoc. 72. Exp. Biol. Med., 107: 56o, 196i. 50. HITZELBERCER, A. L. and GLASS, G. B. J. J. Lab. 73. Clin. Med., 59: 575, 1962. 51. KAKEI, M. Arch. Japan. Chir. 28: 2603, 1958. 74. 52. GLASS, G. B. J., KAKEI, M. and STEPHANSONLIOUNIS, L. Unpublished Observations. (As 75• 76. quoted in Ref. 34). i3. FLORET SIR H., PIoc. Roy. Soc., B 143: 147, 77. 1 955• 78. J. Nat. Cancer GLASS, G. B. JERZY Inst., 13: 54. 101 3, 1953. 79. 55. WEBSTER, D. R., TOOVEY, E. W. and SKORYNA, S. C. Gastroenterology, 35: 31, 1958. 56. JAMES, A. H. The physiology of Gastric Di- 80. gestion and Secretion 141, London, Arnold 1 957• 8,. S7. BARKER, S. A., STACEY, M., TIPPER, D. J. and KIRKHAM, J. H., Nature (Lond.), 184: 68, 1959• 58. voss, j., Zeitsch. f. Physiol. Chem., 197: 42, 8z. 1931. 83. 59. ITO, I., HIROSHE, S. and TAKEUCHI, K. Endocrin. Japon., 6: 59, 1959. 6o. TAYLOR, W. H. Biochem. J., 71: 626, 1 959. 84. 61. SCHAFER, W. G., CLARKE, P. G. and \CURLER, J. C. J. Dent. Res., 38: 121, 1959. 85. 62. KYLE, A. P. and PORTER, R. R. Biochem. J., 73: 75, 1959. 63. TANG, J., WOLF, S., CAPUTTO, R., and TRUCCO, R. E. J. Biol. Chem., 234: 1174, 1959.

1931-32. GLASS, G. B. JERZY, and BOYD, L. N.Y. Med. Coll., 12: I, 1949.

J. a) Bull.

b) Gastroenterology, 23: 636, 1 953. a) J. Nat. Cancer Inst., 13: 989, 1953. JANOWITZ, H. D. and HOLLANDER, F., b) Gastroenterology, 26: 582, 1 954. CAVELLI. (Abst.) Ber. Physiol., 121: 52, 1940. SIMMONS, N. S. J. Dent. Res., 20: 2 55, 1941. ROGERS, H. J. Nature (Lond.), 161: 815, 1948. 1948. KNOX, K. W. and STILL, J. L. J. Dent. Res., 32: 367, 374, 379, 1953. CHAUNCEY, H. H., LIONETI1, F., WINER, R. H. and LISANTI, V. H. J. Dent. Res. 33: 321, 1 954. CONCHIE, J., FINDLAY, J. and LEVVY, G. A. Biochem. J., 71: 318, 1959. WALKER, P. G. Biochem, 51: 223, 1952. MEYER, K. Fed. Proc. 17: 1075, 1958. SALTON, M. R. J. and GHYSER, J. M. Biochem. Biophys. Acta, 36: 552, 1959. BRUMFITT, Wy WARDLAW, A. C. and PARK, J. T. Nature (Lond.), 181: 1783, 1 {p~58. BERGER, L. R. and WEISER, R. S. Biochem. Biophys. Acta, 26: 517, 1957. HOLLANDER, F.,

GRAY, S. J., REIFENSTEIN, R. W., YOUNG, J. C., SPIRO, H. W. and CONNOLLY, E. P. J. Chn. In-

vestigation, 29: 1 595, 1 950. GLASS, G. B. JERZY, PUGH, B. L., GRACE, W. J. and WOLF, S. J. J. Clin. Investigation, 29: 12, 1950. WANG, K. J., GRANT, R., JANOWITZ, H. D. and GROSSMAN, M. Arch. Path., 49: 298, 1950. ADA, G. L. and FRENCH, E. L. Nature (Lond.),

183: 1 740, 1959•

GOTTSCHALK, A. an d PERRY, B. T. Brit. J. Exp. Path., 32: 408, 195 1. WINZLER, R. J. Ci a Foundation Symposium on the Chemistry and Biology of Mucopolysaccharides. 245 Wolstenholme, G. E. W. & O'Connor, M., ed. London, Churchill 1958.

85

Gastrin Release

THE influence of the Pavlov school on research and thought within gastric physiology is truly remarkable. Even today, after the lapse of half a century, the salient features of the current scientific debate can be traced directly to the work of Pavlov and his associates in St. Petersburg (Leningrad). They not only discovered the röle of the vagus nerves in the activation of the HCI glands, but established the importance of the antrumduodenal area. Through ingenious experiments the antrum area was shown to exert both excitatory and inhibitory influences on the HC1 secretion. The mechanisms were believed to be neurogenic. Opposing the idea of the antrum as a reflexogenic zone regulating the secretion of HCI, Edkins advanced, in 1906, his gastrin theory. According to Edkins, the role of gastrin in eliciting gastric secretion corresponds to that of secretin in the activation of pancreatic secretion. It is well known that the propounding of these two hypotheses led to an animated but often somewhat confused discussion, which is, to some extent, still in progress. Numerous physiologists, as well as clinicians, have published observations indicating that no major or even appreci-

Börje Uvnäs*

able reduction of the HC1 secretion results from resection of the antrol mucosa. McCann (1), Wilhelm et al. (2) and Lewis (3) among others reported that the secretory response to a meal declined considerably after antrectomy, but that if fragments of the antral mucosa were retained, the gastric secretion either remained unchanged, or showed only a temporary diminution. Due to the prevalence of negative reports, the gastrin theory fell increasingly into disfavour. Its decline was doubtless hastened considerably by the skepticism of such experienced and esteemed physiologists as Ivy and his associates. Ivy and Farrel (q.) reported that HC1 secretion could be elicited in a subcutaneously transplanted fundus pouch of a dog, on feeding. Since the subcutaneous graft was completely denervated, their observation showed definitely that the HCI secretion was produced by a blood-borne secretory agent. The authors, however, were reluctant to credit the hormonal nature of the active agent. The possibility of absorption of secretagogues from the gastrointestinal contents was not precluded. Some years later, in 1932, another ob-

•From the Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden.

87

I / GASTRIN RELEASE

servation made in his laboratory led Ivy and his group to subscribe to a hormonal theory, but at the same time, led them astray for some years to come. Histamine was extracted from antral mucosa of pigs and identified by crystallization (5). Despite later reports that corpus mucosa of the hog contains even higher amounts of histamine than antral mucosa (6), it was still believed that histamine was identical with the postulated antral gastrin. The identity of gastrin and histamine was rather accepted. Intensive efforts to demonstrate conclusively the existence of a hormonal gas-

tric secretory mechanism led to the observation by Gregory and Ivy (7) that HC1 secretion could be evoked in a subcutaneously transplanted pouch on perfusing a total stomach pouch with liver extract. Since pretreatment of the mucosa of the total stomach pouch with 5 per cent procaine solution blocked the secretory response in the subcutaneous transplant, it was concluded that chemical stimuli were able to liberate a secretory hormone from gastric mucosa. Although members of the Pavlov group had long ago reported that HC1 secretion could be elicited by mechanical stimula-

TABLE I. Control of HCl secretion STIMULATING FACTORS

I

Nervous (cephalic) secretory phase Taste Smell Sight Hearing

Activate HC1 glands (by local gastrin release?) (by potentiation of gastrin effect?)

Conditioned reflexes Vagal impulses

Release gastrin from antrum

Hypoglycemia II Gastric secretory phase Mechanical stimuli in antrum (distension, bulky food, etc.) Chemical stimuli (see Table II)

Release gastrin from antrum (via local cholinergic reflex arcs?)

(Alcohol, caffein, stimulate also the HCl glands directly) III Intestinal secretory phase (quantitative importance unknown) Mechanical stimuli Chemical stimuli

Release gastrin from duodenal bulb?

Chemical stimuli

Stimulate HC1 glands directly after absorption? INHIBITORY FACTORS

Local inhibition of gastrin release by acid pH in antrum (and duodenum?) II

Release of inhibitory factor from duodenal bulb (and other areas of duodenum?) by low intraduodenal pH (z 2 )

III Release of inhibitory factor from duodenum by neutral fats (and other factors?) IV Release of antral inhibitory factor by acid pH in antrum? 88

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tion of the antrum area, it remained for Ivy and his pupils (8) to demonstrate — as recently as 195o — that mechanical stimulation of the antral mucosa could also activate the antral gastrin mechanism. Two completely denervated pouches, one with corpus and one with antral mucosa, were produced in dogs. Distention of the antrum pouch with a balloon caused secretion of HC1 in the corpus pouch. The decisive break-through for the revived gastrin theory came with the papers of Dragstedt and his associates, describing the dramatic influence of autotransplantation of antrum on the HC1 secretion in total stomach pouches, Pavlov pouches and Heidenhain pouches (9,1o). Exceptionally impressive was the observation of profuse hypersecretion (measured as twenty-four hours secretion), occurring on transplantation of antral mucosa into the colon. This coup of experimental surgery finally convinced all gastrin skeptics. Gastrin had come to stay

in physiology — and possibly it will enter pathophysiology as well. During the past decade, numerous elaborate and technically sophisticated investigations have enhanced our knowledge of the gastrin mechanism. A humoral secretory agent has been shown to be released from isolated, denervated or transplanted antral mucosa, and to elicit HCI secretion in isolated, denervated or transplanted corpus mucosa. The main factors known to influence the release of gastrin are brought together in Table I. For illustrative purposes, I have adhered, in the table, to the traditional Pavlovian division of the gastric secretion into three separate phases, the cephalic, the gastric and the intestinal. Such a division of the secretory response to a meal is, however, less justifiable today when — as I will attempt to show — the "three phases" are known to be more intimately interwoven than was previously suspected.

Gastrin Release with Nervous Stimuli According to current theory, there is a fundamental difference between the mechanisms by which the nervous and the gastric secretory phases are initiated and maintained. During the nervous (cephalic) phase, the HCI secretion is thought to be evoked solely by direct vagal impulses to the HCI glands, and during the gastric phase, by blood-borne gastrin released from the antral mucosa by chemical and mechanical stimuli. An assault on this rigid functional separation of the two secretory phases was made as early as 1942, when Uvnäs ( ► i) suggested that vagal impulses were able to release gastrin from the antrum. In acute experiments on cam, he observed that cocainization, resection and other

manipulations with the antral mucosa abolished or markedly reduced the secretory response of the HC1 glands to vagal stimulation. The idea of a nervous influence on the gastrin release met with opposition, the weightiest confutation being the old observation that a Heidenhain pouch does not respond to sham feeding (12). Since resection and denervation of the antrum caused only a minor reduction of the twenty-four hours' secretion and the insulin-induced secretion in total stomach pouches, Oberhelman et al. (1o) concluded that the antrum was not essential for vagus secretory functions and that "vagus secretory function is only slightly impaired if the antrum, which is the whole source of gastrin, is 89

mEQ 040-

Sham feed:rg

4 EXP,

020

2

mEg

3

4 HOURS

Shorn feeding

6 EXP.

080 060 040 020

4 HOURS

mEg

Shorn feeding

3 EXP

0.60j HCI in oNrum pH•LI 04 020

4

mEg

HOURS

4 EXP.

Sham feeding 0 60 040 0.20

2

3

4 HOURS

FIG. r. Secretory responses of Pavlov pouch to shorn feeding. a) Controls with intact gastrointestinal passage. b) Hypersecretion after exclusion of antrum. c) Inhibitory influence of acid pH in antrum on hypersecretion after antrum exclusion. d) Normalization of hypersecretion after resection of excluded antrum.

entirely removed from the body". A similar view was held by Pevsner and Grossman (I 3) on the basis of dog experiments in which insulin hypoglycemia evoked copious HC1 secretion even when removal of the antrum and the entire small intestine had eliminated all the sources of gastrin. In Pavlov pouches, the twentyfour hour secretion did indeed fall after resection of the antrum (1o); the fall, however, was attributed not to a reduced release of gastrin but to the fact that "the presence of the antrum facilitates vagus secretory function" (14). I will return later to the interrelation between vagal impulses and antrum function. Thus the odds did not favour neural control of gastrin release. But now, a few years later, the situation has changed. I 90

need only mention reports on the secretory response of a totally denervated pouch to sham feeding (15), and to teasing (i6); and the disappearance of the response of a Heidenhain pouch to insulin hypoglycemia after cocainization, denervation or antroneurolysis of the antral mucosa (17) . Several other examples could be given, but I have chosen to cite some of our own experiments. Mindful of the early observations on hypersecretion of HCI after exclusion of the antrum-duodenum, we decided to study the effect of this procedure on vagally induced gastrin secretion. The antrum was excluded from the normal gastrointestinal passage by a simple gastrojejunostomy. To our satisfaction, the secretory responses of Pavlov pouches to insulin hypoglycemia were greatly intensified — up to 45o per cent (i8). Since exclusion of antrum-duodenum results in a neutral pH in the excluded area, two possible factors could have been responsible for the hypersecretion: facilitation of gastrin release or disappearance of acid inhibitory mechanisms. Both of these factors were found to be instrumental. Affirming a vagal release of gastrin as the cause of the hypersecretion, the hypersecretory response to insulin hypoglycemia disappeared after resection of the excluded antrum (19). For a more physiological approach, we proceeded to sham feeding experiments. Dr. Olbe, in our department, has devised an excellent technique for sham feeding. Dogs with esophageal fistulas of his type are able to feed themselves and remain healthy and well nourished. We have used such dogs for up to two years. Fig. i illustrates an observation on a Pavlov pouch. Sham feeding elicits a moderate HC1 secretion a). Exclusion of the antrum results in a vast augmentation of the sham feeding response — an increase of more than 600

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per cent b). That this intensification is attributable to gastrin release is indicated by the result of antral vagal denervation c). After this operative procedure the sham feeding response returned to approximately its initial level, as determined before the exclusion of the antrum (zo). As Fig. i.c. attests, we were able to reduce the exaggerated response of the Pavlov pouch more simply than by denervation or resection. When the antral pH was lowered by perfusion with solutions of different ph, it was found that as the pH approached 2.5, the sham feeding response started to decline. At an antral pH of 1.5, the secretory response was markedly depressed. Presumably, at this pH the gastrin releasing action of vagal impulses initiated by sham feeding is greatly inhibited. Woodward et al. (z ► , 22) have previously shown a similar pH dependence of antral gastrin release resulting from mechanical and chemical stimuli. All the observations are consonant with the view that vagal impulses are able to release antral gastrin. Why is it that we — and several others — were suddenly able to elicit secretory responses assignable to gastrin release not only in Pavlov pouches but in Heidenhain pouches as well? The unresponsiveness of a Heidenhain pouch to hypoglycemia and sham feeding had been the very argument used to refute the existence of a vagal control of gastrin release. What, then, had brought about this change of behaviour of the Heidenhain pouch? The answer is that the neutral milieu in an excluded antrum facilitates vagally induced gastrin release, even as it facilitates the release of gastrin in response to chemical and mechanical stimuli. Obviously, the secretory response of the Heidenhain pouch can be demonstrated only within the context of an abnormal situation — exclusion of the an-

trum from the normal gastrointestinal passage. This brings up the question: must a vagally induced gastrin release be regarded as an experimental curiosity, devoid of physiological significance, as has been asserted? The answer to this question depends on whether or not a Heidenhain pouch may be considered a physiological target for secretory stimuli e.g. gastrin. The answer is "No." A Heidenhain pouch is devoid of vagal innervation, and, as will be shown below, the effects of gastrin and vagal impulses on the HC1 cells seem to be mutually supportive. But the main cause of Heidenhain pouch's lack of response to e.g. sham feeding is probably the acid inhibition of gastrin release in the antrum. During sham feeding and insulin hypoglycemia, the main stomach is devoid of food and, in the absence of food's buffering action, an abnormally high acidity will prevail in the antrum-duodenum area, through which the acid must pass. Consequently, the antral gastrin release will be depressed. Furthermore the duodenal inhibitory mechanism may come into play (see below). Thus we may conclude that with an intact gastroduodenal passage, gastrin is unlikely to be released in sufficient quantities to elicit an appreciable HCl secretion in an Heidenhain pouch. Although we can feel reasonably confident, from the observations cited above, that vagal impulses suffice to release gastrin, this does not seem to be the final word on gastrin's role. Back in 1942, in my cat experiments, I observed some kind of synergism between gastrin and vagal impulses to the corpus mucosa. Fig. za depicts an experiment in which vagally induced HCl secretion has been blocked by cocainization of the antral mucosa. When such an inefficient vagal stimulation was superimposed on a subliminal dose of intravenously administered gastrin, a profuse secretion of HC1 re91

NCL II EO/UT

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VOLUME CC

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420 Mt

FIG. 2a. Cat. Gastric secretion during stimulation of vagi before and after cocainization of pyloric mucosa. Following this, extract from cat's pyloric mucosa was injected intravenously; during definite periods the vagi were constantly stimulated.

A. Ø FEEDING W A PAVLOV POUCH DOG WITH ANTRUM AS A TOTALLY ((ENERVATED POUCH AND EXCLUDED DUODENUM B. MECHANICAL STIMULATION OF THE TOTALLY DENERVATED ANTRAL POUCH WITH A 20 et RUBBER BALLOON. C.A+B.

A SØ femdy

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B

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Mechanical antral stimulation

2EØ

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08 06 0.4 02

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FIG. zb. b) Secretory response in

Pavlov

pouch. A. Sham feeding. B. Mechanical stimulation (distension) of a denervated antrum pouch. C. Combination of stimuli A and B.

92

suited. In Fig. zb, a recent sham feeding experiment shows a similar synergism between gastrin release from a totally denervated antrum by distention with a balloon, and vagal impulses to the corpus elicited by sham feeding (2o). Distension of the antrum alone elicits a meagre secretion in the Pavlov pouch. So does sham feeding. A combination of the two stimuli, however, gives rise to a copious secretion in the pouch. Similarly subliminal intravenous doses of gastrin enhance the sham feeding response in a Pavlov pouch dog subjected to antral resection. Grossman (2 3) has reported that mechanical stimulation of corpus mucosa enhances the effect of intravenous gastrin. How are we to explain this synergism between gastrin and vagal impulses? Available experimental evidence does not provide an answer to this question. Since I am partial to simple explanations, I would like to think that gastrin is an indispensable link in the excitation of the HC1 glands. Were this not so, it would be rather difficult to account for the fact that the secretory response of a Pavlov pouch to sham feeding may decline to very low levels or disappear entirely after resection of the antrum (Unpublished observations). Whatever may be the intimate mechanism of the synergism between the actions of gastrin and vagal impulses to the HCl producing mucosa, this synergism is likely to be of considerable physiological significance. I, for my part, have been struck by the relative meagreness of the secretory responses of a Heidenhain pouch to antral chemical and mechanical stimulation when these stimuli have been applied to an isolated antral pouch. Under physiological conditions, such isolated stimulation of the antral mucosa does not occur. Antral stimulation is always reinforced to some degree by vagal impulses

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to the corpus mucosa. Even weak mechanical and chemical stimuli may then suffice to initiate significant HC1 secretion. It should be of interest to know if such a reinforcement could account for the secretagogue effects of drinks and appetizers — if they really have any. However, the weak effect of antral stimulation may

have simple technical reasons. Elwin has shown that on perfusion of antrum with chemical stimuli under a pressure of 20 cm H20, chemical stimuli may elicit a copious secretion. This is not due to mechanical stimulation but possibly due to an increased contact with a gastrin releasing area. (Unpublished observations.)

Gastrin Release with Chemical Stimuli TABLE II. Chemical Stimulation of Gastric Acid Secretion Introduction of different stimuli in an isolated antrum pouch Stimulus

Concentration tested (per cent)

Acid secretion from a denervated corpus pouch (Heidenhain)

PROTEINS

Casein—not digested Casein—different stages of hydrolization Peptone

5 3.3 5

0

++( )

AMINO ACIDS

Glycine Acetyl-glycine DL-a-alanine L-Glutamic acid

5-10 2.1 5 1.5

++ ++

(+)

ALCOHOLS

Methyl alcohol Ethyl alcohol Propyl alcohol (n) Isopropyl alcohol Butyl alcohol (n) sec-Butyl alcohol tert-Butyl alcohol

5-10 4-16 5-10 5-10 5-7.9 5-10 5-10

0

+++

+ () ( +) ( ) ( )

+ +

ESTERS OF AMINO ACIDS

Glycine-ethyl-ester HCI DL-phenylalanine-isopropyl-ester HCI 2-amino-ethanol Acetylcholine Suxamethonium iodide (celocurin) Histamine NaCI

16 16 1-5 0.5 0.5 0.001-0.01 0.9

+ + 0 ++ 0 0 0

+++ =strong stimulation ++ =good stimulation + =weak stimulation 93

GASTRIN RELEASE

It has been known for half a century that liver extracts, meat extracts and protein hydrolysates are especially active as stimulants of the HC1 secretion. These products have recently been found to act as chemical releasers of gastrin from antrum. However, we are still surprisingly ignorant as to which of the simple substances in these products act as stimulants. Elwin, in our laboratory, is working on this problem, and Table II shows the secretory responses of Pavlov pouches to antral perfusion with some known con-

stituents. I will confine myself to some comments regarding this table. Undigested proteins seem to have no effect. Peptone, a partially hydrolyzed product, does have an effect. Some simple substances clearly act as secretagogues, e.g. glycin, acetylglycin, a-alanine, ethyl- and propylalcohol and acetylcholine. The chemical relationship of these substances is food for conjecture, as are the stereodiscriminative properties of the gastrin releasing mechanism.

Gastrin Release with Mechanical Stimuli Ingestion of bulky food (bones, bone dust, paper pulp, etc.) has long been known to stimulate copious gastric secretion. Recent investigations into the excitatory action of mechanical stimulation of antrum distension have disclosed that the excitation stems from the release of gastrin. HCI hypersection resulting from

mechanical stimulation of the antrum in conjunction with vagal impulses (e.g. sham feeding) indicates that mechanical stimulation by food may figure more prominently in the elicitation of HCI secretion during a meal than has been generally assumed.

Local Mechanism of Gastrin Release The chemical structure of gastrin is unknown. Histamine-free polypeptide fractions of extracts from antral mucosa of swine, dog, cat and man contain a secretagogue factor which is assumed to be identical with gastrin (24,25). Gastrin, therefore, is regarded as a polypeptide of rather low molecular weight. No gastrinproducing or gastrin-harbouring glandular cells have yet been identified. Such cells may be located in the basal part of the antral mucosa, since gastrin activity is demonstrable in extracts of the basal three-fifth layer of the mucosa and not in extracts from the submucosa (26). Since gastrin release caused by chemical and mechanical stimuli blocked by 94

local anesthes;a of the antrum (cocaine, procaine etc.), by atropine and by ganglionic blocking drugs, it has been suggested that cholinergic structures are at play in the antral gastrin release mechanism. There is a lack of morphological evidence. We know, however, that the stimuli are effective even after vagal denervation and even after antroneurolysis, i.e., isolation of the antral mucosa and part of the submucosa from the muscular layer, and that gastrin release can still be prevented by pharmacological blockade. Hence, the existence of a submucous or intramucous cholinergic nervous plexus may be postulated. It has been suggested that vagally in-

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duced gastrin release is secondary to peristaltic activity, and is not due to the direct effect of vagal stimuli on the gastrin-containing elements (27). This may be true — though I personally doubt it — as concerns the secretory effect of insulin hypoglycemia, which is accompanied by a fairly pronounced antral peristalsis, but it is not true of the secretory effect of sham feeding. Using small endoradiosondes implanted into the antrum, Olbe, in our department, clearly showed that during sham feeding the antral peristaltic movements diminished or completely disappeared. Similar observations have been reported by Thein and Schofield (15). It

may reasonably be assumed, therefore, that efferent vagal impulses pass to the hypothetical antral cholinergic plexus. Whether the antral mucosa contains specific chemo- and mechanoreceptors for triggering of the gastrin release mechanism, or whether antral stimuli act directly on the gastrin-releasing elements, is thus far not known. The similarity of the release mechanisms activated by neural, chemical and mechanical stimuli is evidenced by the sensitivity of all these stimuli to the antral pH, as mentioned on p. 91. The mechanism by which acidity inhibits gastrin release not been identified.

Duodenal Gastrin The fact that a secretory factor — not identical with histamine — has been extracted from the duodenal bulb (28), as well as the fact that mechanical stimuli (29) and unadsorbable substances, such

as saponins (3o), evoke gastrin secretion when applied to the duodenal bulb, suggests that this area may harbour a gastrin mechanism. Its quantitative significance — if any — remains to be elucidated.

Antroduodenal Inhibitory Mechanisms The discovery of the strong inhibitory effect of antral acid pH on gastrin release recalls early allegations that intragastric and duodenal inhibitory mechanisms control gastric secretion (2,31). Did the inhibitory effects, observed by these investigators to be connected with high acidity, ensue from suppression of gastrin release or from activation of other inhibitory mechanisms? Since additional inhibitory mechanisms have in fact been suggested, I would like to touch upon the matter briefly. The inhibitory effect of high intraduodenal acidity on the HC1 secretion has been repeatedly observed. Most investi-

gators, however, have been skeptical as to the physiological significance of this mechanism, the acidity required to elicit inhibition being considered too high. A fall of acidity below pH 2.5 was necessary for inhibition. It was felt that, due to the buffering action of the duodenal contents, such a low pH would never occur under physiological conditions. Recently, however, we have observed (3 2) that instillation of HC1 into an isolated duodenal bulb pouch suffices to depress the postprandial HCI secretion in a Heidenhain pouch. This observation may point to a physiological röle of this inhibitory mechanism, for it is not unreason95

I / GASTRIN RELEASE

able to assume that secretion of HCl into an empty stomach may give rise to high acidity in the duodenal bulb. The duodenal inhibition is mediated by a humoral agent, since inhibition occurs in totally denervated stomach pouches (33). Further, inhibition of the HCl secretion induced by intravenous gastrin denotes that the inhibitory agents acts on the HCI glands and not on the gastrin release mechanism (34) Of late, a lively controversy has been

under way as to whether or not the antrum area delivers an antisecretory agent. Such an agent is thought to be released when antral acidity passes a threshold level (35,36). Other investigators believe the inhibitory effects of antral acidity to be due merely to suppression of gastrin release (37,38). In my opinion, present experimental evidence does not favour the existence of an antral inhibitory hormone.

Experimental and Clinical Significance of the Antroduodenal Excitatory and Inhibitory Mechanisms Our present knowledge concerning the antrum exclusion resulted in an expected neural and humoral control of HC1 secre- hypersecretion, which occasionally tion is not comprehensive enough to pro- reached inordinately high values. In other vide guidance for a rational surgical or dogs, however, the increase of the HC1 medical therapy of peptic ulcer. Quite secretion was low or negligible. In later obviously the complexity of the antrum- experiments on antrum pouches provided duodenum function with its several, only with cutaneous fistulas, we were able to partially apprehended mechanisms, is determine the pH of the pouch contents. such that exclusions, resections, denerva- We found that the pouches of some dogs dons and other operative measures result showed an acid reaction. In these cases, a in unavoidable disturbances of the bal- rim of HCl-secreting mucosa had been ance between excitatory and inhibitory left in the antrum pouch. When a buffercontrol functions. The consequences for ing solution was introduced into such a the gastric secretory pattern are unpre- pouch, the secretory response to sham dictable. A typical example is the hyper- feeding rose considerably (2o). After the secretion and ulcer disposition occurring introduction of an indicator paper techafter antrum exclusion, a very sobering nique for visualisation of the border beclinical experience in the past and an im- tween acid-producing and non-acid-proportant rediscovery in experimental sur- ducing mucosa, our antrum exclusions gery, leading to the recent successful consistently resulted in neutral pouches studies on the gastrin mechanism. The and a vast augmentation of the HCl secrepitfalls for the experimental surgeon are tion in the Pavlov pouches. The above was only one example of the numerous. I would like to recount the following experience from our own la- surprises that an experimental surgeon boratory. In studying the effect of an- may encounter. Other surprises may lie in trum exclusion on the secretory responses store. In my opinion, the duodenal excitato insulin hypoglycemia and sham feed- tory and inhibitory mechanisms, which ing, we were rather discouraged by the no doubt exist, have been very much variability of our results. In some dogs, neglected by most investigators studying 96

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antrum function. It can hardly be immaterial whether the duodenum is excluded from the normal gastrointestinal passage — as in Billroth II — or is not excluded therefrom — as in Billroth I. Exclusion and inclusion may interfere in different ways with the release both of excitatory and of inhibitory agents. Another reason for the somewhat slow progress in the field of gastric physiology is — I think — the inadequate scientific approach of many experimentalists. Most of the earlier studies have been more qualitative than quantitative. Acidity is still occasionally used as a measure of HCI secretion. Even elementary scientific precepts are not always followed. Supramaximal doses, frequently huge overdoses e.g. of insulin and histamine, are used to

evoke HCI secretion despite the obvious fact that no inhibitory or facilitatory mechanism could be successfully studied — or even detected — under such circumstances. To my mind, undue importance has been attached to results obtained with histamine as a secretory stimulus. I doubt that histamine is a physiological stimulus for HC1 secretion. Available evidence, at any rate, has not convinced me of its physiological significance. The failure of inhibitory mechanisms, e.g. the duodenal, to inhibit histamine-induced HCI secretion should not, therefore, be imbued with too much significance. Today gastrin could well replace histamine in experimental studies of the physiology of gastric secretion.

Gastrin Release and Peptic Ulcer Formation Hypersecretion leading to peptic ulcer has been a frequent sequel of experimental exclusion of the antral area, autotransplantation of antral mucosa and other operative procedures leading to an abnormally high gastrin release (18,20,39). In view of these experimental experiences the question arises: are disturbances in the gastrin mechanism implicated in the genesis of peptic ulcer in man? Theoretically they could be, particularly in duodenal ulcer where secretion of HC1 is one of the factors involved. We have developed, in our laboratory, a technique for gastrin assay on unanesthetized cats with gastric fistulas (24). The technique has a comparatively high sensitivity and reliability. Using this assay technique (4o) Ems and Fyrö recorded a higher content of gastrin in antral mucosa from patients with duodenal ulcer than in mucosa from patients with stomach ulcer (Fig. 3). Whether or not this

observation has any pathogenetic bearing on peptic ulcer remains to be seen. Dragstedt has argued that stomach ulcers may well be due to an abnormally high gastrin release, whereas duodenal ulcers are probAB

a ö

AB

400—

U

300cc

z200— a

DUODENAL GASTRIC ULCER ULCER

n 100-

FIG. 3. Gastrin activity (expressed 'n histamine units) in antral mucosa from duodenal ulcer and gastric ulcer patients.

97

I / GASTRIN RELEASE

ably of nervous origin. Our observations situation may as well be the opposite. do not lend weight to this theory. The One guess is as good as the other.

Summary The history of studies on gastrin is briefly reviewed. It is pointed out that already Pavlov and his associates have established the importance of the antrumduodenal area. The gastrin theory was introduced by Edkins in 1906. Due to the prevalence of negative reports, the gastrin theory fell increasingly into disfavor. The decisive break-through came from the work of Dragstedt and his associates in the early fifties, particularly through the observation of profuse hypersecretion occurring on transplantation of antral mucosa into the colon. We suggested in 1942 that vagal impulses are capable of releasing gastrin from the antrum; thus the hitherto rigid functional separation of the nervous and the gastric phases of secretion has been questioned. Neural control of gastrin release has been confirmed by the studies on the secretory response of denervated pouches in dogs to sham feeding and to teasing, and by the demonstration of the disappearance of the response to insulin hypoglycemia after cocainization, denervation of antroneurolysis of the antral mucosa. Woodward et al. have shown a

pH dependence of antral gastrin release resulting from mechanical and chemical stimuli. Available experimental evidence does not permit at present the explanation of the synergism between gastrin and vagal impulses. Gastrin release also follows perfusion with chemical stimuli and mechanical stimuli such as ingestion of bulky food. It is not known at the present time whether the antral mucosa contains specific chemoand mechanoreceptors for triggering of the gastrin release mechanism, or whether antral stimuli act directly on the gastrinreleasing elements. On purely theoretical grounds it can be suggested that disturbances in the gastrin mechanism may be implicated in the gems of peptic ulcer in man, particularly in duodenal ulcer where secretion of hydrochloric acid is one of the factors involved. We were able to record a higher content of gastrin in antral 7rnncosa front patients with duodenal ulcer, using a gastrin assay technique in cats prepared with gastric fistulae. Further experimental and clinical studies are necessary for evaluation of this problem.

References 1. MCCANN, J. Amer. J. Physiol., 89: 2. WILHELM, C. M., O'BRIEN, F. T. and HILL, F. L. Amer. J. Physiol., 115: 429-440, 1936. 3. LEWIS, E. B. Surgery 4: 692-699, 1938. 4. IVY, A. C. and FARRELL, J. 1. Amer. J. Physiol,

5. 6.

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7. 8. 9.

74: 639-649, 1925.

SACKS, J., IVY, A. C., BURGESS, J. P. and VANDOLAH, J. E. Amer. J. Physiol., tol: 331-338,

1932.

and WILSON, Al. J. J. Physiol. (Lond.) 79: 234-238, 1933.

GAVIN, G., MCHENRY, E. W.

lo.

GREGORY, R. A. and Ivy, A. C. Quart. J. exp. Physiol., 31: 111-128, 1942. GROSSAIAN, Al. I., ROBERTSON, C. R. and IVY, A. C. Amer. J. Physiol., 1S3: 1-9, 1948. DRAGSTERT, L. R., WOODWVARD, E. R., STORER, E. H., OBERHELMAN, H. A. and SMITH, C. A. Ann. Surg., 132: 626-640, 1950. OBERHELMAN, H. A. JR., WOODWARD, E. R., ZUBIRAN, J. M. and DRAGSTEDT, L. R. Amer. J.

Physiol., $69: 7;8-748, 1952. II. uvNÄS, B. Acta Physiol. Scand., 4: suppl., r 3: 1 -86, 1942.

UVNAS

12. JANOWITZ, H. D. and HOLLANDER, F. Proc. Soc. 27. OBERHELMAN, H. A. JR., RIGLER, S. P. and DRAGSTEDT, L. R. Amer. J. Physiol., 190: i91Exp. Biol. Med., 76: 49-52, 1951. 13. PEVSNER, L. and GROSSMAN, M. I. Gastroen395, 1957• z8. Uvxäs, B. Acta Physiol. Scand., iv: 97-101, terology, 28: 493-499, 1955. 14. DRAGSTEDT, L. IL, OBERHELMAN, H. A. JR. WOOD1 945. WARD, E. R. and SMITH, C. A. Amer. J. ysiol Ph 29. NAGANO, K., JOHNSON, A. N. JR., COBO, A., OBERHELMAN, H. A. JR. and DRAGSTEDT, L. R. 171: 7-16, 1952. 15. THEIN, M. P. and SCHOFIELD, B. J. Physiol. Surgical Forum, to: 152-155, 196o. (Lond.) 148: 2 91-305, 1959. 30. IVY, A. C., LIM, R. K. S. and MCCARTHY, J. E. Amer. J. Physiol., 74: 616-638, 1925. 16. GREGORY, R. A. and TRACY, H. J. Amer. J. Dig. 31. SOKOLOV, A., Secretory Mechanism of the Dis., 5: 3o8-32 3, 1960. Digestive Glands, Babkin, B. P. ed. New 17. NYHUS, I.. M., CHAPMAN, N. D., DEVITO, R. V. York, Hoeber, 1944. and HARKINS, H. N. Gastroenterology, 39: 32. ANDERSSON, s. and UVNAS, B. Gastroenter582-589, 1960. ology, 41: 486-490, 1951. 18. UVNAS, B., ANDERSSON, S., ELWIN, C. E. and MALM, A. Gastroenterology, 30: 790-803, 33. ANDERSSON, s., Unpublished Observations, 1956. 1962. 19. ANDERSSON, S., ELWIN, C. E. and UVNÄS, B. Ga- 34. ANDERSSON, S. Acta Physiol. Scand., 50: 105stroenterology, 34: 636-658, 1958. 112, 1960. zo.a OLBE, L. Unpublished Observations, 196z. 35. DUVAL, M. K., JR., FAGELLA, R. M. and PRICE, b OLBE, L., Unpublished Obesrvations, 1963. W. E. Surgery, 49: 569-57 2. 1961. 21. WOODWARD, E. R., LYON, E. S., LANDOR, J. DRAG- 36. JORDAN, P. H., JR., and SAND, Is. F. Surgery, STEDT, L. R. Gastroenterology, 27: 766-785, 42: 40-49, 1957. 1 954. 37• LONGHI, E. H., GREENLEE, II. li., BRAVO, J. L. 22. 'WOODWARD, E. R., ROBERTSON, C., FRIED, W. GUERRERO, J.1). and DRAGSTEDT, L. R. Amer. J. and SCHAPIRO, H. Gastroenterology, 32: 868Physiol., tyt: 64-70, 1957. 38. DRAGSTEDT, L. R., KOHATZU, S., GWALTNEY, J., 877, 1957. 23. GROSSMAN, M. 1. Gastroenterology, 4/: 385NAGANO, K. and GREENLEE, H. B. A. M. A. Arch. Surg., 79: 10-21, 1 959. 390, 1951. 24. UVNAS, B. and EMAS, S. Gastroenterology, 39. DRAGSTEDT, L. R., OBERHELMAN, H. A., JR., EVANS, 40: 644-648, 1961. S. O. and RIGLER, S. P. Ann. Surg., 140: 39625. GREGORY, R. A. and TRACY, li. J. J. Physiol. 404, 1954. (Lond.) 156: 523-543, 1961. 40. EMAS, s. and FYH(i, Is. Preliminary report at 26. FYRÖ, B. and OLBE, L. Unpublished observathe Annual Meeting of Swedish Surgical tions. Society, Stockholm, 1961.

99

Recent Advances in Preparation of Gastrin*

at first sight seem inappropriate to include in a symposium on the pathophysiology of peptic ulcer a contribution bearing the above title; but gastrin is now recognized to play a central role in the stimulation of gastric acid secretion, and it is obviously of fundamental importance to find some means of isolating the hormone so that we can not only determine its structure, but also decide exactly what are its physiological properties. It has come to be generally accepted that gastrin stimulates the oxyntic cell exclusively; but Grossman (I) has obtained evidence that in certain circumstances large doses of a potent preparation of the hormone may have some influence also on pepsin secretion, and in more recent studies, Gillespie and Grossman (z) have shown that gastrin preparations may be capable of inhibiting gastric acid secretion, produced both by gastrin and by histamine. Are these effects attributable to the hormone itself, or are they caused by other substances present in the gastrin extract used, which though potent contained several components? This question — and others like it — can only be answered by IT MAY

R. A. Gregory**

isolating the hormone. Moreover, methods of preparation of the hormone may obviously serve as a basis for its extraction from tissues in order to assay the actual quantities present in different circumstances; and since such studies are now in progress in more than one laboratory, it is obviously of interest to consider by what means the hormone can best be extracted from antral mucosa or other tissues and purified as far as possible, before being assayed. This latter point is by no means unimportant. It is now well recognized that cholincrgic stimuli acting on the fundic mucosa potentiate the response of the oxyntic cell to gastrin. For instance, as Grossman (I) has shown, distension of a denervated fundic pouch will greatly increase its responsiveness to a gastrin preparation, or to gastrin liberated from the animal's own antrum. Nov, if there are present in crude antral extracts substances which stimulate gastric motor activity, such substances might potentiate the secretory response to the gastrin also present, and in this way a spuriously high result be obtained. It has been shown by several

This work was supported in part by a grant from the National Institute of Health (AMt 04786-02) U.S.A. ••Front the Physiological Laboratory, University of Liverpool, England. IOI

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groups of workers, notably Blair, Harper and Lake (3), that there is present in crude extracts from all regions of the stomach and small intestine a stimulant of gastric tone and motility; the nature of this stimulant is unknown. Until some means can be found of excluding this substance from extracts which are to be used for gastrin assays, or it can be shown that this substance does not potentiate the effect of gastrin, it would be wise to express the results of gastrin assays in some such terms as "apparent gastrinlike activity". Another substance which it is of course vital to remove from gastrin extracts is histamine. It is probably true to say that it was, more than anything else, the presence of this substance in simple extracts made from all regions of the alimentary tract, and indeed from every tissue in the body, which for more than forty years obscured the existence of a gastric hormone. Not until 1938, thirty-three years after Edkins (4) had suggested the existence of an antral hormone, was biochemical evidence obtained that a substance having the requisite properties existed in the antrum, and that it was not histamine but of proteinlike character. Edkins' method of extracting gastrin (4,5) was simple in the extreme. He boiled mucosa from various regions of the stomach with dilute HCI, water or solutions of dextrin, peptone and sugar. These extracts he found to be effective in stimu102

lating acid secretion when made from the antral mucosa, although it must be said that his data did not decisively substantiate these claims. Edkins made no attempts to purify or concentrate the active principle present in his antral extracts. There then followed a long period, well reviewed by Lim (6), in which attempts were made, notably by the school of Luckhardt and his collaborators in Chicago, to concentrate and identify the active principle. It is now clear to us, reviewing their work, that what they were concentrating was largely histamine, since at an early stage in their extraction procedures they included a step of deproteinisation which would have removed or destroyed any gastrin present, leaving behind histamine. However, some of their findings caused them to suspect that the active principle in their extracts was not identical with histamine, and it seems likely that, in some cases at least, they may have been dealing with a mixture of gastrin and histamine. The crucial advance in the problem of extracting gastrin from antral mucosa was made by Komarov (7), who was the first to realise that the antral hormone might be of the nature of a protein, so that it could perhaps he separated from histamine in crude antral extracts by the use of a protein precipitant, which would leave behind histamine in the supernatant. He introduced for this purpose trichloroacetic acid and showed that when crude acid extracts of antral mucosa were treated with this substance the precipitate could be virtually completely freed of histamine, but would still show on injection into conscious dogs or anesthetized cats the power to stimulate strongly gastric acid secretion (Fig. 1) so that the active principle could not be histamine but must be of proteinlike nature. Similar extracts made from other regions of the stomach were inactive, while those

GREGORY

made from the upper part of the small intestine showed a very small amount of activity similar to that possessed by antral extracts. In his later papers on the subject (8,9), the chemical properties of the active principle were studied in some detail, and two or three methods offered for concentrating it, notably extraction with alkaline aqueous acetone or methanol. For the first time the chemical properties of gastrin were given some degree of definition, and these studies undoubtedly served as a most valuable basis for the work of those who followed. Komarov not unnaturally made no attempt to assess the completeness of extraction of gastrin from the antral mucosa by his method, and indeed, it now seems that it must have been low; the most recent studies on the extraction of gastrin lead to the conclusion that dilute aqueous acid is a poor reagent with which to extract gastrin from antral mucosa. Komarov's work was followed by a series of valuable papers from Uvnäs and his collaborators in Sweden (Io,I1,12). They employed the same method of initial extraction as did Komarov, namely, boiling the minced mucosa with dilute hydrochloric acid and then precipitating the cooled and partially neutralized extract with trichloroacetic acid. Their papers present several methods by which the crude material thus obtained can be further purified, and valuable information can be found in them regarding the chemical behaviour of the active principle. They utilized a fractional precipitation of the crude aqueous extract at different pH values as the chief means of concentrating the active principle; but this did not effectively separate gastrin from the inert denatured protein with which it is associated in all crude extracts, and which when precipitated by almost any means carries down with it the gastrin at pH values in the region of 7-8 or lower. This was the

chief difficulty which we later encountered in our attempts to isolate the hormone. The only further documented attempts to extract and purify the hormone are those of Harper (13) and Jorpes, Jailing and Mutt(14). Harper, in an abstract of a paper presented to the Physiological Society, stated that he had utilized the ethanol extraction method, introduced by Mellanby, for the preparation of secretion as a means of extracting gastrin from antral mucosa. However, no details were given in the abstract, and the method was never published in full. Jorpes, Jalling and Mutt (14) introduced a novel procedure for the extraction of gastrin which was no doubt intended to exclude at the outset as much inert denatured protein as possible. They extracted the mucosa (which was previously steamed to disintegrate cells and arrest enzyme action) with acid methanol, in which they claimed that gastrin was highly soluble. This extract was then precipitated with ether, and the precipitate subjected to further similar extraction under slightly different conditions. The final material was dialysed at pH 3; the activity remained within the sac. Their preparations were of relatively high, though variable, potency when injected intravenously into anesthetized cats. It seems doubtful from our own later studies whether gastrin is, as they supposed, highly soluble in acid methanol, so that the yield was probably poor, and the bulk of the product was inert protein. When Hilda Tracy and I (t 5) commenced our attempts to extract gastrin from antral mucosa, we began by repeating the methods of Komarov and of Jorpes, Jailing and Mutt. We soon came to the conclusion that neither procedure was satisfactory. Extraction with hot dilute acid, followed by trichloroacetic acid precipitation was technically unsatisfactory, particularly on a large scale, and the 103

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extracts showed little activity when injected subcutaneously into conscious dogs, although they were substantially free from histamine. It seemed clear to us that some means must be found of extracting the antra mucosa in an efficient and clean fashion, which would be usable on a fairly large scale; and we eventually evolved first an extraction of the fresh mucosa with aqueous acetone and picric acid, and then later an extraction with trichloroacetic acid and aqueous acetone, which gave a higher yield. By treatment of either of these extracts with acid ether we recovered an aqueous residue which could be precipitated cleanly and effectively with trichloroacetic acid, giving a crude powder which was very rich in gastrin activity and free from any significant quantity of histamine. The further purification of this crude powder presented a difficult problem. The great bulk of it consisted of inert protein to which the gastrin was very readily bound at pH values lower than about 8.5. We finally evolved a modification of the aniline/acetone precipitation utilized by Greengard and Ivy (16) in their preparation of secretin, and this gave a product virtually free from denatured protein, which was suitable for chromatographic fractionation. For this purpose, we utilized columns of calcium phosphate gel, which one of us had previously used 104

with success in the purification of urogastrone (17). Although the product from the calcium phosphate gel was by no means homogeneous (several components could be detected on paper electrophoresis), it was sufficiently purified to be given without significant side-effects, not only subcutaneously and intramuscularly, but also intravenously to the human subject (i8) (Fig. z). It was free from histamine, and represented a most powerful preparation of the gastric hormone. It was such a preparation which we used for stimulating secretion in a human subject, and which Grossman (i) used in his experiments, to which I have referred earlier. Although this method may fairly be said to have represented a very considerable advance on anything previously described, it nevertheless had very serious defects which became only too apparent to us as our experience of it accumulated. In the first place it was tedious and expensive to perform on any considerable scale, so that it was obviously unsuitable as the basis for any attempt to isolate the hormone in appreciable quantities, i.e. amounts sufficient for structural studies. Furthermore the yield of activity from a given weight of mucosa turned out to be very low. This became apparent to us when in January, 1961, Blair, Harper, Lake, Reed and Scratcherd (19) published a note in the Proceedings of the Physiological Society in which they reported that a simple boiled aqueous extract of homogenized antral mucosa was very rich in gastrin. The crude aqueous extract was centrifuged to remove particulate matter and fat, and then filtered through a highly retentive paper. Only a small proportion of the liquid passed through as a clear opalescent filtrate, and this was then precipitated with zo volumes of acetone, which removed practically all of the histamine originally present.

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The water-soluble white powder thus obtained was shown to be a potent stimulant of secretion when injected intravenously into the anesthetized cat. We repeated their experiments, testing the product by subcutaneous injection into conscious dogs provided with fundic pouches, and it was immediately apparent to us that this was a method of making a most potent, though crude, extract. It showed also that the yield obtained by our own method was very low. This low yield is attributable to the fact that we had not homogenized the mucosa, but only steeped the intact strips in the extracting fluid overnight. It is interesting to note that the method of Blair et al. (i q) is essentially the same as that used originally by Edkins (q.), with the addition of acetone precipitation which excludes almost all the histamine, so that at long last undeniable evidence has been provided that Edkins' simple extracts of antral mucosa must have contained plenty of gastrin, as well as histamine. The finding of Blair et al. made it abundantly clear that in any method intended as the basis for the isolation of gastrin the initial extraction must for reasons of convenience, cheapness and yield, commence with a simple aqueous extraction such as they described. We therefore devoted our attention to finding some means of obtaining from such a crude aqueous extract a suitable powder with which further purification could be carried out, and which could be obtained on the largest possible scale. In collaboration with Dr. M. I. Grossman, who was visiting us, we modified the procedure of filtration, so that it could be performed on a very large scale. This was done by using, not paper, but fine nylon fabric for the filtration, the "filtrate" being a fluid having the appearance of milk, and still containing much fat and protein in suspension. This aqueous extract, or "milk", as we termed it, could

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be made quite easily with suitable equipment from large amounts of mucosa, and the problem then was how to recover from this fluid a material which would be suitable for further purification. We eventually discovered that by merely bringing the fluid to pH 3.5-4 with glacial acetic acid and leaving it overnight, a heavy flocculent precipitate formed, which contained most of the gastrin. This precipitate was collected and extracted with the mixture of trichloroacetic acid and 75 per cent aqueous acetone which we had formerly used to extract the antral mucosa directly (18). This method gave a yield of gastrin from the mucosa which, though not complete, was at least as good as that described by Blair et al. With this material Dr. Tracy and I later carried out studies of the chromatographic behaviour of gastrin on many different columns; and we have now evolved a completely new method which will shortly be published. The final product of this latest method is apparently homogeneous when subjected to paper electrophoresis in acid and alkaline buffers, and also to analytical ultracentrifugation. We believe it to be pure gastrin. Amino acid-analysis shows the presence of the following residues: Aspartic acid, glutamic acid, proline, glycine, alanine, methionine, tyrosine, phenylalanine, and an unidentified substance. This material is far more potent than histamine in stimulating acid gastric secrcI05

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tion, though the time-course is rather different (Fig. 3). The yield of gastrin obtained by this new method is about zo mg from to kilo of fresh mucosa. It is possible to assess approximately the recovery of gastrin in the various stages; and on this basis we would make a rough estimate that the total quantity of gastrin originally present in the fresh hog mucosa in the form in which we have extracted it is probably not more than twice the final yield which we obtain. This would correspond to 4 mg per kilo of fresh mucosa or 4 lug from i gram. The availability of what we believe to be pure gastrin has enabled us to clarify an observation made by Gillespie and Grossman (z) to which I referred at the beginning of this account. They found that in a conscious dog with a fundic pouch secreting steadily in response to either histamine or gastrin, given by repeated small subcutaneous injections, the administration of a single large intravenous dose of their gastrin preparation would completely inhibit for one or two hours or more the preceding secretory response. We have confirmed this observation using our latest preparation, so it seems that the effect is to be attributed to gastrin itself and not to the presence of an inhibitor in th preparation used by Gillespie and Grossman. We hope in due course t

o6

to examine Grossman's earlier observation (t) in which it was shown that large doses of the gastrin preparation which he was using at that time stimulated pepsin secretion. An interesting and important problem which must not be overlooked at this stage is whether the material which we have isolated from antral mucosa is in fact the hormone itself, if by "hormone" we mean the substance which is actually liberated from the antral mucosa into the bloodstream. This point can only finally be settled by comparing in some way the properties of the circulating hormone with those of the substance which we have obtained. It might well be that the substance actually liberated into the circulation consists of the material we have isolated in association with a larger molecule, and that our "gastrin" has been split off from a "parent" molecule in the course of extraction. One method of attacking this problem would be to prepare an extract by the gentlest possible means from fresh antral mucosa, e.g. preparation of a "press-juice", and then to seek to isolate from it, again by gentle procedures, e.g. paper or column electrophoresis, an active fraction, and to compare the nature of this material with that which we have isolated. This has not yet been attempted, and it seems likely that it will prove a somewhat difficult task owing to the physical nature of antral mucosa. However, Anderson and his co-workers (zo) have reported the preparation of material having gastrin activity from the stomach of various species by a method which chiefly utilizes extraction at a high pHvalue (Fig. 4). The material they have thus prepared is not homogeneous, but the gastrin activity appears to be associated with protein material of high molecular weight, and it may be that their preparation contains gastrin in a state in which it normally exists in the mucosa.

GREGORY

Gastrin in Extra-Antral Tissue Edkins (4) reported that a small amount of gastrin activity was present in cardiac mucosa from the hog stomach. This we confirmed (18), and we later discoverd that Lim (6) had also confirmed it earlier. We found no activity whatever in regions of the dog gastric mucosa other than the antrum; but Fletcher has stated that in the fundic mucosa of the ox and the sheep, gastrin activity can be found by his method. In duodenal mucosa Komarov, Uvnäs and Harper, all reported that small amounts of gastrinlike activity were present in extracts made by their method. We and Grossman have made many attempts to extract gastrin from hog and dog duodenal mucosa, without any success whatever; all our extracts have proved uniformly negative. However, Lai (z 1) has recently reported that using the first stage of our 1961 method (18), considerable amounts of gastrin activity can be extracted from fresh human duodenal and jejunal mucosa, and small amounts

from ileal mucosa. An intersting side line in our work on the preparation of gastrin was the use of our 1961 method to extract gastrinlike activity from primary pancreatic tumors and secondary metastases in cases of the Zollinger-Ellison syndrome, in which a severe gastric hypersecretion is associated with the presence of a malignant tumor of islet origin in the pancreas. In the first such case we described (22), we used the picric acid-aqueous acetone method for initial extraction of the tumor, but in our later cases (23,24), we used the improvement on this procedure which I have mentioned above, namely trichloroacetic acid/aqueous acetone; and we now are satisfied that repeated extraction of such tumor tissue or of antral mucosa, with this reagent, after grinding with sand will effect a complete extraction of the gastrin present; so that this method of extraction seems of value as a basis for the assay of gastrin present in antral mucosa and other tissues in different circumstances.

Conclusions In this account I have tried to review briefly the steps by which we have arrived at the present position, in which we are at last in possession of what appears to be pure gastrin, or at least one form of it. By any of the several methods which I have described, potent and more or less purified preparations of gastrin can be made available for physiological experimentation; and there seems to be no reason why in the not so distant future supplies of highly purified or even pure gastrin, suitable for clinical trials, should not be made available commercially as have been secretin and pancreozymin. We may perhaps even look forward to the day when, by some biochemical or even immuno-

logical technique, it will be possible to assay gastrin in the circulating blood, and to locate the "gastrin cells" in the antral nrucosa. These advances, which we may be sure will be achieved in time, will perhaps enable us to make some assessment of the role of the antrum and the hormone it releases in the etiology of peptic ulcer. The elucidation of the structure of gastrin, which will certainly be achieved also, will provide its—as in the case of the posterior pituitary hormones—with a "blueprint" for the manufacture of analogues of the hormone with which to embark on a study of the mechanism by which it stimulates (and in certain circumstances, inhibits) gastric secretion. I07

I / RECENT ADVANCES IN PREPARATION OF GASTRIN

ACKNOWLEDGEMENTS

Figure I reproduced from Komarov, S. A. Proc. Soc. Exp. Biol. Med. 38: 514, 1 938, (7) Figure 2 reproduced from Gregory, R. A. and Tracy, H. J. Physiol. (Lond.) 156: 523, 1961 (18)

Figure 4 reproduced from Anderson, W. R. Fletcher, T. L., McAlexander, R. A. Pitts, C. L., Cohen, R. L. and Harkins, H. N. J. Dairy Science 44: 2218, 1961 (2o). With permission.

References I. GROSSMAN, M. L J. Physiol, (Lond.) 157: 14, 1961. 2. GILLESPIE, I. E. and GROSSMAN, M. I. Unpublished Observations, 1962. 3. BLAIR) E. L. HARPER, A. A. and LAKE, H. J. J. Physiol. (Lond.) 121: 20-21, 1953. 4. EDKINS, J. s., Proc. Roy. Soc. (Biol.) 76: 376, 1905. 5. EDKINS, J. S., J. Physiol. (Lond.), 34: 133144, 1906. 6. LIM, R. K. S. Quart. J. Exp. Physiol., 13: 79-I03, 1922. 7. KOMAROV, S. A. Proc. Soc. Exp. Biol. Med., 38: 514-516, 19g. 8. KOMAROV, S. A. Rev. Canad. Biol., 1: 191-205, 1942. 9. KOMAROV, S. A. Rev. Canad. Biol., 1: 377-401, 1942. 10. MUNCH-PETERSON, J., RONNOW, G., and UVNÄS, B. Acta Physiol. Scand., 7: 289-302, 1944. 11. UVNAS, B. Acta Physiol. Scand. 6: 97-107, 1943• 12. UVNÄS, B., Acta Physiol. Scand., 9: 296-305, 1945. 13. HARPER, A. A. J. Physiol. (Lond.) ,o5: 31P, 1946.

I o8

14. JORPES, J. E., JALLING, o. and MUTT, v. Biochem. J., 52: 327-328, 1952. 15. GREGORY, R. A. and TRACY, H. J. J. Physiol. (Lond.), 149: 70-71, 1959. 16. GREENGARD, H. and IVY, A. C. Amer. J. Physiol, 124: 427, 19 q 38. 17. GREGORY, R. A. J. Physiol. (Lond.) 129: 528546, 1955. 18. GREGORY, R. A. and TRACY, H. J. J. Physiol. (Lond.) 156: 523-543, 1961. 19. BLAIR, E. L., HARPER, A. A., LAKE, H. J., REED, J. D. and SCRATCHERD, T. J. Physiol. (Lond.) 156: I1-13, 1961.. 20. ANDERSON, W. R., FLETCHER, T. L. MCALEXANDER, R. A., PITTS, C. L., COHEN, R. L. and HARKINS, H. N. J. Dairy Science, 44: 22,8, 1961. 21. LAI, K. s. Personal communication, 1962. 22. GREGORY, R. A., TRACY, H. J., FRENCH, J. M. and SIRCUS, W. Lancet, 1: 1045-1048, 1960. 23. GROSSMAN, M. I., TRACY, H. J. and GREGORY, R. A. Gastroenterology, 41: 87-91, 1961. 24. FRIESEN, S. R., TRACY, H. 3. and GREGORY, R. A. Ann. Surg. 155: 167, 1962.

The Osmotic Activity of Gastric Secretion

WHILE it is often thought that gastric secretions are iso-osmotic with plasma, this is by no means invariably the case, and systematic deviations from osmotic equilibrium exist. These deviations are often large enough to be important problems for explanation by any theory of gastric secretion.

Osmotic constituents of gastric juice In gastric juice secreted at a high rate in response to histamine, the major osmolar components are chloride, hydrions, sodium and potassium which between them account for about 97 per cent of the total osmolarity. Less than 10 m Osmols are due to minor constituents such as glucose (2-7 m Osmols), urea (1-2 m Osnols), Ca", Mg", and PO., (1-2 m Osmols) and proteins ( ioo mEq/LHB+O. Even if it be supposed that the non-parietal secretion contained as much as wo mEq/L bicarbonate, the mixture would need to be in the ratio of so ml. of non-parietal secretion for every coo ml. of parietal secretion. This would mean that under maximal histamine stimulation, when the total secretion rate is 300-500 ml./hr., the nonparietal secretion rate might reach 100-i 50 ml./hr. This seems particularly unlikely as it is generally agreed that histamine has little or no effect on the non-parietal se-

cretion rate; our conclusion must be therefore that while admixture plays some role it is very improbable that it can account for the major part of the hypoosmolarity, particularly in those human subjects whose secretion remains hypotonic even at maximal rates. c) The back diffusion hypothesis. As pointed out above, back diffusion of the H'C1- with partial replacement by Na+Clcan account for the development of hypotonicity because of the greater fugacity of the first pair. This theory also predicts the osmotic-secretion rate relationship found by Lifson, Varco and Visscher (5). Equally there seems no reasonable doubt that it plays a rile in producing hypotonicity and raising the sodium concentration of gastric juice. It is theoretically capable of reducing the gastric osmolarity by about 8o m Osmols. This, if it were fully realized, would be nearly enough to account for the maximum hypo-osmolarity, unaided. d) Diffusion of bicarbonate. Hogben's results show the flux of bicarbonate from blood to secretion is even greater than that of sodium. There must normally be a considerable flux of bicarbonate ions across the mucosa into the gastric juice. These will annihilate hydrions as mentioned above. If H'C1- diffusion is countered at least in part by Na+HCOs back diffusion, the reduction of tonicity will be greater than if the back diffusion were only of NaCl. ' - This process is as plausible as the previous one.

113

I / THE OSMOTIC ACTIVITY OF GASTRIC SECRETION

Conclusion We must conclude on present evidence that all of the latter three processes contribute to the hypotonicity of gastric juice, but that diffusional exchange of H' Cl- for Na' Cl- and Na' HCO3 ion pairs is probably the most important. Whether the secretion produced by the gastric glands themselves is hypotonic remains an open question, but it is simpler to suppose that the secretion of the gastric glands is always slightly hypertonic, and that the three dissipative processes tend to remove the hypertonicity at low flow rates and convert it to hypotonicity. A fully satisfactory explanation of Gilman and Cowgill's observations is not available at present. The tendency of gastric juice osmolarity to follow changes

in plasma osmolarity is inherent in the explanations already given, but the reason for a greater osmotic difference, when the plasma osmolarity is low, is not clear. A possible explanation is that most of the osmolar water movement in the gastric mucosa occurs through the intercellular boundaries. A reduction of extracellular osmolarity might be expected to lead to swelling of the mucosal cells, with a consequent reduction in intercellular spaces, and hence reduced intercellular water movement. ACK NOW LEDGEMENT

Figure 1, taken from data of Gilman, A. and Cowgill, G. Amer. J. Physiol. 103: 1 43, 1 933. With permission (to).

References 1. HIRSCHOwn-L, B. I. J. Appl. Pysiol., r5: 933-

2. 3. 4. 5.

6. 7. 8.

9. 10. 11.

938, 1960. National Research Council. Handbook of Biological Data. William S. Spector, ed., 61, Phil., Saunders, 1956. GUDIKSEN, E. Acta Physiol. Scand., 5: 39-54, 1943• HIRSCHDW1T2, B. 1. Amer. J. Dig. Dis., n.s., 6: 199-228, 1961. LIFSON, N., VARCO, R. L. and VISSCHER, M. B. Gastroenterology, r: 784-802, 1943. LIFSON, N., VARCO, R. L. and VISSCHER, Al. B. Proc. Soc. Exp. Biol. Med., 49: 410-415, 1942. GRAY, J. S. and BUCHER, G. R. Amer. J. Physiol., 133: 542-550, 1941 . IHRE, B. Acta Med. Scand., Suppl. 95, 1938. IVY, A. C. and OYAMA, Y. Amer. J. Physlol., 57: 51-6o, 1921. GILMAN, A. and COWGILL, G. R. Amer. J. Physiol., :03: 143-152 , 1 933. COPE, 0., BLATT, H., and BALL, M. R. J. Clip. Invest., 22: 11 I -115, 1943.

114

12. LEE, P. R., CODE, C. F. and SCHOLER, J. F. Gastroenterology, 29: 1008-1015, 1955. 13. HUNT, J. N. J. Physiol., (Lond.) 109: 134-141, 1 949. 14. BANDES, J. HOLLANDER, F. and GLICKSTEIN, J. Amer. J. Physiol., 13r: 470-482, 1940. 15. SLEETH, C. K. and VAN LIERE, E. J. Prov. SO2. Exp. Biol. Med., 36: 57 1-573, 1937. i6. COPE, O. COHN, W. E. and BRENZIER, A. G. JR. J. Clip. Invest., 22: 103-110, 1943. 17. BEITEMEIER, R. J., CODE, C. F. and ORVIS, A. L. Fed. Prov., r4: 119-120, 1955. 18. HOGBEN, C. A. Al. Amer. J. Physiol., :8o: 641649, 195519. TEORELL, T. J. Gen. Physiol., 23: 263-274, 1939. 20. TEORELL, T. Gastroenterology, 9: 425-443, 194721. TEORELL, T. J. Physiol., 97: 308-31 5, 1940. 22. HEINZ, E. Biochim. Biophys. Acta. 6: 434444, 1951.

Gastric Motility'"

THE study of gastric motility may be approached in several ways. The first method is to study gastric contractions, which may be considered as one of the basic manifestations of gastric motor activity. This can be accomplished by direct observation, balloon kymographic recordings, intraluminal pressure measurements, radiologic techniques and electromyography. Another way is to study

G. Vantrappen T. Vandenbroucke S. Verbeke T. Hellemans*

gastric emptying by measuring the time required for evacuation of the stomach, and the amount of food expelled under various conditions. Each approach has its own advantages and disadvantages, and it is only by integrating the data obtained from all methods that some insight can be gained concerning gastric motility.

Techniques of Study of Gastric Contractions Direct observation

Balloon kymographic recordings

It is possible to observe gastric contractions in man through gastroscopy and, rarely, through a gastric fistula (I). In animals, direct observations have been made using transplanted gastric pouches with or without preservation of the vagal innervation and of the vascular supply (2). These methods are subjective, and do not permit quantitative analysis.

The classical system consists of a rubber balloon connected via a catheter to a water manometer, of the spirometcr type, which registers volume changes kvmographically (34). A simple U-tube with a very light floater (5) or an optic manometer (6,7,8) can also be used, instead of the water manometer. This technique is cheap and easy to use. Another advantage

*Supported by a grant from the U.S. National Institute of Health, (AM 03587-o3cM) and from the F.W.G.O., Brussels. **prom the Laboratory of Physiopathology (Dir. Prof. Dr. D. Vanderbroucke), Section of Gastroenterology, University of Louvain Medical School, Louvain, Belgium. II5

1 / GASTRIC MOTILITY

is that small contractions, which may not alter the intraluminal pressure, can be recorded by the balloon system, but not by open-tip catheters or microtransducers. The balloon, which fills the lumen, undergoes a volume decrease, which even for small contractions is sufficient to be registered. Balloon systems also permit the study of changes in the tone of the wall. They are filled with air under a given pressure toll the resistance of the wall does not permit any further inflow of the air. The volume of the balloon is then a measure of the resistance of the wall to the dilating force, in other words of the tone of the wall. These oncometric methods have a number of disadvantages inherent in the use of a balloon and in the dynamic properties of a water manometer. The balloon acts as a foreign body, and as such stimulates motor activity in the gastrointestinal segment under study (g). This probably explains, for example, why the antral motor activity recorded with a balloon in a fasting dog is very similar to the intraluminal pressure recordings obtained after a meal (1o). The balloon offers a greater resistance to the contracting segment than the normal contents. This results in a more forceful and more frequent contractions than under physiological conditions (1 I ). Propulsive peristaltic contractions may displace the balloon, thereby rendering impossible the prolonged study of a given segment. When large balloons are used, the changes registered are the result of the changes in two or more adjoining segments. A compression of one part of the balloon could be compensated by a simultaneous expansion of another segment (4,12,13 ). It is for these reasons that balloons do not permit adequate studies of short segments of the gastrointestinal tract, such as a sphincter. Furthermore, a peristaltic wave will appear longer than in reality, because I16

the wave is being recorded for as long as some part of the balloon is being compressed. Finally, the pressures recorded through balloon systems are very dependent on the shape, length and volume of the balloon used, and of the elasticity of the balloon wall (6,7,12). All the above objections are particularly applicable for large sized balloons and much less so for small ones. But even the very small balloons do not necessarily measure the true intraluminal pressures, rather it is the pressure inside the balloon. A balloon measures volume changes, and these will vary with the pressure within the balloon. If the balloon is very compressible, it could be completely emptied before the maximal amplitude of a contraction is reached. In such cases very powerful contractions are incompletely recorded. If the balloon is under too high pressure, then weak contractions cannot produce a volume change, and consequently are not registered (14). In an ideal system, the volume displaced in the balloon by a given pressure should be equal to the volume displaced in the gut by a contraction creating the same pressure change. This pressure-volume relationship for the alimentary canal is unknown. Water manometers provide further disadvantages in the use of the oncometric method, as their frequency response is much too slow (15), and their pressurevolume coefficient much too great to follow precisely the physiological pressure variations in the gastrointestinal tract. The frequency of the usual water manometer is smaller than 1 cycle per second, and the pressure-volume coefficient easily reaches 0.5 ml. per cm. This is obviously unsatisfactory for a true natural reproduction of physiological pressure changes. Intraluminal pressure recordings Intraluminal pressures can be measured

VANTRAPPEN, VANDENBROUCKE, VERBEKE & HELLEMANS

by the use of open tip catheters, microtransducers, radiotelemetering capsules and motorized pressure capsules. These instruments are designed to reproduce as accurately as possible the true intraluminal pressure changes. For this purpose the entire system, manometer and recording apparatus, has to satisfy definite criteria (16,1 ,18,19,20,21). The pressure changes in the gastrointestinal tract are pressure waves, which, besides a fundamental frequency, also possess harmonic components. The pressure measuring and recording devices must be capable of registering all the harmonic components necessary for an accurate reproduction of the physiological pressure changes. According to Hansen (17), this includes faithful reproduction of the first six harmonics. This frequency response of a pressure measuring and recording system depends on the properties of the catheter used, on the type of conduction medium and on the characteristics of the manometer and the recording instrument. Electric manometers of the strain gauge or capacitans type have a frequency response exceeding Soo cycles per second, and the recording instruments commonly used in conjunction with these manometers also have a high frequency response, above l oo cycles per second. Consequently, the limiting factors in the frequency response of the system are the properties of the catheters and the conducting medium. With water as conductor, the natural frequency of the system will depend on the mass of the conductor, the damping and the pressure-volume coefficient. The mass of the conductor is directly proportional to the length of the catheter: the longer the catheter, the greater the mass, and the less the frequency response will be. The diameter of the catheter determines both the mass and the damping effect. The mass of the conductor is directly proportional to the square of its

diameter, while the damping is inversely related to the cube of the diameter of the catheter. In other words, a narrow catheter has a lower frequency response than the large one. The third important factor is the pressure-volume coefficient. The greater the volume that has to be displaced to produce a given pressure change, the longer the frequency response. Electrical and optical manometers have the advantage of having a small pressurevolume coefficient. These theoretical considerations concerning some of the factors determining the dynamic properties of a pressure measuring instrument are not sufficient to calculate these characteristics. Therefore, the frequency response and other dynamic properties usually have to be determined experimentally. In one method, periodic pressure changes of varying frequencies are produced in the system, and the response is registered. In this manner one can determine how the amplitude of a pressure wave varies as a function of the speed with which this pressure variation occurs. Another method uses the so-called "square wave impact", whereby the system is subjected to a sudden impact and caused to vibrate. The frequency response and damping of the system can be calculated from the recordings of these vibrations. Other factors besides these dynamic properties have to be considered, such as the linearity of the pressure recordings and the absence of hysteresis. It may be emphasized that the intraluminal pressure changes constitute only one manifestation of the gastrointestinal motility. These techniques provide no direct information on other phenomena, such as movements of the intestinal wall or propulsion of the intestinal contents. a) THE OPEN TIP CATHETER. In this procedure, the open end of a catheter filled with water or gas is introduced into the gastrointestinal tract, while the other end 117

I / GASTRIC MOTILITY

is connected with a manometer system. The pressure variations are transmitted through the fluid or gas column to the manometer, and after amplification are registered as pressure curves. This technique has a number of advantages over the oncometric methods. If the measuring and registering apparatus are adequate, the true physiologically occurring pressure changes are measured (22). The pressures can be studied in a very small segment, such as a sphincter. The diameter of the catheters is so small that three or four can be tied together without significant stimulation of the motor activity (Io). It is thereby possible to record pressure changes simultaneously from three or more separate points. This permits one to determine, for example, if a contraction wave is peristaltic, provided the points of recording are separated by a contracted lumen. Finally, the frequency response of an open tip catheter system is much greater than that of a balloon recording system. b)

THE ELECTROMAGNETIC MICROTRANS-

is a tiny pressure sensitive apparatus, which is brought directly into the gastrointestinal canal (23). Pressure variations are converted in situ into electrical charges, which after amplification are registered as pressure-curves. This little device has most of the advantages of open tip catheters. In addition the properties of this system are solely determined by the dynamic properties of the manometer and the recording apparatus; pressureconduction plays no role. DUCER

c)

THE RADIOTELEMETERING CAPSULE

consists of a small cylinder, containing a radiometer with a frequency modulating transistor, a pressure sensitive membrane and a replaceable battery. The intraluminal pressures influence the diaphragm of the capsule, and are transmitted as radiosignals, which are picked up by a frequency modulating receiver (24). The 118

main advantage of this method is that it permits the study of the mobility of those segments of the alimentary tract, such as the distal small bowel and proximal colon, which are accessible only with difficulty by the usual methods. The method is also well tolerated by the patient, and allows one to obtain continuous motility recordings. The great disadvantage of the capsule is that its location and progress are completely dependent on the motor activity of the gastrointestinal tract. Continuous recordings from one given segment and pull through techniques are not possible. d) THE ENDOMOTOR-SONDE COIISISCS of two parts: a microtransducer and an electric micromotor with a traction system (25). The intraluminal pressure-changes influence two pairs of strain gauges, connected through a bridge circuit to an electronic amplifier and a recording instrument. The electric micromotor activates a cogwheel system that allows the capsule to move forwards or backwards along a toothed nylon string. When the patient has swallowed such a string, the motorized pressure capsule can be quickly introduced in the gastrointestinal tract. With this technique, the position of the instrument remains under constant external control, and repeated pulls through the same bowel segment are possible. Radiological Methods With X-ray techniques, the movements of the wall of the alimentary canal and of its contents can be followed. For this purpose, either radio-opaque material is ingested, or small metal pellets are inserted in the gastric wall (lead shot method) (26). Fluoroscopy allows only subjective observations and leaves no permanent document. The danger of radiation exposure limits the period of observation. but this can be remedied in part by the

VANTRAPPEN,VANDENBROUCKE, VERBEKE & HELLEMANS

use of an image intensifier. Radiocinema- Two very small spirals are stitched under tography (27) gives a permanent record the serosa on both sides of the pyloric of the observations made, and allows later canal. An alternating current runs analysis of the data obtained. Cineradio- through one spiral and produces an ingraphy can be combined with balloon duction current in the other spiral, the kymography (z8). The movements of intensity of which is inversely proporthe wall and its contents can be photo- tional to the distance between the two graphed while the balloon manometer spirals. The closure of the sphincter will system registers the contraction waves. intensify the induction current in the secThis method facilitates the interpretation ondary coil. With this device, prolonged of motility tracings, and gives informa- study of the pylorus can be made under tion concerning the nature and function physiological conditions. Electromyography studies the elecof the contraction e.g. if the contractions are evacuating or not. Simultaneous cine- trical activity of the gastric musculature, radiography and intraluminal pressure and registers potential differences between recordings can also be used in such man- two electrodes placed on an exposed ner that both the pressure variations and stomach (32). It is with this technique the radiographic images are being re- that the existence of a pacemaker in the corded on the same document (29,30). vicinity of the cardia was established in This relatively complicated method al- animals. The interpretation of the traclows an exact correlation between mano- ings obtained with intragastric electrodes metric and radiologic data but is very in humans may be complicated by artefacts arising from the friction of the elecexpensive. The inductograph has been used in ani- trodes against the stomach wall. mals to study pyloric contractions (31).

Types of Gastric Contractions The motility tracings vary with the methods used to record the gastric contractions. Recordings made with a balloon placed in the antrum in fasting subjects yield three types of pressure waves (Fig. i) (14, 33,34,35)• Type I has an amplitude of less than 5 cm. of water and a duration varying between i8 and zz seconds. These waves may be rhythmic or not; when rhythmic they occur at a rate of three per minute. Type II is similar to type I except for a greater amplitude, ranging from io to 5o cm. of water. Type III waves consist of a series of rhythmic type II waves superimposed on an increase of

FIG. t. The three types of waves observed in balloon recordings of motility from the pyloric antrum of normal subjects (14)

PRESSURE CHANGE Cm /H20

TYPE I

100-

TYPE II

TYPE IQ

80604020-

A

IAfP

o

t ln,

B

s

19

I / GASTRIC MOTILITY

the basal pressure. A quantitative estimate of the motor activity can be arrived at by dividing the sum of the duration of the phasic activity by the total recording time, and expressing it as a percentage of this recording time (6). At the level of the antrum in fasting subjects, motor activity covers 38 per cent of the recording time (14,36); 23 per cent are type I waves and 15 per cent type II waves. From simultaneous cineradiographic and balloon kymographic recordings (28), it appears that types I and II waves correspond to annular contractions, progressing in a peristaltic fashion over the antrum towards the pylorus. Type I waves come from the contraction of only part of the circular muscle fibers of the gastric antral wall. Type II waves are the result of deep contractions, probaby involving the entire circular musculature. Over 7o per cent of type II waves eject barium through the pylorus into the duodenum, while only 17 per cent of type I waves produce an evacuation. From these observations it is concluded that the primary function of type II waves is propulsion, with mixing as a secondary function, the reverse being true for type I waves. No type III waves were observed in these particular studies. According to Texter and others, the waves recorded with open tip catheters cannot be classified on the basis of their amplitude (13,37). They are differentiated into A and B waves, depending on their duration. Type A waves last for less and type B waves for more than thirty seconds. Using this technique, antral activity in fasting subjects is only 18.7 per cent of the total recording time; 17.1 per cent being A waves and 1.6 per cent B waves. Waves of greater amplitude and shorter duration become more frequent as the recording tip approaches the pylorus. Other measurements made with open tip catheters permitted a classificaI20

GASTRIC PRESSURE WAVES

600

2.

2.6.

51 ,

76-I. 10.1.12.5 1.6.15 15,,17,5 174-20 201225 22625 25-27.5

~7

AMPLITUDC Imm

FIG. 2. Frequency distribution curve of the amplitude of the phasic intraluminal pressure changes recorded in the antrum of normal human beings.

tion of pressure waves based on their amplitude (Fig. 2) (38). According to this data, intraluminal pressure waves are of three types: phasic waves of type I with an amplitude of 1 to 15 mm. of Hg; phasic waves of type II with an amplitude of 15 to 3o mm. of Hg; and tonic, type III waves. Type II waves can be found superimposed upon these tonic pressure waves. The total duration of motor activity in the antrum of fasting normal subjects is 9.5 per cent of the recording time: 3.9 per cent being type I waves, 3.2 per cent type II waves and 2.4 per cent type III waves. Despite the fact that these waves are comparable to those recorded by way of a balloon system, one cannot conclude that they are identical, the techniques of measurement being very different. From simultaneous cineradiography and intraluminal pressure measurements (38), it appears that there is no absolute correlation between the radiological intensity of a contraction and the amplitude of the recorded pressure wave. It happens that deep antral contractions which evacuate the gastric contents result in relatively small pressure changes in the antrum. On the other hand, radiologically weak contractions, producing no emptying of the stomach contents, may give rise to waves of greater amplitude.

VANTRAPPEN, VANDENBROUCKE, ~I min.—.

VERBF.KE & HELLEMANS 10mm Hg „AtiJWv

RAN I

RAN

2

RAN.3

WNW

WOO

FIG. 3. Gastric secretion in Heidenhain-pouch dogs (control series). Each point and column represents the mean of 5 dogs. Bars denote the standard deviations.

Intraluminal pressure recordings indicate that the rhythmic contractions occur with a frequency of three per minute in the body of the stomach, while in the antrum the frequency may change into a rate of six per minute (Fig. 3). Simultaneous fluoroscopy does not confirm this reduplication, but shows that non-evacuating gastric contractions result in a backflow of antral contents towards the oncoming contractions. It is likely that this backflow is responsible for the duplication of the pressure deflection observed. Studies of the electrical activity of the gastric muscle indicate the existence of a pacemaker in the vicinity of the cardia,

from where the impulse is propagated towards the pylorus (32). At regular intervals of eight to ten seconds there is a discharge of potential complexes, which last five to eight seconds. This electrical activity probably constitutes the excitatory mechanism for the peristaltic gastric contractions, which only become visible in the region of the antrum. This stimulating mechanism is autonomous and invariant, but the intensity of the resulting contractions can be influenced by many factors. The most important one is the vagus nerve. Increased efferent vagal impulses increase the forcefulness but do not change the rhythm of the gastric contractions (39) . It has been shown in cats, anesthetized with chloralose, that intermittent efferent vagal stimulation using ►to 5 stimuli per second (the normal frequency of vagal activity) results in weak gastric contractions. Stimulation with 5o stimuli per second for 2.5 seconds potentiates the effect of slower stimulation, which thereafter will result in more powerful contractions for at least five minutes.

The Antral Pump On radiological examination of apparently normal subjects, one may get the impression that the pylorus at times prevents the antral contractions from emptying the barium meal into the duodenum. It should be remembered however that, for passage of barium through the pylorus, it is not only necessary that the pylorus be open but also that the pressure gradient be sufficient between the antrum and the duodenum. Consequently, lack of passage of barium into the duodenum does not prove that the pylorus is closed. Evi-

dence obtained from animal experiments indicates that the normal state of the pylorus is one of relaxation. Studies have been made in dogs, having one balloon placed in the duodenum, one in the antrum and a tube surrounded by a balloon in the pylorus, permitting free passage of contents (40). It could be shown that while the contraction waves pass through the antrum towards the duodenum, the pylorus remains open; that it contracts when peristalsis reaches the pylorus, to relax again afterwards. Similar observations I2I

I / GASTRIC MOTILITY

have been made in cats with the leadshot method (26). Combining the latter techniques with simultaneous pressure determinations in the antrum and the bulb, using open tip catheters and optic manometres, it was found that the pylorus was relaxed for 6o per cent of the recording time, and that the closure of the pylorus coincided with the peak of the pressure rise in the antrum. Recordings made with the inductograph in dogs also show rhythmic contractions of the pylorus, occurring every ten to fourteen seconds (31). No high pressure zone could be shown at the level of the pylorus by most investigators, using open tip catheters or balloons measuring 6.5 mm. in diameter and 1.5 cm. in length (41). Wilbur et al., however, using a balloon i cm. long and 0.5 cm. in diameter, noted the existence at the pylorus of a high pressure zone reaching 13 to 193 cm. of water and extending over 4 to 10.5 mm. (42). From these data one can consider the pylorus as an integral part of the gastric musculature, constituting the terminal portion of the antrum. In the resting state, the pyloric area is relaxed. Gastric emptying is achieved through peristaltic contractions originating about the incisura angularis and usually sweeping all the way to the pyloric sphincter (43). When, on approaching the pylorus, the antral contraction produces an antroduodenal pressure gradient of 2-4 cm. of water, part of the bolus passes into the duodenum, and another portion flows back through the contraction ring. The pyloric sphincter then begins to contract, resulting in a sharp rise of the pressure in the terminal antrum. Until the sphincter is nearly closed, the proximal duodenum is still relaxed. The antroduodenal pressure gradient during this phase is about 20-3o cm. of water. This explains why, for the first three or four seconds there is still a flow of gastric content into the duoden122

um through the contracting pylorus. Finally, the pylorus closes, and the contraction wave dies out at the moment where the antral pressure is maximal. Hereupon follows very quickly a pressure rise in the duodenal bulb, and the contents arc propelled further in the intestine while the pylorus remains closed. Next, the antrum and pylorus relax (43). The fraction of the gastric content, passing through the pylorus with each cycle is very small. Only about i to 3 ml. are ejected into the duodenum with the contractive wave. The main function of the pylorus is to prevent regurgitation of intestinal contents into the stomach when the duodenum contracts (43,44). It also prevents the passage of large food particles into the duodenum. The peristaltic wave proceeds in the stomach as a contraction ring, progressing in the direction of the pylorus and pushing the gastric contents ahead of it. A pressure gradient is created which is a function of the viscosity of the contents, the diameter of the contraction ring, the speed of progression of the peristaltic wave and the size of the compartment distal to the contraction ring (10). When the peristaltic wave approaches the pylorus, it in turn contracts. This creates a narrow canal through which the food must pass. Large particles are held up and return toward the body of the stomach. Similar observations had already been made by Cannon (4546), who followed gastric emptying of large bismuth pills mixed with a bread meal. Each peristaltic wave pushes the pills a little bit further, passes over them and sets them back a little. These pills reach the pylorus but cannot pass it. After all the bread has left the stomach, the pills still remain in the antrum. These experiments indicate that no special mechanism whereby the pylorus would contract to prevent the passage of large food particles has to be postulated. The antrum and the pylor.

VANTRAPPEN, VANDENBROUCKE, VERBEKE & HELLEMANS

us form a narrow tunnel preventing the passage into the duodenum of large food particles and allowing only small amounts of the contents to leave the stomach at a

time. The remainder of the contents are subjected to an oscillating notion which helps in the mixing and liquification of the meal.

Receptive Relaxation of the Stomach The motor function of the proximal half of the stomach muscle differs significantly from that of the distal half. The fundus and body of the stomach have not only weak peristaltic contractions but also show a remarkable capacity of relaxation. A voluminous meal can be accommodated in the stomach without a significant rise in intragastric pressure. Filling of the stomach with a closed abdomen in animals gives rise to only a slight increase in intragastric pressure, just before rupture occurs (47). The pressure rise is greater when the same volume is introduced more rapidly into the stomach. In man, insufflation of 50o ml. of air results in a mean increase of the basal pressure of only 6.3 cm. of water (22). Belching up of that air brings the pressure back to its original level, and all the symptoms caused by the insuf cation disappear. Experiments

with the leadshot method in cats clearly show that this relaxation occurs mainly in the fundus and body of the stomach (z6). Excision of the upper portion of the stomach results in almost complete disappearance of this receptive relaxation. The small pressure rise, noted after filling of the intact stomach, is attributed to the distension of the abdominal wall, due to the increase in intra-abdominal pressure. This rise in pressure upon filling of the stomach is not observed with the abdomen open (z6). According to Cannon, there is a certain degree of relaxation of the abdominal wall itself, induced by the distension of the stomach. This is not observed when equal amounts of air or fluid are introduced into the peritoneal cavity. This reflex relaxation still occurs after section of the vagus and splanchnic nerves.

Gastric Emptying Quantitative data concerning gastric emptying have been obtained in two ways. In one method (48), a radio-opaque meal is given, and gastric emptying is followed by fluoroscopy or multiple X-ray pictures. In this way the emptying time can be determined. In addition the size of the X-ray image of the gastric contents at various intervals can be measured and expressed as a percentage of the initial picture (49,50). When the logarithms of these percentages are plotted against time, å curve is obtained which gives additional information concerning the process of

gastric emptying. However, these measurements are only a rough quantitative estimate of the evacuation of the stomach. A second method consists of emptying the stomach with a tube at a given time after food intake and measuring the amount remaining in the stomach. As a rule, a marker is mixed with the food, and the time necessary for the disappearance of this marker from the stomach is considered as the evacuation time. Classically a fractional test meal method is used. The aspirated samples give an idea of the amount of food still present in the stom123

I / GASTRIC MOTILITY

ach at the time of aspiration. Obviously, only complete aspiration will give accurate information on the gastric contents at a given time, but this procedure interrupts the normal process of gastric emptying. The most complete studies have been done with the serial test meal method (51). Fasting subjects are given a standard meal homogenously mixed with a known quantity of phenol red. After a given time, the entire gastric contents are aspirated. This procedure is repeated in the same person on subsequent days, but the stomach is emptied at different intervals after the intake of the test meal. This provides quantitative data about gastric emptying, assuming that the emptying of a standard meal in the same person is comparable on successive days. A possible error could result from incomplete recovery of the gastric contents. This can be checked by washing out the stomach with water at the end of the aspiration. The presence of significant amounts of phenol red in the rinsing water indicates incomplete aspiration. The volume of gastric juice secreted during the experiment can be calculated from the amount and the concentration of phenol red in the test meal and in the aspirate. If one may assume that the gastric secretions are mixed evenly with the test meal, one can then determine the true fraction of the test meal that has left the stomach. Normally gastric emptying starts a few minutes after gastric filling (46,51,52). Distension of the stomach by food stimulates peristalsis, provided the musculature is in a state of tone and resists distension. Stretching of a flaccid stomach does not cause peristalsis. In the anesthetized cat with intact vagi, peristalsis starts one to two minutes after introduction of a meal (46). Once peristalsis is initiated, section of the vagi is without influence upon gastric emptying. If the vagi are cut before 124

feeding the animal, it takes much longer before gastric emptying starts to proceed. These observations in the animal are comparable to those of Douthwaite and Thorne (5 3) in duodenal ulcer patients given hexamethonium bromide. Under these conditions of acute pharmacological vagotomy, gastric emptying is also delayed. V agal stimuli may be responsible for the accommodation of the tension of the gastric muscle fibers to the volume of the ingested meal, to permit the initiation of peristalsis (54) . Gastric emptying follows, at least in part, an exponential curve (Fig. 4). Experiments with the serial test meal method (51,54), using a liquid meal of 75o ml., show that gastric emptying proceeds increasingly more slowly as the time since ingestion of the meal increases. When the data obtained are plotted on a logarithmic scale with, as ordinate, the volume of the meal remaining in the stomach and, as abscissa, the time, a linear relationship is noted. Gastric emptying can be considered to proceed in an exponential fashion, i.e., a constant fraction of the remaining gastric content leaves the stomach per unit of time. Comparable results are obtained when cereal breakfast food is used instead of a liquid meal (55). Data from radiological studies on the emptying of an opaque test meal are also compatible with an exponential type of gastric emptying (49,50). Information concerning evacuation of solid food is less precise. The time necessary for complete gastric evacuation has been determined by aspiration of the stomach or by the follow-through of a radio-opaque meal. An average solid meal mixed with barium takes from three and a half to four and a half hours for complete evacuation (56,57). This means that at the time of the main meals, the stomach is usually empty. The physical properties of a food substance play at times a more

VANTRAPPEN, VANDENBROUCKE, VERBEKE & HELLEMANS

important role than the chemical composition. In cats, meals consisting of large pieces leave the stomach more slowly than a finely divided food (58). Water intake during the meal has little effect upon the evacuation of solid food. The liquids do

not mix with the solid components of the meal. They run occasionally along the lesser curvature, but usually all around the food, and find their way to the pylorus and duodenum before the solid food (58,59)•

Gastric peristalsis and consequently the rate of gastric emptying is influenced by a number of factors. The volume of the meal (Fig. 4). A two-fold increase in the volume of a radio-opaque meal prolongs the evacuation time but does not double it (48). In other words the gastric emptying is accelerated with increased volume of the meal. This has been studied in greater detail by the use of the serial test meal method. It has already been stated that the evacuation of a 75o ml. liquid meal follows an exponential curve. Intake of a more voluminous meal (125o ml.) results in a more rapid initial emptying, while a small meal has a slower initial evacuation (60). The degree of distension of the stomach is thought to be the controlling factor. After the initial phase, a second period follows during which the evacuation is exponential. The smaller the meal, the more rapid this exponential emptying. In the third or terminal phase of evacuation of a large meal, the last couple of hundred milliliters again leave the stomach rapidly. This probably occurs at a time when the rate of intestinal absorption exceeds that of gastric evacuation, also after a solid meal, because complete aspiration through a tube is impossible under these circumstances. However, radiologic data suggest that normal meals are evacuated from the stomach in a similar fashion (49,50,52 ,55, 57). The osmotic concentration of the meal (Fig. 5). It used to be accepted that

VOLUMEOF MEALREMAINING

Factors Affecting Gastric Emptying 1250 1000 750 400 330 160 100 63 40 25

16

10 0

20 40 60 80 IO TIME IN MINUTES

FIG. 4. The emptying pattern of liquid meals of 125o, 75o and 33o ml. (6o) respectively.

j700 E

600

W å

500

U,

w 400 Z O 300

r å 200 0 0 ° 100 W

9 n

200

800 400 600 CONCENTRATION OF SOLUTE (milliosmols/l)

FIG. S. Relationship between the volume of a 750 nil test steal remaining in the stomach after 20 minutes and the concentration of solute in the meal (Si). I25

1000

I / GASTRIC MOTILITY

the stomach retained its contents until osmotic concentration approached that of the plasma. Any hypo- or hypertonic solution which would reach the duodenum was thought to be regurgitated back into the stomach. The experiments of Hunt appeared at first analysis to support this hypothesis (6i). Hunt determined the changes in freezing point of the stomach contents at variable intervals after feeding test meals of various osmotic concentrations. The hypertonic meal had a depression of freezing point of o.8°C and the hypotonic meal a depression of o.z°C. As the gastric contents were aspirated after longer intervals, the A, and consequently the tonicity of the hypertonic meal decreased, while they increased for the hypotonic meal. After about an hour and a half, the osmotic concentration of both meals became equal. The A of the aspirates was then o.3°C. When a test meal of such concentration is introduced in the stomach, no change is observed. An osmotic concentration giving a A of o.3 °C is still definitely hypotonic in relation to plasma. This concentration is only reached after about an hour and a half, at a time when most of the meal has already left the stomach. Hence one cannot accept the hypothesis that food has to reach isotonicity before leaving the stomach. Nevertheless osmotic concentration does have a definite influence upon the speed of gastric emptying. Hypertonic sucrose solutions have a rapid initial evacuation but a delayed exponential evacuation, resulting in a total gastric emptying (6i). The stronger the concentration of the sugar solution, the more gastric emptying is slowed down. This effect is observed even at low hypotonic concentrations of sucrose. A sucrose test meal of half the osmotic concentration of the plasma leaves the stomach more slowly than distilled water, and this delay is still greater when the concentration is isosmotic with 126

the plasma (6z). Solutions of KC1, dextrose, sorbitol and ammonium sulfate behave like sucrose. On the other hand, test meals consisting of dilute solutions of NaCl, NaHCO3 and urea leave the stomach more rapidly than distilled water (47, 63,64). However, when the osmotic concentration of the latter solution reaches 25o m-osmoles per liter, the speed of evacuation becomes less than for distilled water (47). In other words, all hypertonic solutions, at least above a certain concentration, delay gastric emptying. Ammonium chloride is an exception, and its rate of evacuation from the stomach is independent of its concentrations. Fat content of the meal. Fat is even more effective than hypertonic solutions in delaying the emptying of the stomach (65), fatty acids being more effective than their corresponding triglycerides. Biliary and pancreatic secretions are essential for the inhibition of gastric motility by fat in the duodenum of the rat (66). This could indicate that the depression of gastric motility is not achieved by contact of fat with the wall but rather by an increase in osmolarity of the intestinal contents. Gastric emptying has been studied with test meals containing fat and mixed with barium. The emptying time of the stomach was mainly dependent on the fat content of the meal (56,67). The gastric contractions are not abolished but the peristaltic waves are less forceful (39). The inhibitory effect of proteins and carbohydrates on gastric motility is much less pronounced. Other factors. Unpleasant sensations or emotions decrease gastric motility and slow down the evacuation of a test meal. This was observed in man by direct inspection through a fistula (1). In the dog, unpleasant or painful stimuli also cause a delay in gastric emptying (68). This is not due to pylorospasm but

VANTRAPPEN, VANDENBROUCKE, VERBEKE & HELLEMANS

to the inhibition of antral motility. Dis- results in a decrease in gastric peristalsis tension of the intestine in the dog also (69).

Control Mechanisms of Gastric Emptying Neural and humoral mechanisms control gastric emptying. Receptors are located in the stomach, duodenum and intestine. The most important ones are the duodenal receptors (70,71,72,73,74, 75,76). As a rule the effect is one of depression or even abolition of gastric contractions. There are no data to support the existence of duodenal receptors which activate gastric emptying. Acid in the duodenum delays gastric emptying in the normal and even more in the patient with anacidity (75). The introduction of acid in the duodenum is followed by a brief pyloric contraction, shortly followed by an inhibition of the antrum and the pylorus (76). The latter is an important factor in slowing down gastric evacuation (77) . On the contrary, duodenal drainage results in a marked increase in antral peristalsis and a speeding up of gastric evacuation. Obviously this is the consequence of the removal of an inhibitory factor of gastric emptying. The difference in emptying time between various test meals also disappears if the duodenal contents are continuously aspirated. Indeed aspiration prevents the gastric contents entering into contact with the duodenal receptors, whose function it is to slow down gastric evacuation in accordance with the composition of the meal. This inhibitory mechanism is at least in part transmitted by the vagus. X-ray studies and direct observations in the animal after bilateral vagotomy show at first atonic gastric dilation, decreased peristalsis and delayed emptying (78). Later on, there is only a slowing down of the initial emptying phase. The inhibition of antral

peristalsis, induced by the introduction of acid in the duodenum, is markedly reduced after vagotomy (76). Vagotomy further results in the decrease or the abolition of the inhibitory effect of proteoses, peptones, fats, hyper- and hypotonic solutions (7o,74). The gastric receptors are connected with ascending vagus fibers of slow conduction. Using cervical vagus dissection, it is possible to register the afferent impulses originating from a given area of the stomach. It could be shown that the number of afferent impulses sent out by the gastric receptors upon passive distension or active contraction of the stomach increased in proportion to the increase in intragastric pressure (79). This might represent the peripheral mechanism of immediate satiation of hunger (8o). Other fibers respond to gastric distension, which is not associated with a rise in intraluminal pressure. They give information concerning the size of the stomach, rather than the tone of the wall. Some receptors are sensitive to pH values of the contents below 3, others send out impulses when the pH exceeds 8. This activity probably influences the vagal nucleus, which in turn regulates gastric motility. It is likely that a similar mechanism is also active in the duodenum. Gastric emptying is further controlled by humoral mechanisms (2) . Fat in the duodenum or jejunum liberates enterogastrone. This hormone inhibits the gastric contractions and relaxes the pylorus. Its action has a latent period of three to five minutes and lasts for thirty to forty minutes. Denervated transplanted gastric pouches are also responsive. Local anes127

I / GASTRIC MOTILITY

thesia of the duodenal mucosa abolishes the inhibitory effect of fats. The reduction in the inhibition of gastric motility by fat after vagotomy (74) is attributed to the preponderance of sympathic reflexes, which either interfere with the liberation of enterogastrone or decrease the sensitivity to this hormone. An alternate explanation is that vagal receptors are necessary to liberate enterogastrone. Hunt has proposed the following hypothesis in relation to the duodenal osmoreceptors (81). These receptors would be vesicular structures which are stimulated by deflation. A slowing down of gastric emptying would correspond to a decreased inflow of water into the vesicles. When a solution of high osmotic concentration enters the duodenum, it extracts water from the receptors, which are hereby stimulated, producing a slowing down of gastric motility. The duodenal content is then diluted by duodenal secretions, and the stimulation of the receptors is decreased. Dextrose, sorbitol, calcium chloride, potassium chloride and ammonium sulfate are electrolytes with mono- and bivalent anions and cations. Gastric evacuation is increasingly delayed with increasing concentration of these ions. As the response of the receptor is proportional to the osmotic pressure of

the meal, the receptors may be called osmoreceptors. As mentioned before, ammonium chloride does not affect gastric emptying. It is postulated that the receptor is permeable to ammonium ions (as ammonia) and to chloride ions. Ammonium chloride would therefore not produce an osmotic deflation of the receptors, and the mechanism that inhibits gastric motility would not be activated. Potassium chloride and ammonium sulfate do slow down the evacuation of the stomach. This can be explained, if one accepts that penetrating ions, such as ammonium and chloride, do not diffuse through the membrane of the osmoreceptor when their counter ion is nonpenetrating, such as potassium, calcium or sulfate. NaCI solutions, up to a concentration of 25o milliosmoles, accelerate gastric emptying. At higher concentrations, gastric motility is inhibited. The dilute solutions would be evacuated more rapidly because of a special mechanism, which would accelerate the transport of Na* ions, Cl- ions and water into the receptors. This inflow of water into the receptors would activate gastric emptying. Such a transport mechanism probably becomes saturated at a given osmotic concentration, and further increase of the concentration will then cause a delayed gastric emptying.

Gastric Motility in Peptic Ulcer In patients with duodenal ulcer, the their rhythm are identical in both groups. total motor activity of the empty stomach, There is also no difference in the motility recorded from the antrum with a balloon recordings obtained from the body of the system during pain-free periods, is com- stomach. parable to that of normal subjects (14). When the intraluminal pressure changes The frequency of the various types of are measured with open tip catheters in waves varies. In duodenal ulcer, type II patients with an active peptic ulcer but peristaltic waves are much more frequent in a pain free period, no significant difand type I mixing waves less frequent ferences from the normal are observed than in the normal. The mean amplitude (37). The administration of an acid (o.i and duration of types I and II waves and N HC1) barium meal of zoo ml. to pati128

VANTRAPPEN, VANDENBROUCKE, VERREKE & HELLEMANS

ents with an active duodenal ulcer causes a marked increase in gastric activity of type B and also an increase of the total duodenal activity. This is even more so in patients who experience pain during the evacuation period. In patients with an active gastric ulcer the motility tracings recorded from an empty stomach or during an evacuation period, accompanied or not by pain, are not very different from normal tracings. There is even a tendency for decreased activity in gastric ulcer as compared to

normal. These data are in accordance with fluoroscopic observations of slow motility in gastric ulcer. Duodenal ulcer patients who experience pain during evacuation show a marked increase in antral motility. Nevertheless gastric emptying is often retarded, and this is attributed to a dyssynergy of the antral and pyloroduodenal mechanisms. There are reasons to believe that the above described alterations in motility play a role in the production of ulcer pain (82,83).

Summary The investigation of gastric motility tween the radiological intensity of a conmay be approached either by a study of traction and the amplitude of the recordgastric contractions or by measurement ed pressure wave. of gastric emptying. Each approach has its Studies of the electrical activity of the own advantages, and only by integration gastric muscle indicate the existence of a of the data obtained from all methods can pace maker in the vicinity of the cardia, some insight be gained into this complex from where the impulse is propagated towards the pylorus. Thus electrical field. Gastric contractions can be studied by activity probably constitutes the excitadirect observation, balloon kymographic tory mechanism for the peristaltic gastric recordings, intraluminal pressure record- contractions, which only become visible ings, radiological techniques and electro- in the region of the antrum. This stimumyography. Three types of pressure lating mechanism is autonomous and inwaves can be observed in recordings variant, but the intensity of the contracmade with a balloon placed in the antrum tions can be influenced by many factors, in fasting subjects. Type I wave has an the most important one being the vagus amplitude of less than 5 cm. of water and nerve. The pylorus should be considered as a duration varying between 18 and 22 seconds. Type 11 waves are similar to type an integral part of the gastric musculaI, except for a greater amplitude (10-50 ture, constituting the terminal portion of cm. of water). Type III waves consists of the antrum; in the resting state, the pya series of rhythmic type II waves super- loric muscle is relaxed. The antroduodenal imposed on an increase of the basal pres- pressure gradient is a function of the sure. It is concluded that the primary viscosity of the contents, the diameter function of type II waves is propulsion, of the contraction ring, the speed of prowith mixing as a secondary function, the gression of the peristaltic wave and the reverse being true for type I waves. From size of the compartment distal to the consimultaneous cineradiography and intra- traction ring. Quantitative data concerning gastric luntinal pressure measurements, it appears that there is no absolute correlation be- emptying have been obtained by adminis129

I / GASTRIC MOTILITY

tration of radio-opaque meal or by mea- denal ulcer, the total motor activity of suring the amount of food remaining in the empty stomach is comparable to that the stomach following emptying with a of normal subjects; type 11 peristaltic tube. Normally gastric emptying starts a waves are much more frequent and type few minutes after gastric filling. Disten- I mixing waves less so. Duodenal ulcer sion of the stomach by food stimulates patients who experience pain during evaperistalsis, provided the musculature is cuation show a marked increase in antral in a state of tone and resists distension. motility; gastric emptying is however Gastric emptying follows at least in part often retarded probably due to a dyssynan exponential curve. In human subjects, ergy of the antral and pyloroduodenal an average solid meal mixed with barium mechanisms. In patients with an active takes three and a half to four and a half gastric ulcer, there is a tendency towards a decreased motility. hours for complete evacuation. Gastric peristalsis and the rate of emptying are influenced by a number of ACKNOWLEDGEMENTS factors including the volume, osmotic Figures 1, 4 and 5 are reproduced by concentration and fat content of the meal, as well as emotional and other factors. courtesy of the editor of GastroenterolNeural and humoral mechanisms control ogy (Fig. i) and of J. Physiol. (Fig. 4 gastric emptying. In patients with duo- and 5).

References 1. WOLF, s. and WOLFF, H. G. Human gastric function, New York, Oxford, 1943. 2. FARRELL, J. i. and IVY, A. c. Amer. J. Physiol. 76: 227, 1926. 3. INGELFINGER, F. J. and ABBOTT, W. O. Amer. J. Dig. Dis. Nutr. 7: 468-474, 1940.

4. TERTER, E. C. JR., VANTRAPPEN, G., LIEMER, M. D. and BARBORKA, c. J. Quart. Bull. Northw. Univ. Med. Sch. 32: 281-290, 1958. 5. CHAPMAN, W. P. and PALAZZO, W. L. J. Chn. Invest. 28: 1 517-1525, 1949. 6. HIGHTOWER, N. C. JR.? CODE, C. F. and MAHER, F. T. Proc. Mayo Chn., 24: 453-46 2, 1 949. 7. QUIGLEY, J. P. and GLASSER O. Medical Physics

310-3 i8, Chicago, Year Book, 1944. 8. ROWLANDS, E. N., HONOUR, A. 3., EDWARDS, D. A. W. and CORBETT, B. D. Clin. Sci. 12: 2 99-

06 195.

P. and BRODY, D. A. Amer. J. Med. 13: 73-81, 1952. 10. BRODY, D. A., WERLE, J. M., MESCHAN, I. and QUIGLEY, J. P. Amer. J. Physiol. 130: 791801, 1940. 1I. ABBOTT, W. 0., HAR MINE, H. K., HERVEY, J. P., INGELFINGER, F. J., RAWSON, A. J. and ZETZEL, L. J. Clin. Invest. 22: 22 5-234, 1943. 12. GRUBER, C. M. and DENOTE, A. Amer. J. Physiol. 111: 564-570, 1935. 13. TERTER, E. C. JR. and IKEYA, J. Fed. Proc. 17: 162, 1958. 14. CODE, C. F., HIGHTOWER, N. C. JR. and MORLOCK, C. C. Amer. J. Med. 13: 328-351, 1952. 9.

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and DAVIDSON, M. Methods in Medical Research. Visscher M.B. ed. Chicago, Year Book, 1957. FRANK, 0. Z. Biol. 44: 445-613, 1903. HANSEN, A. T. Acta Physiol. Scand. 19: Suppl. 68, 1949. HANSEN, A. T. Acta Physiol. Scand. t9: 33;343, 1950. HANSEN, A. T. and WARBURG, E. Acta Physiol. Scand. 19: 306-332, 1950.

15. FARRAR, J. T.

16. 17. 18. 19.

20. WOOD, E. H. Science, 112: 707, 1950. 21. WOOD, E. H. Amer. J. Physiol. 163: 762, 1950. 22. LORBER, S. H. and SHAY, H. Gastroenterology

27: 478-487, 1954.

23. QUIGLEY, G. P., BRODY, D. A., MCKAY, B., LANDOLINA, W. C. and MCALISTER, J. H. Fed. Proc. 9: 102, 1950. 24. FARRAR J. T. and BERNSTEIN, J. S. Gastro-

enterology 35: 603-612, 1958.

25. VANTRAPPEN, G., D'HAENS, J., VAN DERSTAPPEN, G., VERBEKE, S., and VANDERIIROUCKE, J. The

motored pressure capsule: A new device for studying gastro-intestinal motility. Read at the world congress of gastroenterology in Munich, May 13-29, 1962. 26. GIANTURCO, C. Proc. Mayo Clin. 8: 784-787, 1933• 27. TEXTER, E. C. ja. Amer. J. Dig. Dis. ns. 6: 983-1001, 1961. 28. SMITH, A. W., CODE, C. F.

and SCHLEGEL, J. F. J. Appl. Physiol. 11: 12-16, 1957. 29. VANTRAPPEN, G., LIEMER, M. D. and TEXTER,

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E. C. JR. Fed. Proc. t7: 167, 1958. 59. NIELSEN, N. A. and CHRISTIANSEN, H. Acta Radiol. (Stockh) 13: 678-685, 1932. 30. VANTRAPPEN, G., LIEMER, M.D., IKEYA, J., TERTER, E. C. JR. and BARBORKA, C. J. Gastro- 6o. HUNT, J. N. and MACDONALD, I. J. Physiol. enterology 35: 592-602, 1958. (Lond.) 126: 459-474, 1954. 61. HUNT, J. N., MACDONALD, I. and SPURRELL, 31. BRODY, D. A. and QUIGLEY, J. P. J. Lab. Clin, Med. 29: 863-867, 1944. W. R. J. Physiol. (Lond.) 115: 185-195, 1951. 32. BOZLER, E. Amer. J. Physiol. 144: 693-700, 62. HUNT, J. N. Guy Hosp. Rep. 103: 161-173, 1945. 1954• 63. APPERLY, F. L. Brit. J. Exp. Path. 7: I1I-120, 33. FOULK, W. T., CODE, C. F., MORLOCK, C. G. and 1926. BARGEN, J. A. Gastroenterology 26: 6o1-6i 1, SHAY, H. and GERSHON-COHEN, J. Surg. Gynec. 34 sMITH, H. W. and TEXTER, E. C. JR. Amer. J. 64. Obstet. 58: 935-955, 1934. Dig. Dis. ns. 2: 318-326, 1957. 65. QUIGLEY, J. P. Arch. Surg. (Chic.) 44: 41435. TEMPLETON, It. D. and LAWSON, H. Amer. J. 437, 1942. 66. MENGUY, R. Amer. J. Dig. Dis. ns. 5: 792Physiol. 96: 667-676, 1931. 80o, 196o. 36. HIGHTOWER, N. C. JR. and CODE, C. F. Proc. 67. ANNEGERS, J. H. and ivy, A. C. Amer. J. PhyMayo Clin. 25: 697-7o4, 1950. siol. 150: 461-465, 1947. 37. SMITH, H. W., TEXTER, E. C. JR., STICKLEY, J. H. and BARBORKA, C. J. Gastroenterology 32: 68. QUIGLEY, J. P., BAVOR, H. J., READ, M. R. and 1025-1047, 1957. BROFMAN, B. L. J. Clin. Invest. 22: 839-845, 38. VANTRAPPEN, G. Unpublished Observations. 1943. Physiology of the diges69. 39. DAVENPORT, H. W. LALICH, J. MEEK, W. J. and HERRIN, R. C. tive tract. Chicago, Year Book, 1961. Amer. J. Physiol. 115: 410-414, 1936. J. 70. CRIDER, J. O. and THOMAS, J. E. Amer. J. Phy40. THOMAS, J. E. and CRIDER, J. O. Amer. siol. 123: 44-45, 1938. Physiol. 111: 124-129, 1935. 71. KEETOx, R. W. Arch. Intern. Med. 35: 68741. ATKINSON, M., EDWARDS, D. A. \V., HONOUR, A. J. and ROWLANDS, E. N. Lancet 2: 918-922, 697, 1925. 72. LUCKHARDT, A. IL, PHILLIPS, H. T. and CARLSON, 1957. A. J. Amer. J. Physiol. 50: 57-66, 1919. 42. WILBUR, D. L. III, KELLEY, M. L. JR., and CODE, C. F. Fed. Proc. 18: 170, 1959. 73. MARBAIX, O. La cellule 14: 249-33 2, 1898. 43. WERLE, J. M., BRODY, D. A., LIGON, E. W. JR., 74. QUIGLEY, J. P. and MESCHAN, I. Amer. J. PhyREAD, M. R. and QUIGLEY, J. P. Amer. J. Physiol. 123: 166, 1938. siol. 131: 606-614, 1941. 75. SHAY, H. Bull. N.Y. Acad. Med. zo: 264-291, 1944. 44. BRODY, D. A. and QUIGLEY, J. P. Gastroenterology 9: 570-575, 1947. 76. THOMAS, J. E., CRIDER, J. O. and MOGAN, C. J. 25029: Amer. J. Physiol. CANNON, W. B. 45. Amer. J. Physiol. to8: 683-700, 1934. 266, 1911. 77. QUIGLEY, J. P., READ, M. R., RADZOW, K. H., The mechanical factors of CANNON, W. P. 46. MESCHAN, I. and WERLE, J. M. Amer. J. Phy-

digestion. London, Arnold, 1911. 47. HUNT, J. N. J. Physiol. 132: 267-288, 1956. 48. VAN LIERE, E. J., sLEETII, C. K. and NORTHUP, 0. Amer. J. Physiol. 119: 480-482, 1937. 49. HF!! FBRANDT, F. A. and TEPPER, R. II. Amer. J. Physiol. 107: 355-363, 1934. 50. HENSCHEL, A., KEYS, A., STURGEON, A. M. and TAYLOR, H. L. Amer. J. Physiol. 149: 107-Ill, 1947. 51. HUNT, J. N. and SPURRELL, W. R. J. Physiol. 1 13: 157-168, 1951. 52. MCCLURE, C. W., REYNOLDS, L. and SCHWARTZ, C. O. Arch. Intern. Med. 26: 410-423, 1920. S3. DOUTHWAITE, A. H., and THORNE, M. G. Brit. Med. J. 1: 111-114, 1951. 54. HUNT, J. N. Modern Trends in Gastro-enterology, Jones, F. Avery, ed. 163-176, London, Butterworth, 1958. 55. CLOUGH, H. D., CARMAN, J. S. and AUSTIN, E. M.

siol. 137: 153-159, 1942. 78. MCSWINEY, B. A. Physiol. Rev. 11: 478-514, 1931.

79. IGGO, A. J. Physiol. (Lond.) ,z8: 593-607, 1955• 80. PAINTAL, A. S. J. Physiol. (Lond.) 126: 2 55270, 1954. 81. HUNT, J. N. Gastroenterology, 41: 49-51, 1961. 82. RUFFIN, J. M., BAYLIN, G. J., LEGERTON, C. W. JR. and TEXTER, E. C. JR. Gastroenterology 23: 252 -264, 1953. 83. TEXTER, E. C. JR., VANTRAPPEN, G. R., LAZAR, H. P., PULETTI, E. J. and BARBORKA, C. J. Ann. Intern. Med. (Chic.) 51: 1275-1294, 1959. 84. GREY, E. G. Amer. J. Physiol. 45: 272, 1918, cited by James, A. H.: The physiology of gastric digestion, London, Arnold, 1957. 85. QUIGLEY, J. P. and BRODY, D. A. Medical PhyJ. Nutr. 3: 1-16, 19 0. sics. Glasser, O. ed. 2: 280-292, Chicago, 56. MAIZE, W. C. D. and scow, K. J. L. Lancet 1: Year Book, 1950. 21-23, 1935. 86. QUIGLEY, J. P. and READ, M. It. Amer. J. Phy57. WILSON, M. J., DICKSON, W. H. and SINGLETON, siol., 1 37: 234-237, 1942. A. C. Arch. Intern. Med. 44: 787-796, 1929. 58. GIANTURCO, C. Amer. J. Roentgenol. 31: 735- 87. VAN UERE, E. J. and NORTHUP, D. w. Gastroenterology 2: 195-20o, 1944. 744, 1934• 131

PART II

EXPERIMENTAL ULCER PRODUCTION AND METHODOLOGY

The Mann-Williamson Ulcer

UNTIL the classic report of Mann and Williamson in 1923 (I ), there were many and diverse opinions about the pathogenesis of ulcer of the stomach and duodenum. The development of ulceration was attributed to emboli or local spasms of blood vessels, alterations in innervation of the stomach and duodenum, mechanical trauma, infection, and variation in secretory levels of acid and pepsin. Experimental methods available at that time had demonstrated that although shallow acute ulcers that healed quickly could be produced easily by a variety of methods, chronic penetrating ulcers, simulating those that occurred clinically, could not be induced by methods then available. Frank C. Mann's interest in peptic ulceration was apparent in 1916, (2) when he published a study of the acute gastric ulcers that sometimes develop after adrenalectomy in dogs. He sustained this interest for the remainder of his career. After a period of several years, during which he and his associates studied the effects of removing the duodenum with and without transplantation of the common bile duct and pancreatic duct to the ileum (3), Mann with Williamson (1), in 1923, published the now well-known re-

Maurice H. Stauffer George A. Hallenbeck*

sults of previous studies and described the operation for production of chronic peptic ulcer that has come to bear their names. The findings of Mann and Williamson had been anticipated by the work of Exalto (4), whose report was published in 1911. In recognition of his contribution, the procedure is often referred to as the Exalto-Mann-Williamson procedure. Exalto had noted that anastomotic ulceration after gastrojejunostomy occurred more frequently in clinical situation when a Roux-en-Y loop was used, than when a simple anastomosis was made employing a short proximal loop of the jejunum. Exalto then performed the Roux-en-Y operation in four dogs, and in three of these, an ulcer developed near the gastrojejunal anastomosis. In the animal in which an ulcer did develop, he noted bile in the stomach, which prompted him to implant the proximal duodenojejunal loop far distally into the cecum. Ulcers developed in all four animals in this second series, and in some instances they were multiple. According to Alvarez (5), an operation similar to the Mann-Williamson procedure was done in 1913 by Langenskiold.

•Fran the Department of Surgical Research, Mayo Clinic and University of Minnesota, U.S.A.

135

II / THE MANN-WILLIAMSON ULCER

Mann-Williamson Operative Procedures Mann and Williamson recounted (Fig. ~ ) that subacute or chronic ulcers occurred just beyond the outlet of the stomach a) in two of ten dogs following a

10 Dogs 2 Ulcers

juices low into the ileum, and second, removal of the duodenum. The last of the three procedures done by Mann and Williamson combined features of both principles, and was the most highly ulcerogenic; however, this procedure was technically difficult, and was associated with a high mortality rate. To simplify the method while retaining its two principles, they devised another procedure, now known as the Mann-Williamson operation, which consisted of the following steps (Fig. 2) : a) division of the duoMANN-W I LLIAMSON OPERATION

31 Dogs 10 Ulcers

b

d 0

0

iv c

Ulcer

FIG. 1. Preliminary procedures performed by Mann and Williamson.

removal of the duodenum and transplantation of the common bile duct and pancreatic duct to the jejunum near a gastrojejunal stoma; b) in ten of thirty-one dogs following transplantation of the common bile duct and pancreatic duct to the terminal part of the ileum, with the duodenum left in its normal position; and c) in eight of ten dogs in which duodenectomy was combined with transplantation of the bile duct and pancreatic duct to the terminal portion of the ileum. From the results of these studies, two methodologic principles seemed apparent in this experimental production of ulcer: first, diversion of the bile and pancreatic

16 Dogs 14 Ulcers

FIG. 2. The Mann-Williamson operation.

denum at the pylorus with closure of the open end of the duodenum by suture; b) division of the first portion of the jejunum, c) end-to-end anastomosis between the distal end of the jejunum and the stomach at the pylorus, and d) end-toside anastomosis between the proximal end of the jejunum and the ileum 25 to 75 cm. from the ileocecal valve.

Observations on the Mann-Williamson Preparation Mann and Williamson (1) used the discreet phrase "functional resection of the duodenum" to summarize the procedure. Thus, in a sense, the duodenum was defunctionalized except for its role 136

in carrying the bile and pancreatic juices into the terminal portion of the ileum distant from the point where the gastric contents enter the jejunum. Subacute or chronic ulcers subsequently developed in

STAUFFER & HAL.LENBECK

fourteen of the sixteen dogs undergoing this operation. Most of the ulcers occurred in the jejunum a few millimeters distal to its junction with gastric mucosa. The ulcers were sometimes multiple, and they were at times present in the pyloric part of the stomach. Grossly, microscopically, and with regard to chronicity and complications, they resembled peptic ulcerations seen in patients. Like ulcers seen clinically, these experimental ulcers sometimes perforated or caused hemorrhage. The findings of Mann and Williamson were rapidly confirmed in many parts of the world, and their procedure soon became the standard method of producing ulcers experimentally. In one large series that was typical of many, Grossman and Ivy (6) reported that ulcers occurred in 98 per cent of 114 animals. The average time of onset of ulceration was seven weeks, and 5o per cent of the animals had died with ulcers twelve weeks after operation. Needless to say, there is considerable impairment of digestion and absorption in the Mann-Williamson dog. For this reason it is important that the diet be liberal in quantity. However, many years

of experience with the Mann-Williamson dog have shown that malnutrition is not the primary cause of these ulcers. Although loss of weight is the rule, and severe malnutrition sometimes occurs, Mann and Williamson (L) in their original series commented that many animals with ulcer lost little weight and, in some instances, maintained virtually normal nutrition. The failure of other procedures of a related kind to produce ulcer, and the long latent period before the ulcer develops in the Mann-Williamson dog contradict the possibility that the ulcer is a result of operative trauma. The greater susceptibility of jejunal mucosa than duodenal mucosa to digestion by gastric contents is not a specific factor, since as early as 1910, Exalto (4) failed to produce anastomotic ulcers in dogs by means of a gastrojejunostomy. Further evidence that unusual sensitivity of jejunal mucosa to ulceration is not the principal factor is found in the duodenal ulcers developed in one-third of the animals in one of Mann's series when the bile and pancreatic ducts were transplanted to the ileum and the duodenum was left in situ (1).

Acid Pathogenesis The most important consequence of the Mann-Williamson operation was the demonstration that acid peptic gastric contents were required for the genesis and continued existence of this type of peptic ulcer. Mann and his associates (7), along with others, used the preparation to study development and healing of peptic ulcers. They soon established the important fact that even large penetrating ulcers healed remarkably quickly if protected from exposure to acid chyme by surgical occlusion of the pylorus,

while the stomach was made to empty into the jejunum by means of a gastrojejunostomy. Indeed, gastrojejunostomy without pyloric occlusion resulted in the healing of sone ulcers (8). In either case, new ulcers usually formed promptly in the jejunum near the new gastroenteric stoma. Observations of this kind supported Mann's hypothesis that peptic ulceration was caused by digestive action of gastric chyme on the mucosa it bathed. He assumed that important factors in deter137

11 / THE MANN-WILLIAMSON ULCER

mining the localization of ulcers were a) mechanical factors related to the manner in which chyme was ejected from the

stomach, and b) the site of first impingement of the "jet" of chyme on the jejunum.

Early Interpretations of the Etiology of the Mann-Williamson Ulcer Mann considered that the deviation of bile, pancreatic juice and duodenal juice from the region of the gastroenteric junction prevented neutralization of chyme, and was the major factor in leaving the digestive power of chyme intact. This concept of the genesis of Mann-Williamson ulcers was accepted by numerous investigators, and led to other types of experiments in which the incidence of ulceration was noted; these included ligation of the common bile duct, establishment of a biliary fistula, ligation of the pancreatic duct and establishment of a pancreatic fistula. For all of these procedures ulcers resulted in some instances, although less frequently than following the Mann-Williamson operation. Mann (7) emphasized that the distance between the ileocecal valve and the point at which the duodenum was joined to the small intestine was important. Ulcers occurred almost invariably when this anastomosis was placed close to the cecum, and they developed correspondingly less frequently the higher it was placed in the ileum or jejunum. Grossman and Ivy (6)

recommended that the duodenum be drained into the ileum a measured 15 cm. from the ileocecal valve. The fact that an ulcer was rarely produced when duodenal contents were diverted into the upper part of the jejunum was considered to be due to neutralization of acidic stomach contents by upward regurgitation of the bile, pancreatic juice and succus entericus. In 1932 Matthews and Dragstedt (9) supported this hypothesis by using the Mann-Williamson preparation with two modifications. They anas tom osed the duodenum to the jejunum only 40 cm. distal to the gastrojejunal stoma and then used a special mechanical valve above this anast0mosis to prevent regurgitation. Ulcers developed in six of ten dogs in which the valve functioned effectively. Skepticism regarding the exclusive role of the neutralization factor was evidenced as early as 1929, when McCann (Io) found that ulcers developed in some dogs after the duodenal segment had been drained into the stomach instead of the terminal portion of the ileum.

Hypersecretion Factor In 1952 Storer and associates (11), of Dragstedt's laboratory, first presented evidence of gastric hypersecretion from Heidenhain pouches in Mann-Williamson animals. They found a two to threefold increase in the amount of hydrochloric acid produced daily. This finding sug138

gested that gastric hypersecretion, in addition to diminution of neutralization, played a role in the causation of ulcers after the Mann-Williamson operation. Other investigators have confirmed this observation. In 1960 Humphreys and associates (1 2) found, by means of pH

STAUFFER & HALLENBECK

studies, and using a Mann-Wiliamson dog preparation with an exterior Mann-Bollman fistula connected to the jejunum just beyond the gastrojejunal anastomosis, an increase in gastric acidity; they noted that it was especially prolonged up to twenty hours. These authors commented that in some studies they observed a pattern not unlike that which follows injections of histamine. In 196o, further data from Dragstedt's laboratory (13,14) provided convincing evidence that the hypersecretion of gastric juice was caused by absence of the usual inhibition that results when acid comes in contact with the duodenal mucosa. These authors (13) performed gastrojejunostomy with the Roux-en-Y technique and placed valves in the jejunal or duodenal segment, or in both. As predicted, a valve in the jejunum prevented regurgitation of alkaline juices to the region of the gastrojejunostomy, and was followed by an increase in the incidence of ulceration, but hypersecredon from a Heidenhain pouch was not observed. Contrariwise, with a valve in the duodenal segment, Heidenhain-pouch secretion was increased, presumably because acid chyme could not reach the duodenum to activate an inhibitory mechanism. Then both valves were used in the modified Mann-Williamson preparation; this prevented regurgitation of duodenal juices into the stoma and also blocked regurgitation of acid into the duodenum, with the result that the incidence of ulceration was markedly increased (1 3) . These studies show why the MannWilliamson preparation is so ulcerogenic. Drainage of duodenal contents into the terminal portion of the ileum allows neither regurgitation of alkaline juices upward as far as the gastrojejunostomy, nor entry of acid chyme into the duode-

num to activate the duodenal inhibitory mechanism. More recent experimental ulcer-producing preparations, such as those of Keefer and associates (is), either defunctionalize the duodenal segment by interposing this organ into the ileum, or remove it entirely, as reported by Brackney and co-workers (16). These studies further indicate the importance of exclusion of the duodenum from contact by gastric contents, one of the basic principles of the Mann-Williamson animal. In the light of present knowledge, it must be assumed that the Mann-Williamson ulcer is the result of at least two factors; first, the diversion of neutralizing secretions from the usual site of ulcer formation, and, second, hypersecretion of gastric juice. Subject to some variation of interpretation, three phases of gastric secretion (cephalic, gastric, and intestinal) have long been recognized. Oliver (17) and later Emerson and associates (18), concluded that vagotomy alone has essentially no effect on the production of the Mann-Williamson ulcer. With the recent renewed interest in the gastric phase of acid secretion, it was obvious that this aspect should be investigated. Baugh and associates (i4), in 196o, found that secretion of gastric juice from Heidenhain pouches in the Mann-Williamson preparation occurred despite removal of the gastric antrum. They concluded that this was further evidence for lack of inhibition of gastric secretion in the Mann-Williamson animal, in which the gastric acid chyme does not come in contact with the duodenal mucosa. It might be concluded that factors operating in the intestine are the most important in the production of the Mann-Williamson ulcer.

139

H / THE MANN-WILLIAMSON ULCER

Summary Studies of ulcers produced by the Mann-Williamson operation provided the first good evidence that acid peptic digestion of a localized area of gastric or intestinal mucosa is the immediate cause of this type of ulceration, a fact that has since been confirmed by a variety of different experiments. Both past and recent studies confirm Mann's original hypothesis that diversion

of neutralizing alkaline secretion from the gastrointestinal juncture is a factor in ulcer production in the Mann-Williamson experimental animal. Pronounced hypersecretion of gastric juice, apparently due to loss of a duodenal inhibitory mechanism, has recently been found to be a potent second factor in the production of this experimental ulcer.

References I. MANN, F. C. and WILLIAMSON, C. S. Ann. Surg., 77: 409-422, 1923. 2. MANN, F. C. J . Exp. Med., 23: 203-209, 1916. 3. MANN, F. c. and KAWAMURA, K. Ann. Surg., ?5: 208-220, 1922. 4. EXALTO, J. Mitt. a. d. Grenzgeb. d. Med. u Chit., 23: 13-41, 1911. 5. ALVAREZ, W. C. A mer. J. Surg., 18: 207-231, 1932. 6. GROSSMAN, M. I. and IVY, A. C. Methods in

Medical Research, Potter, V. R., ed., vol. 1, 263-268. Chicago, The Year Book, 1948. 7. MANN, F. C. S. Clin. North America, y: 753775, 1925. 8. MORTON, C. B. Ann. Surg., 85: 207-238, 1927. 9. MATTHEWS, W. B. and DRAGSTEDT, L. R. Surg., Gynec. & Obst., 55: 265-286, 1932. 10. MCCANN, J. c. Arch. Surg., tg: 600-659, 1929. 11. STORER, E. H., OBERHELMAN, H. A., JR., WOODWARD, E. R. SMITH, C. A. and DRAGSTEDT, L. R.

A.MA. Arch. Surg., 64: 192-199, 1952.

140

I2. HUMPHREYS, J., GRINDLAY J. H. and BOLLMAN, J. L. Gastroenterogy, lo 38: 374-385,

1960.

13. NAGANO, K., JOHNSON, A. N., JR., DRAGSTEDT, I.. R. II, OBERHELMAN, H. A. JR., COBO, A. and DRAGSTEDT, L. R. Gastroenterology, 39: 319-

329, 1960.

14. BAUGH, C. M., BRAVO, J., DRAGSTEDT, L. R., II and DRAGSTEDT, L. R. Gastroenterology, 39:

339-334, 1960.

15. KEEFER, E. B. C., HAYS, D. M., MARTIN, K. A ., BEM, J. M. and GLENN, F. Forum, 5: 288-

S.

2 94, 1955.

16. BRACKNEY, E. L., THAL, A. P. and WANGENSTEEN, 0. H. Proc. Soc. Exp. Biol. Med., 88: 302-306, 1 955. 17. OLIVER, J. V. Arch. Surg., 55: 180-188, 1947. 18. EMERSON, D. M., WOODWARD, E. R., TOVEE, E. B., NEAL, W. B., JR., SIBLEY, J. A. and DRAGSTEDT, L. R. Arch. Surg., 6o: 223-232, 1950.

Experimental Ulcer Production in the Pylorus-Ligated Rat*

Tills paper concerns itself mainly with a critical study of the technique for the production of gastric ulceration in the pylorus-ligated rat. Shay and his associates (i) described an acute ulcerative process in the forestomach of the rat that could be produced simply by pyloric ligation. This technique has been studied extensively by many investigators. The apparent variability in the degree and per cent of ulceration in this preparation, as reported by several investigators, and the influence of different nonspecific measures producing the same physiologic response even led some observers to question the usefulness of the rat in studying gastric ulceration. The development of gastric ulceration in the rat is further complicated by the fact that the stomach

David C. H. Sun Jeanne K. Chen**

of the rat has a nonsecretory portion, the rumen, lined with squamous epithelium, and a secretory portion, the corpus and antrum, lined with gastric glands. It is thus apparent that considerable confusion attends this problem. Several hypotheses have been introduced into the review in an attempt to explain some apparent inconsistencies and gaps in the available data. It is beyond the scope of this paper to review in detail the extensive body of knowledge concerning the various factors influencing the production of gastric ulceration in the pylorus-ligated rat. Many of these factors will be lumped under one category for convenience of the discussion.

Normal Anatomy of the Stomach and Peptic Ulceration in the Rat The gross and microscopic anatomy of the normal rat's stomach has been very well described by Berg (z). Briefly, the stomach of the rat is divided into two main regions by a transverse, firm, raised

ridge (Fig. 1). Proximally, is the forestomach or rumen, and distally a glandular portion. The esophageal orifice is just above the ridge on the lesser curvature of the rumen. The glandular portion is fur-

•Supported in part by the Gastrointestinal Research Foundation, Washington, D.C. * From the Gastrointestinal Research Laboratory, Veterans Administration Hospital, The George Washington School of Medicine, Washington, U.S.A. 141

1 i / ULCER PRODUCTION IN THE PYLORUS-LIGATED RAT

ther subdivided into an antrum (prepyloric region) located on the lesser curvature, and a corpus which comprises a major part of the glandular stomach. Histologically, the forestomach and the transverse ridge are lined with stratified squamous epithelium which is continuous with the lining of the esophagus. The glandular stomach is lined with simple columnar cells which are continuous into the gastric pits. The fundic glands are composed of mucus neck cells, chief cells and large prominent parietal cells. The chief cells are located mainly in the lower portion of each gland. Parietal cells may be seen throughout the entire gland, but are fewer in the basal region. The glands of the antrum are shorter than those of the fundus and are composed principally of the mucous-type cells. The glands are supported by a loose connective tissue stroma, containing a few lymphocytes and eosinophils. The muscularis mucosae and muscularis are continuous with the same structures in the rumen. Lymphoid

tissues are found only in the antrum, and exist in the form of large and small nodules and as diffuse lymphocytic infiltration. One of the important prerequisites to a proper evaluation of the results of procedures designed to produce peptic ulcer experimentally is a knowledge of the frequency of such lesions occurring spontaneously in the rat. Singer (3) reported an incidence of 8 per cent of ulcers in two hundred well-kept control rats, whereas ? icCarrison (4) found no microscopic evidence of disease in the stomach of 1,189 albino rats, and Howes and Vivier (S) found no lesions in seventy-five rats. Multiple dietary deficiency in the rat has been shown by subsequent investigators to produce ulceration in the stomach. The white bread diet used by Singer was certainly inadequate. It is thus necessary to standardize the environmental conditions and diet of the rat in any experiments designed to produce gastric ulcer experimentally.

Mechanism of Gastric Secretion in the Rat The secretion of acid and pepsin is mainly from the corpus, the glandular portion of the stomach. The squamous portion of the rat stomach does not secrete acid. The physiologic evidence for this was provided by the study of Alphin and Lin (6) who showed that a chronic denervated pouch prepared from this portion of the stomach did not secrete acid, even after a meal. The evidence for the existence of the cephalic and humoral phases of gastric secretion in the rat was furnished by Lin and Alphin (7). Sham feeding in a rat equipped with chronic gastric fistula and esophagotomy evoked a slight but definite increase in acid output of the gastric secretion. However, sham feeding had no 142

effect on gastric secretion in rats with bilateral vagotomy. These results agreed with Shay et al. (8) and others (9,1o) that acute vagotomy decreased gastric secretion in the pylorus-ligated rats. The presence of a humoral phase of gastric secretion in the rat was shown by Komarov et al. (ii), when an increase in gastric acid secretion was observed after the introduction of gastric secretagogues, peptones, meat or liver extract into the duodenum or cecum of the rat, and the intravenous administration of gastrin. The evidence for a humoral phase was provided subsequently by Alphin and Lin (6), who found an increase in acid secretion from the chronic denervated

SUN & CHEN

gastric pouch after feeding of Pard into the main stomach. The stomach of rats emptied of food secreted small amounts of acid gastric juice when the animals were under complete narcosis after 24 to 72 hours of fasting (i i) . This spontaneous secretion, or interdigestive secretion, of free acid for several days under fasting conditions was shown by Lin and Alphin (7) to be completely abolished by vagotomy within a

period of two to three months. In addition, when a rat with denervated pouch and another with total stomach fistula were starved at the same time and treated identically, the rat with denervated pouch ceased to secrete any free acid after fourteen hours, whereas the other animal was still secreting free acid thirty-six hours later (6). Thus, the interdigestive phase of gastric secretion in the rat should be considered mainly a "cephalic" phase.

Pylorus-Ligation Technique—Shay Rat Methods The details of the technique have been described previously (1). A slight modification is instituted in this study. Wistar strain male rats were used. The laboratory that houses the colony and the laboratories in which the experiments were conducted are controlled throughout the year at constant temperature and humidity. All animals were healthy, and had been maintained on Rockland Rat Diet Pellets. Each animal was housed singly in a cage with raised bottom of wide wire mesh to insure immediate passage of feces from the cage. Animals which weighed between 120 and 235 grams were chosen for the study. The animals were starved for forty-eight hours and water was permitted ad libitum. Operative Procedure Under light ether anesthesia, the abdomen is shaved, and a mid-line incision is made extending from the xyphoid for about 2 cm. The duodenum is identified, and the junction between the pylorus and the duodenum is picked up gently with a curved glass probe. The pyloric ligature of silk is placed, care being exercised that neither damage to blood supply nor trac-

tion on the pylorus occurs. The abdominal wall is closed with interrupted silk sutures. The abdominal wall is cleansed thoroughly with physiologic saline, dried, and covered with a solution of collodion. The animal is placed in its cage, and receives nothing during the remainder of the experiment. Procedure at Autopsy At the end of the experiment, the rat is anesthetized with ether. The abdomen is opened, and a ligature is placed around the esophagus close to the esophagocardiac junction. Care is taken to remove the stomach before the respiration ceases, because soon after this, the cardiac sphincter relaxes, and the gastric juice may be lost through the nose and mouth. The esophagus, the duodenum and the appropriate peritoneal ligaments are clipped. The stomach is removed, inspected externally, and dried. Before discarding the animal, the chest is opened, and the esophagus and pleural cavity are examined for perforation and fluid. A small opening is made along the greater curvature adjacent to the pyloric ligature, and the gastric juice is drained into a graduated centrifuge tube through a funnel. The stomach is then slit along its entire greater 143

1I / ULCER PRODUCTION IN THE PYLORUS-LIGATED RAT

curvature, stretched moderately by pinning on cork, and the inner surface is examined with a dissecting binocular microscope with a magnification of I o.5x. Specimens for histological study are placed in a solution of formaldehyde. Analysis of Gastric Contents The gastric contents of each stomach are analyzed individually. After centrifuging, the volumes of supernatent and of solids are recorded. If the volume of the solids is over o.6 ml., the animal is discarded because of possible contamination of the gastric contents by food residue or feces. The supernatant is removed, and its free and total acid concentration and total chlorides are determined. Free and total acid are titrated with N/5o NaOH

using Töpfer's reagent and phenolphthalein as indicators. Pepsin concentration is determined by the hemoglobin method of Anson and Mirsky (12), and total chloride by automatic chloride titrator. The influence of time, after pylorus ligation, upon gastric ulceration and secretion was evaluated in this study. Six groups of experiments were carried out. The rats were sacrificed at intervals of 6, 8, 10, 12, 14 and 16 hours after pylorus ligation. In each group, a minimum of fifty rats were done. The influence of environmental conditions was studied in four groups of rat at intervals of 6, 8, Io and 12 hours after pylorus ligation. The rats were kept in a laboratory with changes in room temperature from cold air draft and a low temperature of 6o°F.

Influence of Time after Pylorus Ligation upon Gastric Ulceration Table I and Fig. 2 show the results of influence of time after pylorus ligation upon gastric ulceration. The per cent of dead and/or perforated represents the number of rats in a given experiment which developed perforation of the esophagus and/or stomach. The dead ones are included only if there is evidence of perforation, and of the presence of gastric contents in the pleural or peritoneal

cavity at autopsy. The grade of ulceration signifies the number of ulcers from grade one-plus to four-plus in the rumen, as described by Paul et al. (13 ) . The ulcer index is obtained by the multiplication of the average plus grade of ulceration in a given group by the per cent of the animals showing ulceration in that group. Those animals that developed perforation are classified as grade four plus

TABLE I. Influence Of Time After Pylorus Ligation Upon Gastric Ulceration Hours After Pylorus Ligation 6 Hour 8 Hour 10 Hour 12 Hour 14 Hour 16 Hour IØ

No. of Dead and/or Perf. Rats RATS

NUMBER

PER CENT

50 53 50 57 56 54

0 1 7 19 27 37

0 1.9 14.0 33.3 48.2 68.5

Ulceration NUMBER AVG. GRADE

20 23 21 26 26 17

+ +

+-I-

±± -}-± +++

% dead, perforated and/or ulcerated

Uker Index

40 45 56 79 93 100

40 51 140 225 286 370

sd.. et UI..r.Uen

P.. Cent a R.t. Dead .N/.r P.H.r.ted

100

100

60

300

ulceration. The ulcer index is a more reliable guide to determine ulcer activity than per cent protection or per cent ulceration. The results show a linear relationship between the ulcer index and the time after pylorus ligation in the intervals of eight to sixteen hours after pylorus ligation. The severity of ulceration in the rumen increased with increasing duration of pylorus ligation. Each of fifty-four rats in the sixteen-hour pylorus ligation experiment showed rumenal ulceration with an ulcer index of 379 for the entire group. The maximum ulcer index for any

II

60 000

.0 100

00 -

d Heu.. Atte r

10

It

6

14

Dy!.!.. Lü.tle.

6 e

10 12

pylorus ligation upon gastric ulceration in the rat.

FIG. 2. The influence of time after

group is four hundred. A linear relationship is also observed between the percentage of dead and/or perforated and the time after pylorus ligation in the intervals from eight to sixteen hours after pylorus ligation.

Influence of Time after Pylorus Ligation upon Gastric Secretion Table II shows the results on the influence of time after pylorus ligation upon gastric secretion. Fig. 3 shows the relationship between the rate of secretion in ml./too gm. body weight and duration of pylorus ligation. The rate of gastric secretion increased with increasing duration of pylorus ligation, in the intervals of six to twelve hours. However, the rate

of secretion declined at the fourteenhour period, presumably as a result of dehydration, and fell off rapidly. These findings are in agreement with the work of Donald and Code (io), in which the gastric secretion was drained continuously from the stomach through a gastric cannula. A linear relationship was also observed

TABLE II. The Influence Of Time After Pyloric Ligation Upon Gastric Secretion Body Weight (gm) (Post Starvation)

Rate ml/100 gm

16

16

Heur. After Pyl.ru. Llgatle.

Total Acid Output Total Chloride snEq/100 gm. mEq/L

Hours After Pyloric Ligation

No. of Rats

6 Hour

50

Mean S. D.

167.4 19.0

5.49 1.08

0.65 0.15

162.8 7.1

8 Hour

53

Mean S. D.

170.3 16.1

6.00 0.97

0.70 0.12

158.1 5.2

10 Hour

50

Mean S. D.

174.8 18.8

6.70 1.17

0.77 0.21

155.9 12.4

12 Hour

57

Mean S. D.

169.0 21.2

7.20 1.30

0.77 0.19

157.1 7.9

14 Hour

56

Mean S. D.

174.0 16.6

7.30 1.10

0.73 0.17

155.2 7.9

16 Hour

54

Mean S. D.

159.0 7.0

6.35 2.20

0.53 0.32

148.1 12.4 145

between the total acid output and the duration of pylorus ligation within the six to twelve hour periods. Interesting data were obtained on influence of time on pepsin concentration and will be reported separately.

Hours Alter Pylorus Ligation

FIG. 3. The influence of time after pylorus ligation on the volume of gastric secretion.

Influence of Environmental Changes on Gastric Ulceration and Secretion Changes in room temperature from cold air draft and a low temperature of 6o°F. in the laboratory produced a higher incidence of ulceration in the pylorusligated rats as compared with those stored in laboratory with constant temperature and humidity. In the six-hour pylorusligated experiment, seventeen of the eighteen rats had average grade two-plus ulceration in the rumen of the stomach with an ulcer index of 188. In the eighthour pylorus-ligated experiment, sixteen

FIG. 4. Rumenal ulcerations as seen through the serosal surface.

146

of the eighteen rats had average grade three-plus ulceration with an ulcer index of 266. In the ten-hour experiment of thirty-nine rats, twenty-two were dead and/or perforated, sixteen had average grade two-plus ulceration and only one without ulcer. The ulcer index was 308. In the twelve-hour experiment of nineteen rats, fifteen were dead and/or perforated, three had average grade two-plus ulceration and one without ulcer. The ulcer index was 348. Rats in the laboratory subjected to cold air draft had an ulcer index of 188, 266, 308 and 348 while rats in constant temperature laboratory had an ulcer index of 40, 51, 140 and 225 in the 6, 8, io and 12-hour pylorus-ligared experiments. These results indicate a higher incidence and a more severe grade of ulceration in rats subjected to changes in room temperature. It is thus important to standardize the environmental conditions in pylorus-ligated rat experiment. Pathology Grossly, ulcerations in the rumen are seen through the serosa as blue-black hemorrhagic structures (Fig. 4). When the stomach is opened along the greater curvature and stretched moderately, multiple ulcerations of varied sizes are observed in the rumen, rarely in the body

Esophageal Opening

FIG. r. Internal and external views of rat's stomach showing gross anatomic subdivisions. FIG. T. Inner surface of rat's stomach showing typical lesions in rusten, body and antrum.

FIG. 6. Rumen showing early ulcerative lesion. 90X. FIG. 7. Rumen showing advanced ulcerative lesion. t6X.

I1 / ULCER PRODUCTION IN THE PYLORUS-LIGATED RAT

and antrum of the stomach (Fig. 5). The severity and number of ulcers may vary over a wide range. Some stomachs show only one or two ulcers, while others show many lesions and even perforations. When small, the ulcers appear as tiny, round lesions. The larger ones may be acicular or irregularly shaped areas reaching a size of 5 mm. or more. Ulcers may appear in all parts of the rumen, but when lesions are few, they are found in the region of the apex. Ulceration of the esophagus may occur, and may be severe and extensive. The lower half of the esophagus may appear hemorrhagic, and perforation does occur. Microscopically, the earliest pre-ulcerative lesions seen consist of a tiny area

of necrosis of the stratified squamous epithelium. The subadjacent connective tissue is infiltrated by a few leukocytes. The submucosal blood vessels are congested. The early lesion consists of a subepithelial focus of necrosis with intense polymorphonuclear leukocyte infiltration. The stratified squamous epithelium is lost (Fig. 6). The advanced lesions show an ulcer crater with necrotic debris and leukocyte infiltration at the base. The muscularis mucosae is involved in the necrotic and inflammatory ulcer base, as evidenced by a frayed edge, leukocytic infiltration and necrosis. The submucosa is edematous and infiltrated with scattered polymorphonuclear leukocytes (Fig. 7).

Pathogenesis Ulcerations that develop in the pylorusligated rat appear uniformly in the rumen, less often in the antrum, and rarely in the body of the stomach. The lesions appear when unbuffered juice is present in the stomach in sufficient amounts and for a sufficient period of time. The pathogenesis of the ulcer was shown by Shay et al. (I) to be caused by the accumulation of acid-pepsin within the stomach. The administration of anticholinergic drugs and aluminum hydroxide gel protected the mucosa from ulceration. Madden et al. (14) ruled out distention and interference of blood circulation as the cause of ulceration. Distention alone could not be primarily responsible for the ulceration in the forestomach, since buffering the gastric juice prevented ulceration in spite of distention. The distended and ulcerated forestomach still retained its active blood supply, as evidenced by the findings of staining of the rumen after intravenous administration of benzo sky blue dye, direct observation of capillary blood flow and the relatively lower intragastric pres148

sure than the normal intracapillary pressure. Risley et al. (1 5) found that the introduction of small amounts of acid pepsin mixture (q, ml.) into the stomach of the rats produced no ulceration up to five hours, while introduction of large amounts (8 to 10 ml.) resulted in severe ulceration within an hour. The limiting ridge of the stomach serves to prevent a reflux of gastric juice from the glandular part into the rumen when the volume of juice is low. These findings suggest that the rumenal ulcers in the pylorus-ligated rat may be merely a result of the volume and concentration of gastric hydrochloric acid and pepsin upon the mucosa. The rumen is lined by squamous epithelium and is therefore subjected to the corrosive action of gastric juice. Vagotomy Transabdominal bilateral vagotomy afforded complete protection of rumenal ulceration in the Shay rat preparation (7, 8,9). There was almost complete absence

SUN & CHEN

of secretion in the stomach. Acute uni- new anticholinergic drugs and anti-ulcer lateral vagotomy decreased secretion to agents. In the Shay rat preparation, proone third of normal, and seven of thirteen tective action against rumenal ulcerations animals had tiny ulcerations (8). The in- was afforded by the following drugs: troduction of artificial gastric juice at a atropine sulfate, o.5 to i i.o mg./kg. (2 5, rate comparable to the normal rate of 26); scopolamine methylbromide 0.1 to secretion regularly produced ulcerations 6 mg./kg. (27); propantheline 2.5 to 5.o in rats which underwent vagotomy. The mg./kg. (28); chloropromazine 70 mg./ character and number of ulcers were kg. (29); thephorin 98 mg./kg. (25). similar to those seen in the Shay rat pre- Sodium polyanhydromannuronic acid paration. The depression of gastric secre- sulphate (30) and robuden (3 1) also aftion would thus appear to be the major forded protection against rumenal ulcermechanism whereby vagotomy protects ation in the Shay rat. against the formation of ulcer (16). Urogastrone and Enterogastrone Hypophysectomy and Adrenalectonty Hypophysectomy in rats resulted in a decrease in volume and acidity of the gastric secretion, which were restored by administration of ACTH, cortisone or growth hormone (17) . Removal of adrenals resulted in a decrease in volume and acidity of the gastric secretion and almost complete protection of the Shay rat against rumenal ulceration. Preoperative administration of cortisone and, to a lesser extent, DCA, administration of normal saline and 5 per cent glucose solution increased the gastric secretion with a concomitant rise in gastric ulceration in adrenalectomized rats (18, 19,20). Parenteral administration of ACTH, cortisone, desoxycortisterone and delta-cortisone produced a decrease in gastric secretion with no significant effect on the incidence or severity of ulceration (19,20,21,2 2,2 3). However, Peremans (24) found an increase in the incidence of ulceration after prednisone administration in the pylorus-ligated rat. Anticholinergic Drugs and Similar Agents The Shay rat preparation has been widely used to evaluate the efficacy of

Paul et al. (32) reported on the satisfactory application of the Shay rat to the assay of urine extracts. Risley et al. (33) found similar anti-ulcer activity of urogastrone and enterogastrone in this preparation. An inhibition of gastric secretion was found after administration of each of these extracts. On the other hand, Morris et al. (34) and Benditt et al. (35 ) reported that Shay rat method is of no value in the assay of anti-ulcer property of enterogastrone. The intraduodenal injection of 2 ml. of 0.35 per cent of HCl solution into the pylorus-ligated rat was shown by Gad et al. (36) to prevent rumenal ulceration and a decrease in volume and acidity of gastric secretion as compared with the controls. This duodenal inhibitory mechanism in response to HC1 instillation might represent a release of a hormone sufficient to prevent gastric ulceration. Nonspecific Factors Intravenous administered purified pyrogens from cultures of B. prodigiosus, Pseudomonas aeruginosa and E. typhi possessed marked ulcer-inhibiting action in the Shay rat. There was a concurrent decrease in the volume and acidity of 149

1I / ULCER PRODUCTION IN THE PYLORUS-LIGATED RAT

gastric juice (37). The findings of the protection against ulceration in the Shay rat by the intraperitoneal administration of turpentine, intraperitoneal hemorrhage and intravenous injection of crude urinary extracts were intimated by Haroutunian et al. (38), in that these unrelated and nonspecific measures produced a toxic reaction resulting in decreased gastric secretion. They concluded that the Shay rat preparation is not a good assay animal for evaluating specific anti-ulcer factors. However, if the control group of animals receives the same vehicle and has the same number of animals as the test group, and if all nonspecific factors are taken into consideration, the Shay rat preparation is still a valuable tool for antiulcer assay. Salts of organic acid, potassium acid acetate, fresh bile and commercial bile powder, administered orally possessed a marked preventive action on rumenal ulcer formation in the Shay rat (39,40). Since innumerable factors determine the occurrence and extent of this gastric ulceration, we wish to emphasize again the necessity of standardized conditions when animals are to be used in an assay procedure. It is important to standardize the environmental conditions and diet of the rat. Change in room temperature increased the incidence and severity of rumenal ulceration, and exposure to cold resulted in glandular ulceration. Shortening the preoperative starvation period to twenty-four hours reduced the incidence of rumenal ulceration. Additional starvation and dehydration above that of the control produced a reverse effect and decreased gastric secretion and ulceration. Multiple dietary deficiency in the rat produced an apparent effect of lowering the resistance of the mucosa and ulceration in the stomach (41). Gastric ulceration has also been reported in rats on a specific dietary deficiency (42,43,44,45). 150

The supplementation of an already adequate diet with protein hydrolysates in amounts equivalent to 25 per cent of additional protein produced a marked increase in the resistance of gastric rumen to peptic ulceration in the pylorus-ligated rat (46). The older and the larger the rat, the longer the period of preoperative fasting which is required if ulcer development is to take place. A direct relationship between the weight of the rat and the volume of gastric juice was noted (14, 47). Rats, weighing in the range of 17o to 23o gm. preoperatively, were found to be satisfactory for the ulceration experiment. Males develop gastric ulceration more easily. It is thus advisable to use only one sex in an assay. The incidence and severity of ulcerations in the rumen of the stomach in the pylorus-ligated rat preparation are in direct proportion to the amount of juice and duration of ligation. In an assay procedure for anti-ulcer substance, the duration of ligation should be a minimum of twelve hours. At this level, we observed an ulcer index of 225, which is slightly more than half of the maximum index of 400. The sixteen hour period of pylorus ligation provided us with a 1 oo per cent ulceration in the rat and an ulcer index of 379; however, 68 per cent of the animals were dead or perforated. This might be too severe a test for the anti-ulcer activity of some substances. When the ulcer index in the sixteen-hour pylorus-ligated rat was plotted against the volume of secretion in ml. or units of free acid of the gastric secretion in five hours, a straight line relationship was observed by Risley et al. (15) . They concluded that the severity of ulceration is dependent on the volume and acidity of the gastric contents. We wish to emphasize again the necessity of a standardized condition when

SUN & CHEN

animals are to be used in an assay procedure. For our standard assay procedure for anti-ulcer activity, a minimum of twenty rats should form each test group. For each test group, there is a control group with the same number of animals and, in so far as possible, made up of litter mates. The vehicle used for dissolving the test drug should be similarly used in the control. When the rats are sacrificed at the end of the experiment, they are taken in pairs of one test rat and one control animal. The chest should be opened and the esophagus and pleural cavity inspected for perforation of the esophagus. The data from rats which have significant food-rests or the presence of fecal material should be discarded. The exact nature of gastric ulcerations in the rat is often difficult to evaluate.

Seldom has good evidence for the existence of a chronic indurated ulcer in the rats been produced. In addition, the gastric ulceration produced by varied experimental procedures in the rat does not In any manner resemble the chronic peptic ulcer pathologically in man. However, we do believe that the rat preparation is a useful tool for initial screening of agents capable of protecting the rumenal mucosa against acid pepsin erosion. From a practical standpoint, the maintenance of rats is less expensive than that of larger animals. We believe in the use of this animal preparation, not so much for the study of the ulcerative process per se, but rather to measure quantitatively the many and varied factors that would effect a change in the rate of secretion of the stomach.

Summary This paper concerns itself with a critical study of the technique for the production of gastric ulceration in the pylorus-ligated rat. Voluminous literature on this subject is reviewed. The mechanism of gastric secretion in the rat and the pathogenesis of rumenal ulceration are described. Several hypotheses have been introduced in an attempt to explain some apparent inconsistencies in the available data. The necessity of a standardized condition when this preparation is to be used

in an assay procedure is emphasized. The incidence and severity of ulceration in the rumen of the stomach in this preparation are in direct proportion to the amount of juice and duration of ligation. We believe in the use of this preparation, not so much for the study of ulcerative processes per se, but rather to measure quantitatively the many factors that would effect a change in the rate of secretion of the stomach.

References 1. SHAY, H., KOMAROV, S. A., FELS, S. S., MERANCE, D., GRÜENSTEIN, M. and SIPLET, H. Gastroenterology 5: 43-61, 1945. 2. BERG, B. N. Amer. J. Path. t8: 49-61, 1942. 3. SINGER, C. Lancet 2: 279-281, 1913. 4. MCCARRISON, R. Lancet 1: 1151-1154, 1931. 5. HOWES, E. L. and vIVIER, P. J. Amer. J. Path. 12: 689-700, 1936. 6. ALPHIN, R. S. and LIN, T. M. Amer. J. Physiol. 197:

257-259, 1959.

7. IAN, T. M. and ALPHIN, R. S. Amer. J. Physiol. 192: 23-26, 1958. 8. SHAY, H. KOMAROV, S. A. and GRÜENSTEIN, M. A.MA. Arch. Surg. S9: 2,0-226, 1949. 9. HARKINS, H. N. Bull. Johns Hopkins Hosp.

8o: 1 74-176, 1947. lo. DONALD, D. E. and CODE, C. F. Gastroenterology 2o: 2 98-303, 1 95 2. t 1. KOMAROV, S. A., SHAY, H., RAYPORT, M. and FELS, S. S. Gastroenterology 3: 406-413, 1 944. 151

II / ULCER PRODUCTION IN THE PYLORUS-LIGHTED RAT

and MIRSKY, A. E. J. Gen. Physiol. 16: 59-63, 1932. 13. PAULS, F., WICK, A. N. and MACKAY, E. M. Gastroenterology 8: 774-782, 1947. 14. MADDEN, R. J., RAMSBURG, H. H. and HUNDLEY, J. M. Gastroenterology 18: 119-127, 1951. 15. RISLEY, E. A., RAYMOND, W. B. and BARNES, R. H. Amer. J. Physiol. 15O: 754-759, 1947. 16. ALEXANDER, J. W. and MERENDINO, K. A. Surgery 32: 859-869, 1952. 17. KYLE, J. and WELBOURN, R. B. Gastroenterologia 85: 205-213, 1956. 18. MADDEN, R. J. and RAMSBURG, H. H. Gastroenterology 18: I28-1134, 1951. 19. WELBOURN, R. B. and CODE, C. F. Gastroenterology 23: 356-362, 1953. 20. REMOUCHAMPS, L., PEREMANS, J. and MERCKX, J. Acta gastroenterol. belg. 18: 896-904, 1955• 2I. ROBERT, A. and NEZAMIS, J. E. Proc. Soc. Exp. Biol. Med. 98: 9-12, 1958. 12. ANSON, M. L.

22. BONFILS, S., HARDOUIN, J. P., ROSSI, G., RICHIR, C. and LAMBLING, A. Arch. Mal. App. Dig.

(Paris) 46: 385-399, 1957. and SELYE, H. Ann. endocrinol. 13: 845-848, 1952. 24. PEREMANS, Acta gastroenterol. belg. 2,: 666 5 73+ 958. 25. CAHEN, R. L. and TVEDE, K. 111. Proc. Soc. Exp. Biol. Med. 78: 707-711, 1951. 23. ROBERT, A.

26.

BARRETT, W. E., RUTLEDGE, R., PLUMMER, A. J.

and YONKMAN, F. F. J. Pharmacol. Exper. Therap. ,o$: 305-316, 1953.

27. VISSCHER, F. E., SEAY, P. H., TAZELAAR, A., VELDKAMP, W. and VANDERBROOK, M. J. J. Phar-

macol. Exper. Therap. 110: 188-204, 1954.

28. KOWALEWSKI, K., MACKENZIE, W. C., SHNITKA, T. K. and BAIN, G. o. Canad. M.A.J. 71: 477-

482, 1954.

29. KEYRILAINEN, T. 0., KALLIOMÄKI, J. I..

152

and

Ann. Med. Exp. Biol. Fenn. 35: 431-436, 1957• 30. ROSEN, H., TOWNSEND, P. and SEIFTER, J. Proc. Soc. Exp. Biol. Med. g2: 439-440, 1956. 31. KÜNG, H. L. Gastroenterologla 78: II -16, 1952. 32. PAULS, F., WICK, A. N., EATON, M. and MACK-4Y, E. M. Sci ence 1o3: 673, 1946. 33. RISLEM, E. A., RAYMOND, W. B. and BARNES, R. H. Amer. J. Physiol. 150: 754-759, 1947. 34• MORRIS, C. R. GROSSMAN, M. I. and IVY, A. C. Amer. J. Physiol. 148: 382-386, ,947. 35. BENDITT, E. P. RSNER, J. B. and ROWLEY, D. Gastroenterology 13: 330-335, 1949. 36. GATI, T.1 GELENCSER, F. and SELMECI, L. Experientta 17: 218-219, 1961. 37. MCGINTY, D. A., WILSON, M. L. and RODNEY, G. Proc. Soc. Exp. Biol. Med. 7o: 334-336, 1949. 38. HAROUTUNIAN, L. M., SEGAL, H. L. and MORTON, J. J. Gastroenterology 21: 411-418, 1952. 39. FUNK, C., TOMASHEFSKY, P., SOUKUP, R. and EHRLICH, A. Gastroenterology 20: 625-629, 1952. 40. FUNK, C., TOMASHEFSKY, P., EHRLICH, A. and SOUKUP, R. Science 116: 638-639, 19 2. 41. HOE17FI , F. and DA COSTA, E. PTOC. . Soc Exp. Biol. Med. 29: 382-384, 1,932. 42. MANVILLE, I. A. Amer. J. Physiol. 1o5: 7o, 1933• 43. DALLDORF, G. and }CELIAC, M. J. Exp. Med. GRÖNROOS, M.

56: 391-398, 1 93 2.

44. SHAY, H., KOMAROV, S. A., GRUENSTEIN, H. and FELS, S. S. Gastroenterology 6: 199-212, 1 46. Dig 45. HOELZEL, F., and DA COSTA, E. Am J. . Dis. Nutr. 4: 3 25-331, 1937. 46. SHAY, H., GRUENSTEIN, M., SIPLET, H. and KOMAROV, S. A. Proc. Soc. Exp. Biol. and Med. 69: 369-373, 1948. 47. SHAY, H., SUN, D. C. H. and GRUENSTEIN, M. Gastroenterology 26: 906-913, 1954.

.

Psychological Factors and Psychopharmacological Actions in the RestraintInduced Gastric Ulcer

on gastroduodenal ulcers generally have one or more of the following objectives: a) reproduction of characteristics of the disease of man. (The site, secretory disorders and development characteristic of human ulcers are difficult to reproduce in animals. This objective is rarely achieved.) b) evaluation of specific factors such as secretion and endocrine disturbances. In human subjects ulcers seem to develop almost always by the interplay of several factors. c) development of a pharmacological technique permitting the study of anti-ulcer substances. The restraint-induced ulcer is essentially psychogenic in origin and provides means of studying the second objective. EXPERIMENTS

Serge Bon fils Andre Lambling*

In particular, it permits application of experimental methods in psychosomatic studies. From the pharmacological point of view, the scope extends beyond the field of assessment of compounds to be used in treatment of peptic ulcer (1,2,3). The activity of psychotropic substances can also be tested successfully by this means, and the results obtained reveal findings that are not always in agreement with observations derived from previous studies. The present paper attempts an evaluation of the psychological factors brought about by the restraint-induced ulcer technique; it also describes the psychopharmacological studies pursued by use of this method.

Comparison of Restraint and other Psychogenic Ulcer-Inducing Techniques There are relatively few psychological stimuli capable of inducing gastric lesions experimentally. Chronic psychological conflict Many assays of this type have provided negative results, especially when carried

out during anxiety or experimental neurosis. On the other hand, Sawrey and Weisz (4,5) obtained acute ulcerations by maintaining for a prolonged period (47 out of every 48 hours during one month) a conflict situation based on hunger and thirst and avoidance reactions caused by electric shock.

"Front the Centre de Gastroenterologie, Höpital Bichat and the Unite de Recherches GastroHygiene, Paris, France. enterologiques of the Institut National d'

153

I 1 / RESTRAINT-INDUCED GASTRIC ULCER

Conscious and prolonged feeling of responsibility The well-known experiments of Porter et al. (6) conducted on monkeys eliminate physical drive. Two immobilized animals were exposed to an intermittent electric discharge announced by a signal. Only one of the monkeys had access to a lever permitting it to avoid the stimulus for himself and his partner. The experiment was carried out during six-hour sessions, alternating with six-hour "off-periods", for several weeks. 'When the animals were sacrificed, only the monkey having had access to the levers showed duodenal ulcerations. Prolonged modification of the environment Levrat and Lambert (7) subjected rats to mechanical and to violet-light excitation for 23 hours out of every 24 for periods of four to thirty-four days. The animal's behaviour was characterized by anxiety, overstimulation and a state of hallucination. The gastric ulcerations observed were of an acute type; their frequency was pronounced and accompanied by a high mortality. Physical and psychic restraint It is well known that the rat, an energetic, social animal, who covers a distance equivalent to 20-25 kilometers daily, does not tolerate immobilization very well. The early reports concerning the physiopathological effects of this type of immobilization were contributed by Selye (8) in 1936. In his work, motor restriction was effected either by medullar section, enveloping or by tying the limb; the theory of the general adaptation to stress syndrome was evolved from these experiments. We have devised in our labo1 54

ratories, a technique of spatial restriction which allows variation of the intensity and duration of the stimulus employed (9). This technique, which will be discussed later, has been applied exclusively to the study of nutritional and local psychological factors liable to give rise to lesions of the gastric glandular mucosa (10,1 1,12) . Other techniques have been suggested such as strapping with adhesive tape or confinement in a window grid held together by metal clamps (13) . The technique used to establish complete immobilization is of little importance, as the results are always the same: the erosions occur in the glandular mucous membrane, their incidence ranges from 85 to ioo per cent after twenty-four hours of restraint. Although it appears that the mechanisms of the general adaptation syndrome are not involved, the exact significance and the mechanism of action of the psychological stimulus have not yet been precisely determined. The restraint-induced ulcer technique, like the other psychogenic ulcer techniques raises certain questions: a) the nature of the psychological factor involved, if any; b) process of induction of ulcerations by the psychic stimulus; c) the relative importance of this psychological factor in comparison with other factors engendered by experimental conditions such as nutritional endocrine, vascular, secretory and gastric changes. It is difficult to obtain definite answers to these questions. The following example might be given: experiments employing chronic psychological conflict or modification of the surroundings can elicit serious malnutrition. The resulting state interferes with the first psychological factor, since starvation of itself can induce gastric erosions. As a rule, all psychogenic ulcer procedures present problems of this type. The psychological factor is always accompanied by one or more

BONFILS & LAMBLING

physical actions. These include mechanical or electrical shock procedures, perturbation, immobilization, food-deprivation or malnutrition. Neither Mahl nor Ivy succeeded in inducing gastric lesions by techniques in which physical action was reduced to a minimum. The restraint ulcer technique differs from the' three other types of procedure in several respects: the very rapid occurrence of the gastric lesions (only two hours after the introduction of restraint, some rats de-

monstrate serious ulcerations); a brief food-deprivation unaccompanied by dehydration; a very high and constant incidence of lesions (I ). Thus, there is a contrast between this simple and innocuous stimulus (mortality during experimentation is less than 5 per cent) and the consistency, rapidity and selectivity of the lesion-response in the stomach. Systematic anatomical studies revealed only hepatic stasis and a moderate renal congestion (12,13).

Ulcer Incidence and various Psychological Parameters in the Restraint Technique subjected to a second restraint period (C2). Then, it is sacrificed or released again for forty-eight hours prior to a new assay. In this manner we tested three groups of approximately fifty rats, which were subjected to two, three and four periods of restraint, respectively, before being sacrificed (C2, C3 and C4). After the animal had been sacrificed, an examination of the stomach revealed a Adaptation to restraint by repetition rather complex pattern. Complete healing A new psychological parameter, inure- of the ulcerations — following suspension ment, is brought into play by repeating of the twenty-four hour restraint period the restraint sessions on a given series of — was ensured only after the ninth day animals, while carefully obviating, as far subsequent to liberation of the animal. as possible, the nutritional hazards of However the animals were sacrificed at periods of food-deprivation. We attri- the end of the second restraint period, bute to the adaptation factor the signifi- that is, on the fourth day following the cant decrease in percentage of ulcer-bear- suspension of the first restraint period. ing animals observed following a certain Theoretically, there must be some internumber of repetitions. ference between the various lesions inIn repeated restraint sessions (I 39 fe- duced during consecutive restraint male rats), after an initial twenty-four periods. In fact, macroscopically, it is hour period of restraint (CI ), the animal, possible to trace the progress of the instead of being sacrificed, is released for lesions (fresh acute ulcer, scar-formation, forty-eight hours in a cage with other etc.) (12). On the other hand, on the animals and is permitted a normal feed- fourth day, there is evidence of only to ing. At the end of this time-lapse, it is per cent of severe lesions attributable to

In our laboratory, three types of psychological assay were made. Two of these assays involved imposed action: the search for adaptation to restraint by repetition, and the investigation of consequences of a variation in intensity of the restraint stimulus. A study of the spontaneous behavior of the animal presents the psychological parameter for the third assay.

155

I I / RESTRAINT-INDUCED GASTRIC ULCER

TABLE I. Effects of repetition of restraint procedure on frequency of ulcerations. RECENT ULCERS

Experimental Group

HEALED ULCERS

NORMAL STOMACHS

Number Percentage Variation Percentage Variation Percentage Variation of of animals range of animals range of animals range Animals with ukers with ulcers with ukers

Single testraint procedure in 24 hours

179

86.5

79-91

0

Repeated 1 C2 C3 restraint C4

46 45 48

73.9

54-80

10.9

Nutritional control group (3 fasting periods followed by a single restraint procedure.)

10

70

35-93

0

the first restraint period. It seems probable that the majority of the acute ulcers observed in series Cz developed during the second restraint period. In order to evaluate the possibility of a nutritional factor, we subjected ten animals to three twenty-four hour periods of total food-deprivation at intervals comparable to that of the restraint periods, intermittent with forty-eight hour normal feedings. An actual restraint period was substituted for the fourth fasting period, after which the animals were sacrificed. Ulcer incidence According to the results outlined in Table I, it is apparent that ulcer incidence declines with each repetition of restraint. This phenomenon is statistically significant (p = o.00i). The rise in percentage of healing ulcers also seems to be attributable to repeated restraint, but is less conclusive. This is obviously due to the

3-22 23-50 28-57

13.5

9-21

15-2 11.1 31.3

6-27 5-24 18-44

30

7.65

relatively small number of acute ulcers in series C3. There is a distinct increase in the number of intact stomachs in series C4, only. It is apparent that rhythmic food-deprivation affords no protection against the development of restraint-induced ulcers. Nutrition The body weight loss shown in Table Ia, as a percentage of the initial weight, is computed on groups of ten to thirty animals. The female rats employed had an initial body weight of 140-190 g.

Connnent on the results of the repeated restraint technique The decrease in ulcer incidence attendant to repeated restraint is apparent. It occurs in spite of the development of serious malnutrition, which appears to be responsible for the high mortality in series C3 and C4. This decrease may be ascribed to the intermittent food-deprivation by TABLE IA. Percentage of body weight loss. diminishing organic reactivity through Standard restraint procedure: 9.4% malnutrition or, conversely, by stimulatRepeated restraint procedure: (C2 14.5% ing endocrine, or some other form of (C3 17.8% adaptation. The results obtained in the (C4 19.2% control series are at variance with both Controls (hanger con tractions after final reof these assumptions: compared with straints procedures) 17.9% series C4, the decrease in body weight is 156

■■■■■■■■■■ ..........

--Sz identical (17.9 per cent as compared to 19.2 per cent), whereas ulcer incidence is clear-cut and statistically different (7o per cent as compared to 25 per cent). Our results have led us to postulate the involvement of a psychological phenomenon of inurement to the "imposed restriction" stimulus. Pharmacological studies support this view. Variations in intensity of the restraint stimulus When restraint is applied, two factors must be considered. Firstly, the effect of the actual volumetric restriction imposed and secondly, the motor responses of the rat in its effort to free itself by struggling in its jacket. It is certain that, with repetition, the animal expends energy that may represent an additional drive response which may considerably diminish the importance of the psychological factor, or at least, relegate it to a secondary role. In fact, we were able to demonstrate, when registering the motor activity of 107 rats during restraint, that there is no relationship between the significance of the drive responses and ulcer incidence (1 4,1 5). Volumetric restriction seems to be important therefore, because it entails immobilization. Moreover, when restraint is imposed for a twenty-four hour period, varying the procedure employed does not modify ulcer incidence, provided the animal is completely immobilized. It is not necessary to achieve complete immobilization in order to elicit ulcers. Forced restraint seems to act by means of some other mechanism since it can function as quantitative stimulus (14,15,16). This can be demonstrated by our experiments in which rats are placed in empty enclosures of varying capacity. The general restraint conditions are those employed for the standard twenty-

■■1111■■■■■■ wi.w-2:amonal n cammompser

FIG. t. Diagram to demonstrate the application of various restriction volumes.

four-hour restraint procedure as follows: no previous food deprivation; hydration by physiological saline injection at the initiation of the experiment; and finally, a double check examination of the stomachs which are evaluated according to the "all or nothing" criterion. Standard restraint, in this instance, corresponding to the rat's body volume, 18o nil., is selected as discussed previously (15). Five other restraint types were studied: 36o, 56o, 75o, 1,26o and 7,35o ml. In communal cages the space is divided into compartments by a series of mobile partitions, permitting modification of the width, and by glass rods inserted into the holes of the grid, allowing adjustment of height and length (Fig. i ). The volume of 7,35o ml. is provided by a cage in which the animal has entire freedom of movement. To obtain a volume of 1,26o ml., the dimensions of the cage are reduced to: width, 7 cm.; length, 23 cm.; height, 7.8 cm.; the rat can easily turn around. 76o ml.: width, 5 cm.; length, 21 cm.; height, 7.3 cm.; the animal has some difficulty in turning around; yet it can tamper with the glass rods that form a horizontal ceiling and can shift them in order to free himself if the rods are not secured by a band of adhesive tape. 56o ml.: width 7 cm.; length and height determined by the rat's shape which is molded by the row of glass rods. 36o ml.: width, 4.5 cm.; length and height depend upon the same factors as in the case of 56o (Fig. 2). Paroxysmal liberation responses do not occur in the space volumes of 1,26o and 76o ml. They are insignificant or nonexistent in the other three spaces or at any rate, are much smaller than that of standard restraint. 157

°/p of ulcerations 100 -

80

60

40

20

13

straight line of regression with the following slope: y = (0.086 + 0.014 -!- 0.034) The equation (probit) is: y = 3.81 + 0.004 t x. The confidence limits are more or less small from 4 to 5 per cent corresponding to the points on the line. The existence of a mathematical relationship between the limiting of the volume of the restraint space and the incidence of ulceration affords the following conclusions: a) The liberation response noes not actually play a role in the genesis 79 130 170 270 540 of ulcers, since it does not apply in the Restriction volume (105 %v) case of the two volumes selected; the reFIG. 3. Correlation between restriction volume sults obtained from these two volumes (t/v) and ulcer incidence (control restriction concur with the other points on the line curve). of regression. b) In standard restraint, the stimulus is strictly limited to forced reThe results obtained in 715 animals straint; when allowing a restriction volwere evaluated in terms of the period in ume identical with the size of the rat, the which the trial was made. This was indispensable owing to the fact that the tests were carried out over a period of three TABLE II. Restriction volume and frequency of ulcerations. years. The results of these experiments are Number of Percentage of Restriction Year of shown in Table II. Of the five volumes volume animals ulcerations study nil. selected, the observed incidence of ulcers 137 86.1 1958 180 does not show any statistically significant (standard 85 1959 42 variation (p> 0.0.5). The stimulus elici- restraint) 100 94 1960 279 89.2 Total ted by "force restraint" — always of the same importance — is therefore remark51 47 1958 360 47 44.7 1959 ably stable. 41 1960 27 There is a regular increase in ulcer Total 125 45.2 incidence with decreasing volume of the 29 31 1958 560 cage. The relationship between the two 1959 23 30.4 31 29 1960 phenomena is statistically significant (p= 83 30.1 Total o.00 i) . Fig. 3 clearly shows that by plot31 19.3 1958 760 ting the reciprocal of the volume of the 36 36.1 1959 cage on the abscissa (1/V), against per46 34.7 1960 113 30.9 Total centage ulceration, a linear relationship is obtained, (including the

io5 point (-- -) of the standard restraint). The relationship "ulcer incidence-forced restraint" may be expressed by a 158

1260

1958 1959 1960 Total

57 45 19 123

13.7 25.1 26 20.8

7350

1958

51

11.7

BONFILS & LAMBLING

results are in agreement with the five other spaces studied. c) Restraint offers a stimulus which is measurable according to the space afforded the animal. As in the case of adaption to repetition of restraint, one can for practical purposes eliminate the physical consequences of this stimulus in the genesis of gastric ulcerations. Volumetric restriction appears therefore to act through psychogenic pathways. Spontaneous behaviour of the rat and ulcer incidence In addition to factors such as intensity of stimulus, adaptation and pharmacodynamic actions capable of modifying ulcer incidence, it should be noted that, for a given experimental situation, there are differences in the response of animals; these can be explained only by individual susceptibility. These differences in sensitivity may be due to genetic factors as studied by Sines (17,18), or acquired factors. The age of weaning of the young rat has been found to be most effective acquired factor studied so far (19). We have undertaken a comprehensive investigation of the hypothesis that ulcer incidence depends upon the psychological make-up of the animal. We have attempted to identify the animal's psychological structure by means of a very simple behavioral test performed prior to the initiation of restraint. As it will be shown in the following pages, we have been able to demonstrate that a close correlation exist between certain spontaneous responses of the rat and the genesis of ulcers induced under restraint. The technique employed was based on the method of Lat (2o), who observed the behavior of a rat confined to a high cage for a period of fifteen minutes (21); as shown in Fig. 4, the cage has two compartments of different dimensions, divided

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FIG. 4. Cage with two compartments for the study of the rat's spontaneous behavior.

FIG. 5. Cage with two compartments set in the soundproof enclosure.

159

II / RESTRAINT-INDUCED GASTRIC ULCER

by a lattice partition, and contains a trapdoor raised at the seventh minute. The entire apparatus was placed in an isothermal enclosure, isolated from noise; the animals were observed through a plateglass window (Fig. 5). Behavior was evaluated counting the following responses: a) periods of standing on hind legs; b) washing periods; c) complete immobilization; d) time required to discover that the trap-door opens; e) number of trips through the trap-door (zz). The animals were then subjected to restraint for seven hours, after which they were sacrified. Altogether, seventyone female Wistar rats weighing 130-190 gm. were tested. They had been weaned on the thirtieth day. Based on two criteria of behavior, the number of "standings" and the number of trips through the door in a total of four test periods, the animals can be classified into three groups: Group 1 (active group): the number of "standings" were higher than or equal to forty and the number of trips through the trap-door were higher than or equal to sixteen (thirty-seven rats or 52 per cent). Group 2 (passive group): the number of "standings" were less than forty and number of trips less than sixteen (twenty-one rats or 29.5 per cent). Group 3 (intermediate group): the two types of reactions were conflicting (thirteen rats or 18.3 per cent) as seen in Table III.

At autopsy, 26 out of 71 rats had ulcers (36.6 per cent) — that corresponds to a lesser percentage than that obtained in the basic series (62 per cent). It should be noted that in the latter case, animals were used which had a variable weaning age of less than thirty days. The examination of the pathological material has demonstrated that out of thirty-seven active rats seven were ulcerbearing, that is 18.9 per cent, while out of twenty-one passive rats, sixteen developed ulcers (76 per cent). The intermediate group provided such a small number of rats that no classification was permitted. Using only the criterion of trap-door test results, we find that among the forty-two active rats, seven were bearing ulcers (16.5 per cent). Among the twenty-nine passive rats, nineteen were bearing ulcers (65.5 per cent). The correlation between the spontaneous behavior of the rat and the occurrence of gastric ulcers under restraint seems evident: the passive behavior of some animals coincides with their predisposition to development of gastric lesions. It would have been expected that the most active rats should have suffered from the forced restraint more than the passive rats. In fact, Sines (18) made such an observation with a test which was different from ours. However, by means of yet another test, he arrived at results in consonance with those we have obtained. Keeping in mind our observations and by

TABLE III. Effect of behaviour on frequency of ulcerations. Results

Group 1

Group 2

Group 3

Erect position

40

gastric ulceration

FIG. t. Bolton's experiment. The stages in the production and administration of gastrotoxin.

A) ACTION IN VITRO a) the hemolytic power of rabbits' serum increased after intraperitoneal injection of guinea pig gastric cells. First, an increase in normal rabbit serum hemolysin was observed, but later there was also an increase in "artificial" hemolysin, which is complemented after heating. b) Agglutination of gastric granules, obtained by centrifuging ground mucosa of guinea pig stomach, occurred when these were treated by rabbits' serum. c) Soluble proteins of the gastric cells and mucus were precipitated; filtered gastric cell emulsion contained a protein with which the prepared rabbit serum produced a precipitate. d) Lysis of intact gastric cells was observed in some cases.

but the animal survived. The injection of the rabbit serum also produced shock, to B) SPECIFICITY. a) There was no abwhich some of the animals succumbed, sorption of gastrotoxin by intestinal or with the appearance of gastric ulceration. liver cells or red blood corpuscles in preThe lesion seen comprised mucosal necro- parations showing activity against gastric sis and hemorrhage, usually located at the cells. The reverse was true for hemolysin; curvatures and unequivocally restricted the lytic power was not absorbed by gasto the mucosa, without involvement of tric cells, but was absorbed by liver, intesthe muscle coat or serosa; subsequent tine and red blood corpuscles. b) Hepatodigestion of the necrotic tissue led to the toxic and enterotoxic sera prepared by production of ulcers. Normal serum of similar methods showed some of the efrabbits or guinea pigs had no effect. fects described above; they cannot thereStrongly hemolytic rabbit serum injected fore be truly the specific effects of one into a guinea pig produced hemolysis but toxin such as gastrotoxin. no typical ulcers. Further refinement of the experimental C) INTRINSIC ACTION IN VIVO (I O). The procedure (8) permitted administration experimental production of gastric ulcerof four to five intraperitoneal injections ation invariably led to a series of changes, at seven to ten day intervals. At the end including local hemolysis, precipitation of this time, 5o ml. blood was obtained, of protein as fibrinoid necrotic material, whipped, centrifuged and used the same agglutination and lysis of cells, following day. Ten ml. were injected intraperitone- hyaline change in the area of damage. ally in the guinea pig. Within twenty- These changes were the precursors of four hours, gastric lesions were easily ulceration. The gastric motor activity identified. There was patchy mucosal ne- and the digestive action of gastric juice crosis, with the appearance of punched- (11,12), played a significant role. If these were abolished before the local injection out ulcers twenty-four hours later. Bolton (q) described his findings as of gastrotoxin, the ulcer did not develop as readily. Bolton (13) believed that the follows: 312

SMITH

primary effect was exerted on the capillary wall with cellular changes being secondary; cellular changes were considered a consequence of damage or devitalization, rather than a specific suppression of resistance. D) THE HEALING OF GASTROTOXIC ULCERS was studied (14) after local injection of 2 ml. of gastrotoxin producing a subserous blister in anesthetized animals. The rate of healing of this type of ulcer was not affected by bacteria or feeding of the animal. Although the size of the ulcer was greatest in the presence of hyperchlorhydria, the presence of acid did not

markedly delay the healing of the ulcer. Indeed the ulcer healed more slowly if achlorhvdria were present, as the result for example, of bacterial infection elsewhere in the animal. E) MISCELLANEOUS FEATURES. Serum from an animal treated by repeated injections of cells of heterogenous species could be used for the production of necrotic ulcers of varying depth (15); these experiments were repeated with cats and goats. The ulcer occasionally perforated; the site of the injection for the production of ulcers by the local technique was not important.

Sera, Toxins and Chemical Agents Sera and Toxins Many sera and toxins cause gastric ulceration; it is our opinion that, in most instances, this is the aftermath of cellular damage and release of histamine. The concept of histamine release as a result of injury was advanced by Lewis (16); he suggested that such a mechanism might explain the action of diverse injurious agents in producing common vascular effects and edema; this would be similar to the sensitivity reactions following administration of many proteins and drugs. Various workers, notably Feldberg (17, 18,19,20,2 1), have demonstrated that Staphylococcus or Clostridium welchii toxin, mercuric chloride, bee and cobra venom, lysolecithin and proteins released from the tissues released histamine exerting pronounced effect on the circulation and on the gastrointestinal tract. In the dog (17), after release of histamine by bee or cobra venom, or after large doses of histamine itself, a marked erosive change in the duodenum, intense mucosal

congestion and an increase in the vascularity of the jejunum and ileum could be observed. The villi were distended with blood, and patchy destructive mucosal changes were found. Effects of Horse Serum and Egg Albumin Gastric changes may follow general anaphylaxis produced by the proteins of horse serum and egg albumin. The effect could be restricted by giving intermittent injections of horse serum locally into the wall of the stomach, after preliminary systemic sensitization of both rabbits and dogs (22); healing took one to two weeks for the rabbit; and two to four weeks for the dog. The experiment has also been reversed by giving a local injection first, followed by subsequent systemic sensitization (2 3) . In estimating the role of sensitisation in the production of gastric ulceration, two points require comment. Firstly, the experimental methods require the injection of antigenic material, and the ques313

GASTROTOXIN CHRONIC EFFECTS

ACUTE EFFECTS Local anaphylaxis with mucosal damage via histamine release

a)Antibody formation b) Deplet ion of local hormones c) Lymphoid infiltration

U lcerat ion if acid high

Healing

s

'Gastriti

Gastric mucosaI atrophy

FIG. 2. Acute and chronic reactions to gastrotoxin. Comparable effects might be induced by other proteins, e.g. gastrin.

CHARACTERISTICS OFA HISTAMINE LIBERATOR

/. Depressor response-delayed for /O-?Oseconds ti// histamine enters circulation

2. Consecutive injections ore followed by lessened effects J. Depletion of histamine tissue stores. 4. May mimic the other actions of histamine acid gastric secretion. anaphylaxis triple response etc.

FIG. 3. Features of histamine-releasing agents. The inset depicts at (H) the action of histamine on the circulation, with an immediate depressor response; and at H.L. the delayed one of a histamine liberator.

tion naturally arises of how the antigenic substance may be absorbed in the development of peptic ulcer in man. Demel (24) envisaged the local absorption of protein into the tissues following a breach in the gastrointestinal mucosa. Further contact with heterogeneous, incompletely digested protein might produce local changes comparable with the Arthus phenomenon, which is associated with necrosis, ischemia and edema. Digestion of tissues damaged by this reaction and by the gastric juice should then produce gastric erosion or ulceration. Secondly, although a few injections of a small amount of antigen may lead to fairly striking lesions of the gastrointestinal mucosa (zs,zb), it can be deduct314

ed from principles of immunology that fairly numerous injections, short of general hypersensitivity and anaphylaxis, lead to diminution of local effects and to the acquisition of immunity. Perhaps the commonly experienced failure to induce chronic ulcers by serological methods is related to the development of such an immunity mechanism (admittedly hypothetical) which blocks the "reactivity" to the antigen (Fig. 2). Chemical Agents Release of histamine without cellular damage is widely recognized, following the demonstration by Maclntosh and Paton (27) that many organic bases possess the properties of substances able to liberate histamine from its bound form (Fig. 3). Among the bases found to release histamine were diamines, diamidines, diguanidines, diisothioureas, the diquaternaries and some benzamidines. If injected rapidly into the circulation, all these compounds produce a sudden drop in blood pressure starting twenty-five seconds after the injection, till histamine, freed in the tissues, enters the circulation. These compounds also elicit a triple response on intracutaneous injection, deplete the tissue stores of histamine, and elicit gastric secretion in many species. The most potent of all histamine liberators, discovered to date, is Compound 48/80 (7). This material is obtained by the condensation of alkoxyphenyl ethyl alkylamine with formaldehyde. It stimulates acid secretion in the cat (28) and the dog (29), and is capable of inducing gastric ulceration in rats (3o). In cats (3 i ), repeated injection of compound 48/80 produced gastric ulceration (Fig. 4) of the acute or subacute type; swelling of the rugae, a few erosions, small mucosal hemorrhages and adjacent submucosal infiltration with lymphocytes and plasma cells were observed. But a

small fraction only of the mucosal histamine was released on administration of Compound 48/8o, principally from the glandular mucosa of the body of the stomach.

A

Discussion Much of the histamine of tissues and of organs is present in the mast cells (3 2,33). When tissue histamine is affected by histamine releasing agents, the mast cells throughout the body disintegrate or lose their granules. Examination of a fluorescent histamine liberator under ultra-violet light (34.) showed that it entered the mast cells producing these changes and the histamine releasing effect. Histamine is bound to heparin as a local complex; in the mast cells it is bound in some species to $-hydroxytryptamine as well; hyaluronidase may also be contained in high concentration (35). Proteins, when releasing histamine in course of an anaphylactic reaction, affect mast cells, displacing histamine from its bound form. Proteins or their derivatives can also act on primary injection as histamine liberators in certain species (36,37) (without previous injection to produce a hypersensitive state). Horse serum in cats and rats, and egg albumin in the rat may act in this way without repeated injection and may also induce gastric ulceration. It appears that gastrotoxin might act through a local anaphylaxis or as an "organ-specific" local histamine liberator;

FIG. g. A. Gastric tmtcosa of cat treated with 48/8o, up to 4.5 mg./kg. dosage; gastric ulceration with surrounding diffuse engorgement of the mucosa is evident. B. Photo micrograph of section through edge of the gastric ulcer shown in A. Ulceration penetrates to the level of muscularis mucosae; necrotic epithelium is seen near the periphery, beyond which there is transition to relatively normal mucosa.

both modes of action could lead to damage by the intrinsic release of vasoactive substances within the gastric wall. It is possible that synthetic histamine releasing agents exert their greatest vascular effect on the portion of the stomach where the parietal cells are located: by initiating acid secretory activity at this site, they may increase the local damage. We have therefore undertaken to reinvestigate the production of gastric ulcers by gastrotoxin and other agents in order to determine whether they act via histamine release.

Experiments on Histamine Liberatibn by Gastrotoxin and Horse Serum Methods Compound 48/8o was administered to albino guinea pigs by the serial injection technique of Feldberg and Talesnik (3o),

which is known to produce depletion of the tissue histamine. Blood was obtained for plasma-histamine determination, and tested on the guinea pig's ileum prepar315

Abdominal wall—

Value for Tissue Histamine pg/G

Percentage Reduction after treatment with 48/80

FIG. s. Histamine values for various tissues of the rat are listed (left); the effect of histamine liberation by Compound 48/80 is to diminish tissue histamine as indicated (right); average of three experiments.

ation. The determinations were controlled by extracting the tissues by classical techniques such as Code's (38) modification of the method of Barsoum and Gaddum (39) and by testing with an antihistamine such as mepyramine. Compound 48/80 was given dispersed in a slow release medium consisting of peanut oil plus beeswax, as described by Bruce and Parkes (4o), with antihistamine coverage. For the purpose of sensitization, horse serum was administered subcutaneously, and the antigen repeated fourteen days later. For primary injection in rats without prior sensitization, 1 ml. horse serum was injected, and this was compared with the effects of zoo pg. Compound 48/80. The antihistaminase compound (41) aminoguanidine was injected in i nig./kg. dosage so as to inhibit destruction of the released histamine; this facilitated the determination of the amount of histamine. Gastrotoxin was injected intraperitoneally into guinea pigs and the tissue examined for the presence of gastric ulcers by standard histological methods. Results a) HISTAMINE RELEASE AND GASTRIC ULCERS IN THE RAT. In the Iat, Compound 316

48/80 markedly reduced tissue histamine with the exception of the gastrointestinal tract which showed a much smaller change than other organs (Fig. j). Large amounts of histamine were released into the plasma, and gastric ulcers were augmented by prevention of the enzymatic breakdown of histamine. These effects were duplicated by the action of horse serum, which acts as a primary histaminereleasing agent in the rat without the

C

G FIG. 6. Diagrammatic representation of rat's stomach which normally consists of upper epithelial sac and lower secretory portion. The density of stippling is an attempt to represent umcosal congestion as appearing on colour photographs from 3 animals treated in each group. Same dose as in Table I. A. Normal stomach. B. After horse serum locally subcutaneously. C. After horse serum interperitoneally. D. After horse serum + amittoguanidine. E. After horse serum intraperitoneally (previous injections of Compound 48/80 to deplete labile histamine). F. After Compound 48/80 locally subcutaneously. G. After Compound 48/8o intraperitoneally. H. After Compound 48/8o -{- atninoguanidine.

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agency of prior sensitization; after the subcutaneous injection of I ml. horse serum, the plasma values for histamine were raised, and became still higher when the horse serum was given systematically. The highest values were obtained when enzymatic destruction by histamine was prevented by aminoguanidine (Table I). The effects on the stomach (Fig. 6) closely paralleled the histamine values, the greater the histamine values the higher the incidence of ulceration and erosion. Comparable changes were seen after injections of Compound 48/80. The effects of horse serum on the stomach were lessened if the injection followed those of Compound 48/80, given in a slow-release medium. They were produced most intensely by a treatment of aminoguanidine, prior to the injections of horse serum or Compound 48/80. b) EVIDENCE FOR HISTAMINE RELEASE IN GUINEA PIGS. There is little information available on the effects of histamine liberators in guinea pigs other than that of Mota and Vugman (42), who found little diminution of tissue histamine but described, nevertheless, certain effects which could be attributed to histamine entering the circulation; for example,

Genitalia

Hind paw Ie Value for Tissue Histamine pg/G

Percentage Reduction after treatment with O8/00

FIG. 7. Histamine values for various tissues of the guinea pigs are listed (left); the effect of histamine liberation by Compound 48/8o is to diminish tissue histamine to the small extent indicated (right). (Average of three experiments.)

there was vasodilatation in the ear and prostration with respiratory effects. Compound 48/80, in vitro, released histamine to much the same extent as in the antigen antibody reaction in sensitized tissues (43)• Evidence that there is a release of histamine from the tissues was obtained as follows. Guinea pigs injected intraperitoneally with 4 mg./kg. of Compound 48/8o showed signs of histamine poisoning within fifteen to thirty minutes, and had appreciable amounts of histaminelike substance present in the plasma after aminoguanidine (Table I).

TABLE I. Values for Plasma Histamine

Agent

Histamine (t g./ntl.)

Route of Administration

Normal values

A

B

0.02

0.095

Compound 48/80

Local, subcutaneous General, intraperitoneal General -i- aminoguanidine

0.03 0.8 1.2

0.005 0.24 0.31

Horse serum

Local, subcutaneous General, intraperitoneal General + aminoguanidine

0.02 0.05 0.06

0.005 0.17 0.28

Values for the plasma histamine: A, in the rat after 200 pg. Compound 48/80 and 1 ml. horse serum. B, in the guinea-pig after 4 mg./kg. and 0.5 ml. horse serum repeated after 10 — 14 days' interval (in each case averages of 3 experiments). 317

FIG. 8. Diagrammatic representation of guinea pig's stomach. The density of stippling is an attempt to represent nmcosal congestion as appearing from colour photographs from 3 animals, so treated in each group. A. Normal stomach. B. After Compound 48/80 intraperitoneally. antinoguatridine. C. After Compound 48/8o D. After anaphylactic shock with horse serum intravenously. E. After Bolton toxin (gastrotoxin) intraperitoneally). F. After Bolton toxin (gastrotoxin) intraperitoneally -{- aminoguanidine subcutaneously.

Compound 48/8o was dispersed in a slow-release medium in order to modify the toxic action. The effects of the released histamine, to which guinea pigs are most sensitive, were antagonized by administration of an antihistamine compound, such as mepyramine. It was never possible to obtain marked depletion of histamine as in rats, and there was slight reduction only in the stores of visceral histamine (Fig. 7) ; this may represent all the histamine free to participate in activities requiring its release from bound forms. C) COMPARISON OF THE EFFECTS OF COMPOUND 48/80, GASTROTOXIN AND SENSITIZATION. Compound 48/8o, gastrotoxin and

sensitization reactions with horse serum were compared. The administration of the antigen produced prostration similar to that produced by Compound 48/8o, with less severe effects elicited by gastrotoxin. The antigen antibody reaction led to a considerable fall in the histamine content of various tissues; a smaller reduction in tissue histamine was elicited by Compound 48/8o, with a closely similar one by gastrotoxin (44). The effects of Compound 48/8o on the gastric mucosa were those of erosion and 318

ulceration. The effects were greatly intensified by pretreatment with aminoguanidine. Fig. 8 illustrates the appearance of the stomach after anaphylactic shock, Compound 48/8o and after gastrotoxin. In each case, there is marked engorgement of the gastric mucosa, and the effects have been intensified by the antihistaminase agent, aminoguanidine, which suggests that local release of histamine plays a part in their production. The effects were diminished, or were absent, after prior treatment with repeated injections of Compound 48/80 in a slowrelease medium. Discussion On injection of Compound 48/8o and horse serum into rats, histamine is released into the blood stream with the occurrence of gastric ulceration. The effects are intensified by aminoguanidine, which prevents the enzymatic destruction of histamine. Compound 48/8o may be used to reduce the tissue histamine stores, so that subsequent challenging with horse serum is then ineffective. In guinea pigs the effect on the tissue histamine is not so striking, but this is because the histamine in some tissues, such as the skin and skeletal muscle is not high, the greatest concentrations of histamine being found in the lung and gastrointestinal tract; but only a small fraction seem to be labile, perhaps because of the very great susceptibility of this species to histamine itself. In support of this, Dubos (45), estimated that the guinea pig is at least seven hundred times more sensitive to histamine than the rat. Guinea pigs treated with Compound 48/8o, sensitized with horse serum or given antigen showed typical histamine effects; similar effects were also produced by gastrotoxin. The effect of gastrotoxin was aggravated by the use of amino-

SMITH

guanidine, which prevents destruction of histamine by histaminase. Such a result strengthens the belief that gastrotoxin exerts its effects via the intermediary agency of histamine. After prior treat-

ment with Compound 48/80, injections of gastrotoxin could be rendered ineffective. It may be concluded that gastrotoxin exerts some of its effects as a histamine liberator.

Experiments on Human Gastric Tissue in Vitro The actions of a preparation of human was greater than the mucosal one. Gastro"gastrotoxin" (Smith, unpublished data) toxin released histamine but with an effiand Compound 48/80 (46) were investi- cacy of only one quarter of that of Comgated on human gastric tissue. These were pound 48/80 (Table II). studied in specimens obtained at operaIt is not clear why the submucosa tions on subjects with duodenal ulcer should release more histamine than the and in normal portions of stomachs ex- mucosa, which is more plentifully supcised for lower esophageal malignancy. plied with this substance. Some of this There was no difference in the histamine histamine appears to be more labile, and concentration in the tissue of the two may be located within mast cells; it may groups; one should bear in mind that be distributed around the vessels of the there might be a greater total amount of submucosa, which have to pierce this histamine in the stomachs of the ulcer layer before supplying blood to the mugroup. cosa. iVlast cells are mainly distributed Compound 48/8o released more hista- around blood vessels, and accordingly the mine from the gastric tissue of ulcer sub- histamine release at this site may produce jects than from the control samples. The mucosal circulatory effects (47). release of histamine from the submucosa TABLE II HISTAMINE RELEASE (expressed as percentage of histamine content) in material obtained at operation is compared between (1) 48/80 and (2) gastrotoxin. The histamine release for the body of the stomach only is listed. M =mucosa, S.M.=submucosa, and T.M.=tunica muscularis. ULCER CASES

CANCER CASES

M.

S. M.

T.117.

M.

S. M.

T. M.

(1) 48°80*

8.4

26.8

2.6

9.5

16.1

2.1

(2) Gastrotoxin

2.3

7.1

.6

2.6

3.8

.5

*For the quantitative effect of 48/80 on the pyloric antrum see Smith, 1958 (44); also for statistical treatment of results.

Concluding Comments Various sera and toxins induce gastric ulceration by the release of histamine. It could be argued that such an action is not a dependable means for producing

gastric ulceration, since this is effected via the indirect route of the local stores of histamine. Nor does it provide evidence for a precise physiopathological 319

III / EXPERIMENTAL GASTROTOXIN AND THE COMPOUND 48/.80 ULCERATION

disturbance restricted to the alimentary tract, since the release of histamine may take place from mast cells at other sites. The release of histamine may be causative, but on the contrary, could be secondary to another effect, perhaps a cytotoxic one. In both cases, however, the local effect would be inseparable from vascular damage as hyperemia, edema and final thrombosis. The release of histamine may be necessary after cellular damage to serve such processes as the regeneration or repair of the devitalized tissues (48,49). Much of the recent work on histamine has been concerned with its dynamic aspects, with the changes in the turnover of histamine in the newly formed tissues and with its role in regeneration and repair (Fig. 9). The healing of wounds or ulceration in the stomach may be influenced by tissue hormones, as has been shown in regeneration after hepatectomy in the rat. Regrowth occurs after partial excision, and the organ returns to within normal limits in thirty days; Kahlson found the rate of radioactive histamine formation considerably increased during the initial phase of regeneration (48). Comparable experiments by Boyd and Smith (5o) have shown the importance of skin histamine FIG. 9. An outline of recent work on the dynamic aspects of histamine in the tissues. 1. GROWTH Increased histamine formation in pregnancy. Formative and destructive enzymatic activity is high maternally in foetus 2. REGENERATION "C histidine . "C histamine increased in partially hepatectomised rats 3. REPAIR Experimental wounds

skin ?stomach (experimental ulcer)

320

in the process of repair; after histamine depletion, collagen with poor tensile strength is laid down in wounds at a relatively late phase. Furthermore, the restoration of histamine and of collagen with normal tensile strength take place simultaneously. Is histamine stored in the alimentary tract for the general purposes of regeneration and repair? Its release after gastrotoxin could then be related to the initiation of repair, rather than (or in addition to) the local damage. Though not the earliest to perform such experiments, Bolton elaborated a series of experiments which serve as a model of what might be expected were gastric tissue to become sensitized. Similar experiments await performance using the hormone gastrin as the sensitizing agent. In many diseases, however, sensitivity reactions slowly proceed towards the development of immunity. Such immunity to the proteins of a constituent organ may develop within the same individual. In thyroid disease, for example, immunity to the protein of the gland or to thyroglobulin may be established with later destruction of cells of the thyroid gland and lymphocytic infiltration with fibrosis. A hypothesis is therefore tentatively suggested for similar immunity reactions to "gastrotoxin" (or gastrin); on the one hand, there might be an acute, violent destructive event leading to ulceration, or a much slower chronic reaction (Fig. z) leading to the reduction of acid secretion as a result of gastric mucosal atrophy. It is already known that repeated injections of canine gastric juice may lead to reduction of gastric secretion (51,52) and may later induce gastric atrophy. The release of histamine could be demonstrated more readily in the tissue from ulcer patients than from controls. The submucosa released more histamine than the mucosa. Perhaps this histamine is pre-

SMITH

sent in the mast cells and is more readily released to play a role in the reparative process. It is obvious that care is necessary in deciding the part played by the

tissue histamine in the production of gastric ulceration by gastrotoxin and other agents, if it is equally required for the regeneration of new tissue.

Summary Bolton showed conclusively that gastric mucosa from the guinea pig is capable of causing the production of specific antigens on injection into a rabbit; the rabbit's serum produces gastric hemorrhage and erosions when reinjected into guinea pigs. Thus the early gastrotoxin experiments demonstrated a serological method of damaging gastric mucosal cells and of predisposing to gastric ulceration. Though this is not even remotely analogous to the development of the peptic ulcer in man, cellular damage of this type might follow the release of histamine, as it is known to accompany other hypersensitivity phenomena. The administration of gastrotoxin leads to a series of changes, which are the precursors of ulceration, including local hemolysis, precipitation of protein as fibrinoid necrotic material, agglutination and lysis of cells in the area of damage. It is concluded that gastrotoxin may initiate a vascular or necrotic lesion within the gastric wall, but acid secretion is responsible for the maintenance of the lesion thereafter. The ulcer arising after a single local injection usually heals. The absence of healing probably depends on the perpetuation of both the initiating factor and acidity. Hydrochloric acid action produces an extension of the initial gastrotoxin lesion, as is shown when compared to cases in which acid is absent. Many other sera and toxins produce gastric ulceration. Gastric changes may follow general anaphylaxis induced by proteins present in horse serum and in egg albumen. The effect can be obtained

by local intermittent injection into the gastric wall after systemic sensitization of both rabbits and dogs; it can also be observed if a reverse order of experiments is used. Demel has suggested the local absorption of protein into the tissues f ollowing a breach in the epithelial continuity. Further contact with heterogeneous, incompletely digested protein might then produce local changes associated with necrosis, ischemia and edema. The commonly experienced failure to induce chronic ulcers by serological methods may be related to the development of an immunity mechanism due to diminishment of local effects by numerous injections; such an immunity mechanism may then block the reactivity to the antigen. Release of histamine without cellular damage was widely recognized following the studies of Macintosh and Paton, which demonstrated that many organic bases possess characteristics of histamine liberators. The most potent of these is Compound 48/80 obtained by the condensation of alkoxyphenyl ethyl alkylamine, with formaldehyde. On injection of Compound 48/80 and horse serum into rats, histamine is released and gastric ulcerations occur. In guinea pigs the effect on the tissue histamine, while not so striking, is nevertheless typical. It is concluded that various sera and toxins induce gastric ulceration by the release of histamine. The release of histamine may represent the primary factor, or could be secondary to other effects such as cytotoxic action. In both cases, 32 I

lit / EXPERIMENTAL GASTROTOXIN AND THE COMPOUND 48/80 ULCERATION

the local effect is inseparable from vascular damage such as hyperemia, edema and thrombosis. The release of histamine, in vivo, demonstrated in gastric tissue ob-

tained from ulcer cases occurred more readily than in those removed from normal subjects.

References I. BOLTON, O. Proc. Roy. Soc. (Lond.) ?4: 1 35-147, 1904-05. 2. BARTOSCH, R., FELDBERG, W. and NAGEL, E. Arch. f.d. ges. Physiol, 230: 129-153, 1932. 3. FELDBERG, W. J. Pharm., Lond., 6: 281-301, 1954• 4. PARRATT, J. R. and WEST, G. B. J. Physiol., 5. BER 31ALDO,W. T. Amer. 289, 1 950. 6. SCHACHTER, MELVILLE.

7. 8. 9. 10. 11.

J. Physiol., :63: 28;-

Polypeptides which affect smooth muscle and blood vessels. Oxford, Pergamon, 1960. PATON, W. D. M. Brit. J. Pharmacol., 6: 4995o8, 1951. BOLTON, C. Lancet, 1: 1330-1333, 1908. BOLTON, C. Proc. Roy. Soc., (B), 77: 426441, 1906. BOLTON, C. Proc. Roy. Soc., (B), 79: 533-540, 1907. BOLTON, C. Proc. Roy. Soc. (B): 82: 233-

248, 1909. 12. BOLTON, C. J. Path. Bact., 20: 133-158, 13. BOLTON, C. Proc. R. Soc. Me. , z: 14. 15. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 322

28. SMITH, A. N. J. Physiol. (Lond). r19: 2 332 43, 1953. 29. PATON, W. D. M. and SCHACHTER, M. Brlt. J.

3o. 31.

Pharmacol., 6: 509-51 3, 1951.

FELDBERG, W. and TALESNIK, J. J. Physiol., (Lovd.) :20: 550-568, 1 953. SMITH, A. N. J. Physiol. (Lovd.) 121: 517-

538, 1953.

32. RILEY, J. F. and WEST, G. B. J. Physiol. (Lon), d. rzo: 528-517, 1953. Med., r00: 217-224, 33. FAWCETT, D. W. J. E 1 954. 34. RILEY, J. F. The Mast Cells, Edinburgh,

.xp

35• 36. 37. 38.

1915.

Pt. III 39. (Path.) 54-63, 1908-09. BOLTON, C. J. Path. Bact., 14: 418-431, 1910. 40. BOLTON, C. rit. Med. J., 2: 1288-1289, 1912. LEWIS, T. The blood vessels of the human 41. skin and their responses. London, Shaw, 42. 1927. FELDBERG, W. and KELLAWAY, C. H. Austr. J. Exp. Biol. Med. Sci:, 15: 461-489, 1937. 43. FELDBERG, W., HOLDEN, H. F. and KELLAWAY, C. H. J. Physiol. (Lond.) 94: 232-248, 1938. 44. FELDBERG, W. and KEOGH, E. V. J. Physiol., 45. (Lond.) 90: 28o-281, 1 937 FELDBERG, W. Ann. Rev., Physiol., 3: 671-694, 1941. 46. ROCHA E SILVA, M. Arch. exp. Path. Pharmak., 47. 1 94: 335-350, 1940• SHAPIRO, P. F. and IVY, A. c. Arch. Int. Med., 48. 38: 237-258, 1926. 49. JAHIEL, R. and KRAKAUER, J. Proc. Soc. Exp. Biol. i\Lcd., 79: 54-56, 1952. 50. DEMEL, A. C. Pathologica, IF 128-131, 1923. JAHIEL, R. Arch. d. mal. de !'app. digestif., 51. 28: 627-631, 1938. WALZER, M., GRAY, 1., STRAUS, H. W. and LIV52. INGSTON, S. J. Immun., 34: 91-95, 1938. MACINTOSH F. C. and PATON, W. D. M. J. Physiol. (Lond.) 109: 190-219, 1949.

Livingstone, 1959.

FULTON, G. P., MAYNARD, F. L., RILEY, J. F. and WEST, G. B. Physiol. Rev., 37: 221-232, 1 957• FELDBERG, W. and SCHACHTER, M. J. Physiol., 118: 124-134, 1952. SCHACHTER, M. and TALESNIK, J. J. Physiol., rr8: 258-263, 1952. CODE, C. F. J. Physiol. (Lond.) 89: 257-268, 1937• BARSOUM, G. S. and GADDUM, J. H. J. Physiol.

(Lovd.) 85: 1-1 4, 1935. M. and PARKES, A. s. Lancet, i: 71-72, 1952. SCHAYER, R. W. and SMILEY, R. L. Amer. J. Physiol. 177: 401-404, 1954. MOTA, L. and VUGMAN, L Brit. J. Pharmacol., It: 304-307, 1 956. MONGAR, J. L. and SCHILD, H. 0. J. Physiol., rr8: 461-478, 1952. SMITH, A. N. Brit. J. Surg., 46: 157-163, 1958. DUBOS, R. J. Bacterial and mycotic infections of man. 2nd ed. 182. Philadelphia, Lippincott, 1952. SMITH, A. N. Clin. Sci., r8: 533-541, 1 959. MILES, A. A. and MILES, E. M. J. Physiol. (Lovd.) rr8: 228-257, 1952. KAHLSON, G. Lancer, r: 67-71, 1960. SMITH, A. N. J. Roy. Coll. Surg., Edinburgh, 6: 276-292, 1961. BOYD, J. F. and SMITH, A. N. J. Path. Bact., 78: 379-388, 1 959. LIVERMORE, G. R. JR., and CODE, C. F. Amer. J. Physiol., 168: 605-611, 1952. SMITH, W. 0., HOKE, R., LANDY, J., CAPTTO, U R. and WOLF, S. Gastroenterology, 34: 181187, 1958. BRUCE, H.

PART IV

LOCAL FACTORS IN PEPTIC ULCER FORMATION

Gastric Secretion in Experimental Animals*

THE nervous, cephalic or psychic phase

of gastric secretion is transmitted by the tenth cranial nerves, the vagi. The vagus nerves constitute the entire cranial outflow of the parasympathetic nervous system. The anatomic relationship to the stomach is, as in other parts of the vegetative nervous system, a dual neuron sys-

E. R. W oodward

tem with a central and a peripheral ganglion, the latter located actually in the wall of the stomach. The parasympathetic system is cholinergic, with acetylcholine as the chemo-effector; both the secretory and motor effects of the vagus on the stomach can be duplicated by acetylcholine and other cholinergic drugs.

Role of the Vagus Nervous regulation of gastric digestion was a highly controversial subject throughout the nineteenth century with physicians, relying on clinical observations, presenting the "pro," and physiologists, depending on laboratory experimentation, holding mainly to the "con" view. Although Brodie (1), in 1814, demonstrated that section of the vagi in animals suppressed gastric secretion, his study was not widely accepted. It remained for the classic experiments of Pavlov, reported in 1889, to establish beyond question the röle of the vagus in gastric secretion (2). He prepared a dog with cervical esophagotomy and gas-

tric fistula. When the animal was fed, ingested food escaped through the opening in the neck and did not enter the stomach. Pavlov called this "sham feeding", and it was followed within five minutes by a copious flow of highly acid gastric juice from the gastric fistula. After section of the vagi, the animal "sham fed" as readily as before but no gastric secretory response ensued. This experiment of Pavlov has remained a classic not only because of the basic nature of his discovery, but also because of clarity of concept and simplicity of design. Pavlov's great technical skill as a surgeon was also an important factor in

'Supported by a grant from the National Institutes of Health. •Fron: the Department of Surgery, University of Florida College of Medicine, Gainesville, U.S.A. 325

IV / GASTRIC SECRETION IN ANIMALS

the success of this and other experiments. Pavlov had worked with the German physiologist, Heidenhain, in 1877, and had learned the latter's technique for forming an isolated pouch from the greater curvature of the stomach. Later in his own laboratory, Pavlov modified the vagally denervated Heidenhain pouch with the preservation of vagal innervation by leaving a seromuscular connection between the main stomach and isolated pouch. It was with this preparation that Pavlov performed his subsequent studies on the conditioned reflex. Quantitative studies of the effect of vagotomy on the total secretion of gastric juice, both in the fasting stomach and after the stimulus of food taking, are almost necessarily inaccurate in man. The difficulties in collection led to the development in Dragstedt's laboratory of watertight gastric fistulas, which made possible quantitative collections of gastric juice from Pavlov or Heidenhain pouches or from the totally isolated stomach in animals over periods of many months (3) . In studying the influence of the vagus nerve on gastric secretion, the Pavlov pouch has the major disadvantage of not being supplied by a major vagus trunk but only by a relatively few small vagal fibers traversing the seromuscular bridge between main stomach and pouch. The nervous phase of gastric secretion in this preparation is not prominent. Although a response can be elicited by strong vagal stimulation, as in insulin hypoglycemia or sham feeding, the major share of the daily output of acid gastric juice results from the gastrin mechanism (4)• The production of the totally isolated stomach with preservation of its vagal innervation and blood supply was first described by Dragstedt and Ellis (S) in 193o, and the various modifications of the procedure used in this laboratory at the 326

FIG. 1. present time are described in a more recent publication (6). This method lends itself admirably to a quantitative study of the effect of vagotomy, since the total secretion of gastric juice by the isolated stomach may be collected for long periods before and after division of the vagus nerves to the stomach. The response of the stomach to insulin hypoglycemia makes it possible to check the functional capacity of the secretory fibers in the vagi before vagotomy and the completeness of this operation afterward. Seventeen dogs were prepared with total isolation of the stomach as indicated in Fig. 1. After recovery from the operation, the gastric juice was collected in a rubber football bladder attached to the gold-plated brass or nylon plastic cannula. Each day the hag was emptied, the dog was given an intravenous infusion of Ringer's solution amounting to loo to zoo cc. more than the previous twentyfour volume of gastric juice, and the animal was fed a standard diet of milk, sugar, horsemeat, hamburger, bone meal and brewer's yeast. Free acidity of the twenty-four specimen was determined by titrating with one-tenth normal sodium hydroxide, Toepfer's reagent being used as an indicator. Peptic power was determined by a method described by LeVeen (7). A I:zso dilution of gastric juice in 0.05 normal hydrochloric acid was incubated with a standard solution of egg albumin. The remaining albumin was precipitated with sulfosalicylic acid and determined nephelometrically on the Evelyn colorimeter.

WOODWARD

D-875 TOTAL GASTRIC POUCH DAILY 24-N000. SECRETION

1 00 SO SO

s

40

0 RP Ø

TAAN ST MOR AC IC VAGOT OAIY

I'0I00 0040

TR AN STMOR AC IC VAGOT OMY

TIUC IM DAYS

FIG.

2.

TABLE I. Per Cent Reduction in Average Twenty-Four Hour Gastric Secretion Following Vagotomy in Total Pouch Dogs

Volume

Free Acid

Hydrochloric Acid Output

D-802 D-806 D-812 D-839 D-861 D-865 D-875 D-898 D-912 D-954 D-962 D-996

78% 77 56 60 60 40 53 42 37 46 75 51

— 66% 76 67 54 28 46 82 14 18 43 47

— 92% 90 87 85 57 74 90 46 55 81 74

— 24% 30 47% increase 40 29 72 30 11 33 23 —

Average reduction

56%

49%

76%

32%

Dog Number

Peptic Power

327

D•095 TOTAL GASTRIC POUCH DAILY 24 110UA SECIKTION

KILL INDUS • OWL Man

1111111111111

•

TwOYKK cowiTT vAGOToko

WADI/0 I.

N >a SULLIT

11111110111111111111111111101 4/TT Ø KOTONT

i t~ I KOTONI

TINS IN DAYS

FIG. 3. Vagotomy was performed in twelve of the seventeen animals, by the transthoracic route in eleven and transabdominally in one. In every case there was a profound reduction in the volume, free acidity and hydrochloric acid content of the twenty-four hour secretion as a result of the vagotomy, as exemplified in Fig. 2. Table I shows the per cent reduction of the twenty-four hour secretion. The decrease in volume varied from 37 to 78 per cent, averaging 56 per cent. The decrease in free acidity of the twenty-four hour secretion varied from 18 to 8z per cent, averaging 49 per cent. The reduction in the twenty-four hour hydrochloric acid output varied from 46 to 92 per cent, averaging 76 per cent. This compares closely with the reduction obtained by vagotomy in the patient with duodenal ulcer. In eleven of the twelve animals, vagotomy produced a moderate decrease in the peptic power of the gastric juice. This reduction varied from 11 to 72 per cent, averaging 32 per cent. In one animal, however, there was a 47 per cent increase. 328

In each animal the integrity of the vagus innervation to the gastric pouch was established prior to vagotomy by the use of the insulin test. Essentially the same technique was used as in patients with peptic ulcer, with the exception that 5 or to units of insulin is adequate. In animals with a totally isolated stomach, there is usually a pronounced augmentation in the secretion of gastric juice with the onset of hypoglycemia. Not only the volume and free acidity rise, but also the peptic power of the gastric juice increases markedly. This increase in pepsin concentration, characteristic of "vagus juice," is as diagnostic of a positive response as is the increase in volume and free acidity. In the human being, however, an increase in peptic power has been so inconstant as to be of no diagnostic aid in interpretation of the insulin response. After vagotomy, at least two negative responses to insulin hypoglycemia were obtained in each test animal. The quantitative response of these animals to a standard dose of histamine was reported by Oberhelman and Dragstedt (8) to be markedly reduced. This reduction in hydrochloric acid output varied from 6i to 77 per cent, averaging 69 per cent in the four animals tested. In two animals the effect of partial vagotomy was studied. In one the left vagus nerve was crushed in the neck, and, as seen in Fig. 3, the twenty-four hour secretion of gastric juice continued undiminished, although the peptic power of the juice was slightly reduced. The animal still responded to insulin hypoglycemia in the same fashion as before unilateral cervical vagotomy. After complete transthoracic vagotomy, there was a profound reduction in the volume and acidity of gastric secretion, and the reaction to the insulin test became negative. Four animals succumbed to peptic ulcer in the early postoperative period, two

WOODWARD

from perforation and two from hemorrhage. In each case autopsy revealed a typical, large, penetrating, chronic peptic ulcer. In one animal (D-861), bloody gastric juice indicated the presence of an ulcer. Transthoracic vagotomy was performed, but the animal died of a perforation five days later. In another animal

(D-996), transthoracic vagotomy was performed in similar circumstances. Bleeding markedly diminished, and the juice eventually became relatively clear. However, the animal succumbed to inanition six weeks later, and autopsy revealed a large, healing ulcer with the base extensively filled in with scar tissue.

Relationship between the Vagus and the Gastrin Mechanism The diffuse histological structure of the antral mucosa affords us no clue as to the cytologic origin of gastrin. In contrast to other glandular structures with both endocrine and exocrine secretions, there are no easily apparent cellular complexes in the antrum which might be exclusively responsible for gastrin manufacture. Although the extrinsic nerves to the antrum are not essential for gastrin production, the intrinsic neural plexuses seem to be more important. Zeljony and Savich (9), working in Pavlov's laboratory, applied cocaine to the mucosa of the pyloric antrum, and found that the application of meat extract would no longer stimulate secretion of gastric juice from the body of the stomach. They attributed this to paralysis of efferent secretory nerves from the antrum to the corpus. We utilized a dog prepared with a Heidenhain pouch as well as an isolated antrum (ro) (Fig. 4). Cocaine and other topical anesthetic agents were applied to the antral mucosa by direct instillation. Chemical and mechanical stimuli were then applied, using the techniques outlined above. In addition, gastric secretion was stimulated by meals or by subcutaneous histamine or insulin-induced hypoglycemia. Cocaine, in strengths of 2 -5 per cent,

was found to block completely the effects of both chemical and mechanical stimulation of the isolated antrum; the responses to alcohol perfusion and to balloon distention were also prevented. Stimulation by histamine or insulin hypoglycemia was not inhibited. In more recent experiments (11), xylocaine, pontocaine and nupercaine have been found to produce the same effect, although with less reliability than cocaine. Since these topical anesthetics are quite distinct from cocaine chemically, it seems unlikely that the effect noted was due to anything other than the anesthetic properties of these drugs. Certainly this phenomenon seems to indicate the importance of the intramural nervous tissue of the antrum in the performance of its endocrine function. The history of studies of the relationship between cephalic and gastric phases of acid secretion can be found in the article of Nyhus and Chapman (1 z) in this monograph. Suffice it to say that a considerable amount of evidence has been accumulated to suggest that in addition to chemical and mechanical stimuli, vagal release of gastrin is of significance. The original suggestion of Uvnäs (13) that a "secretagogue agent" becomes liberated from the pyloric region during vagal stimulation remains valid; this agent appears to act in an excitatory capacity 329

VAGUS NERVES SPLENIC ARTERY CUTANEOUS FISTULA

HEIDENHAIN POUCH

PYLORIC ANTRUM

SERO-MUSCULAR BRIDGE

3 WAY CATHETER

FIG. 4. Isolated, vagally innervated antrum in Heidenhain pouch dog. potentiating the direct effect of the vagus upon the parietal cells. In view of these findings, Nyhus et al. (14) have suggested recently a new terminology for the stimulatory phases of gastric acid secretion which distinguishes direct vagal, vagal-antral, local antral and intestinal phases; according to this nomenclature only the direct vagal phase is purely neural, while the vagal-antral phase is neurohormonal, and the local, antral and the intestinal phases are purely hormonal. The vagal-antral phase indicates the effect of vagal stimulation upon the antrum. The relation of vagally induced motility and vagally induced gastrin release is not well understood. It is possible that two types of cholinergic effectors can be distinguished: that mediating gastrin release and that mediating motility. No close relationship has been demonstrated so far between the vagal effects on gastric motility and gastrin release. 330

The significance of vagal release of gastrin in human gastric physiology remains unclear. Attempts to devise new operations for the treatment of duodenal ulcer disease continue; the objective remains to maintain a low recurrence rate after the surgical treatment, as well as to decrease the incidence of undesirable side effects. In several of these operative procedures the antrum is left purposely intact. It will be of importance to follow the long-term results of this method (i5). The significance of vagal release of gastrin in man remains uncertain and careful consideration should be given to technical factors of antral exclusion and antral denervation. Division of the major vagus trunks to the stomach abolishes the nervous phase of gastric secretion, but at the same time will, under certain conditions, enhance the gastric phase. Schmitz, Kanar, Storer, Sauvage and Harkins (r6) and Evans, Zubiran, McCarthy, Ragins, Woodward

EFFECT OF VAGOTOMY ON GASTRIC SECRETION IN HEIDENHAIN POUCH DOG 0 001 HEIDENIAN POUCH WITH VAIN NTACT TO MAN STOMACH

WOODWARD

that this mechanism can result in recurrent peptic ulcer in vagotomized patients. One hundred and fifty-eight duodenal -iJ1r_ . -ulcer patients were treated by vagotomy alone; five subsequently developed benign gastric ulcer, while two had erosive gasFIG. f. tritis. Four hundred and eighty-seven patients were treated by vagotomy and and Dragstedt (17) observed that the se- gastroenterostomy, with twenty-eight cretion of gastric juice from vagus de- marginal ulcers developing as a late comnervated Heidenhain pouches in dogs was plication. In eight of these twenty-eight markedly increased by complete division patients, the basal gastric secretion was of the vagus nerves at the level of the low, and there was a negative gastric selower esophagus (Fig. 5). Since the cretory response to insulin-induced hypostimulation of gastric secretion in a Hei- glycemia, indicating that vagotomy had denhain pouch occurs only in response been complete. These eight patients were to a humoral agent, it is apparent that in found to have high-lying gastroenterossome way division of the vagus nerves tomies, with stasis of a barium meal in the markedly stimulates the release of gastrin antrum. We found three patients who from the antrum. It is probable that the developed benign gastric ulcer following chief factor producing this effect is stasis vagotomy and pyloroplasty for duodenal of food in the gastric antrum, due to a ulcer (19). These patients were found to decrease in the tonus and motility of the have high grade gastric retention, with stomach as a result of the vagotomy. The retained food material mixed with highly hypersecretion of gastric juice did not acid gastric juice. As the stomach was occur in similar animals in which stasis of aspirated, free acid progressively disapfood in the stomach was prevented by a peared until the stomach was empty, previous gastroenterostomy, placed in the when anacidity prevailed. In each of the gastric antrum, or in animals in which the three patients, there was a negative gasantrum had been previously resected. A tric secretory response to insulin hypohigh lying gastroenterostomy failed to glycemia, indicating complete truncal vaabolish this hypersecretion, apparently gotomy. It seems clear from these data because it did not provide adequate that in both the dog and man, complete drainage of the vagotomized stomach. vagotomy of the stomach will result in an Oberhelman and Dragstedt (1 8) found exaggeration of the gastrin mechanism. ZA HOAR COLLECT , OH PERIODS

Summary Vagotomy at the level of the diaphragm reduces gastric secretion in the dog by abolishing the nervous phase of gastric secretion. This is best demonstrated in the Dragstedt pouch, the totally isolated stomach with the vagi preserved. Vagotomy reduces the daily gastric secretion in such a pouch by 75 per cent. Dividing

one vagus has no effect. Vagotomy also reduces pepsin secretion in most animals. The response to insulin-induced hypoglycemia, as well as the sham feeding response, is abolished. Vagotomy also reduces the response to a standard dose of histamine. There is a close interrelationship be331

IV / GASTRIC SECRETION IN ANIMALS

tween the vagus and the parasympathetic system, on the one hand, and the pyloric antrum and the gastrin mechanism, on the other. The intramural parasympathetic plexuses are intimately involved in the formation of the gastric hormone since cocainization or antroneurolysis abolishes the gastrin response. The isolated vagally innervated antrum will produce gastric hypersecretion, which is abolished when the vagi to the antrum are divided. There is less impressive evi-

dence that the presence of the antrum enhances or potentiates vagus stimulation of gastric secretion. Vagotomy of the entire stomach results in an exaggeration of the gastrin mechanism, apparently through prolonged retention of food materials and distention. This effect is obviated by an adequate drainage operation, such as gastrojejunostomy or pyloroplasty, and is also absent if the antrum has been resected.

References I. BRODIE, B. C. Phil. Tr. Roy. Soc. London, 104: Io2-I06, 1814. 2. PAVLOV, I. P. The work of the digestive

glands. London, Griffin, 1902.

3. DRAGSTERT, L. R., HAYMOND, H. E. and ELLIS, J. c. Surg. Gynec. Obstet. 56: 799-801, 1933. 4. WOODWARD, E. R., BIGELOW, R. R. and DRAGSTEDT, L. R. Amer. J. Physiol. 162: 99-109, 1950. 5. DRAGSTEDT, L. R. and ELLIS, J. C. Amer. J. Physiol. 93: 407-416, 1930. 6. DRAGSTEDT, L. R., WOODWARD, E. R., NEAL, W. B., JR., HARPER, P. V. JR. and STORER, E. H.

Arch. Surg. 6o: 1-2o, 195o.

7. LAVEEN, H. H. Proc. Soc. Exp.

Biol. Med.

63: 2 54-259, 1946.

8. OBERHELMAN, H. A., JR. and DRAGSTEDT, L. R. Proc. Soc. Exp. Biol. Med. 67: 336-339, 1948. 9. ZELJONY, G. P. and SAVICH, V. V. Proc. Soc.

Ross. Physicians, St. Petersburg, Jan.-May, 1911-12. 10. WOODWARD, E. R., LYON, E. S., LANDOR J. and DRAGSTEDT, L. R. Gastroenterology. 27: 766785, 1954.

332

I I. WOODWARD, E. R. and SCHAPIRO, H. Amer. J. Physiol. 192: 479-481, 1958. 12. NYHUS, L. M., CHAPMAN, N. D., DE VITO, R. V. and HARKINS, H. N. Gastroenterology, 39:

582-589, 196o.

13. UVNÄS, B.

Acta

Physiol. Scand., 4: Supp. 13,

1942. 14. CHAPMAN, N. D., NYHUS, L. M. and HARKINS, H. N. Surgery, 47: 722-724, 196o. 15. NYHUS, L. M. Gastroenterology, 38: 21-25, 1960. 16. SCHMITZ, E. V., KANAR, E. A., STORER, E. H. SAUVAGE, L. R. and HARKINS, H. N. Surg. Forum. 3: 17-22, 1952. 17. EVANS, SHIRE 0., JR., ZUBIRAN, J. M., MCCARTHY, J. D., RAGINS, H., WOODWARD, E. R. and DRAGSTEDT, L. R. Amer. J. Physiol., 174: 21922 5, 1953. 18. OBERHELMAN, H. A. JR., and DRAGSTEDT, L. R. Surg. Gynec Obstet., ,o1: 194-200, 1955. 19. WOODWARD, E. R. AMA. Arch. Surg., ?7: 2892 93, 1958.

The Nature of Basal Hypersecretion in Man with Duodenal Ulcer

male patients with duodenal ulcer, the mean amount of acid secreted under basal conditions is approximately twice that found in healthy men. Three possible first causes for this hypersecretion can be postulated: a) that there is some supranormal drive on a normal population of parietal cells which secrete the acid; for example a drive similar to that occurring during sham feeding in dogs might operate full force in the fasting state; b) that the parietal cells are hyperexcitable. This could show itself either as an increased rate of secretion from each cell for a given level of stimulation, or as an unduly low threshold for stimulation in a proportion of cells, so that a particular level of drive would excite more cells than usual and so give an abnormally high output of acid; c) that the number of cells in the gastric mucosa is greater than normal and that this is alone sufficient to account for the hypersecretion. The present paper must be regarded as an interim report on this complex problem. So far we think that the data on gastric secretion available to us can be accounted for on the sole assumption that groups of subjects with duodenal ulcer IN

J. N. Hunt A. W. Kay W. I. Card W. Sircus*

have more parietal cells than those without duodenal ulcer. This is contrary to the earlier view of Sircus, who believed that his data also required the postulation of some abnormal secretory drive to the gastric mucosa during basal conditions. We are setting out here the reasoning which has made a change of view necessary. As a result of examining Kay's data (i ), Hunt and Kay (z) came to the conclusion that there was no evidence for any abnormality of excitability of the parietal cells in patients with duodenal ulcer, since the relationship between different doses of histamine and the secretory responses to them, expressed as a percentage of the maximal response to histamine, were similar in five subjects with duodenal ulcer and in five without ulcer. Using the augmented histamine test( ) to assess maximal secretory capacity, and measuring the response in terms of hypothetical parietal component secreted, Hunt and Kay (2) found that basal secretion occupied about 25 per cent of the maximal secretory capacity, both in healthy men and in those with duodenal ulcer. That is, there was no evidence of

*From the Department of Physiology, Guy's Hospital Medical School, London; the Department of Surgery, University of Sheffield, Sheffield, England and the JVestern General Hospital, Edinburgh, Scotland. 333

IV / BASAL HYPERSECRETION IN MAN

any difference between the two groups, TABLE II. Mean basal recovery of acid H+) as percentage of mean recovery after which could have suggested the existence (m.Eq. maximal doses of histamine in men with duodenal of an abnormal drive exciting say 5o per ulcer. cent of the parietal cells in men with Basal recovery Basal 11+ x 100 duodenal ulcer. Maximal H+ Giving an anticholinergic drug to re- (m.Eq./haus) SIRCUS KAY duce vagal drive (3) or cutting the vagi 0-4 8.7 6.9 surgically (4) produced equal reductions 4-10 17.5 15.5 in the parietal response to basal conditions 10 - 00 26.0 30.0 and to histamine (5). Since histamine is believed to act at the periphery more or less directly on the parietal cell, it is now patients with high rates of basal secresupposed that the vagus has a permissive tion. Kay's data (7) however are for action as well as the excitatory action, patients about to be treated surgically, such as apparently operates during sham and therefore represent a selected popufeeding in animals. Thus the fall in the lation. There seem to be two points at response to histamine after a vagal block issue:— a) Are Kay's data consistent with those is ascribed to loss of this permissive action. If the vagus were driving basal secretion of Card and Sircus? b) If the data are concordant, which as well as having its permissive action, cutting the vagus should reduce basal they are, are the different methods of secretion by a larger proportion than it study of the data responsible for the difreduces the response to histamine; where- ferent conclusions reached by the two as histamine-stimulated secretion and basal groups? These points are examined in the secretion fall equally after pharmacolo- following paragraphs. gical or surgical vagotomy (vide supra). Table I shows that the recoveries after It is therefore not necessary to postulate maximal doses of histamine are of the any abnormal stimulatory drive to ac- same order in the two sets of data, and count for these facts. that they increase as the basal secretion Sircus (6), basing his views on the data increases. There is however in Sircus' of Card and Sircus for patients with data a higher proportion of patients with ulcers of varying duration and activity, basal secretion between 0-4 m.Eq./hour. suggested that some special excitation via Table II shows that when the basal rethe vagus was increasing the basal secre- covery of acid is expressed as a percenttion of acid, so that the ratio of basal age of the recovery after maximal doses secretion to maximal histamine secretion of histamine, the two sets of data are was abnormally high, especially in those similar, apparently supporting the view TABLE I. Relation of basal recovery of acid to recovery after maximal doses of histamine in men with duodenal ulcer (m.Eq. H+ per hour) SIRCUS

Basal recovery (m.Eq./hour) 0-4 4-10 10-00

334

KAY

No. of men

Mean recovery after histamine (m.Eq./hour)

No. of men

Mean recovery after histamine (m.Eq./hour)

89 55 22

31 40 49

43 65 41

26 40 59

0

of Sircus that the greater the basal hypersecretion in patients with duodenal ulcer, the larger is the proportion of the total secretory capacity active. The data of Sircus and of Kay are however open to another interpretation. Hunt and Kay did not assess the activity of the parietal cells from the amount of acid recovered, but expressed their results in terms of parietal component secreted, that is as ml. 0.16 N HC1, after making a calculated allowance for the neutralization of acid by the bicarbonate secreted by the nonparietal cells of the gastric mucosa. The secretion of bicarbonate has a proportionately larger neutralizing effect when the secretion of acid is small. Thus a recovery of no acid does not correspond to a lack of secretory activity by the parietal cells, but to a secretion of acid balanced by an equal secretion of bicarbonate. Thus the calculated volume of parietal component is probably a better index of parietal cell activity, than is the amount of acid recovered from the stomach. Having shown that Kay's data and Sircus' data are similar, the analysis will be continued with Kay's data. Comparison of Figs. 1 and 2 will show that the effect of changing the expression of the data from amounts of acid recovered to volumes of parietal component secreted is to increase the values for basal secretion relative to secretion after maximal doses of histamine, especially where basal secretion is low.

0

10

50 40 20 30 MAXIMAL HISTAMINE m.eq. H'/3o min.

FIG. t. Basal secretion plotted against response to maximal dose of histamine in normal persons and patients with peptic ulcer. Kay's data (m.Eq. H'/3o min.).

• 0

100

200 PARIETAL COMPONENT n,l.i30min.

FIG. 2. Basal secretion plotted against response to ntaxhnal dose of histamine. Kay's data (ml. parietal component/3o min.).

Table III shows the basal parietal secretion as a percentage of the parietal response to maximal doses of histamine. With basal secretions up to io m.Eq./ hour, the basal secretion of parietal component is more or less a constant percentage of the maximal secretion, but with higher basal secretion, the mean percentage rises from 24 S.E. ± 1.4 to 36 S.E. 2.4, a change which would be expected by chance only once in one thousand trials. This finding is consistent with the view taken by Sircus that some special

TABLE III. Mean Basal Output of Parietal Component as Percentage of Output of Parietal Component After Maximal Doses of Histamine (Kay's Data) DUODENAL ULCER

Basal recovery (m.Eq./hour) 0-4 4-10 10-00

Number Mean S.E. of mean of men % 46 65 41

20 24 36

±2.0 f 1.4

±2.4

HEALTHY PERSONS

Number Mean S.E. of mean of men °Jo 17 7

20 35

±2.0 ±3.5

335

300

60

IV / BASAL HYPERSECRETION IN MAN

force is acting to drive secretion in patients with duodenal ulcer who have high basal secretions. Against this it may be seen in the last column that healthy persons with basal secretion between 4 and I o m.Eq. H7hour also occupy about 35 per cent of the maximal capacity during basal secretion. So far the data have been classified with reference to basal secretion because this method was used by Sircus. However, as the recovery of the basal secretion is more subject to error than the recovery of secretion after histamine, because of the greater viscosity and relatively small volume of the basal output, it would seem preferable to classify the data according to the responses to maximal doses of histamine. The results of classifying Kay's

data in this way are shown in Table IV. The percentage of the "maximal" capacity occupied by basal secretion is similar at each level of maximal secretion of parietal component. It may be concluded from this reexamination of Kay's data that there is no need at the moment to postulate any abnormal secretory drive to the parietal cells during basal conditions in male patients with duodenal ulcer. A preliminary examination by similar methods of the more numerous data now collected by Card and Sircus has shown no inconsistency with the interpretation expressed above. However a change of view is still possible when data which are technically more reliable are subjected to more detailed analysis.

TABLE IV. The Mean Percentage of the Maximal Response Occupied by Basal Secretion in Terms of Parietal Component (Kay's Data) Maximal secretion (nil//roar)

0-239 240-319 321-399 400-00

Number of men

Basal parietal secretion x 100 MAXIMAL SECRETION PARIETAL SECRETION

52 36 36 28

26.5 25.8 23.1 28.5

Standard error of mean

±1.7

±2.4 ±1.9 ±2.1

Summary Three possible first causes for hypersecretion of acid in patients with duodenal ulcer are evaluated: a) supranormal drive to a normal population of parietal cells which secrete the acid; b) a hyperexcitability of the parietal cells; c) a numerical increase of parietal cells in the gastric mucosa. Data on hyper basal secretion in man with duodenal ulcer obtained in two separate studies (Hunt and Kay; Card and Sircus) are examined. There is no evidence for any excitability of the parietal cells as a result of examining 336

Kay's data; basal secretion occupied about 25 per cent of the maximal secretory capacity, both in healthy man and in those with duodenal ulcer. The data of Card and Sircus for patients with ulcers of varying duration and activity suggested that some special excitation via the vagus was increasing the basal secretion of acid. The recoveries of acid after maximal doses of histamine are of the same order in the two sets of data, and they increase as the basal secretion increases. In Kay's data, the percentage of the "maximal"

HUNT, KAY, CARD Sc SIRCUS

capacity occupied by basal secretion is to the parietal cells during basal condisimilar at each level of maximal secretion tions in male patients with duodenal ulcer. of the parietal component; from this re- A preliminary examination by similar examination of Kay's data it is concluded methods of additional Card and Sircus that there is no need at the moment to data has shown no inconsistency with postulate any abnormal secretory drive the above conclusion.

References I. KAY, A. W. Brit. Med. J., 2: -80, 1953 . 2. HUNT, J. N. and KAY, A. W. Brit. Med. J., 2: 1444-1446, 1954. 3. SEIDFLIN, R. Brit. Med. J., 1: I079-1080, 1961. 4. GELB, A. M., BARONOFSKY, I. D. and JANOWITZ, H. D. Gut., 2: 240-245, 1961.

and DRAGSTEDT, L. R. Proc. Soc. Exp. Biol. Med., 67:3 6-339, 1948. 6. SIRCUS, W. Aetiology of Peptic Ulcer, Peptic Ulceration. Wells, C. and Kyle, J. cd. London, Livingstone, 196o. 5. OBERHELMAN, H. A. JR.,

337

Hypoxia of Vascular Origin in the Development of Gastroduodenal Ulcer Disease

Jacques L. Sherman, Jr. Eddy D. Palmer*

A REVIEW of the investigative work, the is becoming more prevalent, and that the literature and discussions on the etiology recurrence rate is disturbingly high. An of ulcers of the stomach and duodenum answer to the question as to why the over the past century, shows a great pre- normal stomach and duodenum do not ponderance of research effort directed digest by themselves has not been estabtoward elaboration of the role of gastric lished. The reason for the fact that the secretory activity in ulcer formation. mucosa of affected organs is normal at a While it is true that almost every student short distance from an active ulcer has of the subject has stated that attention not been explained by the classical theory. must be given to the mucosa in isolating The observation that ulcers heal in the the cause of ulcer, mucosal factors, in presence of the same "acid-pepsin" milieu fact, remain generally neglected in in- which presumably caused the defect is vestigative work or in studies of patients difficult to fit into the framework of this suffering from this disease. A brief review concept. What is the reason for the conof current experimental literature and of stancy in size of ulcers if they are caused therapeutic recommendations will be by simple mucosal erosion? How can one enough to satisfy the reader on these explain the constancy of anatomic localization in the presence of a generalized points. The classical way of thinking about destructive agent? How is it possible for the ulcer problem has become so stan- deep mucosal biopsy sites to heal spondardized that even the common designa- taneously and quickly in the stomachs of tion of the disease is "peptic ulcer." This animals who are in the process of develis indeed a tribute to the position held by oping acute gastric ulcers (1), if one asthe "acid-pepsin" theory of ulcer genesis. sumes acid peptic digestion as the cause In spite of the general acceptance of of ulceration? this theory and its application to methods A critical look at the available facts, of prevention and therapy of "peptic recognition of the failure to make any ulcer", the facts remain that ulcer disease substantial advance in knowledge and From the Research Division, U.S. Army Medical Service Research and Development Command, Washington; and Department of Gastroenterology, Brooke Army Hospital, Fort Sam Houston, Texas, USA.

339

IV / HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

study of the observations which cannot be explained by classical theory, make it imperative to question the validity of this concept. It is the purpose of this presentation to redirect attention to the role of

the gastrointestinal mucosa in ulcer formation, and to attempt to place into perspective the importance of acid-pepsin factors and mucosal factors in this process.

The Mechanism of Depression of Mucosal Resistance It has been clearly stated that the keystone for a proper theory of ulcer genesis will be found in identification of the mechanism responsible for local depression of mucosal resistance (z). This idea is not a new one, and most proponents of theories of ulcer formation have included this concept in their formulations. Once local defense systems have been overcome, it is likely that the acid-pepsin factor is responsible for the mechanical work of ulcerations. It seems probable that this active ulceration can begin only when there has been depression of mucosal resistance, and that it is restricted to the areas where there has been this depression. The statement, "no acid, no ulcer" may be valid, but more fundamental seems to be the axiom "no local depression of resistance, no ulcer." The search for a mechanism which could lead to this local depression of mucosal resistance may be aided by establishing certain criteria based upon our knowledge of the ulcer process. This mechanism must, first of all, be able to produce focal devitalization of cells of the gastric and duodenal mucosa. This mechanism must have some independence from secretory and motor functions in the gastrointestinal tract. It must be capable of coming under the control of certain systemic and environmental factors such as "stress", emotional and psychic influences, and temperature changes. 340

Since the ulcer crater is a local manifestation of a systemic psychovisceral process, it would follow that the causative mechanism should be the result of abnormal functional activity. Of the three great co-ordinated physiologic systems which might be implicated, endocrine, nervous and vascular, only the latter can meet all of the above proposed criteria, and it meets them specifically. Over a hundred years ago, Virchow (3) stated that ulcer must be considered a vascular trouble which acts through local ischemia, because gastric juice can attack only devitalized portions of the mucosa. He postulated that anemic necrosis and autodigestion followed organic or spastic closure of a nutrient mucosal artery. Rokitansky (4) agreed with this general concept. Many theories of ulcer genesis based upon mucosal ischemia have been proposed from time to time, but such theories (if not the thought behind them) have been short-lived, because there was no anatomic information to permit explanation of localized mucosal ischemia. It has long been known that the blood volume of the total mucosa varies quickly under many physiologic and abnormal conditions. The general response, manifested by alternating mucosal engorgement and blanching, has been considered to depend upon such general reactions as contractile changes in the mucosal arteries (5,6,7)

SHERMAN & PALMER

and the influence of muscularis mucosae contractions on the penetrating vessels. But ulcer genesis could not be explained by such a general mechanism, because ulcers usually occur singly, and they regularly represent sharply circumscribed lesions. Investigators, such as von Bergmann (8), Boles (g), and Ophills (1o), con-

tinued efforts to establish a vascular basis for ulcer, but all faced the same difficulty of attempting to ascribe a focal lesion to general vascular reactions. The work of these students and many others in this field has been reviewed in some detail by Hauser (i 1) and by Ivy, Grossman and Bachrach (12).

Arteriovenous Anastomoses Within recent years, there has been a an arteriovenous shunt arises from each growing appreciation of the significance mucosal artery, either just before or just of normal, native arteriovenous anasto- after the artery pierces the muscularis moses in the circulatory system. These mucosae, following its short course from vessels, connecting arteries and veins, are origin in the submucosal arterial network. present in every organ system so far ex- There is little difference in the concenamined, and are not incidental or acci- tration of shunts in various parts of the dental structures. The volume of blood stomach and duodenum (18), although which can be diverted from a peripheral several observers (19,20,21,22) have concapillary bed is enormous. The anatomic cluded that there is a unique pattern to location of a shunt high above the capil- the spacing of the mucosal arteries along lary level will enable it to influence a large the lesser gastric curvature and the posregion of the peripheral circulation, while terior wall near the cardia. This point of an anastomosis lying immediately proxi- confusion over the arterial supply of the mal to a metarteriole can act discretely stomach is the only major one which has in regulating a much smaller area of the persisted since Hofmann and Nather (23) and DjØrup so beautifully proved the abcirculation. Knowledge of this microcirculation sence of gastric end-arteries. It is created and application of principles of blood by the fact that the mucosal arteries in the flow, based upon the extensive network region of the gastric lesser curvature do of arteriovenous anastomoscs, have aided not arise from the submucosal arterial greatly in an understanding of the way plexus, although they freely communicate in which the vascular system may serve with it, but take their anatomic origin as the sought-for mechanism responsible directly from the right and left gastric for local depression of gastrointestinal arterial chains. mucosal resistance (2,13,14,15,16) . Details Each shunt which lies in the vicinity of of the structure and functions of this sys- the muscularis mucosae operates to cirtem as they pertain to the gastrointestinal cumvent the large mucosal capillary bed, tract will be presented. For a summary of which its mucosal artery normally supcurrent knowledge of shunts throughout plies. The capillary bed supplied by each the body, the reader is referred to Sher- mucosal artery is believed to average man's review (17). about i cm. in diameter. The shunt segNormally in the gastroduodenal wall, ment bridges across to the vein which 341

IV / HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

drains the capillary unit of the artery. In addition to the shunts which lie on either side of the muscularis mucosae, there are many large shunts in the gastric subserosa. De Busscher (19) was impressed with their numbers, and found them especially easy to study. Two anatomic types of shunt exist in the esophageal (24,25,26), the gastroduodenal and the small and large bowel (17, 27) walls—the direct and the indirect. An apparent disagreement over which is the commoner in the gastroduodenum is explained by the misinterpretation by Barlow and colleagues (18) of de Busscher's (19) classification. The direct shunt, the less common of the two, consists merely of a relatively short arteriovenous communication equipped with a pseudosphincteric closing mechanism of muscle cells. The arterial end of the communication is usually a branch of a mucosal artery, although occasionally it arises directly from a main limb of the submucosal arterial plexus. After a short course of variable tortuosity, the arterial segment widens to open directly into a mucosal vein or, occasionally, into a vein of the submucosal plexus. The indirect type of shunt has a distinct, anatomically specialized channel between the arterial and venous sides. When studied histologically, the diameters of the channels vary from 3o-I 5op. Arising from a mucosal artery, the arterial part of the shunt segment carries with it intima, a double muscle layer, and adventitia. There is no elastica. In the specialized intermediate segment, where arterial features predominate, the muscle cells give way to large-layered epithelioid cells, the Quellzellen of Schumacher (28, 29). The course of the intermediate segment, which is easily identified although not followed on ordinary histopathologic sections, may be very tortuous. Its lumen suddenly enlarges as it joins the venous 342

part, and the Schumacher cells disappear. The shunt empties into either a mucosal or a submucosal vein. The specialized segment is surrounded by an adventitial nerve set, which is a continuation of the arteriolar plexus, and which continues on to join the submucosal venous plexus. Nerve fibers in close association with the cells of the shunt segment have been described (30,31). In addition to the shunts, there is found in the gastric and duodenal mucosa an occasional direct terminal glomus body, often close to the mucosal surface. These have only capillary connections, and they do not seem to have the ability to obliterate their lumen. Some confusion has arisen about the place of specialized structures which protrude into the lumen of certain arteries and veins "like cushions." These cushionlike structures are made up of myo-epitheloid cells, and are not necessarily associated with arteriovenous shunts. Arteries containing these structures have been called "Polsterarterien" or "arteres å coussinet," veins with these structures "vene di blocco" or "veines d'arret" ( 1 3,32 ,33, 34) , While making a simple histopathologic study of i12 stomachs resected for ulcer, Herzog (1 3) found arteriovenous shunts near the ulcers and, in addition, described cushioned arteries and blocked veins in the area. The former appeared to have sphincter-like longitudinal cell bundles in their walls, and these had protruded into the vessel lumen "like cushions" to block it. Upon reviewing more than three hundred stomachs resected for ulcer, Herzog (3 2) concluded that in many cases the finding formerly looked upon as endarteritis obliterans in ulcer stomachs actually represented degenerated forms of cushioned arteries. Functional observations of these structures are required before their significance can be determined.

SHERMAN & PALMER

Physiology of Shunts Current understanding of the physiology of gastrointestinal shunts is based largely on observations of the concerted activities of the mucosal capillaries. Knowledge of the operation of individual shunts is very meager. The effectiveness of the gastric and colic shunt systems in diverting blood from the mucosal capillaries is well known from observations on mucosal color changes. Such changes can be studied in the patient with a gastrostomy or a colostomy, and can be seen moderately well through the sigmoidoscope. Observations on color changes during gastroscopy are not valid because of rapid variations in the incidence of the reflected light rays and in their sourceto-mucosa distance. The studies of Wolf and Wolff (3 S ) on influences which can cause sudden blanching and reddening of the gastric mucosa, due to changes in mucosal blood content, are well known and require no elaboration. Although increase in mucosal redness is known to correlate well with increase in mucosal blood flow (36), visual judgment of mucosal color is fraught with interpretational difficulties, and it has been necessary to find a more precise technique for measuring general mucosal capillary flow. Thermal gradientometry fills the need well. It measures blood flow in the capillaries of a specified mucosal area through the degree of cooling achieved when heat is applied to it. Studies by this technique confirm the observation that a sudden increase in gastric and duodenal mucosal capillary flow may result from emotional tension, anxiety and resentment (36,37). There is transitory acceleration of flow with each peristaltic wave, and appetizing stimuli produce an increase within ninety seconds. When gastric mucosal flow is measured during laparotomy, it is found that there is depression at the time of skin incision and a further drop when the

stomach is handled (38). This finding confirms well an observation made on the oxygen saturation of venous blood in the gastric veins during surgery (39,40); blood taken from a gastric vein immediately after the peritoneum was opened was found to have an oxygen saturation of 74 per cent, i.e., its source was capillary. After the stomach had been handled just a few minutes, the saturation rose to 91 per cent, suggesting that now the capillaries were being bypassed. No such change was found in blood drawn simultaneously from an arm vein. As Bentley (4o) pointed out, this variability in gastric venous oxygen saturation is not surprising, because at operation it is common to find that soon after the peritoneum has been opened, the serosal veins of the stomach change from dusky purple to bright red. The reverse effect is not obtained; altering the oxygen content of plasma perfusate does not influence shunt activity in freshly resected human stomachs(41). When the freshly resected stomach is studied at body temperature and is then suddenly cooled, it is found that the shunts are thrown open (41). Similarly, reduction of the pH of perfusion fluid from 7.8 to 7.4 encourages the shunts to open. This latter may prove to be a most significant point in making a correlation with the usual pattern of continuous gastric secretion in the duodenal ulcer patient if, as will be emphasized below, the gastroduodenal mucosa is harmed more by plethora than by ischemia. Secretion of acid by the stomach appears to be accompanied by liberation of equivalent amounts of alkali into the mucosal blood, and Walder (42) has sugested that if alkalinization of mucosal blood should exceed the capabilities of local buffering systems, the arteriovenous shunts would respond by remaining closed. Consequently, the mucosa of the continuously secreting 343

IV / HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

stomach might be maintained in a continuously plethoric state. It seems assured that the shunts are not able to maintain intermediate positions but are either fully open or fully closed. The contraction phase is a brief one (43). The functional diameter of the shunts can be satisfactorily measured by noting the largest size glass spheres which, when introduced into the arterial intake of the organ, appear in the venous outflow. For the present discussion it is important to remember that the gastroduodenum has a system of shunts in the subserosa as well as at the base of the mucosa; therefore, it is possible, although highly improbable, that the functional diameters of the two systems differ—improbable because the functional diameters of the shunts in all organs throughout the body have been found to be about the same. For the gastroduodenum in both man and several species of experimental animals, measured in resected specimens and in situ, the maximum diameter has been reported at from 14ou to 16ou (18,44,45). This proof of the function of shunts during life was provided by Sherman and Newman (44). Although it is clear from thermal gradientometer observations that the majority of gastroduodenal shunts act in concert, an important physiologic question revolves about the degree of paradoxical activity which normally exists among neighboring shunts. Obviously, this is a difficult matter to study. Little progress has been made in its solution. It is known from vivimicroscopic observations that in normal rats not all mucosal arteries are functioning at the same time (46). Some remain patent for a while "and then suddenly collapse." Similarly, not all mucosal veins function at once. The submucosal shunts can be recognized in vivimicroscopic preparations, but only one can be satisfactorily studied at a time. They are often found to be closed, although they 344

open quickly if the animal should pass into shock (46). The rhythmic activity of a shunt is not simply a reflection of that of its arteriole; shunt and arteriole show different rhythmic rates and independent periods of closure (47). Study of the vessels of the gastroduodenum by injection with special masses in situ or after removal of the organ, with subsequent histopathologic or microradiographic examination, has taught a great deal about gastroduodenal vascular physiology (18,48,49,5o). There have been objections to this approach because of the unnatural conditions which necessarily exist, especially conditions created by the pressure required for the injection. The mere fact that it permits comparison of the ability of the intramural vessels to receive injection masses during life, immediately after the extirpation of the organ, and long after death, makes it particularly useful for distinguishing pure anatomy from physiologic anatomy. It is the physiologic anatomy that is important, but this must be studied in connection with the morbid anatomy if its physiologic aspect is to be recognized. The significance of vessel tone, shunt competence, etc., as they may vary from point to point along the stomach wall, can be studied in no other way at the moment, although this is not to say that observations made on these points by this technique arc necessarily valid. The consensus of experienced opinion is that the pressures attained during perfusion studies are not capable of influencing the state of the shunts (41), but there may be some question in the case of injection studies. Perhaps the most basic fact discovered through injection studies is that the injection mass fills the mucosal capillaries completely and evenly when cadaver stomachs are used, but there is almost no filling when fresh surgical specimens are used (39,40). In other words, all of the

SHERMAN & PALMER

shunts are apparently open under the conditions which exist at operation, and all arc closed after death. When stomachs are resected under spinal anesthesia which reaches to T-i or T-_, however, they show upon injection extensive capillary filling throughout the mucosa (39,40), demonstrating again the potency of nervous control over the shunts. Experimentally it has been reported that stimulation of the right vagus nerve decreases capillary filling in the pyloric mucosa of the stomach (5 ► ), although this would not be the expected response, and does not correlate well with the effect of acetylcholine

on the shunt system, discussed below. Blood flow in the colonic mucosa reacts to emotional and pharmaceutical stimuli, as does that in the gastroduodenal mucosa (21), judging by both color changes and thermal gradientometry study. It has, in addition, been shown that a cold stimulus applied to the body surface results in increased mucosal flow, with decrease when the skin stimulus is discontinued (52). When the colon mucosa is infiltrated with procaine, the reflex neurologic influence over capillary flow disappears, although the ischemic effect of intravenous epinephrine is not blocked.

Pharmacology of Shunts Pharmacologic influence over gastric mucosal capillary flow has been studied in some detail. Intramuscular histamine causes a significant and prolonged increase in capillary filling (53) and capillary flow within fifteen minutes (6,46) after a transient decrease (54.), and there is a corresponding decrease in the flow of glass spheres through the shunts from the arterial to the venous side of the gastric circulation (41). Amyl nitrite and glyceryl trinitrate (nitroglycerin) produce variable results. Vasopressin (Pitressin), which has played such a large part in experimental efforts at ulcer production, causes mucosal ischemia (55). By injecting carbon into the aorta of vasopressin-treated rabbits, Crane (56) found that there was no capillary filling in those areas of the gastric mucosa which had responded to vasopressin by developing superficial necrosis, while histologically normal areas of the mucosa showed adequate filling. He seems to have proved that ischemia developed before the ulcers appeared. Berg (57,58) emphasized the

part that vascular innervation plays in governing mucosal damage after repeated injections of vasopressin, having found that sympathectomy afforded protection while vagotomy seemed to render the mucosa more vulnerable. Epinephrine, ergotoxine, tetraethylammonium bromide, pilocarpine and atropine cause depression of mucosal flow, with simultaneous increase in the utilization of the shunts (18,41,59,60,61,62). The action of pilocarpine in this connection is complicated by its production of striking tonic contraction of the gastric muscular coats, with a constricting influence over the vessels which pierce them (46,63). It should be noted that uncertainty regarding the quantitative effect of intramural muscular contraction on mucosal blood flow has interfered seriously with evaluation of pharmacologic influences over the shunts. For instance, Benjamin and colleagues (64) felt it necessary to ascribe the elimination by dicyclomine (Bentyl) hydrochloride of the- usual mucosal temperature fall during 345

IV / HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

hunger contractions entirely to the influence of dicyclomine over muscle tone. Addition of small concentrations of acetylcholine to plasma used for perfusing freshly resected human stomachs causes marked reduction in the perfusion rate and an increase in the total blood flow through the organ. That this is due to the closing of the arteriovenous shunts is known, because at the same time, the passage of glass spheres through the gastric circulation is much reduced (r8). This is important because upon vagus

nerve stimulation, there is a distinct increase of acetylcholine in the venous blood which drains from the stomach and because, it is believed, in ulcer patients there is increased or perhaps constant vagus activity (S4). On the other hand, faradic stimulation of a sympathetic nerve accompanying the left gastric artery causes reduction in total flow through the stomach, with simultaneous increase in outflow of glass spheres, indicating that utilization of the shunts is much increased (i8,41).

Pathology of Shunts Glomus tumors take their origin in arteriovenous shunts of the indirect type and in glomus organs. Only a few cases have apparently been reported for the stomach (65,66,67,68,69,70). De Busscher (r q) and Clara (r 7) have described inflammatory disease of the intermediate segment. Such a change might be expected to produce profound disturbances in the regional capillary circulation. The frequency of and cause for inflammatory disease localized to the shunt apparatus, in the absence of more generalized mucosal or submucosal inflammation, are not known. There may be infiltration of the intermediate segment with lymphocytes and neutrophils. The Schumacher cells may become displaced peripherally. This, plus deposition of a fibrous sheath about the vessel, may, in de Busscher's opinion, result in a permanently open shunt. It is of immense importance that in patients with pernicious anemia, a disease regularly accompanied by chronic atrophic gastritis, the mucosal blood flow remains static, without variations secondary to peristalsis, etc. (71). Here the mucosal capillary net shrinks as the glands 346

atrophy, and one supposes that if the shunts are rendered inactive or are actually destroyed, it is due to the great hyperplasia of the muscularis mucosae, which is always part of the gastric pathology in pernicious anemia. The welldocumented dictum that "active benign duodenal ulcers never develop after pernicious anemia becomes established" has therefore a possible vascular explanation. The situation in cases of chronic atrophic gastritis not associated with pernicious anemia is not known, although a hint is to be found in the report of Kimbel and colleagues (7z). By the use of a swallowed microcounter, they found that the radioactivity picked up from the normal gastric mucosa after intravenous injection of P" is related to the blood content of the mucosa and part of the submucosa. Oscillations in two phases were found, and these could be altered by various pharniacologic preparations, particularly histamine, in a series of patients being treated for polycythemia. In the one patient who had achlorhydria, however, the counts remained uninfluenced by histamine, suggesting that the mechanism controlling vascular dynamics

SHERMAN & PALMER

had been lost as atrophy developed. The mucosal vein beyond a shunt which lies above the muscularis mucosae is subjected to arterial pressure in a vulnerable anatomic location. It may at times be the source of severe gastric hemorr-

hage, according to Menegaux and colleagues (73). They treated a patient who required gastrectomy for hemorrhage and, upon histopathologic examination of the specimen, believed they found evidence of this mechanism.

Hypoxia as the Final Common Pathway for Depression of Mucosal Resistance The concept of a final common pathway may be helpful in attempting to discover the mechanism finally responsible for local depression of mucosal resistance in gastroduodenal ulcer (14,15,16) . A final common pathway is defined as the last mechanism which, when activated in any way, will lead to production of one specific effect. The term also carries the implication that the specific effect can be caused by no other pathway, and that when this pathway is activated under physiologic conditions the specific effect will follow inevitably. The application of such a concept to the problem of the cause of ulcer need not be difficult, although the actual determination of the pathway itself may be laborious. In the earlier discussion of mechanisms, the vascular system was selected as the one most likely to be capable of mediating general and specific stimuli. The final common pathway which, when activated, will lead to focal mucosal devitalization must lie distal to the vascular system. It may be considered to be a distortion of one of the normal functions of the circulation, and hypoxia seems to fit the requirements most closely. A working hypothesis that hypoxia is the final common pathway in gastroduodenal ulcer genesis has been established (14,15,16). That hypoxia does regularly produce devitalization of tissue is accepted by all biologists. The system of

arteriovenous anastomoses can produce local hypoxia in one of two ways. If a shunt is wide open almost no blood can pass through the distal capillary net, and the tissue normally dependent upon this net will suffer from hypoxia due to deprivation of blood. Observations of capillary networks in vivo show that alternate opening and closing of capillaries is a normal phenomenon (74,75). If there is interference with the function of arteriovenous anastomoses, the possibility of prolonged closure of capillaries is great. It is also possible for local hypoxia to be produced by an abnormally long closure of an arteriovenous shunt. In this case, the distal capillaries are plethoric, and flow through these vessels is very slow, due to slowing of the venous drainage. Thus, both ischemia and plethora may produce local hypoxia which can lead to focal mucosal devitalization. The hypoxia theory would remain valid in either situation. There are several reasons to select plethoric hypoxia as the explanation for mucosal damage rather than ischemic hypoxia, which has been favored in the past. First, all workers who have had the opportunity to study gastric mucosa through a gastrostomy have observed erosions to develop only as a sequel to mucosal engorgement. The same phenomenon can occasionally be proved at gastroscopic examination, and in certain 347

IV HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

clinical situations, such as the acute ulcerations which may result from prolonged shock, mucosal destruction is known to begin with a period of mucosal stasis and hyperemia (76,77). There are, to be sure, quickly healing lesions which cannot be considered the same disease as chronic ulcer, but it proves that under these circumstances at least, physiologically attained plethora is more injurious to the mucosa than physiologically attained ischemia. In the case of the well-established ulcer, there is histopathologic suggestion that engorgement with focal hemorrhage continues to act for some time in the periulcerous region (53,78,79). Second, as has been pointed out above, the action of the pharmacologic preparations commonly used to produce acute experimental ulcers, other than vasopressin, is to cause shunt closure. The same is true for vagus activity. Under experimental circumstances, vagus stimulation interrupts arteriovenous shunting, and vagotomy, so effective in the clinical management of ulcer, not only decreases mucosal blood content but also aggravates vasopressin ulceration, the only type of experimental ulcer which is clearly due to mucosal ischemia. The shunts, as pointed out above, are under the control of both nervous and humoral influences. Neurologic vasomotor control of the gastroduodenum, mediated through the vagus nerves, is generally believed to arise in the hypothalamus, with higher connections to scattered areas throughout the anterior half of the cortex. The responsible humoral mechanism appears to be very complicated, even in its generalities, but seems to involve both the pituitaryadrenal axis and the intrinsic upper gastrointestinal hormones. There can be little disagreement that in the ulcer patient there is overt evidence of excessive physiologic activity along both these me348

diating pathways, apart from the vascular repercussions. Lack of appreciation of the extent of this activity can be blamed on the almost exclusive interest of past decades in its small secretory facet. It is necessary to assume, of course, that the physiologic excess which leads to mucosal hypoxia affects different people in different ways—that there is at least a fair amount of individual variation in susceptibility to crater formation under similar conditions of hypoxia. In addition to the simple fact that, throughout all of medicine, individual variation in susceptibility to noxious circumstances is a prominent feature, there is the interesting clinical observation that a number of patients with the other clinical evidences of ulcer disease do not develop a crater. The concerted happenings in the shunt system of the gastroduodenum, as a whole, are obviously insufficient to account for the generation of a focal lesion. Alternating generalized mucosal ischemia and plethora are, as pointed out above, a normal reaction to the stresses of normal living, and if a focal lesion is to develop, a focal area of excess hypoxia or unusual susceptibility to normally occurring physiologic hypoxia must be postulated. In order to determine how excess focal hypoxia might come about, it is necessary that means be found for studying incoordinate activities among the shunts to discover, perhaps, why one or a couple of the shunts should respond with inordinate sensitivity to the influences which cause them to close. The matter of secondary obliterative disease of the arterial vessels which lie in the immediate vicinity of long-established ulcers deserves comment as a probable factor contributing to the chronicity of the lesion in some cases. This is not to be confused with etiology, for anatomic study of chronic ulcers reveals conditions which are much more likely to be effect

SHERMAN & PALMER

than cause. This is known because in the case of new ulcers, injection studies regularly show a striking increase in submucosal vascularity about and extending a little beyond the ulcer base, while in the case of chronic ulcers, there is an ischemic zone about the sides and base of the lesion. The width of the zone correlates well with the amount of fibrosis beneath the crater. It is not so much that the regional arteries have become plugged, as it is that the normal tissues have been replaced by relatively hypovascular connective tissue. The arterio-arterial anastomotic net throughout the gastroduodenal well is so tremendous that there would have to be almost total obliteration of the arterial supply before ischemia could result, and, as already discussed in some detail (z), generalized gangrene rather than circumscribed ulcer would necessarily follow. In thinking about the cycle of ulcer healing and recurrence, particularly the phenomenon which arranges for an ulcer to recur in the same spot occupied by the original lesion, it is important to note that the vascularity of the mucosa around an ulcer behaves very differently from the vascularity of the underlying periulcerous tissue. Thus, although a chronic ulcer is set in tissue which is notably hypovascular, the mucosa at the crater's edge is hypervascular. The criterion of ulcer healing is epithelization—a purely surface healing. It is reasonable to assume, after noting how quickly both chronic benign and cancerous ulcers sometimes become epithelized, that the mucosa can extend to cover large areas without much vascular help from the underlying diseased tissue. It is not known whether new mucosa over an old scar has its normal complement of shunts or not. At any rate, if new mucosa can form over a relatively bloodless ulcer scar, the presence of the local deep ischemia cannot very well be blamed for later break-down of the mu-

coca to form a new ulcer. Perhaps ischemia of the periulcerous zone explains why some chronic ulcers do not heal well, but there is no explanation here in the deep tissues for recurrence of the crater. In the search for answers to the challenging problem of "peptic" ulcer, there has been a great tendency over the past century to emphasize biochemical research and to assume that normal human anatomy is a closed science. For several decades a tremendous amount of research has been carried out on the secretory activity of the stomach, because of the supposition that "peptic" ulcer must be due to acid-pepsin activity. Previous attemps to ascribe a vascular basis for mucosal weakness and ulcer genesis have failed because of lack of knowledge of the detailed anatomy of the microcirculation of the gastrointestinal tract. With the development of information on the extensive system of arteriovenous anastomoses lying above and below the muscularis mucosae, it has been possible to take a fresh look at the problem of the acid-pepsin factor and of the mucosal factors in the development of gastroduodenal ulcers. It seems evident that the acid-pepsin factor is a secondary or passive factor, not strictly concerned with the initiation of the ulcer process. The excavation of an ulcer must be assisted by necrosis and autodigestion, but first there must be focal depression of mucosal vitality. It appears that the native arteriovenous shunt mechanism is capable of producing focal changes in the mucosa by the production of hypoxia. This shunting system is under neural and humoral control, and is very sensitive to general stimuli, such as emotions, "stress", temperature and visceral changes in other portions of the body. It is likely that hypoxia, arising from plethoric capillaries and slow flow, due to 349

IV / HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

abnormally closed local arteriovenous ulcer disease, it is agreed that it meets shunts, can serve as the final common many of the basic criteria for a proper pathway in the ulcer process, by produc- etiologic explanation for some of these phenomena. Further investigation of the ing local devitalization of the mucosa. Although this hypoxia theory cannot anatomy, physiology and pathology of explain all of the observed phenomena the arteriovenous shunt system may clarassociated with the development, chron- ify some of the more obscure problems icity and recurrence of gastroduodenal remaining.

Summary Hypoxia, due to abnormal function of the arteriovenous shunt system, is proposed as the final common pathway in gastroduodenal ulcer genesis. This hypoxia arises from plethora and slow capillary blood flow in focal regions leading to local depression of mucosal resistance. Acid-pepsin digestion of this area of devitalization leads to ulceration. The anatomy and physiology of the native arteriovenous anastomoses of the gastrointestinal tract are reviewed briefly. Each shunt which lies in the vicinity of the muscularis m ucosae operates to circumvent the large mucosal capillary bed which its mucosal artery normally supplies. In addition, there are many large shunts in the gastric subserosa. Two anatomic types of shunt exist: the direct and the indirect. The direct shunt (less common of the two) consists of a relatively short arteriovenous communication equipped with a pseudo-sphincteric closing mechanism of muscle cells. The indirect shunt has a distinct channel between the arterial and venous sides; it opens into either a mucosal or a submucosal vein. The effectiveness of the gastric shunt system in diverting blood from the mucosal capillaries is known from observations on mucosal color changes. Studies by thermal gradientometry confirm the observation that a sudden increase in gastric and duodenal mucosal capillary flow may re350

sult from emotional tension, anxiety and resentment. The mucosa of the continuously secreting stomach might be maintained in a continuously plethoric state. The shunts are not able to maintain intermediate positions, but are either fully open or fully closed. Although the majority of gastroduodenal shunts act in concert, some paradoxical activity exists among neighboring shunts. Intramuscular administration of histamine causes a prolonged increase in capillary filling and capillary flow after a transient decrease. Vasopressin causes nnucosal ischemia. Epinephine and atropine cause depression of nmcosal flow ltaneous increase in the utilizawith simultaneous tion of the shunts. Addition of acetylcholine to plasma used for perfusion of freshly resected stomachs causes marked reduction in the perfusion rate and increase in the total blood flow, due to closing of the arteriovenous shunts. The system of arteriovenous anastomoses can produce local hypoxia in one of two ways: a) if a shunt is wide open, b) by an abnormally long closure. Thus both ischemia and plethora may produce local hypoxia, which can lead to focal mucosal devitalization. Several facts support the postulate of a plethoric hypoxia as the explanation for mucosal damage, rather than ischemic hypoxia. The shunts are under the control of

SHERMAN & PALMER

nervous and hormonal influences. In the excessive physiologic activity in both sysulcer patient, there is overt evidence of tuns.

References T., and vARRo, v. Gastroenterology 32: 119-125, 1957. PALMER, E. D., and BUCHANAN, D. P. Ann. Int. Med. 38: 1187-1205, 1953. vntcHow, R. Virchow Arch. path. Anat. 5: 281-376, 1853. ROKITANSKY, c. Handbuch der Pathologischen Anatomie. Vol. 1: Wien., Braumüller, 155. BENEKE, R. Münch. Med. Wschr. 78: 1831, 1931. JATROU, S. T. Deutsche Ztschr. f. Chir. 159:

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8. VON BERGMANN, G. Berl. klin. Wschr. so: 2374-2379, 1913. 9. BOLES, R. S., RIGGS, H. E. and GRIFFITHS, J. 0.

Amer. J. Digest. Dis. 6: 632-636, 1939. Arch. Int. Med. 11: 469-484, 1913. Handbuch der speziellen pathologischen Anatomie and Histologic. Henke F. and Lubarsch, 0., ed. Vol. 4, Pt. 1, 339-811, Berlin, Springer, 1924-52. IVY, A. C., GROSSMAN, M. 1., and BACHRACH, W. H. Peptic Ulcer, 1088, Philadelphia, Blakiston, 1950. HERZOG, W. Beitt. klin. Chir. 184: 74-84, 1952. SHERMAN, J. L., JR. U.S. Armed I orces M. J. 7: Ioo1-I008, 1956. PALMER E. D., and SHERMAN, J. L., JR. A.M.A. Arch. Int. Med. 1o1: 1106-1117, 1958. SHERMAN, J. L., JR., Amer. J. Gastroenterol. 34: 537-540, 1960. SHERMAN, J. L., JR.2 Normal Arteriovenous Anastomoses, Medicine 42: 247-267, 1963. BARLOW, T. E., BENTLEY, F. H., and WALDER, D. N. Surg. Gynec. Obst. 93: 657-671, 1951. DE BUSSCHER, G., Acta Neerl. morphol. norm. et path. 6: 87-105, 1948. DISSE, H. Arch. mikr. Anat. 63: 512-531, 1904. DJORUP, F. Ztschr. ges. Anat. 64: 279-347, 1922. REEVES, T. B. Surg. Gynec. Obst. 30: 374-385, 1920. HOFMANN, L., and NATHER, K. Arch. klin. Chir. 115: 65o, 1921. CONTI, G., and PASSARELLI, L. Arch. chit. torace 8: 269-286, 1951. MORRIS, W. R. New Zealand M. J. S4: 690, 1955• SPANNER, R. Fortschr. diag. Therap. 1: 1, 1950.

10. oPHÜLS, W. 11. HAUSER, G.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

and PARKS, A. G. Brit. J. Surg. 47: 546-550, 1960. 28. SCHUMACHER, S. Ztschr. mikr. anar. Forsch. 43: 107-130, 1938. 29. STAUBESAND, J. zur Morphologie der Arterio venösen Anastomosen. Capillaren und Interstitium. Stuttgart, Thieme, 1955. 3o. POMPEIANO, o. Arch. kal. anat. e istol. pat. 23: 225-234,1950. 31. DANIEL, P. M. and PRICHARD, M. M. L. Quart. J. Exp. Physiol. 41: 107-123, 1956. 32. HERZOG, W. Beitr. Klin. Chir. :88: 236-246, 1954. 33. STAUBESAND, J. Verh. anat. Ges. f2: 240-2 49, 1 954. 34. STAUBESAND, J. Beirr. klin. Chir. 19o: 367374, 1955. 35. WOLF, S. and WOLFF, H. G. Human Gastric Function: An experimental Study of a Man and his Stomach, 251, New York, Oxford, 1 947. 36. RICHARDS, C. H., WOLF, S., and WOLFF, H. G. J. Clin. Invest 21: 551-558, 1 942. 37. BENJAMIN, H. B., WAGNER, M. and ZEIT, W. Surg. Gynec. Obst. 97: 1 9-24, 1 953. 38. BENJAMIN, H. B., WAGNER, M., ZEIT, W. and AUSMAN, R. Rev. Canad. Biol. ! f: 95-103, 27. BOULTER, P. S.

1956.

39. BARCLAY, A. E. and BENTLEY, F. H. Gastroenterology, 12: 117-183, 1949; Brit. J. Radiol, 22: 62-67, 1949. 40. BENTLEY, H. F. Gastroenterologia (Basel), 75: 31, 1949. 41. WALDER, D. N. Clin. Sci. 11: 59-71, 1952. 42. WAI.DER, D. N. Gastroenterologia (Basel) 81: 66-71, 1 954. 43. CURRI, S. B., and TISCHENDORF, F. Riv. anat. pat. e one. lo: 741-760, 1 955. 44. SHERMAN, J. L. JR., and NEWMAN, S. Amer. J. Physiol. 179: 279-281, 1 954. 45. WALDER, D. N. Lancet /: 162, 19_50. 46. BASU MALLIK, K. C. J. Path. Bact. 7o: 315, 324, 1955. 47. CLARK, E. R. Physiol. Rev. 18: 22 9-247, 1938. 48. BENTLEY, F. H., and BARI.O\V, T. E. Modern trends in Gastro-enterology. Jones, F. A. ed., 309-322, New York, Hoeber, 1952. 49. BARLOW, J. E. Symposium on Visceral Circulation-London, 1951. A Ciba Foundation Symposium, Wolstenholme, G. E. W. ed., Boston, Little Brown, 1953. 50. BOYCE, 11. w., JR. Personal communication. 51. BENJAMIN, H. B. Surg. Gynec. Obst. 93: 672-675, 1951. 351

IV / HYPDXIA OF VASCULAR ORIGIN IN ULCER DISEASE

52. GRAYSON, J. Brit. M. J. 2: 1465-1470, 195o. MILLER, F. B., and HASZCZYC, V. A. A. M. A.

68. KOSTINA, L.

Arch. Surg. 73: 465-468, 1956. Amer. J. Dig. Dis. 16: 2i7-242, 1949• 55. NEDLEL, A. J. Arch. Path. 26: 988-1008, 1938. 56. CRANE, W. A. _J. J. Path. Bact. 67: 379392, 1954. 57. BERG, M. Amer. J. Dig. Dis. 7: 78-81, 1940. 58. BERG, Al. Arch. Path. 33: 636, 6 45, 1942. 59. MASUDA, H., OHARA, M., and KATSURA, S. Tohoku, J. Exper. Med. S7: 119-135, 137-143, 1953. 6o. MASUDA, H. Tohoku J. Exper. Med. 58: 48, 1953. 61. MASUDA H., OHARA, M. and KATSURA, S. TOhoku J. Exper. Med. 58: 49-55, 1953. 62. MASUDA, H., OHARA, Al. and KATSURA, S. Tohoku J. Exper. Med. 58: 57-61, 1953. 63. DE BUSSCHER, G. Acta gastro-enterol. Belg. 9: 545, 1946. 64. BENJAMIN, H. B., WAGNER, M., and ZEIT, W. Amer., J. Gastroenterol. 22: 387-i98, 1954. 65. ALLEN, R. A., and DAHLIN, D. C. Proc. Staff Meet. Mayo Clinic. 29: 429-436, 1954. 66. DEMIN, V. N. Vopr. onkol. 1: 11 3, 1955 (original not seen)

69. MANNIX, A. J., JR., SCHRAFT, W. C. REED, W. P. and ADLE, G. c. Surgery 37: 473-477, 1955. 70. SPANGLER, H. Chirurg. 24: 181-184, 1 953. 71. BENJAMIN, H. B., WAGNER, M., ZEIT, W., PISclorrA, A. V. and AusMAN, R. K. Surg. Gynec.

S3.

54. NECHELLES, H.

67.

KAY, S., CALLAHAN, W. P., JR., MURRAY, M. R., RANDALL, H. T. and STOUT, A. P. Cancer 4:

7z6-736, 1951.

352

1. Tr. Akad. med. nauk USSR, 2,: 16, 1952. (original not seen).

Obst. loo: 566-57o, 1955.

72. KIMBEL, K. H., KINZLMEIER, H. and HENNING, N. Gastroenterologia (Basel) 82: 317-33o,

1 954.

73. MENEGAUX, G., GOUYGOU, C., COURTOIS-SUFFIT, Al. and DETRIE, P. Presse. med. 61: 1328-1 330,

1953•

The Anatomy and Physiology of Capillaries New Haven, Yale University Press, 1936. 75. RICHARDS, A. N. Methods and Results of Direct Investigation of the Function of the Kidney. Baltimore, Williams and Wilkins, 1949. 76. HOGENA, H. G. Arch. mal. app. digest. 42: 122I-I222, 1953. 77. MOYSON F., and DE SCOVILLE, A. Act. chir., belg., (S~ upp• I) 3-144, 1955. 78. AMARANTE, JUNIOR. Arch. mal. app. digest. 44: 1258-1264, 195 Eg M.A. 39: 57-73, 79. KHALIL, H. A. J. yptian 1956. 74. KROGH, A.

The Gastric Antrum and the Regulation of Acid Secretion

INTEREST in the gastric antrum has increased recently. Lack of interest in this portion of the stomach can probably be traced to the operating suite. The standard operations for peptic ulcer disease during the past twenty years have involved excision of the gastric antrum, thus "out of sight, out of mind." In the 1950's, the operative treatment of duodenal ulcer changed markedly. Operations were devised which left the antrum in

Lloyd M. Nyhus Niles D. Chapman*

situ (these procedures must not be confused with the antral exclusion techniques). Therefore, it became more important to develop a complete understanding of all the physiological ramifications of the organ. Because of the clinical overtones, a sense of urgency has been apparent, and rapid progress has been made. It is our purpose to review a few of these concepts which have been or are being developed.

Establishment of the Edkins Theory The existence of a hormone produced by the pyloric gland area of the stomach was first postulated by Edkins (r) in 1906. He was impressed by the distinctive histological structure of the antrum, especially its complete lack of parietal and chief cells. He reported studies wherein intravenous administration of extracts of antral mucosa stimulated gastric secretion; extracts similarly prepared from other parts of the stomach were without effect. Further studies were carried out on animals in which the antrum was separated from the body of the stomach by a rubber diaphragm (2). The introduction of

food substances into the antrum was followed by secretion of acid gastric juice in the body of the stomach. These studies were interpreted by Edkins to indicate the presence of an antral hormone which he named gastrin. Subsequent workers were unable to confirm Edkins' observations and the status of his gastrin hypothesis remained controversial. Finally, Grossman et al. (3), in 1948, demonstrated an antral hormonal mechanism by showing in dogs that mechanical distention of the pyloric portion of the stomach would initiate and maintain the secretion of acid even when

•From the Department of Surgery, University of Washington School of Medicine, Seattle, U.S.A.

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FIG. t. Resection of the antrum reduces the pouch acid secretion to extremely low levels, demonstrating the Immoral nature of the gastric hormone which mediates the gastric phase of digestion.

FIG. 2. Transplantation of the antrum to the colon as a diverticulum produces an augmented release of gastrin. Relief of the mechanical stimulus of distention is followed by a decrease in pouch secretion; return to hypersecretory levels occurs when the distention stimulus is reapplied. (86).

all neural connections between the antrum and parietal cells were interrupted. In the same year, Woodward et al. (4) independently demonstrated the existence of gastrin by the use of dogs with Pavlov pouches. Utilizing quantitative collections of gastric secretions, they were able to show a marked reduction in the output

of acid juice from the Pavlov pouch after antrectomy. Fig. I, taken from a later paper by this group, demonstrates this phenomenon (5). The antrum of the stomach was then moved to different anatomic sites in a series of experiments which further established the hormonal theory of Edkins (Fig.:).

•

6 5

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•

Gastric Production and Release The gastric hormone is the mediator of the gastric phase of digestion. Gastrin is produced in response to specific antral stimuli, i.e., chemical, mechanical, and vagal. The total amount of gastrin produced and released (as measured by the secretory response of the parietal cells) is the net result of antral stimulatory and inhibitory influences. The only known inhibitory mechanism involved in the release of gastrin is regulated by the pH of the antral mucosa. An acid pH prevents the release of gastrin in response to all types of stimuli (6). For man, the critical pH at which gastrin release is prevented is I.5 (7,8). The mechanism by which acid prevents the release of gastrin is not known. Since local neural reflexes appear to be involved 354

in gastrin release, it has been assumed that the antral acid-inhibitory mechanism operates in a like manner (q). Thus the application of cocaine or other local anesthetics (including those not possessing the vasoconstrictive action of cocaine) to the antral mucosa will prevent the release of gastrin (i o). Acid inhibition of gastrin release is a normal physiologic "feedback" mechanism that functions to prevent excess acid secretion (i I) . When quantities of gastrin sufficient to produce a secretory response are released, the response persists only as long as a stimulus is acting to cause release of the hormone. The latent period between the application of the stimulus and the hormonal response is usually a few minutes. Thus, gastrin has a short latency and little

MECHANICAL STIMULATION OF THE GASTRIN MECHANISM

mEa. free HCI X 104 from Heidenhain pouch

persistence of action. In addition to its ability to stimulate directly the secretion of acid, gastrin is capable of influencing the sensitivity of the parietal cell mass to other secretory stimuli. Mechanical and Chemical Release of Gastrin Local stimuli for the production and release of gastrin operate through two mechanisms: a) mechanical distention, and b) secretagogues, or chemical excitants (Fig. 3). Dragstedt and his co-workers (i z) have demonstrated the importance of locally stimulated gastrin release in the daily secretion of acid from a Heidenhain pouch. After antrum resection or exclusion of the antrum from continuity in the digestive tract, there was a 65 to 95 per cent reduction in the twentyfour hour secretion of hydrochloric acid, with return to normal values when the antrum was subsequently implanted in the colon as a diverticulum. Mechanical stimulation of the gastrin mechanism is produced by the bolus of food as it passes through the pyloric gland area. Woodward et al. (13) have found that perfusion of the isolated antrum with an alkaline fluid (isotonic 1.3 per cent sodium bicarbonate) will enhance the gastrin response to a mechanical stimulus. In man, mechanical distention of the antrum is also an adequate stimulus for gastrin release. In subjects who had no basal secretion of acid, distention of the antrum resulted in an increase in the volume and acidity of the gastric juice. Conversely, if the subjects had a high basal secretion of acid, no increase in volume or acidity occurred during distention. This latter finding was interpreted as evidence of acid inhibition of gastrin release (14). Secretagogues in contact with the mucosa of the pyloric gland area also release gastrin. Proteins, their degradation pro-

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ducts and ethyl alcohol are examples of secretagogues that readily produce gastrin release when applied to the pyloric gland area. It is probable that all foodstuffs possess this activity in some measure. The Vagal Release of Gastrin The vagal release of gastrin has been recently reviewed by Woodward and Nyhus (i5). It was Schur and Plaschkes (16) who suggested originally the possibility of a connection between the gastric and cephalic phases of acid secretion. Straaten (17) furthered this postulate in 1 933 with the observation that resection of the pyloric region in dogs led to the decrease of gastric secretion produced by sham feeding. This led him to conclude the necessity of the presence of a part of the antrum for vagal stimulation of acid secretion. On the other hand, Worobjew and Volborth (18) noted the return of cephalic phase of secretion several days after the antral resection. These findings originated a controversy which led to a continuous dispute on the subject. It was stated by Uvnäs that "the cephalic phase of gastric secretion is controlled by a combined neurohumoral mechanism identical in principle with that of the gastric phase. The pyloric region plays a major part in this mechanism" (19). According to Uvnäs, during vagal stimulation, a "secretagogue agent" is discharged from the pyloric region and 355

IV / THE GASTRIC ANTRUM AND THE REGULATION OF ACID SECRETION

exerts an excitatory action, potentiating the direct effect of the vagus upon the parietal cells. Uvnäs's work has been proved to be a significant contribution, although it was criticized in the immediate period following its publication (2o, 21). Lim and Mozer (22) showed that following sham feeding of esophagotomized dogs prepared with gastric fistula, the gastric secretory response was of such a magnitude that vagal phase could not be regarded as the sole cause of it. These authors have suggested "peripheral liberation of ... gastrin" as the explanation of the increase in gastric secretory volume. Lim and Mozer could not demonstrate an augmentation of acid secretion in dogs prepared with denervated fundic pouches, after isolation of an innervated antral pouch and sham feeding. A small amount of secretion was obtained from Heidenhain pouch dogs following sham feeding and insulin hypoglycemia; this, according to Burstall and Schofield (23,24), was produced by gastrin release. In Pavlov pouch dogs after exclusion of the innervated antrum from the acid stream, Uvnäs et al. (25) and Andersson et al. (z6) found an increase in secretion collected over a twenty-four hour period. Because of the maintenance of the continuity between the excluded antrum and the duodenum, local mechanical and chemical stimuli have to be considered in the interpretation of these results. The development of a vagally-innervated, isolated antral preparation (9,13, 27,28) was one of the factors which contributed significantly to the removal of the difficulties surrounding the work on the antrum. A double mucosal bridge is used to divide the innervated antrum from the main portion of the stomach; the pyloric end becomes a fistula brought out to the skin. This eliminates the prob356

lem of local mechanical or chemical effects upon the antrum; the vagal stimuli alone are probably responsible for the variation in gastrin release observed in Heidenhain pouch preparations. Nyhus et al. (6) verified Forrest's (27) earlier suggestion that isolation of the antrum could lead to a considerable increase in base line secretion obtained from Heidenhain pouch dogs. This observation seems to indicate that in the absence of acid inhibition, vagal stimuli bring on a continuous release of gastrin. By employing a Heidenhain indicator pouch and an innervated antral pouch, Oberhelman et al. (28) and Woodward et al.( 3 ) confirmed the röle of insulin-induced hypoglycemia in the liberation of gastrin. According to Thein and Schofield (29) acid secretion from denervated pouches was found to be increased during sham feedings when the innervated antrum was isolated (29). It can be concluded from the above experimental data that vagal stimulation releases the hormone gastrin, and that the two major phases of gastric acid stimulation are interrelated. Antroneurolysis (22,3o) represents the second important experimental method in studies of the vagal release of gastrin; the interruption of intramural nerve connections from the vagus is obtained by separation of the antral mucosa from the submucosa. Thein and Schofield (29) demonstrated that antral motility and acid secretion show a lack of correlation in transplanted fundic pouch preparations. Further studies by Chapman et al. (3i) with antroneurolysis disproved the theory that increased antral motility represents the prime mechanism for gastrin release by vagal stimulation. (Fig. 4). The elucidation of the relationship between vagally-induced motility and vagally-induced gastrin release requires further study. Their' and Schofield (28) observed a decrease in antral motility in

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,»✓✓ fI/i/V)

Free acid in MEQ,

sham-fed dogs. Yet antral hypermotility was noted after a short inhibitory phase (31). In observations on 15o human subjects gastrointestinal motility was found to be uniformly decreased (32). It appears probable that two types of cholinergic effectors exist mediating separately gastrin motility and gastrin release. It seems likely that vagal fibers leading directly to the antral mucosa or the submucous nerve plexus mediate the release of gastrin; however the exact mechanism for gastrin release remains unknown. It has been proved that the release of gastrin by vagal stimulation and that produced by local antral factors are completely separate (6). The question still remains whether vagal gastrin release plays an important role in the stimulatory mechanisms of gastric acid. DeVito et al. (3 3) have shown that a 20 to 8o per cent decrease occurs after antroneurolysis in secretions collected from Heindenhain pouch dogs over a twenty-four period. Neither antroneurolysis nor extrinsic antral vagal denervation seems to modify to a significant extent the gastrin release by chemical or mechanical means (6,34); thus depression in acid secretion after antroneurolysis to the main stomach must be considered to be due to abolition of the vagally induced gastrin production. If this is correct, the total daily gastric acid output produced by vagally induced gastrin release is much greater than has been realized. The increase in acid secretion following isolation of an innervated antrum in a Heidenhain pouch preparation indicates that the antrum is capable of releasing gastrin in the absence of local antral stimuli; this hypersecretion is considered to be due to a continued release of gastrin by vagal stimulation in the absence of acid inhibition. Traditionally, the stimuli for the secretion of acid have been divided into ceph-

Free acid In MEQ

Peristaltic Activity of Normal Antrum during Control Period

YRIIU

0.7 0.5 0.3 0.1 0

Peristaltic Activity of

Peristaltic Activity of

Antroneurolysed Antrum during Control Period

Antroneurolysed Antrum during Insulin Induced Vogol Stimulation

Control Period

10 units Insulin I.V.

2

3 4 Time in hrs.

FIG. 4. Vega! denervation of the antral mucosa blocks the vagal release of gastrin in response to insulin hypoglycemia. The hypermotility of the antrum persists. This demonstrates the presence of two types of efferent cbolinergic nerve fibers to the antrum, one mediating motility and the other mediating gastrin release.

alic, gastric and intestinal phases. Chemical and mechanical stimuli were thought to be the sole mediators of the gastric phase. The experimental data outlined above seem to supply ample evidence that, in addition, a significant vagal release of gastrin exists. It is suggested therefore that the terms cephalic phase, gastric phase and intestinal phase of gastric acid secretion, or vagal, antral intestinal mechanisms, should be dismissed in favor of more specific terms. We have recently suggested a new terminology for the stimulatory phases of gastric acid secretion (6): a) direct vagal, b) vagal-antral, c) local antral and d) intestinal. A portion of the stimulatory effect of the vagi is active only if the antrum is present; similarly, a portion of the stimulatory effect of the antrum is active only if the vagi 357

IV / THE GASTRIC ANTRUM AND THE REGULATION OF ACID SECRETION

are present. The vagal-antral phase indicates the effect of vagal stimulation upon the antrum. The local antral phase encompasses the local effect upon the antrum of chemical and mechanical factors. According to this concept, the meaning of the intestinal phase remains unchanged. The question of an antral inhibitory hormone Some investigators have claimed that when acid comes in contact with the pyloric gland area, an inhibitory hormone is produced, the action of which is to inhibit the secretion of acid (35,36,37,38,

39)• Others have been unable to demonstrate such a hormone. Indeed Dragstedt and his co-workers (39) and Shapira et al. (4o) have repeated the original experiments upon which the concept of an inhibitory hormone was based, and both groups have concluded that the experimental findings can be satisfactorily explained without involving the activity of an inhibitory hormone. The existence of such a hormone remains controversial; preliminary reports of the recent crosscirculation experiments of DuVal and Price (41) and Thompson and Lerner (42) indicate that the question is far from settled.

Gastrin and Histamine The discovery that histamine stimu- sponsible for stimulation of separated gaslates gastric secretion and the further elu- tric pouches during the digestion of a cidation of this action have fostered the meal (47) At the present time no definite conclusuggestion either that gastrin is histamine or that histamine is the final local chemo- sions can be presented concerning the stimulator for any mode of stimulation question of whether endogenous hista(43)• Support for the hypothesis that his- mine has any physiologic röle in acid tamine is a gastric secretory hormone has secretion. Much circumstantial evidence come from studies in man (44) and vari- suggests such a possibility. Histamine is ous animal species (45,46), showing that always present in the gastric mucosa. after feeding of a meat meal, there was Histamine appears in gastric juice regardincreased excretion of endogenously pro- less of the mode of stimulation. Histaduced free histamine along with an in- minase, present throughout the remainder crease in hydrogen chloride secretion. of the gut, is absent from the stomach However, the temporal relationship of (41). Exogenously administered histamithese events was subsequently discovered nase will inhibit the secretion response to be entirely fortuitous. When the meat to a meal (48). Histaminase inhibitors will meal was placed directly into the jejunum, augment the secretory response to any the urinary excretion of free histamine stimulus (49). Atropine will inhibit hispromptly increased but the secretion of tamine-stimulated gastric secretion in all acid was minimal or absent (46). In addi- species (5o). tion, the acid hypersecretion in HeidenUrinary free histamine appears to rehain pouch dogs produced by transplan- sult from the intestinal absorption of histation of the gastric antrum to the colon tamine produced by bacterial decarboxywas not accompanied by increased hista- lation of 1-histidine from the meat meal mine excretion. The evidence suggests in the lumen of the intestine (51). Normthat free histamine in the systemic blood ally, the histamine so produced is inactiis not the humoral agent (gastrin) re- vated by the liver, and cannot stimulate 358

NYHUS & CHAPMAN

gastric secretion. However, after portalsystemic shunting or when an antihistaminase drug is given, this endogenously produced histamine stimulates acid secretion (51,52). That the full story is yet to unfold is illustrated by the finding that meals of fat and carbohydrate, which presumably contain no 1-histidine, also produce increased acid secretion after portacaval transposition (53). The histamine-destroying role of the liver may be necessary to protect the gastric mucosa from continuous stimulation by histamine produced in the bowel. Antihistaminase drugs may augment acid

secretion, not by potentiation of normal stimuli, but by adding an abnormal stimulatory pathway from the intestine. This thesis is supported by the study of Ostrow and his colleagues (54.), who found increased secretion of acid, both basally and after histamine stimulation, in cirrhotic patients with portacaval shunting as compared with cirrhotic patients without portacaval shunting. Loss of this hepatic histamine-inactivating mechanism may be a factor in the higher incidence of peptic ulcer reported in cirrhotic patients after construction of a portacaval shunt (55).

Gastrin and the Reactivity of the Parietal Cell Mass TABLE I Secretory response of the parietal cell mass is dependent upon its size and state of reactivity Factors producing states of high reactivity

Factors producing states of low reactivity

Vagal innervation Parasympathomimetic drugs Gastrin Caffeine Histiminase inhibitors ? Histamine ? Adrenal steroids

Sympathetic innervation Sympathomimetic drugs Nausea, fever etc. Histaminase Enterogastrone Duodenal inhibitor mechanisms

CNS Affective state + or -

1 Secretogenic stimulus

0

= Secretory response

Parietal cell mass Low Degree of Reactivity Small Secretogenic stimulus o = Secretory Response Parietal cell mass High Degree of Reactivity Secretogenic stimulus

0

= Large Secretory Response

Parietal cell mass

359

IV / THE GASTRIC ANTRUM AND THE REGULATION OF ACID SECRETION

There appear to be wide variations in the secretory responses of the parietal cell mass. These changes in secretory response are caused by changes in its reactivity. The reactivity of a tissue is defined as the degree of response of that tissue to a given amount of stimulation. Thus, during states of low parietal cell reactivity, a given secretogenic stimulus produces only a small amount of gastric secretion; conversely, during states of high parietal cell reactivity, the same stimulus produces a greater quantity of secretion (56) (Table I). Factors Producing High Reactivity a) VAGAL INNERVATION AND PARASYNIPATHOMEbIETIC DRUGS. Following vagotomy, the gastric secretory response to all stimuli—for example, histamine, gastrin, and cholinergic drugs—is reduced (57,58). The cause of the decreased reactivity after vagotomy in the response to drugs, which presumably act as direct stimulants to the parietal cell, has not been established. Antia et al. (J9) have suggested that the basal rate of acetylcholine synthesis in the gastric mucosa determines the sensitivity (or reactivity) of the parietal cell to direct stimulants. Accordingly, the decreased responsiveness after vagotomy may be due to the decrease in production of acetylcholine, with loss of its sensitizing or potentiating action. The observation that decreased rather than increased sensitivity follows vagal denervation constitutes an apparent violation of Cannon's "law of denervation" (6o). Acetylcholine and the choline esters, mecholyl and urecholine, stimulate acid secretion. The drug dosage used to demonstrate this effect is important; at low doses they stimulate, but at high doses, they inhibit acid secretion (61). The parasympathomimetic drugs also potentiate the secretory response to all types of stimuli (62). 36o

b) GASTRIN. It has become apparent that gastrin not only stimulates the parietal cell directly to secrete acid but, in addition, increases the reactivity of the parietal cell to other stimuli. During the course of clinical investigations designed to provide a safer operation for duodenal ulcer, vagotomy was added to the von Eiselsberg antrum exclusion operation (63). Clinical experience has subsequently shown that an antral exclusion operation in any form is an unacceptable operation for duodenal ulcer because of a high recurrent ulcer rate. However, the Waddell operation provided a very important physiologic observation in humans: namely, an antrum excluded from gastrointestinal continuity will still produce gastrin. Further studies of acid secretion in humans show that the secretory response of the parietal cell mass to all stimuli is reduced following resection of an excluded antrum (63,64,65,66) (Table II). Noring (67) noted, also in humans, a marked decrease in the sham feeding response following distal gastrectomy (antrectomy). Investigations of the effects of antrum exclusion and subsequent resection in experimental animals have not produced consistent results; some authors report no change (5) or a substantial increase (68) in acid secretion following antral resection. It is significant that in none of the animal experiments were the anatomic relationships of the clinical studies reproduced; hence it is possible that the apparent difference in response between humans and canines can be accounted for without invoking a species difference. Support for this thesis is suggested by the experiments of Langlois and Grossman (69). These investigators found that following resection of a separated antral pouch there was a decreased secretory response to urecholine. Waddell and Williams (7o) have produced evidence in dogs which indicates that the potentiating effect of gas-

NYHUS & CHAPMAN

trin operates by influencing the blood flow through the gastric mucosa.

sympathetic nerves inhibit acid secretion. Completely denervated canine parietal cell pouches were extremely sensitive to histamine, gastrin and parasympathomimetic drugs. In contrast, vagally denervated but sympathetically innervated Heidenhain pouches (parietal cell pouches) were relatively insensitive to the same concentration of these agents that stimulate acid secretion. Attempts to influence gastric secretion by extirpation of the gastric sympathetic nerve supply have not produced conclusive data. Postganglionic sympathectomy rendered dogs more sensitive to histamineinduced ulcers in a study by Lillehei and Wangensteen (73). Others have found that preganglionic sympathectomy influenced neither basal acid secretion nor the secretory response to stimuli in both man and experimental animals (64,74). The sympathomimetic drugs, epinephrine and

C) HISTAMINE AND HISTAMINASE INHIBI-

Prevention of histamine destruction by inactivation of endogenous histaminase will increase the secretory response to all stimuli (49) d) CAFFEINE AND XANTHINE DRUGS. Caffeine and other xanthine drugs are potent stimuli for acid secretion. They also potentiate the secretory response to histamine and alcohol test meals (71). Factors producing states of low reactivity a) SYMPATHETIC INNERVATION AND SYMPATHOMIMETIC DRUGS. While it is well established that the parasympathetic nerves to the stomach stimulate both motility and acid secretion, there is no such unanimity of opinion concerning the function of the gastric sympathetic nerve supply. Gregory and Tracy (72) have produced indirect evidence that the TORS.

TABLE II Secretory response of parietal cell mass before and after resection of an excluded antrum Basal Secretion (mEq./hr.) Author

BEFORE AFTER

Smithwick and Kneisel6a (Avg. of 4 pts.) Waddell" Pt. No. 1 2 3

Broth (mEq./hr.)

(mEq./hr.)

BEFORE

AFTER

BEFORE

AFTER

Insulin (mEq./hr.) BEFORE

AFTER

4.7

0.4

6.1

0.2

20.4

4.9

10.0

2.0

0.3 0 2.5

0.4 0 0.3

1.7 0 6.4

0 0 0.2

7.3 0.4 10.9

0.5 0 3.1

5.0 1.3 3.9

2.6 0 3.1

Basal Secretion (mEq./30 min.) BEFORE

Clark, et al.6' Pt. No. 1 2 3 4 5 Gillespie, et a166 Pt. No. 1 2 3

Histamine

4 4 3 13 2 5.4 0.5 0.4

Maximal Histamine Response (mEq./30 min.)

AFTER

BEFORE

AFTER

0 0 0 8 0.05

2.9 20 3.3

0.06 2.5 1.1

0.4 0 0

2.9 5.4 11.7

0.1 2.0 2.5 361

IV / THE GASTRIC ANTRUM AND THE REGULATION OF ACID SECRETION

l-norepinephrine, definitely inhibit gastric secretion (75). b) PARASYMPATHOLYTIC DRUGS. Atropine and its related belladonna alkaloids inhibit effector organs innervated by postganglionic cholinergic nerves. They are therefore potent suppressors of acid secretion (76), and when present reduce the secretory response to all stimuli. Ganglionic blocking agents also reduce the reactivity of the parietal cell mass to all stimuli (77). C) NAUSEA AND FEVER. Nausea and retching inhibit gastric secretion. The mechanism and the pathways through which these factors operate are not completely known. It is probable that neural and humoral pathways are both involved(78). Elevations of body temperature in man will reduce the secretory response to alcohol test meals (79). d) HISTAMINASE. The intravenous injection (into dogs) of a purified histaminase preparation (diamine oxidase) will decrease the secretory response to histamine, urecholine, mecholyl and feeding. The precise role of histamine in gastric secretion remains to be clarified, and it is not known whether the secretory inhibition produced by these compounds is a result of the diamine oxidase activity or of some other component. However, in view of the other evidence relating to the röle of histamine in gastric secretion, it is possible that histaminase activity may normally operate to influence the level of parietal cell reactivity. e) ENTEROGASTRONE AND DUODENAL INHIBITOR MECHANISMS. It is well established

that these factors can inhibit acid secretion. Therefore, when acting in conjunction with a secretogogue stimulus, the total secretory response will be less than if the secretogogue stimulus were acting alone. Autonomic nervous system and parietal cell reactivity There is general agreement that the emotional state of the individual can produce changes in gastric secretion by direct stimulation mediated over the vagus nerves. However, the available information on the influence of emotions on direct stimuli other than neural is incomplete. Engel and his colleagues (8o) were able to study a fifteen-month-old infant with esophageal atresia and a gastric fistula. It was found that acid secretion and parietal cell reactivity were directly related to the total behavioral activity of the infant. During depressed affective states or sleep, there was a marked decrease in spontaneous acid secretion, and parenteral histamine at this time had little effect on acid secretion. Contrariwise, outgoing affective states were associated with high rates of basal acid secretion and a marked secretory response to histamine. On the basis of these observations, changes in parietal cell reactivity are intimately related to the physiology of the autonomic nervous system. Since the autonomic nervous system is represented at all levels of the central nervous system, it is able to integrate and co-ordinate sensory and environmental events with bodily needs.

Nongastric Sources of Gastrinlike Activity Zollinger and Ellison (81) focused attention on a syndrome consisting of nonspecific islet cell tumors of the pancreas, gastric hypersecretion and intractable 362

peptic ulceration. The islet cell tumors were often multiple and frequently malignant and metastasizing. The association of gross gastric hypersecretion and fulmi-

NYHUS & CHAPMAN

nating peptic ulceration with the islet cell tumor obviously suggested a causal relationship between the tumor and hypersecretion. It appeared that the hypersecretion was of a humoral origin. Insulin and glucogon were considered but soon excluded, as the cause of the hypersecretion.

Recently Gregory et al. (8z) and others (83,84,85) have found an extremely potent stimulant of acid secretion, presumably gastrin, in the islet cell tumors of patients with the Zollinger-Ellison syndrome. The exact nature of this humoral agent has not been identified.

The Role of the Antrum in the Production of Peptic Ulcer Gastric Ulcer a) THE DRAGSTEDT THEORY: Drawing from a large experimental and clinical experience, Dragstedt (86) postulated that gastric ulcers develop primarily as a result of an antral stasis with concomitant hypersecretion of gastric juice of humoral origin. This antral stasis may be secondary to pyloric obstruction following duodenal ulcer disease. In those patients with gastric ulcer independent of pyloric obstruction, Dragstedt suggested that the antral stasis is dependent upon hypofunction of the motor fibers in the vagi. The difference in acid output between patients with gastric and duodenal ulcer (twelve hour night secretion-42 to normal of 58.5 mEq vs. an average of 63 mEq respectively) was the key to comprehension of this concept. If indeed gastric hypersecretion of acid occurs only with retention of food in the gastric antrum, why should we expect increased acid to be present during periods of testing when the stomach is empty? The mechanics of quantitatively collecting gastric content for acid analysis during the digestive phase of secretion in patients with gastric ulcer are essentially insurmountable. We do not have a specific answer to this question—is the environment of the stomach filled with food and other buffering substances, truly acid to

an ulcerogenic degree? Thus, we have a void in our knowledge to detract from full acceptance of the Dragstedt theory as the sole mechanism involved. Dragstedt (87) has summarized corroborative evidence which is of interest: The concept that gastric ulcers are due to a hypersecretion of gastric juice of hormonal origin dependent upon prolonged or excessive liberation of the gastric secretory hormone, gastrin, is supported by the following evidence. A hypersecretion of gastric juice, due to excessive or prolonged liberation of gastrin, can be induced in dogs by the transplantation of the antrum of the stomach into the colon as a diverticulum. This hypersecrction is sufficient in degree to produce typical ulcers in previously normal mucosa. Stasis of food in the stomach, either as a result of pyloric stenosis or gastric atony, can cause a hypersecretion of gastric juice of humoral origin sufficient in degree to produce gastric ulcers in experimental animals and in man. The response of gastric and duodenal ulcers to gastric surgery supports these concepts of their origin. Thus, complete gastric vagotomy has been found effective in the treatment of duodenal ulcers, but not in gastric ulcers. Antrum resection, which abolishes the humoral phase of gastric secretion, is followed by a high incidence of marginal ulceration when done for patients with duodenal ulcer, but is rarely followed by this complication when done for patients with gastric ulcer. Gastroenterostomy which relieves the stasis of food in the stomach is seldom followed by marginal ulceration when done for gastric ulcer, but is frequently followed by marginal ulceration when done for the treatment of duodenal ulcer.

363

IV / THE GASTRIC ANTRUM AND THE REGULATION OF ACID SECRETION

b) JOHNSON THEORY. Johnson of London (88) has divided gastric ulcer disease into three categories: a) prepyloric gastric ulcer; essentially the same disease as duodenal ulcer; b) gastric ulcer associated with pyloric stenosis of duodenal ulcer origin, and c) gastric ulcer associated with deficiency in mucous secretion. According to Johnson, the main defense mechanism in the stomach against autodigestion by the acid-pepsin environment is the mucous layer. Three reasons for breakdown in the mucous barrier are given: a) a prolonged acid attack tends to overwhelm the mucous-secreting cell, thereby exhausting its supply of mucus. This phenomenon occurs most frequently in the presence of gastric retention due to pyloric obstruction; (it is conceivable that mucus exhaustion can be added to the Dragstedt theory of gastric retention due to gastric hypomotility of vagal origin); b) decrease in mucus due to local mucosal anoxia following opening of submucosal arteriovenous shunts in response to adenalin, and c) a long sagging Jshaped stomach allows pooling of buffering gastric content away from the ulcer area in the more proximal stomach, necessitating secretion of greater quantities of mucus to prevent autodigestion. These two theories of gastric ulcer pathogenesis present arguments against a common etiology for gastric and duodenal ulcer. It is of interest that there is a definite overlap in the concept, i.e., gastric retention. Due to the factor of food and mucus-buffering acid secretion in the hypomotile or mechanically obstructed stomach, the acid output due to stimulation of the gastrin mechanism would of necessity have to be phenomenal to break down the normal mucosal defense mechanism. If, however, we accept the Dragstedt theory as a basic mechanism only, and add to it as ancillary postulates, deficient mucous secretion, mucosal anoxia 364

and the J-shaped stomach as suggested by Johnson, a truly understandable mechanism evolves for the production of gastric ulcer. Antral Exclusion The antral exclusion operations of von Eiselsberg (89), Finisterer and Cunha (go), Devine (ui) and Ogilvie (92) were devised to decrease the morbidity and mortality from operative dissection in the region of the pancreas and common duct for posterior penetrating duodenal ulcers. Later reports (93,94) indicated that a prohibitive number of jejunal stomal ulcers developed subsequent to these procedures. Ogilvie (92) found that partial resection of the parietal cell mass added to exclusion of the antrum did not protect against jejunal ulceration. Further attempts were made to leave the antrum in situ, but with recognition of the stimulatory effect of this procedure, operations were devised to core out the antral mucosa, leaving the serosa and musculature of the antrum intact. Unfortunately, this latter operation (95), although satisfactory in preventing stomal ulceration, was attended by another type of complication of the residual antral stump, i.e., necrosis and perforation (96). In our opinion exclusion of the antrum, regardless of modification in technique, is an operative procedure of no clinical value. The physiologic mechanism to explain these poor results may well be found in the work of Woodward and associates (13). These investigators demonstrated that gastrin production by the antrum following mechanical stimulation was markedly potentiated in the presence of an alkaline pH. Antral distention with reflux of alkaline duodenal juice plus alkaline antral secretions can readily occur with the antral exclusion procedure. Kay (97) found that in his

NYHUS & CHAPMAN

patients the antral pH rarely fell below seven after antral exclusion with the "acid cuff." Thus, we have a clear cut physiologic explanation for the exceedingly poor

clinical result of the von Eiselsberg antral exclusion operation, with or without modification.

Summary The story of the gastric antrum has been one of long rejection, followed by an era of acceptance. From the time of Edkins until today, interest in this segment of the stomach has waxed and waned. Through the work of Uvnäs, Grossman, Woodward and many others, the hormonal significance of this "organ" has been delineated. In the past few years, it has become well established that the hormone gastrin is released from the antrum under variable conditions; mechanical distention, secretogogues and alkaline environment all contribute to gastrin release. The exact mechanism of gastrin release is unknown. The vagal release of gastrin is now a fact. There is some question as to the full significance of this; however, it is our belief that it is an important facet in the spectrtan of acid production. A phenomenon of reactivity of the parietal cell mass has been proposed. Factors which seem to cause states of high parietal cell reactivity included: a) Vagal innervation, b) Gastrin, c) Histamine, and d) Caffeine. The concept of reactivity has many ramifications, and it will require intense study. In the clinical patient, the antrum has been impugned as the prime etiological factor in gastric ulcer. Further, all antral exclusion operative procedures have given dismal results in the treatment of duodenal ulcer. These poor results are a

direct effect of the physiologic stimulation of gastrin release as previously listed, i.e., mechanical distension, secretogogues, and the potentiation of an antral alkaline environment. The role of the gastric antrum in the over-all spectrum of gastric secretion is significant. Our knowledge concerning the ramifications of antral function has increased considerably in recent years. The frontier, however, is far from reached. As indicated by the preceding presentation, there are still more questions than answers, e.g., what is the composition of gastrin; and, is there an antral inhibitory hormone. By continued diligence in the experimental laboratory, the answers to these and many other questions will be found. ACKNOWLEDGEMENTS

Tables I and II, and Figure 3 reproduced from Harkins, H. N. and Nyhus, L. M. Surgery of the Stomach and Duodenum Boston, Little, Brown, 1962. Figure 1 reproduced from Woodward, E. R., Bigelow, R. R. and Dragstedt, L. R. Amer. J. Physiol. 162: 99-109, 1950. (5 ) Figure 2 reproduced from Dragstedt, L. R., Woodward, E. R., Oberhelman, H. A. Jr., Storer, E. H. and Smith, C. A. Amer. J. Physiol. 165: 386-398, 1951. (86) Figure 4 reproduced from Chapman, N. D., Nyhus, L. M. and Harkins, H. N. Surgery 47: 722-724, 1960. (31)

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IV / THE GASTRIC ANTRUM AND THE REGULATION OF ACID SECRETION

References I. EDIUNS, J. S. J. Physiol., (Lond), 34: 133-144, 1906. 2. EDKINS, J. S. and TWEEDY, M. J. Physiol., (Lond), 38: 263-267, 1909. 3• GROSSMAN, M. 1., ROBERTSON, C. R., and IVY, A. C. Amer. J. Physiol., 1S3: 1-9, 1948. 4. WOODWARD, E. R., BIGELOW, R. R. and DRAGSTERT, L. R. Proc. Soc. Exp. Biol. ,\led. 68: 473-474, 1948. 5. WOODWARD, E. R. BIGELOW, R. R. and DRAGSTEDT, L. R. Amer. J. Physiol. 162: 99-109, 1950. 6. NYHUS, L. M., CHAPMAN, N. D., DE VITO, R. V. and HARKINS, H. N. Gastroenterology, 39: 582-589, 1960. 7. SCHMIDr, F. R. and HALLENBECK, G. A. A.M.A. Arch. Surg. 77: 26-32, 1 958. 8. GILLESPIE, 1. E. Gastroenterology, 37: 164-168, 1959• 9. LIM, R. K. S. and MOZER, P. Fed. Proc. to: 84, 195 1. 1o. WOODWARD, E. R. The Physiology and Treatment of Peptic Ulcer, Allen, J. ed. 49, Chicago, University ot Chicago Press, 1959. 11. WILHELMJ, C. M., FINEGAN, R. W., and HILL, F. C. Amer. J. Dig. Dis., 4: 457-550, 1937-38. 12. DRAGSTEDT, L. R., WOODWARD, E. R., STORER, E. H. OBERHELMAN, H. A., JR., and SMITH, C. A. Ann. Surg. 132: 626-640, 1950. 13. WOODWARD, E. R., ROBERTSON, C., FRIED, W., and SCHAPIRO, H. Gastroenterology, 32: 868877, 1957• 14. GROSSMAN, M. 1., and WOODWARD, E. R. Gastric Distention as Stimulus of Acid Secretion in Human Subjects 372, International Physiological Congress, loth, Brussels, Proceedings 1956. 15. WOODWARD, E. R. and NYHUS, L. M. Amer. J. Med. 29: 732-737, 1960. ,6. SCHUR, H. and PLASCHKES, S. Mitt. Grcnzgeb. Med. Chir. 28: 795, 1915. Quoted by Noring (66). 17. STRAATEN, T. Arch. klin. Chir., 176: 236-251, 1933. 18. WOROBJEW, A. M. and VOLBORTH, G. W. J. Physiol. USSR, 17: 1281, 1934. Quoted by Noring (66) . 19. UVNAS, B. Acta physiol. scand., 4: supp. 13, 1942. 20. BABKIN, B. P. and SCHACTER, M. McGill Med. J., 13: 117-138, 1944. 21. JANOWITZ, H. D. and HOLLANDER, F. Proc. Soc. Exp. Biol. Med., 76: 49-52, 1951. 22. LIM, R. K. S. and MOZER, P. Amer. J. Physiol., 163: 730, 1950. 23. BURSTALL, P. A. and SCHOFIELD, B. J. Physiol. (Lond)., 120: 383-408, 1 953. 24. SURSTALL, P. A. and SCHOFIELD, B. J. Physiol., (Lond)., 123: 168-186, 1 954. 25. UVNÄS, B., ANDERSSON, S., ELWIN, C. E. and MALM, A. Gastroenterology, 30: 790-803, 1956. 26. ANDERSSON, S., ELWIN, C. E. and UVNAS, B. Gastroenterology, 34: 636-658, 1958.

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27. FORREST, A. P. M. The importance of the Innervation of the Pyloric Antrum in the Control of Gastric Secretion in Dogs, 299300. International Physiological Congress, zoth, Brussels, Proceedings 1956. 28. OBERHELMAN, H. A., JR., RIGLER, S. P. and DRAGSTEDT, L. R. Amer. J. Physiol., 190: 391395, 1957. 29. THEIN, AI. P. and SCHOFIELD, B. J. Physiol., (Lond)., 145: 14P-,5P, 1958. 30. JONES, T. W., DE VITO, R. V., NYHUS, L. Al., and HARKINS, H. N. Surg. Gynec. Obstet., 105: 687-692, 1 957. 31. CHAPMAN, N. D., NYHUS, L. Al., and HARKINS, H. N. Surgery, 47: 722-724, 1960. PIROF. Hand \VOODWARD, E. R. Amer. J. 32. SCHA, Dig. 4: 787-791, 1959• 33. DE VITO, R. V., JONES, T. W., MARTINIS, A. J., NYHUS, L. Al., and HARKINS, H. N. Surg. Forum, 9: 423-417, 1958. 34. WOHLRABE, D. E. and KELLY, W. D. Surg. Forum, 9: 430-433, 1958. 35. HARRISON, R. C., LAKEY, W. Hy and HYDE, H. A. Ann. Surg., 144: 441-449, 1956. 36. JORDAN, P. H., JR., and SAND, B. F. Surgery, 42: 40-49, 1957• 37. LONGHI, E. H., GREENLEE, H. B., BRAVO, J. L. GUERRERO, J. D. and DRAGSTEDT, L. R. Amer. J. Physiol., 191: 64-7o, 1957. 38. WOODWARD, E. R., TRUMBULL, W. E., SCHAPIRO, H., and TOWNE, L. Amer. J. Dig. Dis., 3: 204213, 1958. 39. DRAGSTEDT, L. R., KOHATU, S., GWALTNEY, J. NAGANO, K., and GREENLEE, H. B. A.M.A. Arch. Surg. 79: 10-21, 1 959. 40. SHAPIRA, U., MORGENSTERN, L. and STATE, D. Surg. Forum, to: 143-146, 1960. 41. DUVAL, M. K., JR., and PRICE, W. E. Ann. Surg., 1 53: 410-41 5, 1960. 42. THOMPSON, J. C., and LERNER, fl. J. J. Surg. Res., 1: 117-120, 1961. 43. CODE, C. F. Symposium on London, 1955 Histamine, Ciba Foundation Symposium. Wolstenholm G. E. and O'Connor, C.M., ed., 189. Boston, Little, Brown, 1956. 44. MITCHELL, R. G., and CODE, C. F. J. Appl. Physiol., 6: 387-392, 1953-54. 45. SCHAYER, R. W. and IVY, A. C. Amer. J. Physiol., 189: 369-372, 1957. 46. UPDIKE, E. H., lI, CODE, C. F. and HALLENBECK, G. A. Amer. J. Physiol., 195: 197-201, 1958. 47. IRVINE, W. r., and CODE, C. F. Amer. J. Physiol., 193: 202-208, 1958. 48. GROSSMAN, Al. I., and ROBERTSON, C. R. Amer. J. Physiol., 153: 447-413, 1948. 49. SIRCUS, W. Quart. J. Exp. Physiol., 38: 2533, 1953. 50. JANOWITZ, H. D., and HOLLANDER, F. Amer. J. Physiol., ,86: 373-376, 1956. 51. IRVINE, W. T., DUTHIE, H. L., RITCHIE, H. D., and WATON, N. G. Lancet, 1: 1064-1068, 1959. 52. S1RCUS, W. Quart. J. Exp. Physiol., 38: 97 100, 1953.

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and EISEMAN, B. Surgery, 46: 38-47, 1959. 54. OSrROW, J. D., TIMMERMAN, R. J., and GRAY, S. J. Gastroenterology, 38: 303-313, 1960. S5. CLARKE, J. S., OZERAN, R. S., HART, J. C., CRUZE, K. and CREVLING, V. Ann. Surg., :48: 551-566, 53. SILEN,

and CODE, C. F. Amer. J. Physiol., 177: 425-429, 1954. 75. PETERS, R. M., and WOMACK, N. A. Ann. Surg. 74. FORREST, A. P. M .,

148: 537-550, 1958.

76. GRAY, S. J. Amer. J. Physiol., 120: 637-662, 1 937• 1958. 77. KAY, A. NV., and SMITH, A. N. Gastroenter56. CHAPMAN, N. D. and NYHUS, L. M. Surgery ology, 18: 503-5 17, 1951. of the Stomach and Duodenum, Harkins, H. 78. GROSSMAN, M. I., WOOLLEY, J. R., DUTTON, D. F., and IVY, A. C. Gastroenterology, 4: 347N. and Nyhus, L. M., ed. Boston, Little,

Brown, 1962. 351, 1945. 57. OBERHELMAN, H. A. JR., and DRAGSTERT, L. R. 79. BANDES, J., HOLLANDER, F., and BIERMAN, W. Gastroenterology, 1o: 697-707, 1948. Proc. Soc. Exp. Biol. Med., 67: 336-339, I948. 58. HOOD, R. T. JR., and CODE, C. F. Surg. Forum. 80. ENGEL, G. L., REICHSMAN, F., and SEGAL, H. I.. Psychosom. Med., 18: 374-398, 1956. 1: 73-78, 1951. S9. ANTIA, F., ROSIERE, C. E., ROBERTSON, C., and 81. ZOLLINGER, R. M., and ELLISON, E. H. Ann. Surg., 142: 709-728, 1955. GROSSMAN, M. I. Amer. J. Physiol., 166: 47082. GREGORY, R. A., TRACY, H. J. FRENCH, J. M. 479, 1951. and SIRCUS, W. Lancet, 1: 1045-1048, 1960. 6o. CANNON, W. B. and ROSENBLUETH, A. The Supersensitivity of Denervated Structures. 83. GROSSMAN, M. L., TRACY, H. J. and GREGORY, R. A. Gastroenterology, 4:: 87-91, 1961. 17, New York, Macmillan, 1949. 84. OSBORNE, M. P., BROWN, M. E. and LE COMPTE, 61. GRAY, S. J. and IVY, A. c. Amer. J. Physiol., P. M. Amer. J. Surg., boo: 48-53, 1960. 120: 705-711, 1937. 6z. ROBERTSON, C. R. and GROSSMAN, M. I. Fed. 85. SUMMERSKILL, NV. H. J., CODE, C. F., HALLENBECK, G. A. and PRIESTLY, J. T. Proc. Mayo Proc. 7: 103, 1950. Clin. 36: 611-617, 1961. 63. WADDELL, W. R. Ann. Surg. /43: 520-S3o, 86. DRAGSTERT, L. R., WOODWARD, E. R., 0liF;R3IEl; 1956. MAN, H. A. JR., STORER, E. H., and SMITH, C. A. 64. SMITH\v1CK, Il. H. and KNEISEL, J. J. Rev. Amer. J. Physiol., :65: 386-i98, 1951. Gastroenaerol., 17: 439-457, 1950. 87. DRAGSTEDT, L. R. Gastroenterology, 3o: 20865. CLARK, D. H., KAY, A. W., DUTHIE, H. L. and 220, 1956. GILLESPIE Gastroenterologia (Basel) 89: 28688. JOHNSON, H. D. Gastroenterology, 33: 121290, 1958. 12 3, 1957. 66. GILLESPIE, I. E., CLARK, D. H., KAY, A. W., and 89. VON EISELSBERG, A. Arch. klin. Chir., 50: 919TANKEL, H. 1. Gastroenterology, 38: 361-367, 1960. 939, 1895. 90. FINSTERER, H., and CUNHA, F. Surg. Gynec. 67. NORING, 0. Gastroenterology, 19: 118-125, Obstet., 52: 1099-1114, 1931. 1951. 68. RANGINS, H., RIGLER, S. P., EVANS, S. 0., MC- 91. DEVINE, H. B. Surg. Gynec. Obstet., 40: 1-16, 1925. CARTHY, J. D. and DRAGSTEDT, L. R. A.M.A. 92. OGILVIE, W. H. Lancet, 235: 295-299, 1938. Arch. Surg., 75: 2 30-235, 1957. 69. LANGLOIS, K. J., and GROSSMAN, M. I. Amer. 92. VON EISELSBERG, A. Arch. Kiln. Chir., :14: J. Physiol., 163: 38-40, 1950. 539-544+1920. 70. WADDELL, W. R. and WILLIAMS, H. W., JR. 94. FROMME, A. Arch. Klin. Chir., 196: 281-303, Ann. Surg., 150: 529-537, 1959. 1939• 71. ROTH, J. A., and IVY, A. C. Amer. J. Physiol., 95. BRANCROFT, F. W. Amer. J. Surg. 16: 223-230, 1932. 142: 107-113, 1944. 96. QvIST, G. Brit. J. Surg., 45: 341-344, 1 957-58. 72. GREGORY, R. A. and TRACY, H. J. Amer. J. 97. KAY, A. W. Gastroenterologia, 89: 282-286, Dig. Dis., 5. 308-323, 196o. 1958. 73. LILLEHEI, C. w., and WANGENSTEEN, 0. H. PrOC. Soc. Exp. Biol. Med., 68: 369-372, 1948.

367

The Cytology of Mucosal Regeneration in Experimental Gastric Ulcer

THE purpose of this paper is to review

current knowledge of the cytological aspects of the repair of experimental gastric ulcers, and to incorporate in it some of the results of autoradiographical and histochemical studies that are in progress in the authors' laboratories. The literature that exists on the subject of wound healing and tissue repair is vast, and it is soon apparent to anyone who begins to survey it that the majority of studies have been carried out on skin. This of course is not surprising in view of its ready accessibility for experiment and observation. As far as healing in the alimentary tract is concerned, the stomach has been the organ of most frequent choice by the experimenter, due to the increasingly prevalent problem of human peptic ulcer in this century. Nevertheless many studies have concentrated more upon mechanisms of ulcer production than on those of healing. In general it can be said that, in the sixty years since 1900, physiological and pharmacological knowledge of the stomach has far outstripped advances in cytological knowledge of this viscus, and since all organ activities depend in the ultimate analysis

R. M. H. McMinn F. R. Johnson*

on the behavior of the cells that are grouped together to form that organ, the need for gathering information at the cellular level is paramount. The experimental study of mucosal lesions of the stomach was initiated nearly a century ago by Pavy (1). The literature up to 1950 on the healing of such lesions has been extensively surveyed and documented by Ivy et al. (z), and the interested reader is referred to their encyclopedic work for abundant references. It is clear from work on several mammalian species that small mucosal defects, produced for example by excision or burning, will be repaired with the formation of new gastric glands. From the point of view of comparative pathology, it is perhaps unfortunate that man is almost the sole species to develop gastric ulceration. It is only in seals that any significant number of "natural" lesions in animals has been found, and some of these can be explained by the irritation produced by the ingestion of sharp stones combined with nematode infection (3). While various species have been used for experimental studies, the dog has been one of the most commonly used, and it

From the Departments of Anatomy, King's College, London, and London Hospital Medical College, University of London, London, England. 369

IV / CYTOLOGY OF MUCOSAL REGENERATION

has been generally agreed that there is no essential difference between the healing of acute ulcers in the dog and in man(4). The chronic ulcer in man may also undergo healing in a similar manner; the questions are why it does not always do so, and how it can be made to do so? It will

be convenient to describe our current experiments on the cytology of the healing of mucosal lesions, and then to discuss them in the light of other work on the stomach and in the context of tissue repair in general.

Current Experiments Mucosal defects were produced in the 8o per cent alcohol. After embedding in stomachs of fifty rats and twenty cats, and paraffin wax, serial sections were cut at examined cytochemically and autoradio- 71t, and every twentieth section mounted graphically at varying periods thereafter. and stained with hematoxylin and eosin, The lesion in the cat was made by or with van Gieson stain. These sections opening the stomach on its ventral surface were examined, and from regions of and removing with scissors an area of mu- particular interest serial sections were cosa approximately I.o cm.' in size from mounted for further staining as follows: a) with iron hematoxylin, if showing the greater curvature of the body. In the rat, the stomach was opened on its ventral migrating epithelium, in order to search surface, and an area approximately 0.5 for mitotic figures or, in the case of colcm.' in size in the glandular part of the chicine-treated animals, for arrested metabody on the greater curvature was de- phases. nuded of mucosa with a Volkmann's b) by the Gomori and/or azo dye scoop. The animals were fed on milk on methods for alkaline phosphatase. c) by the periodic acid-Schiff (PAS) the first postoperative day and were thereafter allowed to have their normal diet. method, with and without diastase digesThe animals were killed at periods tion, for mucopolysaccharides (mucin) varying from twenty-four hours to three and glycogen. Gomori's hexamine silver months after operation. Some of the rats method for glycogen was also used. received colchicine by intraperitoneal ind) by the Nile blue sulfate method of jection in doses of 0.1 mg./loo g. body Lillie (5) for zymogen cells. weight six hours before death. These aniThe remaining specimens were frozen mals were injected to 10 a.m., having had for sectioning in the cryostat (freezing access to water only during the previous microtome) prior to carrying out the night, and were not fed for the ensuing technique for the demonstration of sucsix hours. Colchicine in the doses given cinic dehydrogenase (6), a respiratory arrests mitosis in the metaphase stage of enzyme located in mitochondria which the cycle. are particularly large and numerous in parietal cells. Histology and Cytochemistry Immediately after killing the animals, the site of the lesion, together with some normal surrounding tissue, was removed and in most cases fixed at once in ice-cold 370

Autoradiography The radioactive isotope of sulfur, S", was used for visualizing the distribution of the sulfated forms of mucin. Rats re-

MCMINN & JOHNSON

ceived doses of Na_S'O4 at the rate of I µc./g. body weight by intraperitoneal injection six hours before death; two adult cats were given doses of I o mc. The premitotic synthesis of nuclear DNA was visualized in rats by the administration of tritiated thymidine (thymidine-H3) in doses of I sic./g. body weight, four hours before death. An in vitro technique was also used for both rat and cat material; small fragments of mucosa from the margins of lesions of freshly killed animals were incubated for four hours in roller tubes containing 2 ml. Glaxo culture medium No. 199 to which 2 sic. of isotope had been added. The autoradiographs were prepared by the technique of Heatley et al. (7), using Kodak AR Io stripping film, counterstaining if required being carried out with PAS and/or hematoxylin.

The Mechanisms of Repair In the above experiments, the long-term results (after three months) indicated that the site of the mucosal lesion had become repaired with the formation of new glands that closely resembled those of the normal gastric mucous membrane, although they were somewhat less deep (Fig. 1). The glands contained mucus, parietal and zymogen cells, and were embedded in a connective tissue stroma. The muscularis mucosae was the only element of the mucosa that showed no evidence of regeneration. These findings are in accord with those of many previous workers, and the mechanism by which the mucosal repair is achieved will now be considered under the general headings of epithelial, connective tissue and muscular reactions.

Epithelial Reactions During the first few days of the healing of a full-thickness mucosal defect, epithelial cells migrate from glands at the wound margin over a floor of accumulating granulation tissue. Some of these marginal glands become considerably dilated, giving rise to cystlike structures similar to those noted in experimental and pathological lesions at other levels of the alimentary tract. It has been suggested (8) that in the stomach they are due to obstruction produced by a reparative process, and similar cystic dilatations of glands have been found at the sites of gastroenterostomy stomata (q), at wound margins in the ileum (to) and in colitis cystica (i i) . During the second week and later, the cells dip down into the underlying connective tissue that is now maturing, and in this way new glands are formed. It must not be imagined, however, that the various cellular elements in

the normal gland, such as parietal and zymogen cells, become mobilized so that they can be found at some stage lying on the surface of a mound of granulation tissue. In fact, such cells are never found in such a situation, for the components of the glands that are immediately adjacent to the margin of the lesion appear to undergo a process of dedifferentiation. At the same time, mitotic activity in these marginal glands increases. These various processes—migration, mitosis and dedifferentiation—are fundamental to wound healing in all types of epithelia, and require detailed consideration, our own results being particularly pertinent to the last two categories. Migration The precise mechanism that induces migration of epithelial cells when a gap 371

IV / CYTOLOGY OF MUCOSAL REGENERATION

is produced among them is not known either for gastrointestinal or any other variety of epithelium. Presumably the mere lessening of lateral pressure that ensues, following a solution of continuity, acts as some sort of stimulus (12), but a number of other factors present themselves for consideration. If cells are to move, they must have freedom to do so, and in the case of the single-layered epithelia of the alimentary tract, this implies release from their attachment to the underlying basement membrane and rearrangement of the boundary membranes that adjacent cells present to one another. Most epithelia display considerable interdigitation of cell membranes, and gastric epithelium is no exception. Changes might also be expected in the desmosomes and terminal bars. It is to be hoped that in the future electron microscopy will shed some light on the way in which the various modes of cell attachment become modified during repair. Migrating epithelia in general have been said to produce pseudopodia that assist in locomotion (13), but it is not known whether this applies to gastric epithelium. In skin wounds, it has been suggested, migrating cells can exert a fibrinolytic influence on clots that might otherwise impede their progress (14, 15 ). While fibrinolysis and its possible mechanisms have been reviewed recently by Sherry et al. (16), its råle in cell migration remains speculative. In his well-known review of wound healing, Arey (17) concluded that as far as epidermis was concerned, the chief factor responsible for the extension of epithelium over a denuded area was the ameboid movement of the cells, an explanation that was first advocated by Klebs (18). The mechanics of cell arnehism have been reviewed by Allen (iv), and in the absence of specific studies on gastric cells it can only be assumed that the various factors mentioned may be ap372

plicable to them. After making artificial ulcers in the stomachs of rabbits, rats and guinea pigs, Williams (zo) noted that migration of epithelial cells had begun within twentyfour hours, and most other epithelia also appear to be capable of beginning their migration within this period (21). Our own findings in the stomach of the rat and cat are in agreement. Williams also found that, contrary to current beliefs, the migrating epithelium did not necessarily require a mound of granulation tissue over which to move; some cells grew over necrotic material, but their subsequent fate was not noted. The cells of migrating epithelium are relatively flat, compared with the normal columnar shape of surface cells, and contain only very little PAS-positive material. In the cat, whose surface cells contain considerable quantities of histochemically detectable glycogen, scanty glycogen granules can sometimes be detected; there is certainly no dramatic increase in glycogen content as appears for example in regenerating skin (2 2) or esophageal epithelium (23). The transitional epithelium of the urinary bladder has unexpectedly shown no increase during regeneration (24), but the whole question of the histochemical detection of glycogen has been brought more sharply into focus by the papers of Kugler and Wilkinson (25,26, 27). These workers have established that of the two fractions in which glycogen exists in tissue cells—acid-soluble and protein-bound—only the acid-soluble fraction is detectable by present histochemical methods. Considerable quantities of glycogen can be present in cells in the protein-bound fraction, and yet give no histochemical reaction. Kugler and Wilkinson concluded that of the several methods available, staining by Gomori's hexamine silver method, following fixation in icecold 8o per cent alcohol, gave the best

FIG. 1. Cat. Three months. Site of wound margin, showing the glands of the undisturbed nattosa (with underlying musctdaris mucosae seen here as a light band), and on the right, new glands H. & E. x 32. FIG. 2. Cat. Five days. Epithelium, growing towards the left over accumulating granulation tissue, showing a nucleus in mitosis. Van Gieson. x 750.

Plakt=

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i

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FIG. 3. Rat. Five days. Migrating epithelial cells (detached from underlying granulation tissue) showing a typical arrested metaphase. H & E x750. FIG. ¢. Rat. Five days. Autoradiograpb after injection of thymlidine-H', showing a labeled nucleus in migrating epithelium. Counterstained PAS d hetnatoxylin. x you.

1

FIG. 5. Cat. Five days. Autoradiograph from part of a wound margin that had been cultured in a medium containing thymidine-H`, showing two heavily labeled nuclei in a sheet of migrating epithelium. Part of a labeled connective tissue cell nucleus is seen under the epithelium on the left. Counterstained PAS & henlatoxylin. x7S0.

IV / CYTOLOGY OF MUCOSAL REGENERATION

results, and it may be that some reappraisal of existing observations on cellular glycogen is needed in view of their interesting findings. The suggestion has been made by Levander (28) that connective tissue cells are capable of undergoing metaplasia into gastric epithelium and thus assisting the epithelial coverage of the lesion that occurs by migration. His conclusions were based on studies of embryonic subcutaneous implants in rabbits, but other workers have not reported such metaplasia in adult stomachs. Mitosis It is well established that the epithelium of a full-thickness mucosal defect is made good not merely by migration of cells but also by an increase in mitotic activity. In the normal stomach, mitotic activity is almost entirely confined to the mucinproducing cells of the isthmus and neck regions of the glands (29,30,31), but in the dedifferentiated cells of glands adjacent to wound margins, nuclei in mitosis can be found at all levels of the glands, even at their bases. Apart from noting its occurrence, most workers have not made any precise analysis of the extent of this increase, but Hunt (32) has provided some details from lesions in the rat stomach. He found a) that on the first day of regeneration mitosis was increased in a 2 mm. zone adjacent to the wound, b) that by the second day the increase was confined to a zone too p. wide adjacent to the wound, and c) that the mitosis occurred primarily in deep foveolar cells and in undifferentiated basophilic cells, while after the third day, when an increased number of mucous neck cells was present, there was also an increase of mitosis in them. An analysis comparable with the above is not available for any other species. 374

In our own experiments in the rat and cat, the study of many serial sections from specimens of lesions between two and seven days old revealed that apart from abundant mitoses in the marginal glands, some nuclei in mitosis could be found among the cells that were in process of migrating over the accumulating granulation tissue (Fig. 2). Nuclei in all phases of the mitotic cycle have been observed in such migrating cells, and although mitoses in these situations were not as numerous as in the marginal glands, their presence is both emphasized and confirmed in the colchicine-treated animals in whom typical arrested metaphases could be found (Fig. 3). Further proof of the capability of migrating cells to undergo division was obtained from the autoradiographs with thymidine-H3. In the rats that had received the thymidine by injection, labeled nuclei were found not only in the neck region of undisturbed glands remote from the wound and scattered throughout the length of the dedifferentiated glands adjacent to the wound, but also in epithelial cells overlying the accumulating and organizing granulation tissue (Fig. 4). Similar labeling occurred in the cat material that had been cultured (Fig. 5), and although no grain counts were carried out, it was clear that in these in vitro preparations the labeling was much heavier over the nuclei of migrating cells than over those in the glands, whether normal or at the wound margin. This peculiarity is being investigated. It is thus clear that mitosis is contributing to the coverage of the denuded area, not only by helping to make good the loss of cells from glands by migration, but also by adding cells to the migrating sheet itself. It is the common belief, derived in the past from the study of skin wounds, that mitosis is not seen in epithelial cells when they are migrating (17), but in recent years we have accumulated considerable

MCMINN & JOHNSON

evidence of the occurrence of such a the tenth day, there was abundant mitophenomenon in several epithelia. Those of tic activity that resulted in the heaping up the urinary bladder (3 3) and gall-bladder of epithelial cells at the margin, but there (34) show the greatest number of mitotic was no migration over the wound surface. nuclei in migrating cells (35), but occa- It was only in wounds made after the sionally mitoses have also been noted dur- tenth day that migration occurred, and it ing the repair of rectal (36) and small began at this time regardless of how much intestinal lesions (21). In terms of area time had elapsed since the injury. Weiss covered as a result of mitosis in migrating and Matoltsy comment that "this rules sheets of cells, the contribution is pro- out any of the immediate traumatic efbably very small, but at the same time fects of wounding as being causally reshould not be underestimated. In normal lated to migration", and they also found skin, some workers in the past have ques- that when fragments of skin and cornea tioned whether the number of mitoses with wounds were cultivated on a plasma found in epidermis was sufficient to ac- clot in vitro, there was no migratory decount for the periodical renewal of the lay at any age. It is thus possible that a whole thickness of the stratified epi- humoral mechanism is at work, and a sigthelium, but subsequent calculations have nificant contribution to wound healing shown that they are in fact adequate (37). will have been made when the causal facThis emphasizes the fact that the finding tors in migration and mitosis have been of even one or two mitotic figures in a found and can be separately controlled. line of epithelium indicates a degree of It is well established that gastric erocell division greater than may be imagined sions can be repaired very rapidly. Grant at first sight; they represent merely those (39) found that in cats the introduction nuclei that were undergoing division of 5o or 6o per cent alcohol or i per cent either at a single moment of time, i.e., at acetic acid into the body of the stomach the death of the animal, or in the case of caused a variable amount of desquamaanimals that have received colchicine or tion of surface cells, and that replacement thymidine-H° over a period of only a few occurred within only a few hours, withhours. In the course of a few days, there- out any apparent increase in mitotic acfore, considerable increases in the cell tivity. The repair of the desquamation population may occur, and while, as men- that follows sublethal doses of irradiation tioned above, the contribution in terms of is also rapid (40), the new cells arising area covered may be small, the mere fact from the normal mitotic foci in the upper that mitosis can proceed in the abnormal parts of the glands, provided, of course, environment of a moving sheet of cells is that the dose has not been large enough an indication that at least some of the to destroy them. Scott et al. (41) studied essential metabolic activities of the cell the coagulation necrosis that can occur in are not disturbed. the dog stomach due to the heat of a gasIt is obvious that both migration and troscope bulb. These defects also became mitosis have a part to play in epithelial rapidly repaired, but Scott and his corepair, and the report by Weiss and authors drew attention to the importance Matoltsy (38) on the healing of skin and of recognizing that such lesions could corneal wounds in chick embryos, is of be caused by the gastroscope, and might great interest, revealing some degree of be misinterpreted. Following the applicaindependence between the two activities. tion of eugenol to the surface of the muThey found that in wounds made before cous membrane of vagally denervated 375

IV / CYTOLOGY OF MUCOSAL REGENERATION

(Heidenhain) pouches in the dog (42), the surface cells, as far down as the necks of the glands, were shed but replaced within four hours, apparently by migradon of foveolar cells. Here, in contrast to ulcer healing, an occasional parietal cell was observed to participate in the resurfacing. While some increase in epithelial mitotic activity has been postulated during mucosal repair in other regions of the alimentary tract, the assessments have usually been made by visual impressions only, and have not been supported by nuclear counts. A study by illcMinn and Mitchell of ileal lesions in the cat using colchicine (Io), indicated, on statistical analysis of nuclear counts, that the apparent increase in mitosis was not in fact significant. The cells of the intestinal crypts normally display the highest mitotic index of any epithelium in the body (with the possible exception of the endometrium during post-menstrual repair), and it seems that the presence of defects in small intestinal mucosa does not serve as an added stimulus to the amount of mitotic activity that is already occurring. This of course contrasts with other epithelia (including epidermis), which have a lower mitotic index and in which there is an undoubted increase during the repair of a full-thickness defect. There is need for more detailed analyses of both small and large intestinal lesions from the point of view of epithelial mitosis, as well as for more information from the stomach. Dedifferentiation, Redifferentiation and New Gland Formation It has already been mentioned that the sheet of epithelial cells that advances over the floor of the lesion does not contain parietal or zymogen cells. It has also been shown by several workers that the glands in the immediate vicinity of a mucosal 376

lesion seem to lose these special varieties of cells, and come to contain many more mucin-producing cells than is usual. In our own experiments, staining for mucin has shown that the number of mucous cells increased, the nearer the wound margin was approached, and by the third day most, if not all, of the cells in the marginal glands showed a greater or lesser degree of mucin secretion. The cells in the glands that were at the wound margin were seen to be in continuity with those migrating over the wound floor, and a gradual lessening of PAS-positive material could be traced from gland to advancing epithelial margin, as has been noted by Hunt (32). The use of the Nile blue sulfate method for zymogen cells and of the succinic dehydrogenase technique for labeling parietal cells confirmed the concomitant decrease of these cells as the mucous variety increased (Figs. 6 and 7). There was no histological evidence to suggest that zymogen or parietal cells were lost from the glands by extrusion, and it is usually held that embryologically they develop from the mucous neck variety. It seems therefore that the zymogen and parietal cells dedifferentiate into mucinproducing cells. It has been shown, biochemically, that the stomach secretes not only one but several kinds of mucin (43), and it can be demonstrated by autoradiography with sulfur-35 that the mucous neck cells of normal glands and the mucin-producing cells of marginal glands incorporate the isotope (Fig. 8). It may be concluded that dedifferentiation is into the mucous neck and not the surface variety of mucin-producing cell. There is abundant evidence that in experimental lesions, the layer of migrating epithelium that overlies the accumulating granulation tissue dips down into it, thus forming the precursors of new glands. In most species this epithelium is derived

FIG. 6. Cat. Three days. Dark staining zymogen cells, prominent in undisturbed glands on the right, are absent front glands (left) that are adm jacent to the wound margin. Nile blue sulfate. x 80.

FIG. 7. Rat. Three days. Parietal cells (black) prominent in undisturbed glands, are absent front glands (left) that are immediately adjacent to the wound margin. Succinic dehydrogenase. x 80.

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FIG. 9. Rat. Three months. New glands front the center of the original wound area, showing the reappearance of parietal cells (black). Succinic dehydrogenase. x 12o.

IV / CYTOLOGY OF MUCOSAL REGENERATION

from the glands at the wound margin. In 1928, Ferguson (44) had noted that in dogs following the removal of large areas of mucosa, there was very extensive glandular regeneration, whereas in the rabbit there was not. The possible explanation for the discrepancy lies in the manner of creating the lesions and in the slight anatomical differences in the mucosa of dogs as compared with other species. Longmire et al. (45) found that in the dog the muscularis mucosae was firmly adherent to the underlying submucous layer, and that when attempting to strip off the whole thickness of the mucous membrane, the muscularis mucosae was invariably left behind, with the bases of gastric glands adhering to it. In the stomach of the cat, however, Longmire et al. found that the plane of cleavage lay below the muscularis mucosae. Thus, in the dog, epithelization of gastric lesions produced by stripping was assisted by the remains of glands all over the floor of the wound, whereas in the cat it could only occur by inward growth from the intact mucosa at the wound margins, as it does in rabbits and rodents (zo). The mode of epithelization in dogs was reaffirmed by Finckh and Milton (46); our own work and that of Gunter (47) in cats is in agreement with the views of Longmire et al. The precursors of the new glands are developing within a week of creating the lesion, and by the end of two weeks wellmarked tubular invaginations can be found near the original wound margin, although the central area of the lesion (depending on its original size) may still be unepithelized. New gland formation occurs not only when lesions involve the mucous membrane alone, but also after more penetrating injury involving almost the whole thickness of the gastric wall (48). The method of making the lesion, whether by excision, burning with a cau378

tery or heated bar, etc., does not appear to affect the long-term end result. The cells that comprise the new glands in the first instance are mucin-secreting, and have sometimes been described as pyloric or colonic in type. Our autoradiographic studies, using radiosulfur, have shown that, as in the dedifferentiated marginal glands, all the cells produce the sulfated variety of mucin. Subsequently, however, typical surface, zymogen and parietal cells appear. Presumably this can only occur by differentiation of the mucous cells, but there are some discrepancies in the literature about the time of appearance of these specialized secretors, and indeed, about whether they reappear at all. It seems clear that inadequate histological technique has often been the reason for these differences of opinion. Gunter's observations on the cat (47) were limited to a period of ten days after injury, which was not long enough for differentiation to occur. Myhre (49) and Hunt (32) found parietal and zymogen cells in the rat at three to five weeks, while Williams (so) noted them in the guinea pig at three months, though they were not apparently present by forty days (zo). In the dog Ferguson (44) did not detect zymogen granules at the end of three months, but in the same species Milton et al. (s I) recorded the first appearance of parietal and zymogen cells within four to five weeks; by the end of twenty-four weeks, the new glands were indistinguishable from normal. Lippman and Longmire (s z) found parietal cells in new glands at the margin of jejunal mucosa grafted to the mucosa-stripped stomach of dogs at ten weeks. Confirmation that the new parietal and zymogen cells in the dog are in fact secretory is now available. Milton et al. (5') stripped off approximately seven-eighths of the gastric mucosa, and supplemented their histological studies by histamine

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MCMINN & JOHNSON

tests to estimate the secretory activity of the parietal cells. Within the first three weeks after operation, the response to histamine was much below normal, but subsequently there was a slow increase, and although by six months the level of secretion was not quite normal, it appeared to be still rising. Lippman et al. (53), after stripping off 90 per cent of the mucosa in the dog, found that histologically gland reformation was completed within eleven weeks, but that secretory function lagged for eight to twelve months, and even then did not reach a nomal level. Secretion of enzyme appeared to return quicker and more completely than that of acid; vagotomy and antrectomy did not alter the rate of regeneration but suppressed the level of secretion. Our own studies provide a cytochemical basis for the above. The histochemical test for succinic dehydrogenase revealed that some parietal cells were present in almost all the new glands after six weeks in both species studied, and by the end of three months, they were abundant (Fig. 9). Only in the very centre of the original wound area were these cells rather scanty, but they were nevertheless present. The Nile blue sulfate method showed that zymogen cells had also appeared (Fig. i o) in the new glands within two months, while mucin-secreting cells were now confined to the upper parts of the glands and the gastric surface. The distribution of radioactive sulfur was normal, i.e., absent from the surface epithelium but present in the neck regions of the glands. It would appear therefore that not only are glands re-formed, but a complement of cell types closely resembling the normal in both distribution and function is reconstituted. Since at some stage of their development, all the cells that make up the new glands secrete sulfated mucin, the concept of Ferguson (44), Gunter

(47) and Hunt (32) that mucous neck cells can transform into parietal and zymogen cells is substantiated. Furthermore, some of the mucous cells lose their ability to secrete sulfated mucin as indicated by autoradiography, and thus assume the character of surface cells. Whether the nonsulfated secretion produced by such cells is identical with that produced by normal surface cells has not been proven. Many cytological problems of extraordinary interest are posed by this dedifferentiation and redifferentiation ("modulation") of gland cells. The stimulus that invokes, say, the transformation of a cell, with the remarkable property of concentrating hydrogen ions to a strength of over one million times that in plasma, into one secreting mucin, is quite unknown, and the internal reorganization required in that cell to bring about such a change must be very extensive. It would be intriguing to know whether a parietal cell from an `old" gland usually reverts to a parietal type in the new, or whether the renaissance results in the formation of a different type. That an occasional aberrant type can arise is indicated by the "intestinalization" of mucosa that can occur, not only in man, but also in the experimental animal. One of our three month rat specimens showed typical intestinal crypts and villi, covered by epithelial cells with striated borders and goblets, at the site of the lesion. Blomquist (54) in his human series noted that this type of metaplasia was more frequent in the vicinity of ulcers than elsewhere, and Arey and Bothe (S5) concluded from their survey of the subject that it was never a feature of the normal stomach. Skoryna et al. (48) have also reported some evidence of squamous metaplasia in rat lesions. If various stages in these transformations could be captured by the electron microscopists and correlated with histochemical studies, a great deal of use379

IV / CYTOLOGY OF MUCOSAL REGENERATION

ful information on the synthetic activities of cells should accumulate. As far as the alimentary tract is concerned, the new formation of glands following experimental mucosal lesions is not confined to the stomach. In the small intestine regeneration of crypts and villi

has been observed in the duodenum (56), jejunum (57) and ileum (Io), but in the large bowel there is evidence to show that glands re-form rather less commonly than at higher levels or even not at all ( 36,58,59,60,61).

Connective Tissue Reactions The experimental gastric ulcer pro- rived from mononuclear cells of the blood duced by excision provides a typical ex- or system that have reticulo-endothelial ample of healing by granulation. The immigrated into the area. A number of area denuded of mucosa becomes covered studies (66,67,68,69) suggest the latter, by blood and fibrin clot, and underneath but conclusive proof is awaited. One aspect of the connective tissue this, there is a gradual accumulation of granulation tissue, consisting of blood problem that is pertinent to gastrointesticells, connective tissue cells and vascular nal ulceration is the role of contraction. endothelium. From about the fifth day Within a few days of artificial ulcer proonwards, signs of new fibrous tissue form- duction in any part of the gut, the original ation are evident, and in the ensuing area of the wound has become very much weeks, this new tissue matures into gland- reduced, not so much by the ingrowth of supporting stroma that closely resembles epithelium as by movement of the whole the normal but may contain more colla- mucosa bordering the lesion. At the gen fibres. It would be out of place here present time, several groups of workers to consider in detail the various aspects are investigating contraction in skin of new fibrous tissue formation(62,63,64, wounds (70,71,72,7 3,74,75,76,77,78,79,80, 65), since there is no evidence to suggest 81,82), but this matter has received no atthat the process is different in the stomach tention in the alimentary tract, and the from anv other sites. Suffice it to say mechanism of the movement is obscure that neither the fibers nor ground sub- (see Muscular Reactions, below). It is imstance of connective tissue can be formed portant to appreciate that the mobilization without the presence of the cells known of skin or mucous membrane that occurs as fibroblasts, which are apparently capa- in the first hours or days after wounding ble of inducing in their immediate vicinity may have a different causation from the the physicochemical changes necessary contraction due to the activity of maturfor fiber production. Mitotic activity in ing fibrous tissue at later stages. fibroblasts is prominent from the second Recent work on skin wounds has corday onwards, and for some days there- related some of the histochemical with after, depending on the size of the lesion, the biochemical aspects of repair (83), and the proliferation of these cells is em- but again little information is available phasized in animals that have received from alimentary wounds. Our own excolchicine or thymidine-H'. The question periments in rats and cats have drawn currently at issue is whether the fibro- attention to possible species differences blasts are fibrocytes that have been ac- with regard to the role of alkaline phostivated in situ, or whether they are de- phatase in the formation of new connec38o

MC,MINN & JOHNSON

Live tissue ( 21,23,34,36). A number of workers have put forward evidence that in skin and other organs in rodents, there is a strong histochemical reaction for this enzyme in areas of fibrous tissue proliferation, and in skin this increase has been confirmed biochemically (84,85). In the stomach and rectum of the rat, we have found a similar phosphatase reaction histochemically, but in the stomach of the cat, as in skin (86), esophagus (23), ileum (21), rectum (36), gallbladder (34) and urinary bladder (z 1) of this species, we have been unable to obtain a positive reaction under similar conditions of histochemical technique. While this apparent absence of the enzyme still requires to be substantiated biochemically, our findings

suggest that in the cat, phosphatase is unlikely to play a significant part in the production of either the cells or fibers of new connective tissue. However, its association with the manufacture of other tissue elements in the cat, such as bone or hair, is not questioned. The examination of skin wounds( 87 ) and gastric ulcers (88) in man indicates that phosphatase is present in newly forming connective tissue. Although the biochemist can describe what the enzyme is doing from a chemical point of view, the precise reason for the presence of the enzyme in a particular biological context—and there are many of them—is, more often than not, a matter of pure speculation at the present time.

Muscular Reactions In the making of excision ulcers in the gastrointestinal tract, the muscularis mucosae is usually removed together with the rest of the mucosa. There has been general agreement that these smooth muscle fibers never show any regenerative reaction (2,21). In contrast to this view, Jones (89) concluded from a study in dogs of transactions of ileum and of areas of gut into which polyvinyl sponge had been implanted, that there was evidence of sprouting and mitosis in visceral muscle. However, there was a lack of uniformity in his results, for he noted that while defects in the muscularis externa were repaired by fibrous tissue only, the muscularis mucosae appeared to re-establish continuity. This latter finding by Jones is contrary to all other accounts concerning the muscularis mucosae. In our own experiments on various parts of the alimentary tract, including the stomach, no regenerative activity in any part of the muscularis has ever been observed. Certainly condensed fibrous tissue under

new glands in a healed site can simulate the muscularis mucosae very closely, but staining with iron hematoxylin and picrofuchsin (van Gieson) has always confirmed the absence of muscle fibers. Injury to the muscularis externa is repaired by fibrous union. It has been stated that incised wounds and surgical anastomoses in the alimentary tract leave very little sign of such union(9o), but whether this occurs by migration alone, or is assisted by any degree of proliferation is not known. It is recognized that some of the newly-formed capillaries of granulation tissue eventually acquire an arteriolar or arterial structure by becoming invested with a coat of smooth muscle cells. However, there is as yet no agreement on the mechanism by which this is brought about. The work of Clark and his co-workers on transparent ear chambers in rabbits (91,92,93) has suggested that undifferentiated connective tissue cells could arrange themselves around the vessels, and become transformed into smooth 381

IV / CYTOLOGY OF MUCOSAL REGENERATION

muscle that was contractile and that acquired a vasomotor nerve supply from nerve fibers that grew into the area. In contrast, Florey (94) considered that the connective tissue cells did not become contractile, nor were they arranged circumferentially around the vessels but longitudinally. He thought it most likely that muscle cells from pre-existing arterioles migrated along the new vessels, but pointed out that this has not yet been demonstrated. The part played by muscle in reducing the size of an intestinal lesion is not clear. Localized contraction of the muscularis

externa induced by the trauma of making the lesion has been suggested but never proved by direct measurement. The fibers of the muscularis mucosae are predominantly longitudinal in direction, so that contraction on their part could be expected to increase the size of the lesion. However, the lesion never increases in area for this reason, although some degree of retraction of the muscularis mucosae no doubt assists in the "tumbling over" of the edge of the mucosa, so that the glands nearest the original wound margin come to lie parallel with the wound surface.

Concluding Comments Quite apart from the various changes that occur in the size, shape and metabolic activities of epithelial cells during the repair of an experimental wound, the end result is the culmination of what must be a highly co-ordinated interplay between epithelium and connective tissue. In the stomach, an almost normal mucosal architecture is restored, and this implies that the epithelium that has formed the new glands has "known when to stop", as far as its invasion of the underlying connective tissue is concerned, and that the connective tissue for its part has been amenable to this intrusion. It is interesting to note that changes similar to those found in repair have been observed following the implantation of hydrocarbon carcinogens into the gastric mucosa of rats (95). The abnormalities found included the replacement of parietal and zymogen cells by mucous cells, dilatation of glands and cyst formation, and the presence of undifferentiated cells with mitotic nuclei at the bases of glands. The ultimate aim behind all experimental gastric ulceration is the elucidation of the mechanism of human disease processes, and while much 382

still remains to be learned about activities at the cellular level, the information obtained from experimental lesions can provide useful pointers to the lines along which future study of human material might be directed. Hitherto, there have been virtually no attempts at cytological, as opposed to general histological, comparisons between the margins of experimental ulcers and those of human lesions. The problem of what inhibits the healing of some gastric (and duodenal) ulcers in man is not likely to be solved until we know how acute and chronic ulcers differ cytologically. There is no doubt that the epithelium can grow out from the margins of chronic ulcers, but it is not known why further progress may be halted. When the chronic ulcer does heal, Ivy et al. (z) consider that it does so more slowly than the acute, with obvious scarring, and that the former is more likely to show areas of intestinal-like epithelium. They also indicate that while new gland formation is still possible, the glands are more irregular in form than in the acute lesion, and that the gland cells tend to remain undifferentiated or mucoid in

MCMINN & JOHNSON

type. In a recent survey, however, Evans (96) states that new glands do not form. It would appear particularly pertinent to examine the extent of mitotic activity at the margins of the two types of ulcer —acute and chronic—not only by ordinary histological means but also, for example, by culturing fragments of fresh specimens obtained by biopsy or operation with colchicine or thymidine-H'. In this way the potentiality of the epithelium for regeneration might be assessed, which would help to determine to what extent the failure of epithelization in chronic ulcers is due to deficiencies in the production of new epithelial cells. If it transpires that production is not affected, then cells are being lost through failure to survive, and here various connective tissue factors, such as vascularity and permeability of ground substance affecting cell nutrition, may be involved. It is not yet known whether the chronicity of an ulcerative lesion is determined within the first few days of its existence or only after some weeks. The very early phases of repair—say the first ten days—may well be critical in determining the end result. It is probably immaterial whether or not

new glands form; what must be achieved if repair is to be adequate is complete epithelial coverage of the lesion. The observation (97,98) that peptic ulcers in the human stomach almost invariably occur in the pyloric gland area stresses the need for more experimental work on this type of mucosa. A further field that might be explored more deeply is that of the effects of drugs, hormones and other factors on gastric repair. Most studies, e.g. of the effects of ACTH and cortisone, have been carried out on skin and related connective tissue, and little direct work on the stomach has been reported (42,48,49,50,99); the paper by Ehlers(99) refers to much of the relevant steroid literature. A whole battery of all kinds of microscopical and microchemical techniques must be brought to bear on the problem of epilthelization. It is the detection of the earliest deviations from the normal that will be of the greatest service to an understanding of ulcer pathology, for only then will it become possible to direct logical attempts to reverse such changes before they become irreversible.

Summary The results of some histochemical and autoradiographic studies on the repair of experimental lesions in the stomach of the rat and cat have been incorporated in a review of the cytological aspects of mucosal repair and of tissue repair in general. Acute experimental lesions of the whole thickness of the mucosa in the body of the stomach are capable of healing with the formation of new glands, resulting from downgrowths of new epithelium into the maturing granulation tissue that accumulates in the floor of the ulcer.

Mitotic activity in the epithelium occurs not only at all levels in the glands at the margin of the ulcer, but also in the surface epithelium adjacent to the ulcer, and in the epithelium that advances over the granulation tissue. Studies with colchicine and with thymidine-H have served to emphasize the occurrence of mitotic activity in these sites. Histochemical studies have confirmed that the glands at the margin of the lesion lose their complement of parietal and zymogen cells, presumably by dedifferentiation, and become capable of secreting 383

IV / CYTOLOGY OF MUCOSAL REGENERATION

mucin, that can be shown by autoradiography with radiosulfur to be sulfated, like that of mucous neck cells. In the first instance, the new glands are composed of these mucous cells, but parietal and zymogen cells later differentiate.

Emphasis is laid on the need for further cytological, as opposed to general histological, studies of both experimental and human lesions as a contribution towards solving the problem of the unhealed chronic ulcer.

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3rd ed. London, Longmans, Green, 1958. 13. FORBUS, W. D. Reaction to Injury. Patholo gy for Students of Disease, Vol. 2, London, Bailliere, 1952. 14. CLARK, E. B. and CLARK, E. L. Amer. J. Anat., 93: 171-219, 1953 15. GILLMAN, T., and PENN, J. Med. Proc. (Joannesb.), 2: 121 - 186, 1956. 16. SHERRY, S., FLETCHER, A. P. and ALKJAERSIG, N. Physiol. Rev., 39: 343-3 82, 1 959. 17. AREY, L. B. Physiol. Rev., 16: 327-406, 1936. 18. KLEBS, E. Arch. exp. Path. Pharm., 3: 124156, 1874-75. 19. ALLEN, R. D. The Cell. Bracher, J. and Mirsky, A. E., ed., 135-216. New York, Academic Pr. 1961. 20. WILLIAMS, A. W. Brit. J. Surg. 41: 319-326, 1 953-54. 21. JOHNSON, F. R. and MCMMINN, R. M. H. Biol. Rev., 35: 364-412, 1960. 22. BRADFIELD, J. R. G. Nature (Lond.), 167: 4041, 1951. 23. MCMINN, R. M. H. and JOHNSON, F. R. J. Embryol. exp. Morph., 6: 288-296, 1958. 24. MCMINN, R. M. H. Ann. Roy. Coll. Surg. Engl., 26: 245-260, 1960. 384

25. KUGLER J. H. and WILKINSON, W. J. C. J. Histocm. he Cytochem., 7: 398-402, 1959. 26. KUGLER, J. H. and WILKINSON, W. J. C. J. Histochem. Cytochem., 8: 195-199, 1960. 27. KUGLER J. H. and WILKINSON, W. J. C. JJ. Histochem. Cytochem., :9 498-503, 1961. 28. LEVANDER, G. Virchows Arch. Path. Anat., 329: 184-213, 1956. 29. STEVENS, C. E. and LEBLOND, C. P. Anat. Rec.,

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Surg., 45: 76-8o, 1957-58. 35. MCMINN, R. M. H. and JOHNSON, F. R. Nature (Lond), 178: 212, 1956. 36. MCMINN, R .M. H. and JOHNSON, F. R. J. Embryol. exp. Morph. 6: 509-517, 1958. 37. PINKUS H. Physiology and Biochemistry of Skin, Rothman, S., ed., 584-60o, Chicago, University of Chicago Press, 1 954• 38. WEISS, P. and MATOLTSY, A. B. Develop. Biol., 1: 302-326, 1959. 39. GRANT, R. Anat. Rec., 9t: 175-185, 1945. 40. BRECHER, G., CRONKITE, E. P., CONARD, R. A. and SMITH, W. w. Amer. J. Path., 34: 105119, 1958. 41. SCOTT, N. M., JR., PRESTON, J. A. and PALMER, S. E. Gastroenterology, 32: 708-716, 1957. 42. JANOWITZ, H. D., WEINSTEIN, V. A., SHAER, R. G., CEREGHINI, J. F. and HOLLANDER, F. Gastroenterology, 34: 11-2o, 1958. 43 • JERZY GLASS, G. B., RICH, M. and STEPHANSON,

L. Gastroenterology, 34: 598-615, 1958.

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1952. 46. FINCKH, E. S. and MILTON, G. W. J. Path. Bact., 8o: 143-145, 1 96047. GUNTER, G. S. Gastroenterology, 1 5: 708-717, 1950. 48. SKORYNA, S. C., WEBSTER, D. R. and KAHN, D. S Gastroenterology, 34: I-lo, 1958.

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49. MYHRE, E. Arch. Path., (Chic.), 62: 30-36, 1956. 50. WILLIAMS, A. W. Brit. J. Surg., 48: 564-572, 1961. 51. MILTON, G. w., MAXWELL, G. A. and FINCKH, E. s. Brit. J. Surg., 47: 562-566, 1961. 52. LIPPMAN, H. N. and LONGMIRE, W. P., JR. Ann. Surg. 140: 86-92, 1 954. 53. LIPPMAN, H. N., SHELDON, D. B., and LONGMIRE, W. P., JR. Surgery, 4o: 212-222, 1956. 54. BLOMQUIST, H. E. Acta chir. scand., 111: 465-474, 1956. 55. AREY, L. B. and BOTHE, IL T. Surg. Gynec. Obstet., 9o: 86-90, 1950. 56. FLOREY, H. W. and HARDING, H. E. J. Path. Bact., 40: 211-218, 1935. 57. MANN, F. C. and BOLLMAN, J. L. J.A.M.A., 99: 1576-1582, 1939. 58. O'CONNOR, R. J. Brit. J. exp. Path., 35: 5455f 9, 1954. R.R. J. Brit. J. Surg., 44: 93-97, 195659. 0 00R, NNO

Surg., 148: 145-152, 1958. 76. WATTS, G. T., GRILLO, H. C. and GROSS, J. Ann. Surg., 148: 153-160, 1958. 77. BILLINGHAM, R. E. and MEDAWAR, P. B. J. Anat., 89: 114-123, 1955. 78. BILLINGHAM, R. E. and RUSSELL, P. S. Ann. Surg., 144: 961-981, 1956. 79. VAN DEN BRENK, H. A. S. Brit. J. Surg., 43: 525-550, 1955-56. 80. cUriourrSoN, A. M. Surg. Gynec. Obstet., 1o8: 421-432 , 1959. 81. JOSEPH, J. and TOWNSEND, F. J. J. Anat., 95: 403-410, 1961. 82. JOSEPH, J. and TOWNSEND, F. J. Brit. J. Surg., 48: 557-564, 1961. 83. DUNPHY, J. E. and UDUPA, K. N. New Engl. J. Med., 253: 847-851, 1955. 84. FELL, H. B. and DANIELLI, J. F. Brit. J. exp. Path., 24: 196-203, 1943. 85• DANIELLI, J. F., FELL, H. B. and KODICEK, E. Brit. J. exp. Path., 26: 367-376, 1945. 86. JOHNSON, F. R. and MCMINN, R. M. H. J. Anat., 92: 544-550, 1958. 6o. LUMB, G. and PROTHEROE, IL H. B. Lancet, 269: 87. FISHER, I. and GLICK, D. Proc. Soc. Exp. Biol. 1208-1215, 1955. Med. 66: 14-18, 1 947. 61. HIGHTOWER, F. Ann. Surg., 147: 775-780, 1958. 62. JACKSON, D. S. New Engl. J. Med., 259: 814- 88. FODDEN, J. H. Gastroenterology, 23: 372-390, 820, 1958. 1953. 63. JACKSON, D. S., FLICKINGER, D. B. and DUNPHY, 89. JONES, D. s. The Healing of Wounds; A Symposium on Recent Trends and Studies, J. E. Ann. N.Y. Acad. Sci., 86: 943:947, 196o. Williamson, M. B. ed., 149-167. New York, 64. EDWARDS, L. c. and DUNPHY, J. E. New Engl. Blakiston, 1957. J. Med., 259: 224-2 33, 1958. 65. EDWARDS, L. C. and DUNPHY, J. E. New Engl. 90. WILLIS, R. A. The Borderland of Embryology and Pathology. London, Butterworth, J. Med. 259: 275-285, 1958. 1958. 66. MAx1NNOw, A. A. Arch. Exper. Zellforsch., 91. CLARK, E. R., CLARK, E. L. and WILLIAMS, R. C. 5: 169-268,1927-28. Amer. J. Anat., 55: 47-77, 1934. 67. CAMERON, G. R. Pathology of the Cell. Edin92. CLARK, E. R. and CLARK, E. L. Amer. J. Anat., burgh, Oliver, 1952. 66: 1-49, 1940. 68. HARTWELL, S. W. The Mechanisms of Healing in Human Wounds. Springfield, Ill. 93. CLARK, E. R. and CLARK, E. L. Amer. J. Anat., Thomas, 1955. 73: 21 5-2 50, 1943. 69. ALLGÖWER, M. The Cellular Basis of Wound 94. FLOREY, H. W. General Pathology, Florey, H. ed., znd ed., 378-409. London, Lloyd-Luke Repair. Springfield, Ill., Thomas, 1956. Ltd., 1958. 70. ABERCROMBIE, M., FLINT, M. H. and JAMES, D. w. J. Embryol. exp. Morph., 2: 264-274, 95. GRANT, R. and IVY, A. c. Gastroenterology, 29: 199-218, 1955. 1954• 71. ABERCROMBIE, M., FLINT, M. H. and JAMES, D. 96. EVANS, R. w. Peptic Ulceration. .A SymW. J. Embryol. exp. Morph., 4: 167-175, 1956 posium for Surgeons, Wells, C. and Kyle, J. ed. 37-55, Edinburgh, Livingstone, 1960. 72. ABERCROMBIE, M. and JAMES, D. NV. J. Embryol. exp. Morph., 5: 171-183, 1957. 97• 01, M., OSHIDA, K. and SUGIMIURA, s. Gastroenterology, 36: 45-56, 1959. 73. ABERCROMBIE, M., JAMES, D. W. and NEWCOMBE, J. F. J. Anat., 94: 170-182, 1960. 98. MARKS, I. N. and SHAY, H. Lancet, 1: 1107'111, 1959. 74. JAMES, D. W. and NEWCOMBE, J. F. J. Anat., 95: 247-255, 1961. 99. EHLERS, P. N. Arch. klin. Chir., 293: 120-166, 75. GRILLO, H. C., WATTS, G. T. and CROSS, J. Ann. 1 959-60.

385

PART V SYSTEMIC FACTORS IN PEPTIC ULCER FORMATION

Role of the Central Nervous System in Peptic Ulcer Development

Joseph Bald*

(I) was the first to suggest H. Meyer (6) established that the blood that affections of the nervous system may vessels and musculature of the stomach give rise to the formation of peptic ulcer. are under the control of the sympathetic In 1841 he described the acute and chronic and parasympathetic innervation, and the form of peptic ulcer, as well as the equilibrium of both assures the normal changes of the gastric wall, and expressed function of the stomach. Eppinger and the opinion that these processes accede to Hess (7) pointed out that parasympathetic the primary alterations of the brain or its impulses may surpass sympathetic innercovering membranes. According to Roki- vation, and they called this state "vagotansky (r ), the irritation brought about tonia". As a consequence of this condiby the diseases of the brain or its mem- tion, spastic contraction of the gastric branes is transmitted to the stomach by musculature occurs, and the secretory the vagus nerve. Hoffmann (2), Arndt activity of the stomach increases. Such (3), Mogilnitzky (4) and Korst (S) re- individuals respond to small doses of piloported their observations concerning the carpine with spastic contractions of the nature of the affections of the nervous gastric musculature and increased secresystem which may lead to the formation tion. On the other hand, atropine abolishes of peptic ulcer of the stomach, duoden- spasm of the musculature and decreases um, jejunum and esophagus. secretion. The pharmacological school of Hans ROKITANSKY

Neurogenic Theory of Peptic Ulcer Von Bergmann postulated, in 1913, the neurogenic or spasmogenic theory of the origin of peptic ulcer (8). According to this concept based on earlier work of Rokitansky and others, the disturbance of the functional equilibrium of the autono-

mic nervous system represents a hereditary trait. The abnormality is characterized by an excessive vagal stimulation, which disturbs the functional balance of the autonomic nervous system, and results in stigmatization. Individuals afflict-

•From the First Institute of Pathological Anatomy and Experimental Cancer Research, Medical University, Budapest, Hungary.

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ed with this disorder exhibit, besides abnormal gastric function, some other changes such as a moderate degree of hyperthyroidism, slight exophthalmus and sparkling eyes, increased perspiration and dermographism. The familial tendency to peptic ulcer formation has been frequently observed. A clinical example of such a tendency has been reported by Kidd (q), who found a pair of forty-one year old twins, suffering from duodenal ulcers, which perforated in both during the same hour. Harvey Cushing (i o) was responsible for important observations concerning the genesis of peptic ulcer. He investigated the relation between peptic ulcer and interbrain lesions. He collected eleven cases of intracranial affection accompanied by lesions of the upper gastrointestinal tract. In ten cases, these were primary or metastatic tumors of the brain, and in one case an aneurysm of the basilar artery. Gastric autopsy findings ranged from acute hemorrhagic erosions of the gastric mucosa and esophageal or gastric perforation, to extensive esophageal or gastric malacia. Cushing described cases of brain tumors in which the growth was successfully removed, but the patient died during the postoperative period due to perforation of a peptic ulcer. Grant (r r ) reported two cases of duodenal ulcer associated with brain lesions. The theory of Rokitansky has been abandoned for a long period of time in favor of the concept of Virchow, who regarded peptic ulcer as a local process of the gastric wall. Only with the development of experimental pathology has the neurogenic theory of peptic ulcer gained a wider acceptance. Schiff (r z) demonstrated that unilateral injury of the optic thalamus or cerebral peduncle in dogs and rabbits results in peptic ulcer formation. The unilateral transection of the pons, medulla oblon390

gata or spinal cord produces equally a peptic ulcer development. Schiff has expressed the opinion that all these lesions interfere with the nervous mechanism regulating the size of the lumen of the gastric blood vessels. Brown-Sequard (13) discovered that injuries of the cerebral cortex are followed by the formation of chronic and perforating ulcers of the stomach. According to Ebstein (r4), production of punctured unilateral lesions of the corpora quadrigemina in rabbits, or injection of chromic acid into these areas of the brain, result in hemorrhagic changes in the gastric mucosa. Burdenko and i\'logilnitzky (i 5) have stressed the fact that following the damage of midbrain, development of hemorrhagic erosions, acute and cicatrized gastric ulcers can be observed. Watts and Fulton (r 6) have reported their studies on monkeys; they came to the conclusion that lesions of the hypothalamic area, and especially of the supra-optic nucleus and tuber cinereum, are far more prone to produce profound gastrointestinal changes, including gastric and duodenal erosions, bleeding and even perforation, than lesions of other parts of the nervous system. Hoff and Sheehan (r 7) observed hemorrhagic erosions in the stomach of monkeys following production of discrete lesions of the hypothalamus. Oberling and Kållö (1 8) produced an injury of the basal ganglia in the brain of dogs and rabbits, which resulted in hemorrhagic erosions and peptic gastric ulcer formation. Alterations occurring in the brain of dogs with Eck fistula, fed on meat, have been ascribed to encephalitis, and it has been stated that the condition is transmissible. Kålle) and Korpåssy (r 9) have demonstrated that the intracerebral inoculation with brain emulsion induces peptic lesions in the stomach of a dog, resulting in the death of the animal, but that the encephalitis is not transmissible.

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Pavlov (2o,21) was the first to throw some light on the functional relationships of the higher nervous centres on the basis of his studies of the conditioned reflexes. Pavlov demonstrated that the cerebral cortex affects gastric secretion, and demonstrated the psychic phase of the secretion. He and his pupils proved the existence of a functional relation between the cerebral cortex and the internal organs. In continuation of the studies of Pavlov, Bykow and Kurzin (22) have investigated the relationships existing between peptic ulceration and cerebral cortex, and developed the corticovisceral theory of peptic ulcer formation. The theory explains the genesis of peptic ulcer by the interrelation which exists between the human organism and environment, and between the nervous system and milieu interne. The theory of Bykow and Kurzin may be summarized as follows: collectors of nervous stimuli, the exteroceptors respond to the stimuli of the environment, while the interoceptors supply the information on the condition of the body and internal organs. The cerebral cortex uninterruptedly registers the impulses arising from different organs. It is possible that these impulses are not always perceived, i.e. they do not reach the level of consciousness. However, this does not diminish their importance. The impulses originating in the internal organs, transferred through the visceroceptors, bring about a certain condition of the cerebral cortex which is altered by the stimuli carried to the cortex by the exteroceptors. This double origin of impulses creates new factors in the integration of the organic functions which are encompassed by corticovisceral dynamics. The formation of peptic ulcers is regarded as a result of a pathological integration of the relation between cerebral cortex and stomach; the abnormal cortical impulses reach

the organ, and the organ stimuli are carried to the cortex and aggravate its pathological function. Besides the role of nervous and humoral mechanisms in peptic ulcer formation, one should consider the constitutional types, occupations and living conditions and a number of other factors. Therefore, the interpretation of the peptic ulcer development as being a consequence of purely local alterations cannot be accepted. Neither can the opinion that peptic ulcers arise solely as a result of disorders of the vegetative nervous system be supported. The disease originates undoubtedly from the disturbance which results from the two information systems of the cerebral cortex, i.e. exteroceptive and interoceptive system or as a result of the abnormal interoceptive "signalization". It is conceivable that excessive function and exhaustion of the ganglion cells of the cerebral cortex result in the release of subcortical centres from the control of the highest co-ordinating system, with resulting "chaotic" function. This state is followed by the formation of certain foci of irritation. In the transmission of irritation, acetylcholine seems to play an important part. Al'pern (z 3) has demonstrated an elevated acetylcholine level in the serum of patients suffering from peptic ulcer. It may be postulated that the subcortical foci of irritation induce contractions of the gastric musculature and blood vessels, which result in primarily functional, but later also organic alterations. Peptic ulcers can be seen to develop as a sequela of abnormal centrifugal impulses, of which the stomach is the target organ. Changes in the trophicity can be observed in this process. As a result the resistance of the mucous membrane decreases, and the digestive power of the gastric juice asserts itself. According to the opinion of Soviet investigators in the genesis of peptic ulcer, 391

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the primary importance should be attributed to the abnormal function of the cerebral cortex. The derangement of the functional equilibrium of the autonomic nervous system and changes in the mechanism of the humoral regulation represent secondary manifestations. The postulate that the primary alteration is vested in changes of the regulating mechanism of the cerebral cortex has led to the application of sleep therapy in the treatment of peptic ulcer. Protracted sleep appears to be capable of restoring the impaired regulating mechanism of the cerebral cortex and its controlling effects on the vegetative functions of the organism. The theory of Bykow and Kurzin is useful in understanding the evolution of peptic ulcer disease, because it takes into account the psychical factors, as well as the effects of environmental factors. Cushing once stated that it is well known to clinicians that individuals which carry out excessive mental activity without recreation are particularly inclined to peptic ulcer development. The symptoms appear to decrease if such patients are subjected to mental and physical rest, and they reappear with return to previous levels of activity. The importance of emotional and psychic factors in the development of peptic ulcer has often been stressed, although it is difficult to supply an adequate anatomical explanation for these effects. Nevertheless, a gradual

transition from functional alterations to changes which may be proved anatomically or histologically appears to be evident. In the great majority of cases with gastric and duodenal ulcer, such lesions can be found in the brain or in the nerve routes leading to the stomach. Undoubtedly a great significance should be attached to these changes in the genesis of peptic ulcer, which by no means can be regarded as a local process. Rössle (24) expressed that it appears to be a secondary disease. He pointed out in 1913 that peptic ulcer often accompanies some other disease such as appendicitis, cholecystitis or other inflammatory processes in the abdominal cavity which result in fibrous tissue (scar) formation. He suggested that reflex irritation brings about the second disease i.e. peptic ulcer. In analyzing the corticovisceral theory of Bykow and Kurzin, Gubergric (25) expressed doubts as to the significance of the role of cerebral cortex in the etiology of all cases of peptic ulcer. In the following demonstration, we have attempted to prove that in the majority of peptic ulcer cases, anatomical and histological lesions can be found either in the central or peripheral nervous system. If one examines the central and peripheral nervous systems in all autopsies in which peptic ulcer occurred, convincing evidence becomes available.

Cerebral Hemorrhage and Peptic Ulcer Hemorrhage occurs in the brain in two forms. One is the massive hemorrhage or apoplexy, the other one a punctate or petechial bleeding. Both forms appear to be related to the development of peptic ulcer. Brown-Sequard and Lepine (z6) were first to study the erosions and ulcers of the upper alimentary tract which may 392

follow cerebral apoplexy. Charcot (27) and Rössle have observed meningeal hemorrhage, which are also followed in some cases by the development of a gastric ulcer. Lepine described in 1895 a case in which a patient succumbed following a massive bleeding from a gastric ulcer; post-mortem examination revealed

FIG. u. Autopsy specimens from a 78 year old patient demonstrating cerebral hemorrhage (A) and a prepyloric peptic ulcer (B).

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an egg-sized hemorrhage which had penetrated into the ventricle. According to Lepine, the cerebral hemorrhage was primary and the hemorrhage from gastric ulcer secondary. Hart (28) reported in 1913 a case of a fatal gastric hemorrhage which he named a "neurogenic" hemorrhage. Autopsy on a fifty-nine year old man who died following hematemesis showed four peptic ulcers in the stomach. The basal ganglia on the left side had been destroyed by a hemorrhage which penetrated into the lateral ventricle. In a further publication, Hart (29) gave an account of two similar cases in which peptic ulcers developed following brain hemorrhage. Hart (3o) suggested that the formation of peptic ulcers in consequence of cerebral hemorrhage occurred on the basis of a reflex vasoconstrictor effect, followed by ischemia and digestion of the gastric mucosa. Another possibility which should be considered is that following vasoconstriction of the gastric vessels, vasodilatation occurs with development of stasis. Owing to the stoppage of blood circulation, gastric juice affects digestion of the gastric mucosa or the wall. In our own material (31,32,33,34) we have examined 240 cases of peptic ulcer, and found that in a third of these instances a cerebral hemorrhage could be held responsible for the development of the ulceration. In 12 per cent of these cases a massive brain hemorrhage was demonstrated, and in 20 per cent petechial hemorrhages were present (35)• As a consequence of apoplexy, peptic ulcers can be located in the stomach, duodenum or esophagus (Fig. 1). A few days after a massive brain hemorrhage, a single or multiple circumscribed area of stasis can be observed in the epithelium. In ten to twenty-one days a peptic ulcer can be found. If the ulcer is superficial, the healing occurs quickly. A deep peptic ulcer heals with a stellate cicatrix. Per-

foration of an acute peptic ulcer is not uncommon. In one of our previous publications (36), we have expressed the opinion that the ulcers which follow apoplexy are predominantly acute, and although they may perforate, the healing occurs spontaneously, and transformation into a chronic stage is not observed. One of the most important factors of a peptic ulcer appears to be a change from an acute ulcer into a chronic ulcer. Since the time of our earlier publications, we have rarely encountered chronic ulcers in consequence of a cerebral hemorrhage. A peptic ulcer can develop as a result of a massive hemorrhage localized in the basal ganglia. Moreover an ulcer may develop even in cases in which the massive hemorrhage has an atypical localization. In these cases considerable diagnostic difficulties exist, and only an autopsy examination can demonstrate that brain hemorrhage has preceded the peptic ulcer development. As a result of a massive brain hemorrhage, one may find several peptic ulcerations. Another condition which can be observed is a massive necrosis of the duodenum (Fig. 2) . In this case, in an area of the duodenum measuring 6 by 2 centimetres, the mucosa, or the underlying portion of the duodenal wall, becomes necrotic. Undoubtedly such a process can result in a perforation. A peptic ulcer resulting from a massive cerebral hemorrhage occurs most commonly in an advanced age, usually between the ages of 5o to 7o. Although the occurrence of peptic ulcer following brain hemorrhage is a relatively common finding, not all cases of brain hemorrhage are associated with peptic ulcer. This can be explained in part by the fact that a certain number of those individuals who suffered a cerebrovascular accident die within a short period of time. According to the early data of Jones (37), 37.7 per 393

V / ROLE OF THE CENTRAL NERVOUS SYSTEM

cent of the patients die following apoplexy in the first twenty-four hours, and about twice as many by the end of the first week. According to Winkelman and Eckel (38), the shortest period of survival follows pontine and medullary hemorrhage. Newbill (39) has studied the longevity of patients following cerebrovascular accidents. He included in this group brain hemorrhage, thrombosis and embolism. Since hemorrhage is the most common among these conditions, the statistical data of Newbill demonstrate that following cerebrovascular accidents 21.6 per cent of the patients die in the first twenty-four hours. The survival period was the shortest following lesions occurring in the brain stem, and the longest if the process was located in the hemispheres. In zo per cent of peptic ulcer cases, punctate hemorrhages were found in the hypothalamus and the medulla. These occurred under the ependyma of the third ventricle, below and above the hypothalamic sulcus, in the nucleus reuniens situated in the massa intermedia, in the paraventricular nucleus, the supraoptic nucleus and in the tuber cinereum (Fig. 3). Similar petechial hemorrhages have been detected in the medulla oblongata in the ala cinerea and in the dorsal motor nucleus of the vagus. These hemorrhages take place around the capillaries and veins and can be found between the vessel wall and the perivascular limiting membrane of glia (Fig. 4). Nevertheless, petechial bleedings can penetrate between groups of ganglion cells. Most hemorrhages were of recent character, but some were observed to be surrounded by glia cells; in such locations, cells containing hemosiderin have been found. Punctate hemorrhages of the brain were observed in association with peptic ulcer in young adults, zo to 3o years old. A number of cases in which petechial 394

bleedings were detected underwent an operation or died due to perforation of an ulcer which was of the acute type. We have concluded that the cerebral bleedings were primary, and the peptic ulcer followed the hemorrhage. The possibility that the punctate hemorrhages developed as a result of some circulatory disturbance or toxic effect cannot be excluded. Vonderahe (40) described in 1939 similar punctiform hemorrhages in cases of peptic ulcer. Although he has observed petechial hemorrhages in the hypothalamus ithout evidence of a peptic ulcer, such alterations never occurred in the medulla oblongata without being associated with a peptic ulcer. On the basis of these findings, Vonderahe expressed the opinion that punctiform hemorrhages in the medulla oblongata are more characteristic for peptic ulcer development than petechial bleedings in the hypothalamus. The relation between the stomach and the central nervous system has been studied by Neubürger (41,42). It appears that a reciprocal relationship exists: on the one hand the nervous system exercises an effect upon the stomach, and on the other hand the gastric function influences the central nervous system. Neubiirger has collected data on nonalcoholic cases of Wernicke's disease. He has observed that nonalcoholic Wernicke's disease occurs in the phase of progression of malignancy, especially in the final stage of gastric cancer. In such cases the corpora manaillaria and corpora quadrigemina were found to be particularly affected. In a subsequent publication, Neubürger has reported that chronic gastritis can be observed in association with Wernicke's disease. According to Neubürger, the alcoholic form of Wernicke's disease can be differentiated from the nonalcoholic form by the fact that in nonalcoholic cases hemorrhages preponderate, and serious parenchymal lesions are present.

FIG. 2. Autopsy specimens front a patient who ~. died following massive duodenal necrosis (B) which developed in consequence of a cerebral ili►. • hemorrhage in the region of the right basal ganglia (A).

FIG. 3. Autopsy specimens from a 31 year old man. (A) Left side punctate hemorrhages under the ependyma of the third ventricle, below and above the hypothalamic sulcus. (B) Perforated juxtapyloric peptic ulcer in the duodenum.

FIG. g. Section through the floor of the fourth ventricle demonstrating petechial perivascular hemorrhages beneath the floor (x so). A peptic ulcer of the stomach was present in this Individual.

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Vonderahe quotes the views of Wolff (43), who has attributed cerebral vasodilatation to direct afferent stimulation through the vagal and sympathetic routes. Thus, efferent stimuli arising in the brain may be of importance in the genesis of peptic ulcer. Such impulses are elaborated in massive and petechial hemorrhages. On the other hand, efferent stimuli originating from a peptic ulcer, and especially from peritoneum (in a case of perforation) may proceed to the brain and produce a vasodilatory effect. Dalgaard (44,45,46,47,48,49,50,5i ) has carried out in recent years a careful investigation of the neurogenic origin of peptic ulcerations. He has studied the alterations of the central nervous system which may be held responsible for the genesis of peptic ulcer in a large number of cases. Dalgaard's material included traumatic lesions of the brain, tumors with and without operative procedure, cases of infectious diseases, hemorrhage,

Curling's ulcers following burns, effects of intoxications and the corticosteroid therapy. He came to the conclusion that among these conditions, the vascular lesions of the brain represent the most frequent cause of peptic ulcer development. Acute ulcers can be observed as early as twelve hours after, but usually they can be detected a few days following the cerebrovascular accident. Dalgaard (5 t) has found that among 208 cases of acute peptic ulcer, cerebral vascular lesions were present in 32 per cent; in chronic peptic ulcer, 16.6 per cent were associated with cerebrovascular lesions. Our original impression was that cerebral hemorrhages are not associated with the development of a chronic ulcer, but with acute lesions only; however we became convinced by the accumulating evidence that chronic peptic ulcers do occur as a result of a cerebral hemorrhage.

Encephalomalacia and Peptic Ulcer Twenty per cent of peptic ulcers associated with nervous system lesions develop in consequence of softening of the brain (Fig. 5). Peptic ulcers following encephalomalacia occur mostly as seguela of arteriosclerosis, concomitant with an advanced age; however, embolism of a cerebral artery, with resulting encephalomalacia, leads to peptic ulcer development in young individuals as well. The site of brain softening is often the nucleus caudatus; the malacia of the head of the nucleus, especially, is frequently associated with peptic ulcer. Often large softenings, which include the caudate nucleus or the island of Reil, are complicated by peptic ulcer development. The relationship of certain localizations of encephalomalacia to peptic ulcer development has 396

been demonstrated by repeated observations of the association with lesions arising in these areas. Staemmler (52) described a case of a forty-two year old man who died a few hours following a gastric resection for a peptic ulcer. Autopsy demonstrated bilateral calcifications in the pallidum. Calcified concrements were found in the vessel walls; in consequence, the ganglion cells and medullated fibers were destroyed. It is well known that the calcification of the vessels of the pallidum, followed by symmetricla softenings, occur in consequence of carbon monoxide poisoning, however this etiological possibility could be excluded. Since the patient was an alcoholic, Staemmler believed that the brain lesion was attributable to this con-

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dition, and that it did not relate to the presence of the peptic ulcer with the central nervous system lesion. Overhof (53) has made a similar observation: a fifty-one year old male patient with an eight-year history of a peptic ulcer underwent gastroenterostomy, and died in consequence of a severe anemia. Post-mortem examination revealed bilateral softenings in the putamen and the pallidum. The wall of the blood vessels contained calcified concrements, and compound granular cells were found in the brain tissue in consequence of softening. Overhof considered the possibility of a carbon monoxide poisoning, but finally concluded that the alterations of the brain tissue were due to anemia. Scherer (54) observed a fatal hemorrhage in a peptic ulcer case in which autopsy demonstrated bilateral softenings of the pallidum. Histologically,

calcification of the arteries could be detected. All these observations seem to prove that softenings of the lenticular nuclei are in relation to the peptic ulcer. We have observed five cases of bilateral softenings in the lenticular nuclei which were associated with the presence of a peptic ulcer. It is our belief that softenings of the pallidum or putamen may result in peptic ulcer formation. Erbslöh (55) and others regard the bilateral pallidum necrosis as a sequela of anemia. In our opinion, in the coexistence of encephalomalacia and peptic ulcer, the encephalomalacia represents the primary lesion. Likewise the bilateral softenings of the pallidum, although their etiology remains undetermined, should be regarded as the primary alterations, and development of a peptic ulcer as a secondary effect.

Meningitis and Peptic Ulcer Inflammatory processes of the brain covering membranes may also be of etiological significance in the genesis of peptic ulcer. Hartung and Warkany (56) described a case of duodenal ulcer occurring in the course of meningococcus meningitis. Arteta (57) drew attention to the association of pneumococcus meningitis and peptic ulcer development. In our own material, we have observed acute peptic ulcers in consequence of an acute leptomeningitis, as well as in the course of a tuberculous meningitis. Dalgaard has observed cases of esophagomalacia and gastromalacia following acute and tuberculous meningitis, brain abscess and encephalitis. Much more characteristic is the occurrence of a peptic ulcer in the course of pachymeningitis. Charcot (27), Rössle (24) and Freisinger (58) et al. have suggested that hemorrhagic internal pachy-

meningitis may represent a causative factor in peptic ulcer development (Fig. 6). Bleeding occurs in the course of hemorrhagic internal pachymeningitis on the inner surface of the dura at certain intervals, and these hematorcas are from time to time organized. Occasionally one can follow the whole course of the disease, which is characterized by simultaneous presence of stellate scars in gastric epithelium and peptic ulcer. At the autopsy of a fifty-three year old man who died following the operation for a chronic peptic ulcer, we observed hypertrophic Pacchionian bodies on both sides of the superior sagittal sinus, producing deep depressions on the inner surface of the skull. The bodies were thickened by an accumulation of fibrous connective tissue. Large, chronic peptic ulcers can also be observed in cases of thickenings of the dura mater (Fig. 7). On rare occasions 397

V

/ ROLE OF THE CENTRAL NERVOUS SYSTEM

the dura may be five times thicker than normal. In this case a large chronic peptic ulcer was present, measuring 7 by 3.5 centimeters, the longer diameter being at right angles to the lesser curvature. The

ground of the ulcer was formed by the adherent pancreas. We have found the same changes in three other cases, and therefore it would be difficult to regard them as accidental findings.

Presence of Parasites in the Brain and Peptic Ulcer Parasites embedded in the brain have also been observed in association with a peptic ulcer (Fig. 8). Admittedly they are not easy to find; the best method appears to be to fix the brain in formalin, and then to dissect it in frontal slices. Using this method we have detected Cysticerci in the brain tissue of two cases of a chronic peptic ulcer. A brief summary of findings is given below: CASE i . A sixty-three year old male was admitted with a diagnosis of a chronic peptic ulcer of the pyloric region with penetration into the pancreas. Gastroenterostomy was carried out, and the patient died shortly afterwards. A hazel nut-sized brown area was found in the left putamen on autopsy. Histological studies revealed cholesterol crystals and cells filled with hemosiderin. In the

center, a scolex was located with suckers surrounded by calcified detritus. Rostellar hooks could not be detected. The parasite was classified as Cysticercus bovis. CASE 2. A spherical corpuscle, measuring 8 mm. in diameter, was found in the cerebral cortex on the convex surface of the left occipital lobe at the posterior end of the interparietal sulcus. The corpuscle was located immediately under the leptomeninges, at the same distance (of 5 cm.) from the longitudinal fissure and the left occipital pole, and was enclosed by fibrous connective tissue. Histological sections showed lymphocytic and plasma cell infiltration and foreign body giant cells. The Cysticercus could be recognized within the capsule in the midst of calcified material.

Peripheral Nerve System and Other Lesions Associated with Peptic Ulcer Among the peripheral lesions associated with peptic ulcer, we observed most frequently the lesions of the vagus nerve. Chronic peptic ulcers were observed in cases in which calcified lymph nodes were matted together with the vagus nerve. Malignant tumors, which infiltrate the stem of the vagus as well as enlarged lymph nodes, may cause compression of the nerve fibres. Besides the lesions mentioned in the preceding pages, other changes have been encountered in association with the de398

velopment of peptic ulcer. Single observations exist on cysts or calcifications of the choroid plexus which appear to be of etiological significance. We have previously described thrombosis of the superior sagittal sinus and the accompanying softening of the right frontal and parietal lobes which occurred in a threeyear old girl in whom four peptic ulcers were found in the duodenum. Schlumberger (S9) investigated the occurrence of peptic ulcer in children. There were seven cases of acute duodenal

FIG. 6. Autopsy specimens front a 45 year old patient who died following hemorrhagic internal pachymeningitis (4); two peptic ulcer scars are evident in the stomach (B). FIG. 5. Autopsy specimens demonstrating softening of the right caudate nucleus (A) and a perforated peptic ulcer of the anterior gastric wall; an ulcer scar of the posterior wall of the stomach is also visible (B).

FIG. 7. Autopsy specimens from a p year old man. (A) Transverse section of the dura demonstrates considerable thickening (right); transverse section through dura (left) is shown for demonstration of normal thickness. (B) Large chronic peptic ulcer measuring to x s cm. was found in the same individual.

V / ROLE OF THE. CENTRAL NERVOUS SYSTEM

ulcer, two cases of gastromalacia and one case of multiple acute gastric ulcer among 251 consecutive autopsies of infants and children. All these conditions were associated with the following cerebral lesions: three cases of tuberculous meningo-encephalitis, two cases of bulbar poliomyelitis, two cases of edema and anoxia, one case each of intraventricular hemorrhage, of sinus thrombosis and of microcephaly. The question arises how often does the peptic ulcer occur as a result of these lesions of the nervous system. According to the data of Greiss (6o), peptic ulcer is not commonly found in association with the pathological conditions of the nervous system. Out of 138 cases of peptic ulcer Greiss found seven cases (5 per cent) which followed meningitis, encephalitis, cerebrovascular accident or myelitis. On the other hand, Krech (61) observed that

in serious cerebral diseases, peptic ulcer occurs in 12.7 per cent of the cases, while in 42.7 per cent it is associated with milder or serious alterations in the nervous system. Hart has observed an incidence of 17.4 per cent of diseases of the brain in his series. Vonderahe reported pathological alterations in the brain in 21.6 per cent of his peptic ulcer material. The question of the incidence of nervous system lesion in peptic ulcer cases can be correctly answered only after a comprehensive examination of the central and peripheral nervous system in all cases of ulcerations of the upper alimentary canal. The fact remains that in the records of most cases of peptic ulcer, the data on post-mortem examination of the brain or results of histological studies are lacking.

Summary Pavlov has demonstrated that the cerebral cortex and psychic processes affect the gastric secretory process, and he was the first to recognize the psychic stimulation of the secretory flow. Bykow and Kurzin developed the corticovisceral theory of peptic ulcer formation. According to this theory the genesis of the peptic ulcer is explained on the basis of a pathological integration of the relationships between the cortical perception, the environment and the internal organs. The conflict of exteroceptive and interoceptive stimuli and exhaustion of the cerebral cortical function leads to an abnormal (chaotic) function of subcortical centers. Peptic ulcers develop from pathogenic

400

centrifugal impulses reaching the stomach. Evidence has been submitted that in the majority of peptic ulcer cases studied, discernible lesions can be demonstrated either histologically or anatomically in the central or the peripheral nervous system. Massive and puncti f orm hemorrhages appear to be the most frequently found lesions. Less commonly encountered are: encephalomalacia, meningitis, parasites embedded in the brain tissue, and lesions of the peripheral nervous system. The use of routine autopsy protocols without detailed examination of the brain is insufficient for assessment of the incidence of cerebral lesions in peptic ulcer cases.

BALG

References 1. ROKITANSKY, c. Handbuch spez. path. Anat. Vol. II, 195. Vienna, Braumüller and Seidel, 1942. 2. HOFFMANN, C. E. E. Virchows. Arch. Path. Anat. 44:52-365, 1868. 3. ARNDT, R. Deutsch. Med. Wschr. 14: 83-85, 1888. 4. MOGILNITZKY, B. N. Virchows. Arch. Path. Anat. 257: 109-118, 192 5. 5. KORST, L. Ztschr. ges. Neurol. Psychiat. 107: 553 1953• 6. MEYER, H. H. Med. Klin. 8: 1773, 1912. 7. EPPINGER, H. and HESS, L. Z. klin. Med. 68: 205-230, 1909. 8. VON BERGMANN, G. Deutsch. Med. Wschr. 65: 1069-1073, 1939. 9. KIDD, C .w. rit. Med. J. /:9-45o, 19 3 8. 10. CUSHING, H. Surg. Gynec. Obstet. 55: 1-23, 1932. 11. GRANT, F. C. Ann. Surg. ror: 156-166, 1935. 12. SCHIFF, M. De va motoria baseos encephala inquisitions experimentales. Frankfurt, M. Bockenkeim, 1845. 13. BROWN-SEQUARD, C. E. Progres ,Med. 4: 136, 1876. 14. EBSTEIN, W. Arch. exper. Path. Pharmakol. Leipz. 2: 183-195, 1874. 15. BURDENKO, N. and MOGILNITZKY, B. Ztschr. ges. Neurol. Psychiat. 103: 42-62, 1926. 16. WATTS, J. w. and FULTON, J. F. Ann. Surg. rot: 363-372, 1935. 17. HOFF, E. C. and SHEEHAN, D. Amer. J. Path. ii: 789-802, 2935. 18. OBERLING, CH. and KALLO, A. C. R. Soc. Biol., (Par), toe: 832-833, 1929. 19. KALLO, A. and KORPASSY, B. Z. Ges. Exp. Med. 77. 533-537, 1931. 20. PAVLOV, L P. The Work of the Digestive Glands. 2nd Eng. ed., trans. by W. H. Thompson. London, Griffin, 191o. 21. PAVLOV, 1. P. Conditioned Reflexes. An Investigation of the Physiological Activity of the Cerebral Cortex. London, Oxford, 1927. 22. BYKOW, K. M. and KURZIN, 1. T. Kortiko-viszerale Pathogenese der Ulkuskrankheit. Berlin, Volk and Gesundheit, 1954. 23. AL'PERN, D. E. Acetilcholin v krovi pri jazvcnnoj bolczni. Tezisü dokladov i vüsztuplenii na konferencii po lecsebnomu pitamlu, 1950. 24. RÖSSLE, R. Mitt. Grenzgeb. Med. Chir. 25: 766, 1913. 25. GUBERGRIC, M. M. Therapeut. Archiv. 2:: 3, 1949• z6. LEPINE, R. Revue Med. 15: 51 4, 1895. 27. cHARcor, quoted by Mogilnitzky, Virchow Arch. Path. Anat. 2f7: 1o9-118, 1925. 28. HART, C. Frankfurt. Z. Path. r3: 242-248, 1913. 29. HART, C. Med. Klin. to: 363-365, 1914. 30. HART, C. Mitt. Grenzgeb. Med. Chir. 31: 350-380, 1919.

31. HALO, J. Math. u. Naturwiss. Anz. d. Ung. Akad. d. Wiss., 6o: Part II; 545, Budpest, 1941 . 32. BALG, J. Magy. Tud. Akad (Biol Orv) 2: 1 57, 1951 • 33. BALO, J. Acta Morph. 1: 167-181, 1951 (R). 34. BALO, J. Schweiz. Z. Path. Bakt. 21: 56162 1958. O, j Deutsch. Med. Wschr 67: 479-482, 35. BAL. 1941. 36. BALO, J. Wien. klin. Wschr. S4: 326-327, 1941. 37. JONES, A. E., Brain 28: 527-555, 1905. 38. WINKELMAN, N. w. and ECKEL, J. L. J. Nerv, Ment. Dis. 61: 593-602, 1925. 39• NEWBILL, H. P. J.A.M.A., 114: 236-237, 1940. 40. VONDERAHE, A. R. Arch. Neurol. Psychiat. 4/: 871-912, 41. UÜGE, R K. Virchows Arch. Path. Anat. 298: 68-86, 1936. 42. NEUBÜRGER, K. Z. ges. Neurol. Psychiat. ,6o: 208-225, 1938. 43. WOLFF, H. G. Physiol. Rev. 16: 545-596, 1936. 44. DALGAARD, J. B. J. Forensic Med. 4: 120, 1957. 45. DALGAARD, J. B. Acta Path. Microbiol. Scand. 41: 1-18, 1957. 46. DALGAARD, J. B. J. Forensic Med. 5: 16, 1958. 47- DALGAARD, J. B. Acta Path. et Microbiol. Scand. 42: 313-323, 1958. 48. DALGAAR I, J. B. J. Forensic Sci. 4: 412, 1959. 49. DALGAARD, J. B. Gastroenterology, 37: 28-34, 1959. 5o. DALGAARD, J. B. Acta neurochirurgica (Wien). 7: 1, 1959. 51. DALGAARD, J. B. A.M.A. Arch. Path. 69: 359370, 1960. 52. STAEMMLER, M. Beirr. Path. Anat. 71: 503513, 1922-23. 53. OVERHOF, K. Virchows Arch. Path. Anat. 287: 784-789, 1932-33. 54. SCHERER, E. J. Ztschr. f. d. ges. Neur. u. Psych. tso: 6 2-639, 1934. 55. ERIISLÖH, F. Handbuch der speziellen patologischen Anatomie und Histologie Lubarsch, Henke, Rössle, ed. Vol. 13, Part ll, 1501, 1958. 56. HARTUNG, C. A. and WARKANY, J. J.A.M.A. 110: 1101-1103, 1938. 57. ARTETA, J. t.. Brit. Med. J. 2: 580-582, 1951. 58. FREISINGER, F., LAPIS, K. and BALG, J. Orvosi Hetilap 91; 801 Hungarian 195o. S9. SCHLUMBERGER, H. G. A.M.A. Arch. Path. 52: 43-66, 1951. 6o. GNEISS, FR. Zur Statistik des runden Magengeschwures. 1045. Kiel, M.D. Thesis, 1879. 61. KRECH, USE, Beziehungen des Ulcus ventriculi, der hämorrhagischen Erosionen des Magens, der Gastro-und Osophagomalazie zu Veranderungen des Zentralnervensystems. M.D. Thesis, University of Erlangen, 1922.

401

Neurogenic Factors in Experimental Peptic Ulceration

David A. Brodie*

THE neurogenic production of experi-

cusses the work of Wolf and Wolff on mental gastric ulcers has been an interest- their fistulous subject, Tom, in this coning field of study, since ulcers can be pro- nection. It was shown that when Tom duced seemingly without harming the was subjected to acute or chronic periods animal. This situation is often considered of emotional stress, he had an increase of to produce the counterpart of the "emo- gastric acidity. Studies by Shay and cotional" or "psychogenic" ulcer in man. workers (3) demonstrated that emotional While the exact interplay of "emotions" stress in a duodenal ulcer patient increased and gastric activity is not clear, it has total acid output to over four times that been demonstrated that various brain of basal levels. Eichhorn and Tracktir areas are profoundly concerned with gas- (4) concluded from a study of gastric trointestinal activity, and it is assumed secretory activity in patients with hypthat emotional disturbances act through notically-induced emotions, that there these areas to derange gastric function. was no simple relationship between emoEliasson (1), in his review of the central tions and gastric secretion, but rather that control of digestive function, indicates an interaction of emotion with anxiety that electrical stimulation of both cortical levels altered acid secretion. Thus, it and subcortical areas can increase gastric would appear that emotional stress can be acidity; but the studies of the effects of a factor in the etiology of peptic ulcer, emotion on gastric secretion have shown but the importance of its röle is still unthat the alterations in gastric activity are certain. much more variable and unpredictable In the experimental ulcer situation, anithan those produced by electrical stimula- mals can be subjected to a number of tion of a particular brain area. situations which alter "emotionality," or There have been several studies in the produce "anger" or "fear." When gastric human which demonstrated that emotion or duodenal lesions occur in these situacan increase gastric acidity, and on this tions, it would appear that the central nerbasis, "emotional stress" has come to be vous system is involved, but whether considered as an important factor in the there is an "emotional" change is difficult etiology of peptic ulcer. James (2) dis- to assess in an organism that cannot ver"From the Merck Institute for Therapeutic Research, West Point, U.S.A. 403

V / NEUROGENIC FACTORS IN EXPERIMENTAL ULCER

balize. Nevertheless, it is possible to induce gastric ulcers in animals with various conflict situations or with restraint procedures which support the notion of an important central nervous system factor in the etiology of these lesions. However, the mechanism through which the central factors alter gastric function is still not clear. Selye (5) first reported that exposure of rats to many different types of noxious stimuli, such as immobilization, injection of drugs, exposure to cold or surgical trauma, produced a similar syndrome involving widespread tissue damage, including clearly defined gastric ulcers. These stimuli were classified as nonspecific stresses, since they altered the function of

several systems. It was postulated that certain of these factors produced emotional disturbances in the animals which increased adrenal function, and one of the consequences of this was the appearance of a "neurogenic" ulcer. Since certain behavioral situations cause what appears to be "emotional" changes in animals, such as fear or rage, this area of research has been extensively investigated. Porter and co-workers (6) have investigated several behavioral conflict situations in monkeys, and have used these behavioral methods to induce a high incidence of gastric and duodenal ulcers in monkeys. This technique appears to be the closest experimental analogy to clinical peptic ulcer.

Methods of Induction of Neurogenie Ulcers Although there are a host of nonspecific stressers which rapidly induce gastric ulcers in rats, the one most often used is that of immobilization or restraint of the animal. This procedure does not subject the animal to any observable trauma, yet a reliably high percentage of ulcers can be induced in twenty-four hours by this method. One of the differences in the work of various investigators is the manner in which the animals are immobilized. In Selye's original work, the rats were restrained by sectioning the spinal cord, wrapping the rat in a towel or binding the legs together with adhesive tape (5). French workers have used an elaborate method (7), which involves anesthetizing the rat, putting it into a wire screen with the limbs protruding through the wire and taped together, and then suspending the "caged" rat from a burette clamp. Sines (8) restrained rats by wrapping them in a plaster of Paris bandage. Brodie 404

and Hanson (q) placed wire window screen around the rats and held it in place with staples. It appears that the method of restraint is unimportant, since similar lesions occurred with each of the various techniques, and the incidence of the ulcers after twenty-four hours of restraint was always 85 to wo per cent. The lesions produced by acute restraint stress have the following characteristics: tney occur only in the fundus (acid secreting) portion of the rat stomach, never in the rumen (nonglandular) portion; they are lesions of the gastric mucosa and do not penetrate into the muscularis mucosae, and they heal completely without scarring in four to seven days (9,10). A study of food residue of the stomach of restrained rats indicated that restraint also caused the stomach to empty more rapidly (q). A detailed study of the histology of the restraint-induced lesions was reported by Bonfils et al. (i i ). They found that the lesions were erosions of the gas-

BRODIE

tric mucosa which were surrounded by an area of local edema. There were also numerous capillary pits (areas of intense vasodilatation) in the gastric mucosa. Although there was a great deal of hemorrhage in the stomach, perforations never occurred. Behavioral techniques for the production of neurogenic ulcers have been described in rats by Sawrey and Weisz (12,13). These workers used a conflict situation in which the rats were shocked when they obtained food and water. This schedule produced rumen ulcers and fundic hemorrhages in rats in approximately thirty days. Porter and co-workers (6) have developed techniques for the production of neurogenic ulcers in monkeys. These workers used a variety of behavior stress situations among which were conditioned "anxiety," conditioned "punishment" and conditioned "avoidance" situations in rhesus monkeys. Some animals were run on combinations of the behavioral stresses. Eleven of nineteen monkeys tested in this type of situation died or became moribund, and were sacrificed in two to seventeen weeks from the beginning of the experiment. Each of the eleven monkeys showed some pathology in the gastrointestinal tract, and four of these animals

had extensive hemorrhages and/or erosions of the gastric mucosa, and five animals had duodenal ulcers. Since it was difficult to pin-point the type of behavioral stress which produced the gastrointestinal lesions, a test was designed based on a conditioned avoidance (6,14). This experiment, a "yoked-chair" avoidance procedure, has become famous as the "executive-worker" monkey experiments. In this experiment, four pairs of monkeys were trained in an avoidance procedure in which only one animal of each pair (the executive monkey) could delay the shock for itself and its partner (the worker monkey). The shock was delayed if the monkey pressed a lever every twenty seconds during a six-hour avoidance session, which was signaled by a red light. The avoidance session was alternated with a six-hour off period. After a training period, the shock rates during the avoidance sessions never exceeded two per hour. Three of the four "executive monkeys" died within twentyfive days, and one was sacrificed in a moribund condition in forty-eight days. All executive (experimental) monkeys had gastrointestinal lesions, while none of the "worker" (control) animals had gastrointestinal complications.

Effects of Neurogenic Stress on Gastric Secretion The increasing use of neurogenic ulcer techniques has led to research designed to study the mechanisms related to the gastric pathology. In human ulcers, it is a clinical dictum "no acid — no ulcer" so that much of the work on the mechanism of neurogenic ulcers has been directed toward study of the effect of stress on gastric acidity.

Several investigators have studied the effect of vagotomy (9,15,1 6) on restraint ulcers, and it has been repeatedly demonstrated that bilateral vagotomy reduces the incidence of these lesions. Studies on the effects of anticholinergic agents, which can be considered to produce chemical vagotomy, on ulcer incidence (1 7) added support to this observation 405

25

CHRONIC FISTULA RATS

J

w

20

VOLUME

PYLORUS LIGHTED RATS

VOLUME

15

Z 10 •

5

5

0

since it was possible to abolish restraint120 TO-AL ACO kt-r.ti TOTAL ACID induced lesions by the use of atropine or 100 scopolamine methylbromide. These stu- Jr 80 E _ dies suggest that gastric acid secretion c 60 plays a role in the production of restraint -J ulcers. The first investigation of the ef✓ 40O Ifects of restraint on gastric secretion was 20 done by Menguy (16) in the pylorus0 ligated rat. Although he confirmed the fact that vagotomy prevented restraint ulloo FREE ACID FREE ACID cers in rats, he found that restraint signi- 80 ficantly decreased free acid output in a six-hour pylorus-ligated rat. Thus, Men- E60-guy postulated that acid-pepsin digestion R40- • W N was not basic to the production of re- ig 20•---- RESTRAINED •~— UNRESTRAINED straint ulcers, and the protection pro0 0 vided by vagotomy was due to an altera0 4 8 12 16 20 24 4 8 12 16 20 TIME PERIOD-HOURS TIME PERIOD-HOURS tion in gastric blood flow or delayed gastric emptying. FIG. 2. The effect of restraint on gastric secreSince the pylorus-ligated rat prepara- tion in chronic fistula rats and pylorus-ligated rats. Values for the chronic fistula rats are tion secretes highly acid gastric juice, it averages obtained from the sante chronic fistula seemed to us that it would be valuable to rats studied in each time period hi both the reand unrestrained state, and from groups study the effects of restraint on gastric strained of to pylorus-ligated rats studied at each time secretion in the chronic fistula rat which period. The volumes of gastric juice are cumuhas not had operative trauma immediately lative values, but free and total acid values are obtained at each individual four-hour prior to restraint, and to compare this averages collection period. with the effects of restraint in pylorusligated rats (18,19). Stainless steel cannulas were implanted juice was collected from the chronic fisin the rumen portion of the stomach of tula rats. In order to minimize dehydrasixteen male Holtzman rats (average tion, a subcutaneous injection of 0.9 per weight 196 gm.). One week after the cent sodium chloride solution, equal to operation the rats were starved for twen- the volume of gastric juice secreted, was ty-four hours, and then gastric juice was given to the chronic rats every four hours. collected over a twenty-four hour period The pylorus-ligated rats were given a from the unrestrained animals (Fig. 1). standard subcutaneous injection of 4 ml. Six days later the same rats were starved of saline solution every four hours. Fourfor twenty-four hours, then restrained in hour collection periods were used to obwire screen, without prior anesthesia, and tain gastric juice from the chronic fistula gastric secretion was again collected over rats over a twenty-four hour period, and a twenty-four hour period. Pylorus- a group of pylorus-ligated rats was autopligated rats (average weight, 163 gm.) sied at each of the six collection periods. were prepared by the method described The four-hour collection period samples by Shay et al. (14). Gastric secretion was of gastric juice were measured for volstudied in groups of ten pylorus-ligated ume, free acidity and total acidity. Acidrats, both restrained and unrestrained, ity measurements were carried out on a during the same time intervals that gastric Beckman Zeromatic pH meter by titra406

O cm 1 FIG. t. Unrestrained chronic gastric fistula rat illustrating method of attaching spring-covered polyethylene tube with collecting test tube. The rat is free to move in the limits of its cage while gastric juice is collected. The inset shows size of catheter and connector.

2

3

4

BRODI Ii

tion with N/zoo sodium hydroxide to pH 3.5 for free acid and pH 8.5 for total acid. The changes produced in the volume of gastric secretion are illustrated in panels a and d of Fig. z. In the group of chronic fistula rats, the rate of gastric secretion after the first collection period averaged 3.o ml./4 hours in the unrestrained rats and 2.3 ml./4 hours for the restrained animals. The restraint procedures significantly reduced the volume of gastric secretion at all time periods studied, as shown in Table I. In contrast to the increasing volume secreted by the chronic rats, the volume of gastric secretion in the unrestrained pylorus-ligated rats decreased after twelve hours. Restraint of the pylorus-ligated rats produced a significant reduction in the volume of secretion for the first three and the final collection periods.

Gastric acid secretion in the chronic unrestrained rat was characterized by a steady increase during the twenty-four hour test period (Fig. zb, 2c). When the chronic animals were restrained, both free and total acidity increased significantly, and free acid remained at significantly higher levels for the entire twentyfour-hour test period, while total acid was significantly increased for the first five collection periods (Table I). The greatest increase in acidity was found during the first four-hour period, immediately following the initiation of restraint. The pattern of acid secretion in the pylorus-ligated animals was quite different from that of the chronic fistula rats (Fig. 2e, 2f). Both free and total acid values of the unrestrained pylorus-ligated rats decreased during the experimental

TABLE I CHRONIC GASTRIC FISTULA RA TS' Volume (ml) Free Acid mEq./1 TIME PERIOD

Difference R to U 3

4 Hours 8 Hours 12 Hours 16 Hours 20 Hours 24 Hours

-1.4 -2.1 -2.9 -3.8 -4.3 -4.8

p Value

Difference R to U

< .005 .05 > .05 > .05 > .05 > .05 > .05

P Y LORUS-LIGATED IM TS2

TIME PERIOD

(Hours)

Unrestrained

4 8 12 16 20 24

483.1 763.8 785.0 481.8 338.4 168.0

Restrained 354.7 481.6 621.8 383.2 304.6 219.0

Difference -128.4 -282.2 -163.2 - 98.6 - 33.8 + 51.0

'N =16 rats per group, p Value obtained by t test, paired comparisons (df =15). Values based on cumulative volumes. =N =10 rats per group, p Value obtained by t test (df =18). 4.08

p Value

.05 >.05 > .05

BRODIE

gastric acid concentration, then the pro- logical changes. The average free acid tection reported with vagotomy and anti- output was found to rise during the cholinergic drugs would be related to the second week in all monkeys. Only two prevention of this increase in acidity. monkeys were found to have a sharp rise Data in support of this idea have been ob- in acid concentration, which peaked in tained from preliminary experiments in the second week and declined during the this laboratory which showed that atro- remaining two weeks of the test. No corpine sulfate, in doses that significantly re- relations could be found between acid duced restraint ulcers (17), abolished levels, urinary steroid values or lever free acid secretion in the restrained chro- pressing rates. On autopsy two of the nic fistula rat. monkeys had duodenal erosions. These Since the behavioral stress was active two animals had levels of free acid and in producing gastrointestinal lesions, the showed a progressively increasing avoidgroup from Walter Reed Hospital carried ance lever pressing rate. These studies indicate that the effects out a study of gastric secretion in monkeys trained on the same conditioned of chronic behavioral experiment is not avoidance procedure. Dr. Edwin Polish simply an extension of the acute situation. has kindly given me permission to sum- The etiology of these lesions appears to marize their observations (21). The stu- have increased acidity as an important dies carried out by Polish and co-workers factor, but these increases may occur were divided into an acute and a chronic after the stress period rather than during phase. Monkeys were trained to press a the noxious stimulation. Unpublished lever once every twenty seconds to avoid work by Hawson and Brodie confirms a foot shock during a six-hour avoidance the effect of acute avoidance training on period, which was signaled by a red light. gastric secretion in rats. These workers After training was complete, the animal found that chronic fistula rats trained in was tested during one six-hour avoidance an avoidance situation showed marked desession and a six-hour postavoidance pression of gastric acidity during the period. During this time, gastric juice was avoidance session. collected through a gastric cannula, and Although the studies on gastric secrethe contents were analyzed for free and tion indicate that stress alters gastric acid total acidity. Three of six monkeys were secretion, it is not necessary, as James found to have free acid in their gastric points out (2), that this effect is mediated juice. The avoidance session suppressed through the vagus nerve. Gray (22) has gastric secretion in these monkeys, and reviewed the effects of endocrine changes there was a rebound of free acid in the on the gastrointestinal tract, and has indicated that gastric acidity can be inrest period. Nine gastric fistula monkeys were creased by adrenocortical stimulation. It placed in the conditioned avoidance situa- has also been shown that one of the prime tion for a chronic study, and gastric effects of neurogenic stress is to stimulate samples were collected three days of the adrenal secretion. Thus, it is possible that week. The test was run six hours on, six neurogenic stress produces ulcers via a hours off, for a four-week period, at the hormonal pathway, via the hypothalamicend of which time the animals were sacri- pituitary adrenal axis, as well as through ficed, and the viscera examined for patho- central vagal stimulation.

409

v/

NEUROGENIC FACTORS IN EXPERIMENTAL ULCER

Concluding Comments The two neurogenic ulcer techniques, restraint and behavioral stress, that have been discussed clearly have a central nervous system component, since no drugs are given to the animal, and there is no surgical intervention to induce the lesions. In the case of restraint, the secretion of highly acid gastric juice appears to be an important factor in the induction of the gastric lesions. However, since vagotomy is only partially effective in preventing the ulcers and there is histological evidence of marked vascular changes, it seems possible that a vascular factor (gastric blood flow or vascular tone of mucosal blood vessels) may be important in the etiology of the

restraint ulcer. In the behavioral situation, an increase in gastric acidity is also a factor in the chronic situation. However, in this case the increase in acidity occurs much later than with restraint, and acid increases during the nonstress periods rather than during a period of noxious situation as with restraint. In this situation it appears that the central nervous system activity may be mediated through hormonal as well as nervous mechanism. It is possible that suppression of gastric acid could be due to epinephrine and/or norepinephrine release, and the rebound of acid due to increase in adrenal steroid output.

Summary The effect o f neurogenic stress was studied in rats which were restrained and in monkeys which were subjected to behavioral stress. Chronic gastric fistula rats were prepared by implanting stainless steel cannulas in the rumen portion of the stomach of male Holtzman rats, and gastric content was collected in both the unrestrained and restrained state. Gastric secretion in acute pylorus-ligated rats, unrestrained and restrained, was studied at the same time. Volume of gastric content, free and total acidity and free acid output were significantly lower in the chronic fistula rats as compared to the pylorusligated rats in the initial four-hour collection period. A study of twenty-four-hour gastric content in chronic fistula rats showed that restraint produced a significant decrease in volume, a significant increase in free and total acid concentration

410

and no change in free acid output, while the restrained pylorus-ligated rats had a significant decrease in volume, no change in free or total acid concentration and a significant decrease in free acid output as compared to control values. This study suggested that an increase in acid concentration is an important change produced in gastric secretion by restraint stress. In monkeys in a conditioned avoidance situation, gastric samples were collected in acute and chronic tests. In the acute test, the avoidance situation suppressed gastric secretion, and there was a rebound of free acid in the rest period. In the chronic test, the average free acid output rose during the second week, and in two monkeys that had duodenal lesions on autopsy, there were elevated levels of free acid.

BRODIE

References I. ELIASSON, S. V. Amer. Physiol. Soc., Washington, D.C. 2: 1163-1172, 1960. siology of gastric di2. JAMES, A. H. The phy gestion. London, Arnold, 1957. 3. SHAY, H., SUN, D. C. H., OLIN, G. and WEISS, E. J. Appl. Physiol., 12: 461-467, 1958. 4. EICHHORN, It. and TRACKTIR, .3. Gastroenterology, 29: 417-421, 1955. 5. SELVE, H. Nature (Lond). 138: 32, 1936. 6. PORTER, R. W., BRADY, J. V., CONRAD, D., MASON, J. W. GALAMBOS, R. and MCK, RIOCH, D. Psychosom. Med., 20: 379-394, 1958. 7. ROSSI, G., BONFILS, S., LIEFOOGHE, G. and LAMBLING, A. C. R. Soc. Biol., (Par). 150: 21242126, 1956. 8. SINES, J. O. J. Comp. Physiol. Psychol., 52: 61 5-6 1 7, 1959. 9. BRODIE, D. A. and HANSON, H. M. Gastroenterology, 38: 353-360, 1960. 10. BONFILS, S., ROSSI, G., LIEFOOGHE, G., and !AMBLING, A. Rev. Franc. etudes din. et biol., 4: 146, 1959. I1. BONFILS, S., RICHIR, C., POTET, F., LIEFOOGHE, G. and LAMBLING, A. Rev. Franc. etudes din. et biol., 4: 888, 1959. 12. SAWREY, W. L. and WEISZ, J. D. J. Comp.

Physiol. Psychol., 49: 266-270, 1956. 13. WEISZ, J. D. Psychosom. Med., l9: 61-73, 1957. 14. BRADY, J. V., PORTER, R. W., CONRAD, D. G. and MASON, J. W. J. Exp. Anal. Behay., l: 69-72, 1958. 15. BONFILS, S., LIEFOOGHE, G., ROSSI, C. and LAMBUNG, A. Arch. Mal. App. Digestif, 48: 449459, 1959. 16. MENCUY, R. Amer. J. Dig. Dis., 5: 91I-916, 1960. 17. HANSON, H. M. and BRODIE, D. A. J. Appl. Physiol., 15: 241-294, 1960. 18. BRODLE„ D. A., MARSHALL, It. W. and MORENO, O. M. Physiologist, 4: 14, 1961. 19. BRODIE, D. A., MARSHALL, R. W. and MORENO, O. M. Amer. J. Physiol. 202: 812-814, 1962. 20. SHAY, H., KOMAROV, S. A., FELS, S. S., MERANZE, D., GRUENSTEIN, M. and SIPLET, H. Gastroenterology, 5: 43-61, 1945. 21. POLISH, E., BRADY, J. V., MASON, J. W., THATCH, J. and NIEMECH, W. Gastroenterology 34: 193-201, 1962. 22. GRAY, S. J. Amer. J. Dig. Dis., 6: 355-371, 1961.

411

Effects of Corticotrophin Release Produced by Pitressin and Pitocin on Gastric Secretion

to be well established that hypothalamus produces a chemical mediator concerned with corticotrophin (ACTH) release (1). It has been also suggested that the presence of hypothalamic centers is essential for ACTH release (z). Although the recent studies have demonstrated the presence of the hypothalamic ACTH releasing factor in the peripheral blood (3), it appears logical to assume that the transport along the hypothalamic-hypophysial tracts and/or through the hypophysial portal vessels represents the preferred route. Considerable evidence has been accumulated pointing to the presence of a substance in extracts of median eminence and posterior pituitary that is capable of causing ACTH release (4,5). Certain experimental data lead to the belief that the ultimate mediator of ACTH release is vasopressin (6,7); however, convincing evidence suggests that it differs from vasopressin (5,8,9). In view of the demonstration of Karplus and Peczenik (io), that the amount of oxytocic and melanophoreexpanding substances in the cerebrospinal fluid was increased by electrical stimulation of the tuber cinereum, the posterior pituitary hormones have also been assumed to discharge, at least in part, into IT APPEARS

Keizo Ishihara*

the ventricular fluid and to reach from there the adenohypophysis, via portal vessels. Already Cushing (11) observed in surgical patients that pituitrin and pilocarpine, when injected into the cerebral ventricle, had a similar action in producing widespread vasodilatation, sweating, vomiting and lowering of the body temperature, suggesting a central autonomic stimulation predominantly of the parasympathetic division. Cushing has pointed out that intraventricular injection of pituitrin had a profoundly different effect from that following the intravenous administration. Regarding pitressin and pitocin, however, he observed no comparable effects following administration of pitressin and pitocin when one milliliter of the commercial preparations was injected intraventricularly to three patients who had previously shown a marked reaction to similar doses of pituitrin injected into the ventricle. Dodds and associates (i 2) showed that subcutaneous or intravenous injection of large doses of posterior lobe extracts produced ulcerative lesions in the stomach of rabbits; the active extract inhibited the gastric secretory response to histamine. Dutton and Ivy (13) have administered pituitrin sub-

'From the Department of Surgery, Gunma University School of Medicine, Maebashi, Japan. 413

V / EFFECTS OF CORTICOTROPHIN RELEASE

cutaneously to dogs hourly for two weeks in doses adequate to cause almost complete anuria, without observing occurrence of ulcers. Our previous reports (14,15) have shown mat cinchophen causes a rise in neurosecretion of the hypothalamus, and that pituitary stalk section suppressed ulcer production by this compound. In contrast with histamine and insulin, cinchophen produced gastric juice very rich in pepsin after a latent period of three hours, reaching a peak in five to six hours. This response was blocked by bilateral adrenalectomy as well as pituitary stalk section, but not by denervation of the stomach; contrary to insulin response, histamine response was not altered by these operations. These observations have suggested the existence of humoral mechanism transmitted through the hypothalamicpituitary-adrenal pathway. Gray and associates (16) have demonstrated an increase in gastric secretion of hydrochloric acid (HCI) and pepsin following prolonged administration of adrenocorticotrophic hormone (ACTH) in man. A single injection of 40 IU of crystalline ACTH (Parke-Davis) to dogs produced a consistent increase in the concentration and hourly ouptut of acid, pepsin and chlorides in gastric juice of intact, vagally denervated, and antrectomized animals( 17). This response to ACTH characteristically began after a latent period of three to four hours, reaching a peak in five to six hours, and was abolished by bilateral adrenalectomy. It is noteworthy that the gastric response to ACTH closely resembled that of cinchophen. Porter and associates (18) have shown in the cyclopropane-procaine-anesthetized monkey that electrical stimulation of the anterior hypothalamus at the level of the opic chiasma evokes a gastric HC1 response in 3o to 6o minutes, with return to 414

the prestimulation secretory levels within three hours. This response was presumably mediated by the vagus nerves and could not be demonstrated following vagotomy. The stimulation of the posterior hypothalamus at the level of the manuniliary bodies or tuber cinereznn produced a delayed rise in acidity, reaching a maximum at three hours and declining to the prestimulation levels in five hours; this could be demonstrated after vagotomy but not following adrenalectomy. The above investigators have also found that insulin caused a prompt and delayed response which could be abolished by vagotomy plus adrenalectomy. These observations appear to suggest a hypothalamic source for the stimulation of gastric secretion through vagal and adrenal mechanisms. However, Zukoski et al. (19) have demonstrated that electrical stimulation of the anterior and posterior hypothalamus had no effect on gastric HCl secretion in nonanesthetized dogs; and the stimulation of the median eminence area produced a marked increase in adrenal corticosteroid secretion without producing an increased HC1 secretion. In an attempt to elucidate the pathogenesis of peptic ulcer, the experimental cinchophen ulcer was selected. The method appeared to us suitable because of its high incidence, the close resemblance of the lesions produced to the typical peptic ulcer in man, as far as location and appearance are concerned, and the tendency of these ulcers to penetration, perforation and bleeding. As mentioned, our previous studies have suggested the existence of humoral mechanism transmitted through the hypothalamic-pituitary-adrenal axis (14). On the basis of these findings, our research was directed toward shedding light on the humoral mechanism involved. It is the purpose of this paper to describe the ef-

ISHIHARA

fects of posterior lobe preparations, in- anesthetized dogs, on gastric secretion. jected into the lateral ventricle of non-

Materials and Methods MATERIALS AND METHOD6

Partial gastric pouches were prepared in fifteen adult, male, mongrel dogs weighing 12 to 18 kg. and maintained on a diet consisting of waste meals from the University Hospital. The pouch operation was performed aseptically under sodium pentothal anesthesia using the standard supra-umbilical approach. Two types of partial gastric pouches were prepared: the vagus-denervated pouch of Heidenhain and the vagus-innervated pouch of Pavlov. Pituitary stalk section and hypophysectomy were carried out eight to ten days after pouch operation through a transbuccal approach. In each case, an attempt was made to sever the pituitary stalk under direct vision, and as close to the hypothalamus as possible. After stalk section, a barrier of film plate, 2 mm. X 5 mm. in size, was inserted between the cut ends of the stalk in an attempt to prevent vascular regeneration. The operation result was ultimately verified by microscopic sections. At the time of pituitary operations, the left parietal bone was trephined at a point located in the middle of the distance between orbita and external auditory foramen and 0.5 cm. lateral from the median line for the purpose of a later intraventricular injection. About one week afterwards, a polyethylene tube, o.8 mm. in diameter, was introduced into the left lateral ventricle through a small slit in the dura mater, made at the center of the trephined hole using a sharp knife. By means of the tube, 0.5 ml. of test materials was substituted for ventricular fluid very slowly after being warmed up to 37°C.

These procedures could be easily performed without the aid of anesthesia. Subarachnoid injection was carried out in a similar manner. For cisternal injection, the cerebromedullary cistern was punctured through the occipital scalp utilizing a lumbar puncture needle. Intravenous injection was carried out into a subcutaneous vein of the leg; subcutaneous and intramuscular injections were made into the hip. Following recovery from operation, the animals were trained to stand in a frame to facilitate collection of gastric juice into test tubes through a rubber catheter. In each test, gastric juice was collected every thirty minutes, from one hour before to eight hours after stimulation; its acidity was determined by using Beckman's pH-meter; pepsin concentration was estimated by the hemoglobin method. A series of experiments were performed on each dog. Pitressin (Parke-Davis vasopressin), pitocin (Parke-Davis oxytocin), and atonin-O (Teikokuzoki oxytocin), were injected intraventricularly in doses of 0.5 IU per kilogram body weight and of 4 IU per kilogram body weight on intravenous administration. Syntocinon (Sandoz synthetic oxytocin), and synthetic lysine vasopressin (Sandoz) were injected intraventricularly in doses of o.5 IU per kilogram body weight. Pitressin and pitocin were subjected to descending paper chromatography in nbutanol/acetic acid/water (5: 1: 4) at 15°C for fifteen hours using a sheet of Toyo Filter Paper No. 5o and stained with ninhydrin. Elutes were obtained from three zones indicated in Fig. i. On 415

PITRESSIN

0

FRACTION I

MOG

0.5

0.4

0 0

Fr III 9• Fr II

B. P.

0 0

0.3

0 0

0.1

Fr I

,L. D

1.

1 0.2

3—

Br.

FRACTION II

FRACTION III

D Rf Pisressi

u

Pilocin

L

FIG. I. Localization of fractions 1, 11, and 111 in paper chromatograms of pitressin and pitocin. The spots were visualized by ninhidrin spray.

testing blood pressure, breathing and myocardiograms using urethane-anesthetized rabbits, fraction II proved to be as active as pitressin, while fractions I and III were found to be far less active (Fig. z) on comparison of blood pressure, breathing and myocardiograms.

Time sec.

FIG. 2. Kymograph records showing effects of 2M/kg of pitressin (A) and its fractions 1(B), 11(C), and 111(D) on myocardiogram (MCG), blood pressure (BP.) and breathing (Br.) in a urethane-anesthetized rabbit.

HISTAMINE-LIKE COMPONENT OF PITRES-

was prepared according to the method of Swingle et al. (2o). Inactivation of pitressin and atonin-O was carried out following the method of Birnie (21), and using cell-free extracts of rat liver tissue. ADRENALIN AND NORADRENALIN were injected intraventricularly in doses of 0.025 mg./kg. body weight and in doses of o.I mg./kg. on intravenous administration, respectively. PILOCARPINE was used as a single dose of 0.2 mg./kg. body weight intraventricularly or intravenously. SIN

ACETYLCHOLINE was injected intraventricularly in doses of o.5 mg./kg. body weight and intravenously in doses of 5 mg./kg. body weight, respectively. HISTAMINE was injected subcutaneously in doses of 0.01 mg./kg. ACTH-depot (Schering) was injected intramuscularly as a single dose of 1, z, and 5 IU/kg. body weight. Two ml. of to per cent gelatin solution was given similarly as control. One half ml. of physiologic saline or pH-4 phosphate buffer solution was injected into the ventricle as control.

Results Experiments on five intact dogs prepared with Heidenhain pouches CONTROL SERIES. Basal secretion was found to be less variable throughout the whole experiment of eight-hour duration.

416

The mean gastric secretory volume was 3.o ml./hr., the mean pH was 2.6, and the mean pepsin concentration was found to be go units /ml. (Fig. 3, I). The control animals which received intraventricular injection of saline or the buffer solution

Ie

BASAL SECRETION

showed no rise in gastric secretion (Fig. 3, 111). Subcutaneous injection of histamine has produced rapidly a striking inI, crease in volume and acidity, reaching a peak in thirty minutes, without augmenting pepsin concentration (Fig. 3, 11). Ine tramuscular injections of z and 5 IU/kg. of ACTH produced a definite rise in acid-pepsin output and the gastric secretory volume after a latent period of 1* three hours, reaching a peak at six hours (Fig. 3, /V). One IU/kg. body weight PILOCARPINE proved to be the smallest effective dose. 1° a2mCks Gelatin vehicle failed to elicit this response. EFFECTS OF PITRESSIN. Intraventricular injection of 0.5 IU/kg. body weight has produced a definite rise in pepsin and acid output, as well as gastric secretory volume, after a latent period of three hours, 11 reaching a peak of six hours (Fig. 4, /); ADRENAL! NE Ø administration of 0.05 IU/kg. body weight proved to be ineffective. Intravenous injection of 4 lU/kg. caused rapidly an extreme pallor of mucous membrane of the mouth and orifice of pouch, tachypnea and bradycardia for 0 five minutes or so, occasionally accom2 3 4 2 3 0 panied by vomiting, defecation and uri> Tao nation. The pouch continued to discharge FIG. 3. Gastric intraluminal pressure study. only a small amount of mucous juice for The upper three tracings are pressure tracings; the lower one is a pneumographic tracing. three hours; a depressive effect on pepsin Note the tonic waves (type III) in channel 1, and acid secretion values was evident recorded from the body of the stomach, and (Fig 4, 11). Subarachnoid and cisternal the phasic waves in channel 2 and 3, recorded 5 cm distance in the antrum. The rhythm of injections of IU/kg. body weight failed at 3-4 per minute in channel 2 changes into a to increase gastric secretion appreciably. rhythm of 7 per minute in channel 3. EFFECTS OF PITOCIN AND ATONIN-O. Intraventricular injection of 0.5 IU/kg. Fraction I administered into the ventricle body weight caused a gastric secretory showed a gastric secretory activity simiresponse equal to that produced by pitres- lar to pitressin (Fig. 4, 111) and fractions sin (Fig. 4, V). Intravenous injection of 4 II and III proved to be lacking this activIU/kg. body weight produced a prompt ity (Fig. 4, IV and VI). acceleration of intestinal movements, but Treatment with cell-free liver extracts neither pallor of mucous membranes nor abolished the gastric secretory activity rise in gastric secretion was observed. produced by pitressin and atonin-O. Chromatographic fractionation of the Administration of a histamine-like three fractions obtained from pitressin component of pitressin had no effect on and pitocin gave the following results: gastric secretion.

Ii

10

9

0 0 7 1

0 1

4 9

M

41 7

7

8

Is

EFFECTS OF SYNTHETIC LYSINE VASOPRESSIN AND SYNTOCINON. Intraventricular in-

jection of 0.5 IU/kg. body weight failed to elicit a gastric secretory response (Fig. 4, VII and VIII). Both synthetic products were found to be almost homogeneous chromatographically; their Rf values were 0.28 and 0.48 respectively.

Ov

m

PITRESSIN :Fromm I

N PITRESSIN :Freston 0

a

147 a

4

1e

a

EFFECTS OF ADRENALIN AND NORADRENALIN. Intraventricular injection of 0.025

mg./kg. body weight produced a definite rise in pepsin and acid output and gastric secretory volume after a latent period of three hours; the activity reached a peak at six hours (Fig. 3, VII); intravenous injection of 0.1 mg./kg. body weight produced a marked depression of gastric secretion similar to pitressin (Fig. 3, VIII). The intraventricular injection of noradrenalin produced a similar rise in gastric secretion, but the intravenous injection had no effect on gastric secretion. EFFECTS OF PILOCARPINE. IntraVentrlCUlar injection of 0.2 mg./kg. produced an

onset of salivation, hyperperistalsis, vomiting, defecation and urination in approximately five minutes; these symptoms disappeared in thirty minutes without increasing gastric secretory response (Fig. 3, V); the intravenous injection induced the symptoms earlier and more intensively, and produced a marked increase in volume and acidity lasting for two hours, but no augmentation of pepsin concentration was observed. (Fig. 3, VI). EFFECTS OF ACETYLCHOLINE. Intraventricular injection of 0.5 mg./kg. body

weight induced vomiting five minutes later without increasing gastric secretion. After intravenous injection of 5 mg./kg., pronounced vomiting and hyperperistalsis were observed, but gastric secretion failed to rise throughout the experiment. Experiments on four intact dogs prepared with Pavlov pouches CONTROL SERIES. Basal secretion was 418

v PITOCIN

Ø

0

0

0

iiiiIlRIi♦'1 11 SYNTHETIC VASOPRESSIN

a

ae,

aF

4

0L

0 0

2 å 4 e e 7 6 0 I Time 0r

2

7 4 e

FIG. 4. Effects of Pitressin on gastric secretion in Heidenhain pouch dogs. Each point and tofuntH represents the mean of 5 dogs. Bars denote the standard deviations.

somewhat variable, showing mean volume of 3.1 ml./hr., mean pH of 1.7, and mean pepsin concentration of about 7o units/ ml. EFFECTS OF PITRESSIN. Intraventricular injection of o.5 IU/kg. body weight produced a typical rise in gastric secretion similar to that observed in the Heidenhain pouch dogs. EFFECTS OF PILOCARPINE. IntraVentriCUlar or intravenous injection of 0.2 mg./kg.

body weight produced the symptoms and gastric secretory response similar to that observed in the Heidenhain pouch dogs.

O

7

1SHIHARA

Experiments on four stalk-sectioned dogs prepared with Heidenhain pouches In four of the eight stalk-sectioned animals, histological examination of the hypothalamic-pituitary complex revealed morphological changes which indicated the success in division of the stalk and interposition of a barrier. There was some doubt about the success of operation in the remaining four animals, and these have been excluded from the series. CONTROL SERIES. Basal secretion was less variable than in previous experiments, showing mean volume of 2.8 ml./hr., mean pH of 2.7, and mean pepsin concentration of approximately 6o units /ml. EFFECTS OF PITRESSIN. Intraventricular injection of 0.5 IU/kg. body weight failed to elicit the gastric secretory response observed in the intact animals. EFFECTS OF ATONIN-O, ADRENALIN AND

likewise, proved to have no effect on gastric secretion. EFFECTS OF ACTH. Intramuscular injection of 5 IU/kg. body weight produced a gastric secretory response, equal to that produced in intact animals. NORADRENALIN,

Experiments on two hypophysectomized dogs prepared with Heidenhain pouches CONTROL SERIES. Basal secretion was less variable, showing mean volume of 2.4 ml./hr., mean acidity of pH 5.8, and mean pepsin concentration of about 33 units /ml. PITRESSIN, ANTON IN-O, ADENALIN AND NORADRENALIN intraventricularly proved

to have no effect on gastric secretion. EFFECTS OF ACTH. Intramuscular injection of 5 IU/kg. body weight produced a gastric secretory response, similar to that produced in intact animals.

Discussion The results of this study demonstrate that intraventricular injection of 0.5 IU/ kg. of commercial pitressin produced a significant rise in pepsin and acid output as well as gastric secretory volume from the Pavlov and Heidenhain type pouches in nonanesthetized dogs. The secretory response occurs after a latent period of three hours, reaching a peak at six hours. The response has failed to occur when pitressin was injected into a leg vein, even when the dose was increased eight times; only in the latter case, an extreme pallor of mucous membranes, tachypnea and bradycardia have appeared soon after injection and a small amount of mucous juice was released from the pouch, indicating splanchnic effects rather than vagal ones. The pattern of gastric secretion after intraventricular administration of pitressin resembled closely that following

administration of ACTH and cinchophen, but it differed from that of histamine and insulin (15). Intraventricular injection of pilocarpine produced lacrimation, salivation and vomiting in thirty minutes, but had no stimulating effect on gastric secretion; after intravenous administration, these manifestations set in earlier, were more intensive and were accompanied for two hours by a rapid discharge of gastric juice which was low in pepsin content. The similarity of the reactions to pituitrin and pilocarpine injected into the ventricle has led Cushing (I I) to postulate that pituitrin administered intraventricularly stimulates the hypothalamic nuclei of a parasympathetic center. The experimental data observed in this study on pitressin do not lend support for this suggestion. The similarity of the response to pitressin and ACTH was apparent in both 419

V / EFFECTS OF CORTICOTROPHIN RELEASE

the timing and the magnitude of pepsin secretion in the intact animal. However, these responses proved to be profoundly different in stalk-sectioned and hypophysectomized animals. Although commercial pitressin has been shown to contain minute amounts (about 5 ,ug. per Io/IU pitressin) of ACTH contamination, the dose of pitressin used in the present experiments was much too small to contain an effective amount of ACTH. Equally, a histamine-like component which has been shown to be present as a contaminant in commercial pitressin (2o) proved to have no effect on gastric secretion. Hence the results of this study indicate that pitressin-induced gastric secretion was not due to exogenous ACTH, but to an endogenous one. It is highly conceivable that the gastric secretory response following pitressin administration into the ventricle was induced by a chemical mediator reaching the adenohypophysis through the pituitary stalk, presumably via hypophysial portal vessels, rather than through the systemic circulation; the response was observed after such a minute dose of intraventricular administration that a dose eight times higher was not effective when the intravenous route was used. The response was blocked by pituitary stalk section and hypophysectomy but not by denervation of the stomach. Interpretation of pituitary stalk section has been rendered difficult by species differences and the variability of the operative result. Rothballer and Skoryna (z 2) observed in the dog that pituitary stalk section caused extensive infarction of the pars distalis. The present data which were obtained only from animals with a complete stalk section and have shown a moderate reduction in basal secretion in stalk-sectioned dogs indicate some impairment of pituitary activity. Hence some effect on pituitary ACTH 420

release should be expected to follow pituitary infarction subsequent to division of the stalk. Pitocin and atonin-O proved to have the same effect on gastric secretion as pitressin has, while synthetic products, syntocinon and synthetic lysine vasopressin, failed to do so. This implies that the gastric secretory activity was due to some active principle present as an impurity in posterior lobe preparations. This active substance was successfully separated by means of paper chromatography. The spot containing the gastric secretory activity of pitressin and pitocin could be clearly seen to be isolated from those spots which correspond respectively to the main spot of synthetic lysine vasopressin and syntocinon in paper chromatograms. The gastric secretory activity was lost after incubation with cell-free liver extracts, which have been previously shown to inactivate enzymatically the posterior lobe hormones (2 i) . It appears Justifiable to conclude that the gastric secretory activity, common to the effects of posterior lobe preparations, was due to the specific peptide fraction which was probably identical with CRF (corticotrophin-releasing factor) separated from vasopressin by Saffran, Schally and Benfey (5). Similarly to the origin postulated for vasopressin and oxytocin, it appears likely that the CRF is elaborated within hypothalamic neurones. The results of this study suggest that CRF originating in the hypothalamus may be liberated, at least partially, into the ventricular fluid; from there it reaches the adenohypophysis via portal vessels, and induces the gland to release ACTH, responsible for stimulation of gastric secretion. It appears also reasonable to suggest that CRF is responsible for gastric secretion following cinchophen administration. Our previous studies (1 .4.) have demonstrated that cin-

IS II IH ARA

chophen causes a rise in neurosecretion of hypothalamic neurones. However, there is no conclusive evidence that cinchophen ulcer production is related to an increased ACTH release. It is of interest

to note that intraventricular injection of adrenalin has a similar effect on gastric secretion to that of pitressin; the mechanism involved in this action awaits further study.

Summary Injection of pitressin preparations into to ACTH present as a contaminant in the lateral ventricle of nonanesthetized conmzercial pitressin preparations, but to dogs in doses of 0.5 1U/kg. body weight ACTH released in response to pitressin produced a significant rise in pepsin and administered into the ventricle. acid output in dogs prepared with HeiPitocin and atonin-O were shown to denhain and Pavlov gastric pouches after have an effect on gastric secretion similar a latent period of three hours; this effect to pitressin, while synthetic lysine vasoreached a peak at six hours. When pitres- pressin and syntocinon failed to elicit sin was injected into a leg vein, a dose such a response. eight times higher caused vasoconstriction Paper chromatographic studies disand discharge of a small amount of mu- closed the presence of a specific peptide cous gastric juice, without stimulating fraction containing an active principle, pepsin and acid secretion. The pattern of probably identical with corticotrophingastric secretion induced by pitressin was releasing-factor. It is postulated that the similar to that following ACTH and cin- CRF is elaborated within hypothalamic chophen administration, but it was differ- neurones and liberated into the ventricuent from that of histamine, insulin and lar fluid: from there it appears to reach pilocarpine. the adenohypophysis through the pituiPitressin-induced gastric secretion tary portal vessels. The stimulation of closely resembles that following ACTH gland causes an increase in ACTH release, and cinchophen administration in timing which appears to be involved in the gasand magnitude of pepsin secretion, re- tric secretory response to cinchophen. gardless of intact nerve supply to the ACKNOW LEDGMENT gastric pouches. The effect of pitressin, The author is indebted to Drs. A. Fanas well as that of cinchophen, was abolished by pituitary stalk section and hypo- champs and W. v. Orelli, Sandoz Rephysectomy in sharp contrast with the search Laboratory in Basle, for a generous effects of ACTH administration. This supply of synthetic lysine vasopressin response to pitressin cannot be attributed used in the experiments.

References 1. HARCus, c. w. Brit. Med. J. 1: i39-342, 1948. 2. HUME, D. M. and WITTENSTEIN, G. J. Proceedings, First Clin. ACTH Conference, J. R. Mote, ed., 177-183, Philadelphia, Blakiston, U.S.A. 3. BRODISH, A. and LONG, C. N. H. Endocrinology 71: 298-306, 1962.

4. ROYCE, P. C.

and SAYERS, G. Proc. Soc. Exp.

Biol. Med., zo3: 447-450, 1960.

and BENFEY, B. C. Endocrinology, S7: 439-444, 1955. 6. MCCANN, S. M. Endocrinology, 6o: 664-676, 1957. 5. SAFFRAN, M., SCHALLY, A. V.

7. HEARN, W. R., WEBER, E. J., RANDOLPH, P. W.

42I

V / EFFECTS OF CORTICOTROPHIN RELEASE

8. 9. 10.

1 i. 12. 13.

14.

42 2

and BARKS, N. E. Proc. Soc. Exp. Biol. Med., 107: 515-517, 1961. SCHALLY, A. V., SAFFRAN, M. and ZIMMERMANN, BRIGETTE Biochem. J. 70: 97-103, 1959. NICHOLS, B. JR., and GUILLEMIN, R. Endocrinology, 64: 914-920, 1 959. KARPLUS, 1. P. and PECZENIK, o. Arch. Ges. Physiol., 225: 654-668, 1930. CUSHING, H. Proc. Nat. Acad. Sci., USA, 17: 163-170, 1931. DODDS, E. C., HILLS, G. M., NOBLE, R. I.. and WILLIAMS, P. C. Lancet, 228: 1099-1100, 1 935. DUTTON, D. and IVY, A. v. (Quoted from Ivy, A. C., Grossman, M. I. Bachrach, W. H., Peptic Ulcer 317. Philadelphia, Blakiston, 1950). ISHIHARA, K., KAWAFUCHI, J. KANO, K and ISHIZAKA, I. Okajima Folia Anat. Jap. 28:

477-488,1956.

15. ISHIHARA, K. an d ISHIZAKA, I. Gunnla J.

Med.

Sci., 7: 59-69, 1958.

16. GRAY, S. J., BENSON, J. A. JR. and REIFENSTEIN, R. W. Proc. Soc. Exp. Biol. Med., 78: 338342. 1951. 17. VILLARREAL, R., GANONG, W. F. and GRAY, S. J.

Amer. J. Physiol. 183: 485-494, 1955.

18. PORTER, R. WV., MOVIUS, H. J. and FRENCH, J. D.

Surgery, 33: 875-88o, 1 953.

19. ZUKOSKI, C. F., LEE, H. M. and HUME, D. M. Surg. Forum, 12: 282-285, 1961. 20. SWINGLE, W. W, BRANNIK, I.. J., ARRETT, W. LEBRIE, S. J. and PARLOW, A. F. Proc. Soc. Exp. Biol. Med., 91: 223-226, 1956. 21. BIRNIE, J. H. Endocrinol ogy, 52: 33-38, 1 953. 22. ROTHBALLER, A. B. and SKORYNA, S. c. Anat.

Rec., 136: 5-25, 1960.

5-Hydroxytryptamine (Enteramine, Serotonin) and the Gastrointestinal Tract*

ALL vertebrates, with the possible exception of Cyclostomata, contain in their gastrointestinal mucosa a particular system of cells, which includes in their cytoplasm specific granules characterized by several distinctive chemical and physicochemical properties and by peculiar requirements for their histological fixation. It is known as the enterochromaffin cell system (EC system). So far, it seems justifiable to attribute to the EC system all epithelial cells of the mucosa which; a) show granules capable of reducing the ammoniacal silver nitrate solution (argentaffinity), after a simple formol fixation; b) turn yellow or brown following treatment with bichromate or with other oxidizing agents (chromaffinity); c) are capable of coupling in an alkaline medium with diazonium salt; d) exhibit a yellow fluorescence in Wood's light; e) give, after mild oxidation, with thioindoxyls at alkaline pH levels, reddish thioindigoid dyes. Alcohol, acetone and all the usual histological fixatives lacking formol are unsuitable for preservation of the above distinctive histochemical characteristics of the specific granules.

Vittorio Erspamer**

Besides the vertebrates, typical EC are present in the gastrointestinal mucosa of ascidians and of Amphyoxus lanceolatus. The reader interested in analytical data on the distribution of the EC system in vertebrates is referred to the review article by Clara (r) . For a better comprehension of the limits and the significance of the EC system attention should be drawn to some points: a) it is quite possible that in some mammalian species (more precisely in those containing 5-hydroxytryptamine in their mast cells, such as rat and mouse), the mast cells in the intestinal wall under normal or (more likely) pathological conditions give the histochemical reactions peculiar for the typical EC. In fact, the negativity or positivity of these reactions in the mast cells seems to be only a matter of concentration of 5-HT in their granules. b) the distribution of the EC system in the different sections of the gastrointestinal tract is quite irregular, and differs sharply from one animal species to another. c) typical EC, indistinguishable from the EC of the gastrointestinal tract, may

*Supported in part by a grant from the Rockefeller Foundation, New York. "From the Instituto di Fannacologia, Ospedale Maggiore, University of Panna, Panna, Italy. 423

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

be found in the mucosa of other hollow crease in the number of preenterochroorgans (urinary bladder, urethra, pros- maffin argyrophil cells, to the extent that tate, oviduct, upper respiratory tract) the sum of both cellular types remains and, what is more important, within the constant (3). endocrine tissues, such as pancreatic islets The gastrointestinal mucosa of some and thymus. It seems difficult to presume vertebrate species seems to contain only an external secretion for these cells. authentic EC; in other more numerous The first appearance during embryonic species, both types of cells are found; in life of EC in the gastrointestinal tract still other species (e.g. Cyclostomata), occurs in the eleventh week in man, in only preenterochromaffin argyrophil cells the sixth to eighth week in oxen, on the are present. Similarly to EC, preenterosixteenth day in mice, and on the tenth chromaffin argyrophil cells occur outside to sixteenth day in chickens. Typical EC the gastrointestinal tract; they are found differentiate, in vitro, in organotypic cul- in the mucosa of the urethra, prostate and tures of intestine of chick embryos ex- bronchi of several mammalian species, inplanted before EC are known to differ- cluding man. It is evident that wherever entiate during the incubation period (2 ). EC exist, there is a basic condition for This demonstrates that 5-HT is elabor- the development of typical "argentaffinoated by the EC themselves from the L- mas" i.e. 5-HT-secreting carcinoids; it is tryptophan available in the cultural medi- similarly obvious that preenterochromum. In addition to the typical EC, the affin argyrophil cells may give origin to gastrointestinal mucosa of vertebrates "argyrophil carcinoids" (provided that usually contains other granular cells the neoplastic tissue maintains its original which are indistinguishable from the true characteristics), lacking the histochemiEC, both in their morphology and in their cal reactions specific for the true EC and unspecific staining properties, but do not containing no 5-HT. It is now generally exhibit the specific histochemical reac- accepted that the EC of the gastrointestions, and have no 5-HT in their granules. tinal mucosa owe their peculiar histoThese cells have been called pre-entero- chemical properties to their content of chromaffin argyrophil cells in view of 5-HT. Evidence in favor of this assumptheir capacity to stain black with silver tion is abundant and decisive (4,5). Sandin the presence of an external reducing ler and Snow (6) have tentatively sugagent such as formaldehyde. gested that whereas "argentaffin" carciStrong experimental evidence for the noids secrete 5-HT, "argyrophil" carciexistence of a strict correlation between noids may secrete 5-hydroxytryptophan true EC and preenterochromaffin argyro- (5-HTP), the precursor amino acid of phil cells of the gastrointestinal mucosa, 5-HT. However, so far we have no eviin the sense of a possible transformation dence that normal preenterochromaffin of one cellular type into the other, is argyrophil cells (argyrophil cells of the afforded by the observation that follow- human biliary tract, of the large intestine ing reserpine treatment, which causes a of the cat, of the gastrointestinal tract of 5-HT discharge from the gastrointestinal Teleostei etc.) may store or secrete 5mucosa, there is a remarkable reduction HTP. in the number of EC with a parallel in-

424

ERSPAAIER

Occurrence and Distribution of 5-Hydroxytryptamine in the Gastrointestinal Tract of Vertebrates TABLE I. The 5-HT content in the gastrointestinal tract of man and the common laboratory animals 5-HT content (in itg base per g wet tissue) Animal species Man

Stomach 0.7fb 0.9°

0.60

Small intestine proximal middle distal 3.7

Monkey (Papio hamadryas)

1.2-1.5g

2.1

Dog

1.6-2.3g 5.2

2.4

0.52 0.45

1.6

Cat Rabbit

5.7°

0.9

4.9*

0.9

1.7(85)

1.65

1.4

1.85

2.5*

1.6

1.8

1.5

1.55* 2.8 (4,5)

1.2

3.2

6.2 3.3 7.2

1.15

3.9

4.3

2.2

3.7

2.0 6.2 5.0

1.7* 1.2

0.8 0.53

1.8 3.3

Large Intestine

2.9

0.9

0.9

Guinea pig

3.7

Caecum Appendix

3.4

1.5

0.7

2.25*

2.7 (4,5) 3.5 (97) 1.95 4.5

0.9 0.8

0.45* 0.8 (87) 0.7 (4,5)

Hamster

1.7

3.7°

0.9

0.9

0.85

0.7

1.3*

Rat

0.5 1.8

3.9g 2.1g

2.9 5.6

1.6 5.5

3.3 3.2

4.75

6.0* 3.8 88) ) 5.9 (89

0.64

2.15 2.2

4.5 (87) 8.7*

1.9g

25.06

2.5

Mouse Marmot

5.2 8.8 0.2

2.8

2.5 2.8

Pigeon

2.6

1.6

1.1

1.46

Hen

1.7

(4,5)

3.1 0.6

4.9

0.9

1.1

1.3

4.5

0.6g

2.0

3.4

1.0

1.7°

0.7

0.8

1.0

Frog

0.3

1.2

Toad

2.2

1.6

2.0*

4.1 (4,5) 1.9* 1.5

*

(4,5)

f - gastric fundus a - gastric antrum p - pylorus g - glandular stomach * - M.B. Nobili (unpublished observations) 425

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

Tables I and II tabulate the content and rabbit, hedgehog and hamster, the small distribution of 5-HT in the different sec- intestine of the guinea pig, hog, sheep and tions of the gastrointestinal tract of man chicken, and the large intestine of the and several other vertebrates, including rat, horse, cow, marmot and pigeon. In the commonest laboratory animals. Data man, large amounts of 5-HT are present have been obtained mainly by bio-assay, not only in the duodenal mucosa, but also and unless otherwise stated, they refer to in the mucosa of the large intestine. It extracts of the whole gastrointestinal wall. appears that the above data have too Some observations can be made on the often been neglected or underestimated basis of the data tabulated: in the interpretation of the physiological a) There seems to be a definite exception significance of gastrointestinal 5-HT. If to the general rule that the number of one assumes that 5-HT is intended for EC in a given section of the gastrointes- internal secretion, the varying distributinal tract correlates with the 5-HT con- tion is of no importance. If however, one tent of the same section; in the case of the supposes that it is intended for the local mouse, the stomach contains a relatively regulation of secretion and motility of small number of EC and an enormous the gastrointestinal tract, the different disamount of 5-HT. There are sound rea- tribution of 5-HT in the alimentary canal sons to believe that most of this 5-HT is of various species is difficult to explain. stored in the mast cells of the mucosa and In a given species, the 5-HT content of submucosa. Thus it is quite possible that the intestines may vary considerably dein animal species whose mast cells con- pending upon the diet and the bacterial tain 5-HT (rats and mice), the 5-HT flora in the intestinal lumen. By adminisfound in gastrointestinal extracts origi- tration of radioactive tryptophan, and nates both from the EC cells and the mast through the study of the daily excretion cells. This should be kept in mind when of 5-hydroxyindoleacetic acid (5-HIAA), interpreting changes in the 5-HT content it has been calculated that under normal of rat and mouse gastrointestinal tract conditions the half-life of intestinal 5-HT evoked by drugs and other agents. is approximately four to twelve hours b) The distribution of 5-HT in the differ- (7) . However, when tryptophan is adent segments of the gastrointestinal tract ministered in excess (especially orally), varies conspicuously from one animal a quantity of 5-HT corresponding to species to another. The part which con- that present in the intestines may be protains more 5-FIT is the stomach of the duced within three to four hours. BertacTABLE. II. The 5-HT content in the gastro-intestinal tract of some large domestic mammals (3°) 5-HT CONTENT (IN

BLG

BASE PER G WET TISSUE)

A nimal species Rumen Ox Calf Sheep Hog Horse 426

0 0 0

Stomach Reticulum Omasum Abomasum 0 0 0

0 ?

0.5

1.6 1.2 3.2 l

Small intestine proximal distal 5.10 3.6 4.1 4.0

0.72

1.6 3.0 2.0 0.5

0.32

Large intestine proximal distal 1.2 3.7 2.5 0.28

0.49

4.9 4,5 2.9 0.53

1.0

ERSPAMER

cini and Nobili (8) have calculated that gastrointestinal mucosa of the rat, may synthetize 5-HT at a date of lug./g./hour under a tryptophan load. As far as it concerns the subcellular localization of 5-HT in the gastrointestinal mucosa, it has been found that the amine is contained in a granular fraction which is rich in adenosine triphosphate (ATP), the molar ratio 5-HT: ATP being 2 .4-3 (9). Little is known about the physiological mechanisms which regulate the release of 5-HT from the gastrointestinal mucosa.

It has been claimed that elevation of the intraluminal pressure and/or increase of the motility of the intestines increases the amount of the 5-HT released both into the intestinal lumen and into the venous blood. Although from a theoretical point of view this is quite possible, there is as yet no decisive evidence that EC discharge part of their 5-HT into the intestinal lumen under strict physiological conditions. On the other hand, it is firmly established that EC can discharge their specific amine into the blood stream.

Action of 5-Hy droxytryptamine and 5-Hydroxytry ptophan on Secretion of the Gastrointestinal Tract Salivary secretion

found that both the intravenous injection of 20-1 zoug./kg. of 5-HT base and the intravenous infusion of 5-20 ug./kg./min of 5-HT caused a complete or partial inhibition of the secretory response of the log submaxilliary gland to electrical stimulation of the chorda tympani, as well as to intravenous infusion of pilocarpine. The inhibitory effect lasted three to fifteen minutes, and was sometimes preceded by transient potentiation of salivation. Denervation of the gland did not alter the effect of 5-HT. There was no definite correlation between reduction of salivation and the observed decrease in blood flow through the gland.

DOG SUBMAXILLARY GLAND. According to Petrucelli et al. (1o), single injections of 50-iooug. of 5-HT into the common carotid artery (via the cranial thyroid artery) caused a potentiation of salivation produced by continuous electrical stimulation of the chorda tympani. 5-HT in this dose range produced no salivation by itself. The intravenous administration of iproniazid further increased the potentiation observed with identical doses of 5-HT. The intravenous administration of reserpine in single doses, which in itself produced salivation, also potentiated further 5-HT after cessation of reserpineinduced salivation. Moreover, it appears Gastric secretion that 5-HT potentiates the sialagogue effect of acetylcholine by subminimal inMAN. Almost all research workers (15, fusion. Petrucelli and co-workers suggest 16,17,18,19) observed that 5-FIT and 5that 5-HT, while not strictly necessary HTP inhibit volume and acidity of both for neural transmission, may facilitate spontaneous and histamine-induced gastransmission at the level of parasympa- tric secretion, while at the same time inthetic ganglia and/or neuroeffectors. The crease the production of mucus. Followresults of Caldarera et al. (1 2) are at com- ing the intravenous infusion of 1.5-15 plete variance with the above. They pg./kg./min. of 5-HT, maximum effects

427

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

appeared after five to twenty-five minutes, previous dietary history of the animal. In and secretory response wore off within dogs which were not starved completely fifteen to twenty minutes after the infu- but were given a fatty meal in the twelve sion was discontinued (18) (rise of pH hours preceding the experiment, 5-HT values by 0.5-1.5). 5-HTP infused intra- elicited a much more striking inhibitory venously for five minutes at a rate of response, and all acid secretion was aboli o-18 ug./kg./min. produced a more in- ished; whereas in dogs in which the period tense and sustained reduction of gastric of starvation was prolonged to thirty-six secretion. The pH of the gastric juice hours, 5-HT did not inhibit, but in rose by 3.5, and the inhibitory effect several cases stimulated the acid secretion. 5-HT given after bilateral cervical vagolasted up to sixty minutes 09). The only results at variance with these tomy was found to be ineffective in infindings are those of Cali and Cordova hibiting histamine-stimulated secretion. (13,14), who found that intravenous This has been considered as evidence in 5-HT (2 mg.) displayed in man a stimu- favour of the suggestion that 5-HT might lant action of gastric secretion, which exert its effects on gastric secretion by was inhibited by antihistaminic drugs, reflex stimulation of vagal receptors in and therefore did not appear to be medi- the gastric wall. ated through liberation of histamine. These observations were confirmed by DOG. Black et al. and Smith (20,21) ex- several research workers. Hammond (22) amined the effect of 5-HT on gastric observed that in dogs receiving 5-HT insecretion in dogs anesthetized with chlor- travenously in doses which stimulated inolose and urethane, starved for at least testinal motility, no gastric secretion entwelve to twenty-four hours and prepared sued; stimulation of gastric secretion by with a gastric fistula and a ligature at the reserpine was not affected by the simulpyloric-duodenal junction. They found taneous injection of 5-HT. White and that intravenous infusions of 5-HT (at a Magee (2 3) found that intravenous inrate of z.5-zoug./kg./min. for 45-90 min.) fusion of 10-30 ug./kg./min. 5-HT indid not stimulate any gastric secretion creased by zoo per cent the secretion of whatsoever, but caused the production of mucin from the pyloric mucosa of the an alkaline juice rich in mucus. During dog; this effect was regularly reduced by this infusion, the arterial plasma pH fell, the administration of i mg. atropine intraunlike the plasma pH during histamine venously. The action of 5-HT was not infusion. When an acid gastric secretion dependent on increased motility since it was stimulated by a continuous intra- was present in the everted pouch, and was venous infusion of histamine (e.g. 5 pg./ not abolished by hexamethonium, which kg./min.), concurrent infusion of 5-HT decreased motility. Finally, Haverback et (15 ug./kg./min.) for thirty minutes was al. (24) noted that whereas exogenous found to inhibit the secretion. Recovery 5-HT in a dose up to 4 ug./kg./min. had from the inhibition usually began within no significant effect on gastric secretion sixty minutes after stopping the 5-HT of acid, a dose of 16 pg./kg./min. caused infusion. Infusion of 5-HT at the start the Heindenhain pouch to secrete juice of histamine-stimulated gastric secretion which was less acid. failed to prevent the onset of secretion Results obtained with the administradue to infusion of histamine. However, tion of the precursor amino acid, 5-HTP the effect of 5-HT on histamine gastric were on the whole in accordance with secretion was greatly influenced by the those obtained with 5-HT; however an 428

ERSPAMER

important partial discrepancy was observed, concerning the effect of 5-HTP on the histamine-induced gastric secretion. Black et al. (25,26) found that the intravenous infusion of doses of 5-HTP as low as 5-20 pug./kg./min. produced, after a latent period of fifteen to thirty minutes, a marked fall in secretory acid output elicited by histamine. Occasionally 5-HTP caused an initial stimulation of acid gastric secretion for a short time. The effect reached a maximum when 5HTP was given two hours after histamine stimulation. Bilateral cervical vagotomy markedly reduced the inhibitory effect of 5-HTP. In dogs prepared with Heidenhain pouches and with simple gastric fistulae, Haverback et al. (27), failed to observe inhibition of the secretory response to histamine (2 pg./kg./min.) with single intravenous doses of 5-HTP as high as 20-25 mg./kg. At this dosage level, the amino acid inhibited not only spontaneous gastric secretion (from 20 ml. and 93 mEq to 2 ml and o mEq per liter/4o min) but also secretion induced by insulin hypoglycemia or urecholine infusion (3.5 Jug./ kg./min.). According to Haverback and his co-workers 5-HTP does not directly affect the mechanism of acid formation, but probably interrupts the pathway for the transmission of stimuli to gastric secretion. Of considerable interest is the observation of Black et al. (25) that in previously starved anesthetized dogs, feeding of trytophan (300-1000 mg.) before the start of an experiment led to a fall in acid output between one and three hours after the onset of histamine-stimulated secretion. GUINEA PIG. Given to nonanesthetized guinea pigs in intraperitoneal doses of 1 to zo mg./kg., 5-HT reduced the concentration of free acid and raised the concentration of Na in the gastric secretion induced by histamine. Similarly to other inhibitors of gastric secretion, 5-HT caused the re-

placement of Cl ions with Na ions, leaving unaffected the concentration of potassium. Small doses of 5-HT increased, and larger doses decreased, the peptic activity of the gastric juice (28). RAT. Both 5-HT (4-8 mng./kg. of the base, subcutaneously) and 5-HTP (io-zo mg./kg.) produced a significant depression in volume, acid and pepsin output of the interdigestive gastric secretion in the rat, as well as a depression of gastric secretion stimulated by urecholine (15,27). Doses of 5-HT lower than z o mg./kg. were ineffective. Following 5-HTP administration, inhibition of gastric secretion occurred after a considerable latent period. In order to explore whether the inhibition of gastric secretion observed following HCl perfusion of the small intestine was due to a release of 5-HT by the upper intestine, gastric secretion in the pyloric-ligated rat was studied by Resnick and Gray (29), after the administration of varying quantities of 5HT into the portal vein. It was found that infusion of 5-HT (2-zo ,ug. 5-HT base in one-hour experiments, and 4-180 pg in three-hour studies) did not alter either gastric pH or secretory volume. Similarly, neither partial depletion of intestinal 5-HT by reserpine, nor use of 5-HT antagonists, prevented the suppression of gastric secretory function caused by HC1 perfusion of the small intestine. CAT. The effect of intravenous 5-HT (1-6 mg. base/cat) on the secretion of gastric pouches was variable, partially depending upon the integrity of the vagus nerves. In the presence of intact vagi, spontaneous secretion was but little affected by 5-HT (production of a small amount of nonacid juice); histamine-induced secretion (40 pg. histamine/min.) was generally reduced in both volume and acidity. When vagus nerves were sectioned, nonacid spontaneous secretion was more abundant for a period of ten 429

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

minutes; reduction of histamine-induced secretion was preceded by an increase of secretion. When vagus nerves were cut at the cardias instead of at the neck, an immediate reduction in both volume and acidity of the histamine-induced secretion was constantly encountered (30) . Pancreatic and biliary secretion Rats given daily injections for three days of 2 mg./kg. of 5-HT base presented a slight decrease in the lipase and protease activity of the pancreatic juice, whereas the amylase activity remained unchanged (3 i ). In dogs with total pancreatic fistulas, 5-HT (o.z mg. base/kg. intravenously) reduced both food-stimulated and non-stimulated flow of pancreatic juice. 5-HTP (25 mg./kg., intravenously) produced similar but slower and less pronounced effects. The flow rate of secretin-stimulated pancreatic juice was not affected by 5-HT and 5HTP. These drugs decreased bicarbonate levels of pancreatic juice, but did not affect pH or amylase activity (32). In man intravenous infusion of 4 ,ug./kg./ min. 5-HT for sixty minutes had no significant effect on external pancreatic secretion (volume, bicarbonate concentration and amylase concentration of the duodenal aspirate), but partially inhibited the bile pigment flow (i6). Continents on the effects o f 5-HT and 5-HTP on gastrointestinal secretions It appears from the preceding data that 5-HT and 5-HTP exert on the secretions of the alimentary canal a prevalently depressive effect. The problem arises as to whether or not the pharmacological action elicited by hexagonous 5-HT or 5HTP is indicative of a possible physiological function exerted by strictly endogenous 5-HT. In other words, consider430

ing for the sake of simplicity the stomach only, does the 5-HT produced and stored within the gastric mucosa contribute to a local regulation of the gastric secretion, or does the 5-HT circulating in the blood display the same function? In the writer's opinion evidence so far available is insufficient for an answer to the above questions. However the question deserves the following comments: a) There is no direct proof whatsoever for saying that a local release of 5-HT by the EC of the gastric mucosa occurs at the time of gastric secretion, and that it may exert any influence upon this secretion. In fact, no changes in the 5-HT content of the stomach have been described which could be referred to the phases of the secretory activity of the organ. The suggestion that reserpine may stimulate gastric secretion through local release of 5-HT is hardly tenable, because 5-HT, in contrast to reserpine, inhibits gastric acid secretion, and because reserpine is active even after pretreatment with 5HTP which is a potent inhibitor of gastric secretion (24,27). b) Black et al. (25) affirm that 5-HT levels in portal blood are higher in fed (0.24-0.39 pg./ml.) than in starved dogs (o.13 Eig./ml.). These observations, which would indicate a release of 5-HT by the gastrointestinal mucosa in connexion with presence of food in the alimentary canal, await confirmation before they can be accepted. In fact, keeping in mind the rate of blood flow through the portal vein, excess 5-HT liberated by the intestines of fed animals would be enormous, far beyond any reasonable expectation. Caution is more than ever justified since Johnsen et al. (3 3) failed to detect in man any relationship between meals and the peak in the diurnal variation in 5-HT metabolism, as inferred from the urinary excretion of 5-HIAA, which showed a maximum in the three-hour period on either

ERSPAMER

side of noon. It should be added that in- for one to three hours at a rate of up to creased liberation into the blood of 5-HT 20-50 pg./rat/hour failed to alter either produced by food would neither prove gastric pH or secretory volume (29)). nor disprove that the release of 5-HT is Changes in gastric secretion were accoma consequence or a cause of secretory panied by changes in gastrointestinal moprocesses. tility, and by a more or less conspicuous c) Although the 5-HT content of the involvement of the vascular system. So gastric mucosa is generally maximal in far, information is lacking about the bethe glandular portion of the stomach, havior of gastric secretion following ineven the nonglandular portion may con- tra-arterial injection at a close distance or tain large amounts of the amine. In infusion of physiological doses of 5-HT. Arneiurrts catlts, a fish belonging to the e) As stated previously, Black et al (z5) Teleostei, EC are localized only near the observed that feeding of tryptophan to pylorus, while completely lacking in the dogs, after a latency period, produced a glandular portion of the stomach. These reduction in histamine-stimulated gastric species differences in the gastric distribu- secretion. The confirmation of these retion of EC and 5-HT are not in favor of a sults appears to be of the greatest interest, direct local action of 5-HT on gastric and it is important to follow up the urisecretion. nary excretion of 5-HIAA following d) Accepting the view that 5-HT may separate feeding of L-tryptophan and Dexert its physiological action on the gas- tryptophan. It is evident that the demontric secretion via the blood, then it should stration that endogenous 5-HT originatbe stressed that the intravenous doses of ing from dietary L-tryptophan is caexogenous 5-HT required to produce pable of influencing gastric secretion changes in the gastric secretion were al- would offer strong support to the hypoways considerably high (in the rat doses thesis that 5-HT is involved in the physioof 5-HT base infused into the portal vein logical control of this secretion.

Action of 5-Hydroxytryptamine and 5-Hydroxytryptophan on the Motility of the Gastrointestinal Tract MAN. Single intravenous injections of 5-HT produced a sharp rise in intestinal tone and the disappearance of mixing waves, studied by the balloon-kymographic method in normal volunteers. In 25 per cent of the subjects, doses as low as 0.4 mg. 5-HT base had a pronounced effect; however, in another 15 per cent doses up to 1.5 mg. were ineffective, and in yet another 10-15 per cent 5-HT produced a decrease in tone. The stimulant effect appeared thirty seconds after the injection, and disappeared within three to four minutes, followed by a period of hypo-

motility and refractoriness to 5-HT. The increase in tone was accentuated by prior administration of monoamine oxidase inhibitors, inhibited by anticholinergic drugs and BAS (benzyl analogue of serotonin), and left unchanged by hexamethonium. 5-HT in doses up to 13 mg. of the base failed to elicit any intestinal response when administered intrajejunally to normal volunteers. However, twelve cirrhotic patients and two patients with nontropical sprue showed a typical response when 5-HT was instilled into the jejunum (34,35)• 431

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

It has been suggested that 5-HT stimulates intestinal motor activity through cholinergic nerves at a site distal to the ganglionic synapse (35), and that hypersensitivity to oral 5-HT seen in certain pathological conditions may be due to a deficit in monoamine oxidase (34). In patients with ileostomies, the intravenous injection of i mg. 5-HT base produced an initial spasm of the intestine, followed by rhythmic contractions which lasted several minutes and then by a period of mechanical inactivity. The electrical activity of the ileal bud just distal to the intestinal segment containing the balloon was characterized by an initial continuous burst of action potentials, followed by inhibition of electrical activity and then by intermittent bursts of action potentials. The effect of 5-HT was not inhibited by atropine, morphine and promethazine, but was blocked by large doses of LSD (3 mg. I.V.) (3o). The above observations have been substantially confirmed by experiments in which 5-HT was given by intravenous infusion, at rates of z-IO pg./kg./min. for 7-18 minutes. It was seen, however, that 5-HT, while stimulating the musculature of the jejunum and the ileum (in half of the cases the amine elicited contraction waves lasting two to three minutes, in the other half spasms), frequently reduced tonus and spontaneous movements of the stomach, and constantly inhibited the movements of the large intestine. Inhibition lasted ten to twenty minutes. BOL (z mg. orally) effectively counteracted the effects of 5-HT; atropine (I mg. I.V.) was inactive. Tachyphylaxis developed during the continuous I.V. infusion of 5-HT irrespective of doses, but within ten minutes after cessation of the infusion the bowel regained its former sensitivity (15,18). The effects of intravenous 5-HTP (threshold too-zoo pg./kg. administered 432

over a period of five minutes) differed from that of 5-HT in that the amino acid provided a more sustained stimulation of the small intestine, with no tachyphylaxis. The stimulant effect of 5-HTP was inhibited not only by BOL but also by atropine, and the amino acid increased motility in doses that neither produced flushing, nor altered respiration, blood pressure, pulse rate and mental status. In contrast to the small intestine, stomach and colon were again inhibited by the drug (15,19). The incapacity of 5-HT to stimulate all segments of the alimentary canal was confirmed by Debray and Besancon (36) in intubated patients, using an electronianometric method. Rapid I.V. injection of 0.2-0.45 mg 5-HT base was followed by an evident stimulation of the motility of the duodenum, the jejunum and the middle small intestine in 83, 63 and 47 per cent of the experiments, respectively. The effect lasted three to six minutes. The ileum responded only in 15 per cent of the experiments, and the large intestine never. There were striking differences from one patient to another even in the response of the more sensitive intestinal loops. A second injection of 5-HT administered one to five minutes after the disappearance of the response to a first injection was nearly always ineffective. In spite of the above rather ambiguous results, Debray and Besancon suggest that 5-HT may play a physiological role in controlling the motility of the duodenum and the jejunum, more precisely in provoking movements which cause an evacuation of these segments: this was suggested on the basis of the analogy existing on the one hand, between motor responses to 5-HT and certain prolonged sequences of spontaneous waves, and on the other hand, between the physiological refractory state seen after spontaneous contractions and the tachyphylaxis seen after

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5-HT injections. DOG. 5-HT in intravenous doses as low as 4 pg./kg./min. of the base was a potent stimulus to intestinal motility. Response usually started after four to five minutes, but tachyphylaxis developed during continuous infusion, irrespective of the dose. Within ten minutes after cessation of the infusion, the bowel regained its former sensitivity. The action of 5-HT was not blocked by atropine, hexamethonium or mepyramine. Surprisingly enough, it was potentiated by LSD (lysergic acid diethylamide) (24). 5-HT administered by injection into a terminal artery of the colon was as potent as acetylcholine in its contractile effect upon the intestinal musculature, the threshold dose varying from 0.001 to 1 /lg. However, even with 1-10 ,ug. doses, the effect was short-lasting, not exceeding 30-90 seconds. Atropine was one hundred times more active towards acetylcholine than against 5-HT. Effect of 5-HT was enhanced by inhibition of monoamine oxidase, and was blocked by both BAS, (benzylanalogue of serotonin and prochlorperazine. Hexamethonium was ineffective. Sleisenger et al. (37,38) suggest that 5-HT, although not directly acting via a cholinergic pathway, may be a modulator of the cholinergic system. When administered by single intravenous injection, the threshold dose of 5-HT was o.1 mg. 0.2-0.3 mg. elicited maximal response consisting of spasm and reinforcement of contractions, lasting three to four minutes. BOL (3-7 mg.) was an effective antagonist. The intestinal villi were stimulated in their rhythmic automatic activity both by intra-arterial and intravenous 5-HT (4-4o ,rig. base), as well as by direct application of the drug on the exposed mucosa (4 ug./ml.). Simultaneously, there was an enhancement of the peristalsis of the intestinal muscle layer and a constriction of

the capillaries. The effect lasted for about ten minutes, and was reversible. It was blocked by LSD and chlorpromazine, but not by atropine (39). Tyce et al. observed (40) hypermotility of the intestinal tract occurring in dogs above the level of an intestinal obstruction, and being present also after hepatectomy or Eck fistula; the hypermotility was accompanied by marked increase (up to Soo per cent!) of the 5-HT content in the pyloric mucosa, and by slight increase in the duodenal mucosa. No changes were appreciable in the 5-HT content of jejunal or ileal mucosa, nor in the blood and brain 5-HT levels. Similarly, the urinary excretion of 5-HIAA was normal. CAT. Similarly as in the dog, an increase in intestinal motility could be produced by intra-arterial administration of amounts of 5-HT as low as 1-2 ug./kg. in the cat. The onset of the action began two to three minutes after the injection and lasted two to four minutes (41). RABBIT. Like the isolated intestine, the rabbit intestine in situ was poorly sensitive to 5-HT. In fact, to obtain a clear stimulant action on the intestinal tone intravenous doses as high as 0.2 mg./kg. 5-HT base were required, the threshold dose being 1 o pg./kg. Even at enormous dose levels (5 mg./kg., I..), the tonus increase lasted less than one minute, and tachyphylaxis appeared with subsequent doses. The spasmogenic activity was abolished by atropine and Banthine, but not by LSD (42). As in guinea pigs, 5-HT is also released in rabbits from the intestinal wall into the lumen during peristalsis. The amount released by the caudal portion of the distal colon was barely 10 per cent of that released by the small intestine. At partial variance with the guinea pig, in the rabbit, 5-HT applied either outside or inside the intestine (1-10 ug./ml.), stimulated pro433

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

pulsive activity after an initial spasm, al- species, and results obtained in the guinea though the stimulant effect was less ra- pig constitute the basis for the theory pidly obtainable if 5-HT were applied which considers 5-HT to be a hormone participating in the physiological regulainside (43) tion of peristalsis. RAT. Both 5-HT (I o mg./kg., s.c.) and 5-HTP (100 mg./kg., s.c.) first increased The influence of exogenous 5-HT and (by 15-2O times during the first thirty 5-HTP on peristalsis and the influence of minutes) and then reduced the number peristalsis on liberation of endogenous inand weight of feces produced. a mg. 5- testinal 5-HT have been thoroughly inHT or Io mg./kg. 5-HTP was just effec- vestigated by Bulbring et al. and by other tive and o. a mg. or i mg./kg. was ineffec- research workers on both isolated and in situ intestinal loops. The main results tive (44). In studies of the intestinal transit, Brit- were as follows: a) 5-HT (0.5-Io eeg./ml.) introduced tain and Collier (44) found that whereas 5-HTP, up to loo mg./kg. doses, failed to into the bath in which an intestinal loop influence significantly the distance travel- was suspended, produced a transient led by a 5 per cent suspension of charcoal muscle contraction followed by abolition in 1 o per cent acacia, I o nig. /kg. 5-HT of the peristaltic reflex (47,48,49). This reduced this distance by 30-5o per cent, predominant inhibitory effect was conprobably owing to a spasm of the pyloric stant for the small intestine, whereas as sphincter, since the effect disappeared if a rule the colon ascendens showed incharcoal was allowed to enter the duo- creased propulsive activity (49)• 5-HT denum before 5-HT was given. added in low concentrations (0.005-0.02 Lish et al. (45) obtained partly diver- Eig./ml.) to the fluid outside the intestine gent results, especially from a quantitative restored slight peristaltic activity after point of view. First of all, a consistent this activity was abolished by cooling. If inhibition of intestinal transit, with or the peristaltic activity was depressed but without significant stimulation of gastric not abolished by cooling, 5-HT in the emptying, was observed already after bath sometimes abolished it (5o). subcutaneous administration of doses of The transient or persistent facilitation 5-HT as low as 0.1-I mg./kg. (per os the of peristalsis produced by 5-HT applied amine was one hundred times less active); to the serosal surface has been interpreted secondly, 40 mg./kg. 5-HTP produced a as being due to sensitization of the muscle definite inhibition of both gastric empty- to acetylcholine (47), or to facilitation of ing and intestinal transit, the threshold transmission at synapses involved in the dose of the amino acid active on the dis- peristaltic reflex arc (5o); the inhibition tribution of 0.2 per cent phenol red being of peristaltic reflex has been attributed to 0.4 mg./kg. ganglionic block (4.7,9.9). It is of interest MOUSE. As in the case of rats, 5-HTP that, on external application, both BOL produced a conspicuous increase in the (z-bromolysergic acid diethylamide) and number and weight of feces. The ED 50 LSD blocked the emptying phase of the by the intraperitoneal route was 25 ug./ peristaltic reflex in the guinea pig's lower mouse, the ED 100 was 300 ug./mouse ileum similarly to 5-HT. b) Introduction of 5-HT into the lu(46). GUINEA PIG. Very important work has men of the isolated guinea pig intestine been devoted to the study of the influence stimulated peristalsis in concentrations of of 5-HT on intestinal motility in this 10-° to 10-0. The threshold of the intra434

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luminal pressure required to elicit peristaltic waves was lowered, the contractions became more frequent and a larger volume of fluid was propelled (51). Lembeck (49), for example, found that the frequency of peristaltic waves was increased after infusion of 5-HT into the lumen at rates of 1.7 and 4.8 ,ug./min., by 90 and zoo per cent respectively. Stimulation of peristalsis was observed in isolated strips of both the ileum and the colon. During irregular muscular activity of the latter, 5-HT applied in the lumen abolished nonpropulsive contractions and produced co-ordinate waves; however, higher concentrations of 5-HT caused an initial contraction and stimulation of peristalsis followed by depression (43). ii 1ucosal application of 5-HT was highly effective in removing the block of the peristaltic reflex produced by morphine, Dibenzyline, procaine and 5-HT applied to the serosa, but less effective in removing the block produced by atropine and hexamethonium, and completely ineffective in abolishing the block caused by BOL or LSD (47). c) When peristalsis was recorded from loops of intestines with normal blood supply in situ, it was observed that in the absence of spontaneous activity at low intraluminal pressure, both intra-arterial (o.z -o.z pg.) and intravenous (4-lo pg.) injections of 5-HT, as well as those given into the lumen (zoo-zoo pg.), caused the appearance of short bursts of peristalsis. Similarly, slow intra-arterial infusion of 5-HT (o.z to z pg./min.) and 5-HTP (o.z to 2.4 ug./min.) caused vigorous peristalsis at pressures zo mm. below the normal threshold. However, tachyphylaxis occurred readily with all three routes of administration, and high doses of 5-HT always caused a diminution of fluid transport. Desensitization was more evident in vivo than in vitro (52).

d) Small amounts of 5-HT (o. -0.2 pg. in 3o min.) were found to be released into the fluid flowing through the lumen of an isolated or in situ loop of the small intestine at an intraluminal pressure subthreshold for the initiation of peristaltic waves. When the intraluminal pressure was raised and the fluid was actively pushed out by peristalsis, the amount of 5-HT increased two to eight times (51). A further increase occurred in the presence of iproniazid, but the most striking increase (up to two thousand times the normal) was observed following intraluminal or intraarterial administration of 5-HTP. It should be emphasized that 5-HTP produced only a small increase in the mucosal 5-HT content (5z). At a constant filling pressure, the 5-HT release was much greater during bursts of peristaltic activity than during the intervening periods of rest. However, a three-fold increase of 5-HT release also could be observed in the absence of any muscle contraction when the pressure was raised (53). The amount of 5-HT released into the lumen during peristalsis of the oral portion of the guinea pig distal colon (6.3 ni. pg./min.) was of the same order of magnitude as in the small intestine (5.7-9.6 m. pg./nmin.), whereas the amount releasd from the caudal portion was considerably smaller (1.6 m. pg./min.). Sympathetic nerve stimulation reduced the amount of 5-HT on the average by only 3o per cent, whereas pelvic nerve stimulation caused no significant change (43) e) Peristalsis was abnormally active in reserpine-treated animals, and occurred at low thresholds. It declined gradually but did not stop in spite of extremely low mucosal 5-HT level (z-lo per cent of the normal) and very small amounts of released 5-HT. Peristalsis did not stop in these animals even when BOL was introduced into the lumen (52). 435

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

According to Bülbring and Lin (51 ) the nervous tissue, and D receptors are and to Lembeck (49) 5-HT might stimu- probably in the muscle (54,55)• CHICKENS. The 5-HT content of the late intestinal motility mainly by sensitizing the sensory receptors in intestinal small intestine was estimated comparamucosa, which are sensitive to pressure tively in conventional, contaminated and and trigger the peristaltic reflex. In addi- in germ-free white Leghorn chicks at tion, 5-HT might also stimulate chemo- four weeks of age; it was found that receptors whose fibres converge on the germ-free animals showing poor tonus of same motor ganglion cells on which the the intestinal tract and distension of the fibres from pressure receptors impinge. caeca had a higher 5-HT content in their The stimulation of both types of recep- gut, especially in the ileum (ii pg. versus tors would be additive, and the reflex 6.1 ,egg./g.), than conventional animals would thus be triggered off more readily. possessing elevated tonus of the intestinal However, on the basis of results listed musculature (56). FISH. Fish afford another very impresunder (e), Bülbring herself (52) has recently advanced the opinion that 5-HT sive example of species differences in the might not be of primary importance in sensitivity of the gut to 5-HT. In fact, initiating and maintaining peristalsis. whereas concentrations of 5-HT as low Two kinds of tryptamine receptors as 0.005 pg./ml. were sufficient to stimuhave been described in the guinea pig late the isolated intestine of Pleuronectes ileum, namely the M receptors which can and Labrus, concentrations one to two be blocked with morphine and the D hundred times higher were barely effecreceptors which can be blocked by Diben- tive on the intestine of Squall's Raja and zyline. The M receptors are probably in Myxine (S7).

The Role of 5-HT in the Physiological Control of Intestinal Peristalsis Because the main sites of production and storage of 5-HT in the mammalian organism are the enterochromaffin cells of the gastrointestinal mucosa, and because 5-HT in several species has a powerful stimulating action on intestinal motility, it has been suggested that the chief physiological function of 5-HT, or at least one of its functions, is to control motor activity of the gut. Smith et al. (58) and Lembeck (49) went even further, since they believe that the effects of 5-HT outside the gastrointestinal tract are always unphysiological, because they become apparent only after administration of exogenous 5-HT or 436

following hyperproduction of 5-HT by malignant argentaf inomas. The hypothesis of a local function of 5-HT within the gastrointestinal mucosa soon after its release from the enterochromaffin cells is at first sight very attractive, and it has been recently substantiated by the important results reported in detail in the preceding pages. On the basis of these results, Bülbring (52) and Lembeck (49) maintain that the formation of 5-HT by the enterochromaffin cells could be part of the physiological mechanism required for peristalsis; more precisely they state that 5-HT has a physiological modulating function in peristalsis.

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The results and conclusions of Biilbring et al. are undoubtedly of great importance. However, once again extreme caution seems to be necessary in generalizing results obtained in one or two animal species or in transferring experimental data obtained on isolated intestines, or even on intestinal loops in situ, to the gastrointestinal tract of the intact animal. In fact, if we assume that the 5-HT which enhances the peristaltic reflex has a local origin, then it is rather difficult to give an acceptable explanation for the conspicuous differences in the distribution of the enterochromaffin cell system and of 5-HT along and outside the gastrointestinal tract of the various vertebrate species. What could be the meaning of the enormous accumulation of enterochromaffin cells and 5-HT in the intramural portion of the pancreatic duct of the rabbit? What could be the reason for the predominant accumulation of 5-HT in the stomach of mice, hedgehogs and dogs, in the small intestine of guinea pigs and chickens, and in the large intestine of rats, horses and cows? What, finally, is the significance of the enterochromaffin cells in the pancreatic islets of some mammals and in the thymus of some birds? Moreover, if the 5-HT introduced into the lumen of an isolated guinea pig intestine produced a stimulation of peristalsis in very low concentrations, the same 5-HT required a decidedly higher dosage to produce short bursts of peristalsis when introduced into a loop of the guinea pig intestine in situ, and required a dosage far beyond any physiological limit to influence the motility of the intestine in the intact animal. In fact, the threshold oral dose capable of accelerating intestinal transit in rats was 4 mg./kg. of the base, and doses of 5-HT up to 10-15 mg. given intrajejunally to normal volunteers failed to elicit any intestinal or systemic response.

This is not all. In man only the upper part of the small intestine seemed to be stimulated by 5-HT, whereas stomach, ileum and large intestine were not affected by the amine, or were even inhibited. Yet all sections of the gastrointestinal tract contain enterochromaffin cells and 5-HT, and it is not easy to conceive that the same substance can display different functions in the different segments of an unique apparatus. Finally, nobody has so far brought any decisive evidence demonstrating that distension or hypermotility of the intestines, kept within physiological limits, actually produced in the intact animal a local discharge of 5-HT from the enterochromaffin cells into the interstitial liquid of the mucosa, which evidently represents an essential condition for the stimulation of neuronal elements. It is evident that the above findings and considerations are not in favor of the hypothesis that 5-HT has a general significance as a local hormone modulating peristalsis. Of course, 5-HT could also act on the gastrointestinal tract via the blood. But, frankly, it would be rather surprising if the 5-HT released from the intestines had to return to the same intestines, after having been exposed to severe enzymatic attacks and to large losses, to display there its main physiological action. At any rate, if there are experimental results which demonstrate that stimulation of intestinal motility can be produced in some mammalian species by physiological amounts of 5-HT injected intravenously or intra-arterially, there are other results which show that in other species, parenteral 5-HT acts on peristalsis only at very high dosage levels, considerably exceeding those required to produce other biological effects, e.g. antidiuresis in rats. Furthermore there is as yet no proof 437

V / 5-HT ANI) THE CASTRO-INTESTINAL TRACT

that distension of the intestines or hyperperistalsis produces a discharge of 5-HT into the blood. Johnsen et al. (33) failed to observe any relationship between meals (i.e. motor and secretory activity of the bowels) and the peak in the diurnal variation in 5-HT metabolism, as inferred from the diurnal variation in the urinary excretion of 5hydroxyindoles; Bertaccini and Erspamer (59) were unable to detect any increase in urinary 5-HIAA excretion in rats given by mouth 4 ml./loo g. paraffin oil, in spite of evident distension and hyperperistalsis of the intestines; Paasonen et al. (6o) found that cholinergic drugs and cathar-

tics did not increase the urinary excretion of 5-HIAA in man; finally, several research workers (61,62,63) concordantly demonstrated that by chronic diarrhea and cholitis ulcerosa urinary 5-HIAA was within normal limits. The obvious conclusion is that before the hypothesis of a general significance of 5-HT in controlling intestinal motility can be accepted, additional experimental evidence is required, obtained, if possible, in representatives of all vertebrate classes, because all vertebrates contain enterochromaffin cells and 5-HT in their gastrointestinal mucosa.

Ulcerogenic Action of 5-Hydroxytryptamine and 5-Hydroxytryptophan 5-HT and 5-HTP, when injected in lesions produced by 5-HT and 5-HTP, sufficiently large doses, caused in rats and whereas iso-5-HT, promethazine, thenamice the appearance of hemorrhagic mu- lidine, Antistin and papaverine were concosal erosions in the stomach and the siderably less active (65,68,69,70,71,73, colon sometimes evolving, after prolong- 74). Histamine enhanced the pathological ed treatment, into chronic ulcers and effects of 5-HT (75). In mice, 5-HT failed to produce gastric hemorrhage scars (64,65,66,67,68,69,70,71). Following two subcutaneous doses of when administered to vagotomized ani15 mg./kg. 5-HT, 95 per cent of the rats mals or to animals pretreated with iproexhibited ulcers in the stomach, and 50 niazid (64). It is possible that, as with per cent in the colon; following two doses renal lesions, gastric lesions produced by of 30o mg./kg. 5-HTP the percentages 5-HT heal with restitutio ad integrum in were 40 and o, respectively (69,70). spite of continuous administration of the Ulcerations were particularly numer- drug. In fact, MacDonald et al. (76) and ous both in the glandular and squamous Davidson et al. (77) failed to detect ulcers epithelium of rats in which the cardias in the gastrointestinal tract of rats treated and the pylorus were ligated at the time of up to 3S3 days with daily subcutaneous the first injection (three injections of 40 doses of 7 mg. 5-HT base/rat, or daily mg./kg. 5-HT base each, in twenty-four intraperitoneal doses of 200 mg./kg. 5hours) (72) . HTP. Tryptamine (500 mg./kg. x 2), tryptoAs far as the pathogenesis of gastric phan, iso-5-HT (100 mg./kg. x 2) and iso- 'ulcers is concerned, several research 5-HTP (300 mg. /kg. x 2) did not display workers (66,67,70) maintain that 5-HT any ulcerogenic effect (70). LSD, UML, acts directly through its vasoconstrictor chlorpromazine, chlorbenzoxamine and action with consequent focal ischemia of atropine effectively prevented gastric the gastric mucosa. Nikodijevic and 438

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Trajkov (68) suggest, on the contrary, that the ulcerogenic action of 5-HT is the result of the histamine-liberating properties of 5-HT rather than of a direct effect on the rat gastric mucosa; this was postulated on the basis of the striking protective effect afforded by antihistaminic drugs possessing no anti-5-HT properties. However, if one accepts this point of view, it is rather difficult to understand why 5-HT displays a clearcut antagonistic effect on the histamineinduced gastric secretion. Two main problems have been raised by the discovery of the ulcerogenic action of 5-HT in rats. The first concerns the possible intervention of the amine in the pathogenesis of gastric ulcers produced by reserpine; the second problem, a more important one, concerns the possible röle of 5-HT in the pathogenesis of gastric ulcers in man. The hypothesis that reserpine and reserpine-like drugs cause gastric hemorrhage by a mechanism which involves the liberation of 5-HT is based on the similarity of the lesions produced by the two drugs, on the fact that some antagonists (monoamine oxidase inhibitors, atropine, hexamethonium, antacid mixtures) are capable of protecting the rat gastric mucosa against the ulcerogenic effect of both 5-HT and reserpine, and finally on the circumstance that reserpine is a powerful 5-HT liberator (61,64). However, there is decisive evidence against the existence of any important participation of 5-HT in the production of gastric ulcers by reserpine. In fact: a) reserpine produces in dogs and rats a consistent and pronounced gastric secretion; 5-HT is either inactive or reduces the volume and acidity of gastric juice (22 ,2 4,27). b) 5-HTP potently inhibits gastric secretion in the dog; reserpine is capable of stimulating gastric secretion, in spite

of prior administration of 5-HTP, i.e. in spite of the presence of large amounts of 5-HT originating from the precursor amino acid (27). c) BOL, which effectively counteracts the ulcerogenic effect of 5-HT, gives no protection against the ulcers produced by reserpine (78). d) MAO inhibitors markedly inhibit the production of gastric hemorrhagic erosions caused by reserpine, but do not show any visible protection against the ulcerogenic action of 5-HT (79,8o). e) the doses of 5-HT and 5-HTP required to produce gastric ulcers or erosions are enornious, far beyond the limits of physiological significance; on the other hand, amounts of 5-HT released by reserpine from the gastric mucosa are minimal, and this is especially so in the rat whose gastrointestinal 5-HT is released by reserpine with unusual difficulty and incompleteness. It should be added that peripheral and central actions of reserpine are so complex and the active biogenic compounds released by the drug so numerous, that it seems futile attempting to ascribe to excess or defect of a single substance effects which are probably due to the rupture of the equilibrium existing among a number of different amines and other active compounds as well. Concerning the possible role of 5-HT in the pathogenesis of gastric ulcers in man, the problem is valid only for gastric ulcers seen in patients with carcinoid tumors, provided of course, that it is definitely proved that the incidence of ulcers is actually greater in patients with metastasizing carcinoids showing a typical "carcinoid syndrome", than in patients suffering from other neoplastic diseases of the gastrointestinal tract. It is indeed conceivable that the enormous amounts of 5-HT released by the argentaffinoma (the daily release of 5-HT 439

V / 5-HT AND THE GASTRO-INTESTINAL TRACT

may attain or even surpass 1000 mg., as products. The histamine-releasing activity of 5judged from the urinary excretion of 5HIAA) might cause or facilitate the func- HT has been known for several years, tional and anatomical alterations of the and it has been recently demonstrated that gastric mucosa which are at the root of also high doses of 5-HTP (50-100 mg./kg. intravenously) are capable of releasing in ulcer formation. According to Sandler et al. (81), it is vivo 5-HT from rabbit platelets (82). likely that the incidence of gastric ulcers Moreover, Pernow and Waldenström will be higher in those rare patients in found that out of twenty-two cases of which the carcinoid tissue secretes 5-HTP metastasizing malignant abdominal carcinoid tumors, seven presented an ininstead of 5-HT. 5-HTP and 5-HT might display their creased urinary excretion of histamine, ulcerogenic effect either directly, through with figures ranging from 27 to 6800 their capacity for changing vascular ug./24 hr. It seems well possible that these caliber, as well as secretion and motility, amounts of histamine may facilitate the or indirectly through their ability to re- appearance of gastric ulcers in predislease histamine and/or other active tissue posed individuals.

Blood 5-HT Levels and Urinary Excretion of 5-HIAA in Gastrointestinal Diseases Table III presents some data on the blood 5-HT levels and the urinary excretion of 5-HIAA in gastrointestinal diseases. The carcinoid syndrome has been deliberately omitted owing to the exhaustive literature existing on the topic, which cannot be profitably summarized within the narrow limits of this article, and because "carcinoidosis" is not properly a gastrointestinal disease but a general syndrome produced by neoplastic argentaffin tissue, wherever this tissue might develop and grow. Tabulated data show that the only pathological conditions in which a clearcut change in the 5-HT metabolism may be seen are the nontropical sprue, and the syndrome ensuing the resection of large segments of the gastrointestinal tract. In nontropical sprue, alteration of 5HT metabolism is probably an indirect consequence of the basal biochemical defect of this syndrome which consists of poor utilization of serine with increased 440

urinary excretion of p-hydroxyphenylacetic acid. Elevated blood 5-HT and urinary 5-HIAA levels returned to normal during remission of the disease induced by a gluten- and gliadin-free diet (62,83). The drop in blood 5-HT and urinary 5-HIAA levels seen following resection of large segments of the intestinal tract must be ascribed to the reduction of the 5-HT secreting tissue. Experimental evidence supporting this view has been afforded by Bertaccini (84) and by Erspamer and Testini (7). Finally, emphasis should be laid on the fact that neither pathological changes in the motility of the gastrointestinal tract, in the sense of hypermotility (chronical diarrhea, ulcerative cholitis), nor pathological changes in the gastric secretion, both in the sense of hyperchlorhydria and of achlorhydria, were accompanied by signs of increased or decreased production or release of 5-HT. These observations

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TABLE III. Blood 5-HT levels and urinary 5-HIAA excretion in diseases of the gastro-intestinal tract BLOOD 5-HT

Pathological condition Platelet 5-HT (µg/mg platelet protein)

Normal subjects

0.17-0.7(81) 0.42±0.11(83)

Gastritis

Superficial} mild J severe

-

Atrophic gastritis

-

Gastric ulcer

-

Duodenal ulcer

-

Gastric carcinoma

-

Chronic diarrhea and ulcerative colitis

-

0.16-0.31`61) -

Regional enteritis

Serum 5-HT (µg/ml)

0.21(9') -

-

-

Whole blood 5-HT (µg/ml)

0.13±0.025(63) 0.11(91)

2_9(61.83.9") 1.7 5. 5(92) 3.8±1. 47(63)

-

2.59±1.34

-

3.36±2.43

0.12(9)

2.76±1.47

0.076

2.38±1.56

-

0.12-0.13 -

2.48±1.22(63) 1.5-4.3(81) 1.2-6.2(62)

-

-

0.49-0.71

-

Nontropical sprue

0.28-1.57

-

0. 75 -1.30(93)

-

-

-

-

-

Gastric recent resection remote Extensive resection of small & large bowel

1.4-3.8(91) 8.4-12.4

0.23-1.08(631

0.098(f')

0.064

0.03-0.05(61)

2.71±1.20(6J)

3.84±2.04

Intestinal lipodystrophy

Achlorhydria

Urinary 5-HIAA (mg/day)

0.01(93)

8_27(83). 11-26(63) 5.8-12.4(6» normal' 92) 3.08±2.06(63)

0.35 -1.06(81) 0.15(9')

Segmental intestinal resection

-

normal

-

normal(93)

Intestinal sterilization with oxytetracycline

-

-

-

normal(12) 441

V / 5-HT AND THE

GASTRO-INTESTINAL. TRACT

are obviously rather against than in favor of the hypothesis which attributes to 5HT a role in the physiological control of

motility or secretions of the gastrointestinal tract.

Summary The characteristics of the enterochroma ff in cell system in vertebrates are briefly outlined. The negativity or positivity of histochemical reactions peculiar for the EC system appears to be dependent on concentration of 5-HT in the granules. The distribution of the EC system in different sections of the gastrointestinal tract is irregular and differs between species. Typical enterochroma ff in cells can be also found in the nmcosa of the urinary bladder, prostate, upper respiratory tract and in the pancreas and thymus. The action of 5-HT on gastrointestinal secretions and motility in various species is discussed. In man an inhibition of gastric secretory volume and acidity of both spontaneous and histamine-induced gastric secretion has been almost universally observed following the administration of 5-HT and 5-HTP; mucus production was found to be increased. A single intravenous injection of 5-HT produces a sharp rise in intestinal tone and the disappearance of mixing waves. The stinmlatory effect appears thirty seconds after injection and disappears within three to four minutes; it is followed by a period of hyliomotility. It appears that there is not sufficient evidence at the present time in favor of

the hypothesis that 5-HT has a universal significance as a local hormone regulating peristalsis; certain experimental data point against this possibility. The postulate that reserpine and reserpine-like drugs cause gastric hemorrhage by a mechanism which involves the liberation of 5-HT is based on the similarity of the lesions produced by the two-drugs in rodents. However, several experimental data supply sufficient evidence against the existence of any participation of 5HT in the production of changes produced by reserpine. Concerning the possible role of 5-HT in the pathogenesis of gastric ulcers in Mall, the problem is valid only for lesions seen in patients with carcinoid tumors. It is conceivable that the enormous amounts of 5-HT released by the argentaffinoma may facilitate the functional and anatomical changes leading to ulcer formation. 5-HTP and 5-HT might exert either a direct ulcerogenic effect or act as a liberator of histamine and/or other active tissue products. The histaminereleasing activity of 5-HT has been known for several years, and it is possible that the amounts liberated may facilitate the development of a peptic ulcer in predisposed individuals.

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52 4, 1960. TO. PETRUCELLI, L. M., BULLE, P. H. and OSKOUI, M. Fed. Proc., 20: 3,8, 1961. 11. CALDARERA, C. M., RIVA SANSEVERINO, E., and URBANO, A. Boll. Soc. Ital. Biol. Sper., 37:

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and HIDEG, J. Arch. Int. Pharmacodyn., :18: 62-69, 1959. TYCE, G. Al., STOBIE, G. H. C. and BOLLMAN, J. L. Fed. Proc., 20: 252, 1961. LEMBECK, F. 5-Hydroxytryptamine Lewis, G. P. 147-152. London Pergamon Press, 1 957. GEORGES, G., D. Al. HEROLD, Therapie, (Par), 13: 56-61, 1958. LEE, C. Y. J. Physiol., (Lond), 152: 405-418, 1960. BRITTAIN, R. T. and COLLIER, H. O. J. J. Physiol., (Lond), rot: 14-15, 1958. LISH, P. M., CLARK, B. B. and ROBBINS, S. L. Amer. J. Physiol., 197: 22-26, 1959. ERSPAMER, V. Z., GLASSER and MANTEGAZZINI, P. Experientia, 16: 505, 1960. BÜLBRING, E. and CREMA, A. Brit. J. Pharmacol., 13: 444-457, 1958. GINZEL, K. H. Arch. Exp. Path. Pharmak., 232: 287-288, 1957-58. LEMBECK, F. Pfl uegers Arch. Ges. Physiol.,

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1083-1085, 1961. 13. cALt, G. and CORDOVA, c. Progr. Med. (Nap), 14.

(Par), 53: 525-541, 1961.

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45. 46. 47. 48. 49.

265: 567-574, 15158. 50. BELESLIN, D. and VARAGIC, V. Brit. J. col., 13: 266-270, 1958. 51. BÜLBRING, E. and LIN, R. C. Y. J.

Pharma-

Physiol., 140: 181-407, 1958. 52. BÜLBRING, E. and CREMA, A. J. Physiol. (Lond) 146: 29-53, 1959. 53. BÜLBRING, E. and CREMA, A. J. Physiol. (Lond), 146: 18-28, 1959. 54. GADDUM, J. H. and PICARELLI, Z. P. Brit. J. Pharmacol. 12: 323-328, 1957. 55. LEVY, J. and MICHEL-BER, E. J. Physiol. (Par), 48: 1051-1084, 1956. 56. MACDONALD, R. A. Amer. J. Med., 21: 867878, 1956. 57. VON EULER, U. S. and ÖSTLUND, E. Acta physiol, scand. 38: 364-372, 1957. 58. SMITH, A. N., NYHUS, 1.. M., DALGIESH, C. E. DUTTON, R. W., LENNOX, B. and MACFARLANE, P. S. Scot. Med. J. 2: 2 4-38, 1957. 59. BERTACCINI, G. and ERSPAMER, V. Unpublish-

ed Observations.

60. PAASONEN, Al. K., PELTOLA, P. and VANHAKARTANO, P. A. An. Med. Exp. Fenn., 38: 220-226, 196o. 61. HAVERBACK, B. J. Discussion of the paper

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presented by Benditt, E. P. Gastroenterology, 4o: 338-343, 1961. KOWLESSAR, O. D. WILLIAM, R. C., LAW, D. D. J. and SLEISENGER, M. H. New Engl. J. 259: 340-341, 1959.

63. SCHMID, E., ROSENBUSCH, U., HEINKEL, K. SCHWEMMLE, K., and SCHÖN, H. Gastroenterologia (Basel), 96: 275-290, 1961.

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64. BLACKMAN, J. G., CAMPION, D. S. and TASTIER, F. N. Brit. J. Pharmacol., 14: 112-116, 1959. 65. HAVERBACK, B. 3. and BOGDANSKI, D. F. Proc.

Soc. Exp. Biol. Med. 95: 392-393, 1957.

66. HEDINGER, C. H., and VERAGUTH, F. Schweiz. Med. Wschr., 87: 1175-1176, 1957. 67. GARRET[, D. and MISSERE, G. Clin. Patol. Sper.

3: 323-3 28, 1955.

68. NIKODIJEVIC, B. and rRAJKOV, T. Biochem.

Pharmacol. 8: 169, 1961. Hely. Physiol. Pharmacol. Acta, 15: 83-84, 1957. 70. WILHELM!, G. and SCHINDLER, W. Arch. Exp. Path. Pharmakol., 236: 49-51, 1959. 71. LEVIS, S. and BEERSAERTS, J. Arch. Int. Pharmacodyn., :26: 359-364, 1960. 72. SKORYNA, S. C. and WEBSTER, D. R. PrOC. Roy. Coll. Phys. & Surg. Can. 32: 47-48, 1963. 73. NICODIJEVIC, B. and VANOV, S. Experientia, r6: 464-465, i96o.

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84. BERTACCINI, G. and NOBILI, M. B. Brit. J. Pharmacol., r7: 519-525, 1961. 85. RESNICK, R. H. and GRAY, S. J. Gastroenter-

ology, 4t: 119-121, 1961.

86. GARVEN, J. D. Brit. J. Pharmacol., r 1: 66-7o,

1956.

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Pharmacol., 15: 532-539, 1960.

89. RESNICK, R. H., SMITH, G. T. and GRAY, S. J. Amer. J. Physiol. 201: 571-573, 1961. 90. ROSENBERG, J. C., DAVIS, R. MORAN, W. H. and ZOMMERMANN, B. Fed. Proc., :8: 503, 1959.

91. SCHMID, E., SENG, I., HENNING, N. and HEINKEL, K. Gastroenterologia (Basel), 93: 2352 55, 1960. 92. BRUMMER, P. and JAUROLA, A. Acta med.

74. RADOUCO-THOMAS, C., LATASTE-DOROLLE, C. ROGG-EFFRON, C., VOLLITER, G. I. MEYER, M. CHAUMONTET, J. M. and LARUE, D. Arznei-

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1958. 77. DAVIDSON, J. A., SJOERDSMIA, LOOMIS, L. N. and UDENFRIEND, S. J. Clin. Invest., 36: 15941599, 1957. 78. LA BARRE, J.

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Scand. 162: 397-400, 1958. and JAUROLA, A. Acta med. Scand. :62: 397-400, 1958. DE CORRAL SALETA, J. Abstr. Commun, 225-226, Int. Phy. Congress. Bruxelles 1956. DANIEL, E. E., HONOUR, A. J. and BOGOCH, A. Gastroenterology, 39: 62-73, 1960. ERSPAMER, V. and TESTINI, A. J. Pharm. Pharmacol., 1 r: 618-612, 1959. GARVEN, J. D. Brit. J. Pharmacol, t r: 66-70, 1956. PERNOW, B. and WALDENSTROEM, Amer. J. Med., 23: 16-25, 1957.

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97. 98.

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104. PAGE, I. H. Physiol. Rev. 38: 277-355, 1958.

The Relationship of the Major Endocrine Glands to Experimental Peptic Ulceration*

experimental work has been performed in recent years in order to clarify the relationship of the major endocrine glands to peptic ulceration. The assessment and clinical interpretation of the results are unfortunately difficult and complex for the following reasons. First, peptic ulceration does not occur spontaneously in experimental animals. Artificial preparations must be employed, and the two most commonly used, the MannWilliamson dog and the Shay rat, are anatomically rather abnormal. Second, while we know and can measure most of the attacking forces in gastric juice, we know virtually nothing of the defensive mechanisms of the mucosa, and are unable to measure powers of resistance to peptic digestion. The physicochemical complexities of mucus render uncertain the value of the few observations that have been made on it. Third, species differences are always important, especially in hormonal studies. For example, the naturally occurring glucocorticoid in the rat is corticosterone, while in the dog it is cortisol. Alterations in hormonal environMUCH

James Kyle Stewart D. Clarke John T. Ward A. 0. Adesola Richard B. Welbourn**

ment may affect not only secretory activity but also mucosal structure differently in different species. Fourth, the major endocrine glands constitute a closely integrated and delicately balanced system. Changes in the hormone output of one gland may alter the activity of the others, and these secondary changes may themselves be reflected in the gastric secretion. Furthermore the hormones may have some effect on the intrinsic hormones of the alimentary canal and more particularly on gastrin. Fluid and electrolyte balance may be altered by hormonal changes. These difficulties in the experimental elucidation of the causes of peptic ulceration must always be borne in mind. As a result of them, many investigators have confined their work to studying gastric acid production (being an indirect measure of the likelihood of peptic ulcer formation) after the administration of a hormone or the ablation of an endocrine gland. Ulcer studies in man have produced supporting evidence. Unfortunately the confusion resulting from the in-

'Supported in part by a grant from the Linen Industry of Northern Ireland Research Grant. From the Departments of Surgery, the Universities of Aberdeen, Scotland, and of Sheffield, England; Queen's University, Belfast, Northern Ireland; and Lagos University Hospital, Ibadan, Nigeria.

445

V / ROLE OF THE MAJOR ENDOCRINE GLANDS

herent difficulties of the problem has been more confounded by man-made variations in hormone dosage and timing and in nomenclature. Many doses administered have been at the upper limit of the pharmacological range, and can bear little relationship to any naturally occurring physiological or pathological process.

Time schedules have varied widely and significantly, and there has frequently been a complete failure to differentiate gastric ulcers (representing a failure of mucosal resistance) from duodenal ulcers (representing a triumph of acid pepsin digestion).

The Adrenal Glands Little is known of the influence of the adrenal medulla on gastric secretion. Both adrenalin (epinephrine) and noradrenalin have such profound effects on other body systems that any alterations which they might produce in the stomach would be extremely difficult to evaluate. Medullectomy in the rat produces no alteration in gastric secretion (1). Consequently, the adrenal medulla is usually disregarded in adrenal gland studies. A few days after adrenalectomy structural alterations are seen in the gastric mucosa of the rat (2). The zymogen cells become smaller and degranulated; changes in the parietal cells are less marked. Our own studies in rats show that the thickness of the gastric mucosa is significantly reduced two to three weeks after bilateral adrenalectomy. The mean thickness in ten adrenalectomized rats was 0.39 mm. compared with o.44 mm. in ten control animals. Studies of gastric secretion following adrenalectomy have shown that acid production is significantly reduced in the Shay rat (3,4). Pepsin production is also diminished (2). Adrenalectomy does not, however, protect the rat from developing ulcers in the stomach. Fifteen out of twenty-four of our adrenalectomized animals had small, superficial ulcers mostly in the glandular part of the stomach. The administration of optimal doses of cortisone to intact dogs results in an in446

crease in the thickness of the gastric mucosa and in the number of parietal cells. When a total dosage of 28 mg./kg. is given over a period of three to seven days there is a 27 per cent increase in mucosal thickness and a 5o per cent increase in the parietal cell count (5). A shorter period of hormone administration is ineffective. Larger doses may reduce the thickness and density of the parietal cells, and a similar result may be observed in rats (6). Secretory studies in intact animals follow a similar pattern. In the intact rat, it is very difficult to raise the normal rate of gastric secretion by any method. In our experiments with Heidenhain pouches in dogs, when moderate amounts of cortisone were given for ten days (75-10o mg. per day to io kg. dog), there was a marked increase in the volume and acidity of gastric secretion (7). Methyl prednisolone (12 mg. per day by mouth for ten days) and aldosterone (0.2 mg. intramuscularly each day for ten days) facilitated acid secretion in a similar manner, both under basal conditions and with histamine stimulation. Cortisone increased the maximal response to histamine to a supranormal level (Fig. I). All three hormones very slightly decreased the concentration of pepsin in gastric juice. Sodium and potassium concentrations also fell, and the viscosity of the basal secretion was reduced. Since cortisone and aldosterone

THE EFFECT OF CORTISONE ON THE GASTRIC SECRETORY

produced very similar results, it is probable that their effects are not directly related to their individual glucocorticoid and mineralocorticoid characteristics. Larger doses of cortisone (30o mg.) do not give any increase (8). Prednisone produces similar results(9). It however increases pepsin production as does hydrocortisone, although the latter response is greatly affected by changes in dosage (to). The results of pepsin estimations may also vary significantly according to the time that has elapsed between applying the stimulus and collecting the juice. The zymogen cells are unable to secrete pepsin at high rates for a prolonged period. When an adrenal inhibitor (SU 4885) is given daily for two weeks to dogs, there is a significant reduction in acid concentration. The volume and pepsin content of the juice are not altered, and a single injection is ineffective (11). A single injection of amphenone, however, inhibits acid secretion in rats (12). Both these structural and secretory investigations illustrate the importance of correct and comparable dosage and timing. During the past twelve years there have been many reports of the development or exacerbation of peptic ulceration in patients receiving cortisone, its analogues or corticotrophin. However, many of the diseases for which these hormones were

TOTAL FREEWCI SECRETION IN M.EQ

RESPONSE TO INCREASING DOSES OF HISTAMINE TOTAL FREE HCI 2.6

cor tisone

2.4 2.2

20 I.e 16 1.4 1.2

control

r0 0.e 0'6 dosage of histamine base

0.4 0.2

.....,unimonw nnnullllllllllllllllllllllllll I III III I IIIIIIIII

--2

TIME IN HOURS

FIG. 1.

being given may themselves have contributed to the disintegration of the gastric or duodenal mucosa. In rats cortisone in moderate dosage (0.025 mg./Gm. weight thrice weekly) delays healing of ulcers produced by thermocoagulation (13). In dogs with stress-induced ulcers, cortisone (1.5 mg./lb. B.W.) does not hamper the healing of the gastric lesions, but does delay recovery of the duodenal ulcer cases (14). In Mann-Williamson dogs, low dosage cortisone treatment prolongs life, but does not significantly reduce the incidence of ulceration (15). In the latter respect, it differs from corticotrophin, which reduces ulceration. It would appear that corticotrophin evokes some other beneficial hormonal response in addition to that of the glucocorticoids. After adrenalectomy in the rat, we found that cortisone would restore secretion not only to normal but to levels above those usually encountered in this species (3). It is possible that the normal

TABLE I. Effect of adrenalectomy and of replacement therapy with cortisone (10 mg./day for 3 days) and desoxycortone (2 mg./day for 2 days) on gastric secretion and ulceration in the pylorus-ligated rat. Both hormones cause a significant increase in gastric secretion and incidence of ulceration after adrenalectomy.

Experiment Control, normal Adrenalectomy Adrenalectomy + Cortisone Adrenalectomy -I- Desoxycortone

No. PERCENTAGE WITH ULCERS of Mean Volume Rats pH nal. /100G./hr. Glandular Ruinen 21 23 5 8

1.3 2.6 1.2 1.9

0.6 0.3 0.9 0.6

23 53 100 50

34 4 40 60

447

V / ROLE OF THE MAJOR ENDOCRINE GLANDS

adrenal gland secretes a gastric inhibitory I). With cortisone the ulcers were deep, factor. By contrast, the mineralocorticoid hemorrhagic and mainly in the glandular desoxycortone only restored the volume part of the stomach. Histamine produced of secretion, not its acidity. Volume and similar angry lesions. The ulcers in the acidity were both restored to normal in desoxycortone treated group were smaller adrenalectomized animals by histamine in and superficial, more like those seen in a dosage of 5.5 mg./kg. b.w. This dos- control rats. It is unlikely that the severe age has no effect in normal rats, but the ulceration present after giving cortisone adrenalectomized animal is very sensitive is due solely to the increase in intraluto histamine, and this sensitivity must minal pressure resulting from an increased apply to the parietal cells. By human volume of secretion. The same type of standards the dosage, however, is rela- ulceration was seen in intact rats receiving cortisone and corticotrophin, which fail tively enormous. The administration of cortisone (10 to raise the volume of secretion above mg./day for three days) and desoxycor- normal. In the large doses employed, these tone (z mg. twice) produced a significant hormones would appear to have an injurincrease in the ulceration within the stom- ious effect upon the glandular part of the achs of the adrenalectomized rats (Table stomach.

The Pituitary Gland In considering either ablative or addi- is a reduction in the number of parietal tive experiments on the pituitary gland, cells. The mucosa becomes thinner, the it must always be remembered that when change being greater in the glandular the anterior lobe of the gland is involved part of a rat's stomach than in the squaseveral or all of the subservient endocrine mous-lined rumen where, however, it is glands will be affected, and that they in still detectable. f Iucosal regeneration turn may significantly alter gastric func- after injury is retarded (13) in the rat, tion. but delay in healing is less marked in the There is little definite evidence that the dog (23). A normal histological picture posterior lobe of the pituitary normally can be partially restored by giving somainfluences gastric secretion, although totrophin, but hydrocortisone is more large doses of its extract can slightly de- effective. press secretion in cats (16). In dogs secreThere is a considerable reduction in tion remains unaltered (1 7) when poster- gastric acid formation following hypoior pituitary extract is given, but section physectomy in the rat (4,20), in the cat of the pituitary stalk—which cuts off (16), and in the dog (24). In hypopituinerve impulses to the posterior lobe—de- tarism in man, there is usually a marked creases gastric secretion (18). Removal decrease in gastric secretion (22,25). of the posterior lobe alone from a dog is Administraiton of corticotrophin for said, however, to increase secretion (19). some days increases gastric secretion in In most experiments, hypophysectomy dogs and monkeys (7,26,27), both the removes both lobes of the gland. It causes basal and histamine-stimulated acid secreconsiderable involution of the gastric tion being increased. Pepsin production is mucosa (zo,21,zz). The zymogen cells be- not constantly affected, while the viscocome smaller and less granular, and there sity of gastric mucus is diminished, the i 448

KYLE, CLARKE, WARD, ADESOLA & WELBOURN

latter response being more marked with corticotrophin than with cortisone. Similar observations have been made in man (28). As well as their unknown effects on mucosal resistance, hormones may affect acid and pepsin formation in different ways, and there is evidence that the secretory functions of the stomach can behave in an independent manner (29). The normally high secretory rate of the rat's stomach is not increased by giving corticotrophin (4). Corticotrophin's influence on uropepsin excretion is disputed (3o); perhaps the supply of this enzyme may be quickly "washed out" of the zymogen cells, which are unable to manufacture it at a rapid rate in response to continued stimulation. Thyrotrophic, gonadotrophic hormones and somatotrophin have no effect on uropepsin output. After hypophysectomy in the rat, corticotrophin will restore acid secretion, if given in the correct dosage and for a sufficient length of time. One unit of corticotrophin daily for seven to fourteen days is much more effective than two units daily for four days, and produces a result comparable to that obtained after giving Io mg. of cortisone daily for only three days. Somatotrophin, I mg. per day for eight days also fully restores secretion. In Mann-Williamson dogs, corticotrophin lessens the incidence of peptic ulcers,

and is more effective in this respect than cortisone (I$). Corticotrophin causes ulcers in the glandular part of the stomach of the normal rat (3 I) similar to those produced by cortisone (32). In pylorus-ligated rats, the usual site of ulceration is the rumen. In our own experiments with them, small superficial ulcers were seen in the glandular part of the stomach after hypophysectomy; they were much less severe than those encountered in control animals after six hours (Table II). They resembled the superficial erosions seen after adrenalectomy. Hypophysectomy conferred a significant degree of protection on the rumen, even in those animals receiving doses of corticotrophin or cortisone sufficient to restore secretion, both volume and acidity, to normal. This is all the more surprising in view of the thinning of the mucosa caused by hypophysectomy. The ulcers which were produced in the glandular part were smaller, less deep and less hemorrhagic than those seen following comparable experiments in adrenalectomized rats. It would appear that some nonadrenal noxious factor is removed or rendered ineffective by hypophysectomy; the diminished endocrine secretions of the thyroid and gonads may be involved, providing an environment less favorable to acid pepsin attack or more favorable to mucosal resistance.

TABLE II. Effect of hypophysectomy and of replacement therapy with cortisone (10 mg./day for 3 days) and corticotrophin (1 unit/day for 10 days) on gastric secretion and ulceration after hypophysectomy in the rat. The hormones restore secretion but removal of the pituitary protects the rumen against ulceration.

Experiment Control, normal Hypophysectomy Hypophysectomy + cortisone Hypophysectomy + corticotrophin

No. of Mean Volume Rats pH ml./100G./hr. 17 15 8 9

1.3 2.7 1.3 1.4

0.7 0.2 0.6 0.6

PERCENTAGE WITH ULCERS

Glandular

Rumen

28 47 63 78

33 0 0 0

449

V / ROLE OF THE MAJOR ENDOCRINE GLANDS

The Thyroid Gland Thirty years ago investigations of the effects of the hormones from the thyroid gland and gonads were popular, but for over two decades gastroenterologists evinced very little interest in these glands. Before the commencement of modern endocrinology, it had been shown that an aqueous extract of thyroid increased gastric acid production in Pavlov pouch dogs and the effect was unaltered by vagotomy ( 33 ). The exact composition of the extract is unknown. Early clinical studies indicated that in hyperthyroidism, gastric acidity was frequently reduced (34). Recent work has shown that this reduction is greatest in middleaged women (35). Thyroid-stimulating hormone does not increase the output of uropepsin in man (36). In rats, thyroidectomy produces a slight diminution in the size and number of granules in the zymogen cells. There is a significant decrease in the volume of gastric juice and in pepsin output, although the concentration of pepsin remains unaltered. A combination of thyroidectomy, gonadectomy and adrenalectomy frequently produces structural and secretory changes comparable to those seen after hypophysectomy (2) . In the dog thyroidectomy appears to have little

effect on gastric secretion although it may render the latter more sensitive to administered hormones (37). Recent experiments in rats have shown that the administration of thyroxine reduces acid secretion. Following the injection of 20 erg. thyroxine daily for seven days, the volume of juice and the output of acid is reduced to about 6o per cent of normal (35). The pituitary and adrenal glands do not decrease in weight, indicating that these glands are not being inhibited. Gastric secretion can also be diminished by feeding thyroid extract to rats (38), but in this type of experiment the exact dose of active hormone absorbed must be doubtful. In the dog oral thyroid extract, thyroxine and tri-iodothyronine all decrease acid formation; subcutaneous trjiodothyronine is even more effective. Correlative studies indicate that it is not the elevation of the metabolic rate which causes the decrease in gastric activity (36). The excess of circulating thyroid hormone may interfere with the concentration or action of the adrenal hormones around and in the parietal cells in the gastric mucosa. Alternatively, it may alter the structure of the mucosa; this possibility requires further investigation.

TABLE III The effects of single injections of calcium lactate and parathormone and of repeated injections of parathormone and vitamin D on the volume and acidity of gastric secretion in the dog. CONTROL

Experiment

Dose

No. of Dogs

Mean Vol./Hr.

Mean pH

TEST

Mean Vol./Hr.

Mean

pH

Calcium Lactate

13 mg./kg. I.V.

5

2.0

6.8

1.3

6.3

Parathormone

20 u. s.c. once

4

1.8

7.1

2.3

5.8

Parathormone Vitamin D

20 u./day s.c. for 7 days 400,000 u./day s.c. for 18 days (terminal)

5

1.0

6.8

2.7

4.0

1

1.5

7.5

3.8

1.5

450

KYLE, CLARKE, WARD, ADESOLA & WELBOURN

The Parathyroids In humans with primary hyperparathyroidism, peptic ulceration is common. Surgical treatment reduces the basal secretion of acid but not the maximal histamine response (39). Very large doses of parathyroid extract in dogs may cause calcium deposition and necrosis in the depths of the fundic glands (40), but these doses are not physiological. Long ago it was shown that an aqueous extract of the parathyroids increased gastric secretion in dogs (3o). A more refined preparation of the hormone increases acid secretion in denervated pouches, but not in Pavlov pouch dogs (41). In humans parathormone increases the volume and total acid and pepsin output (42). Our own experiments have confirmed these findings and show the importance of correct dosage and timing (Table III). When four dogs with Heidenhain pouches were given single subcutaneous injections of parathormone (2o units/kg.), there was no alteration in basal gastric secretion. When, however, zoo units were given daily for seven days to five dogs, three of them showed an increased output of acid. In all cases the serum calcium level was elevated. Intravenous calcium lactate (i 3 mg. Ca/kg.) as a single injection into each of five dogs did not alter

basal or histamine-stimulated secretion. The daily injection of vitamin D (400,000 units I.M.) for eighteen days likewise failed to stimulate secretion until the animal was dying, at which time there was a sudden unexplained outpouring of highly acid juice. How abnormal parathyroid function affects gastric secretion is unknown. Presumably the parietal and other gastric cells require an optimum concentration of calcium in the fluid surrounding them. The above observations show that histamine can make the parietal cells function normally in spite of changes in the concentration of circulating calcium and parathormone. In man the association between multiple endocrine adenomas and severe peptic ulceration has recently been discovered (43,44). The parathyroids, pituitary and adrenal glands and pancreatic islets are generally involved. To account for this bizarre syndrome, an abnormal gene has been suggested (45). How this gene acts is completely unknown, and a plea is here made for careful secretory studies and parietal cell counts in any further examples of the syndrome which are encountered.

The Gonads Little detailed work has been done in recent years on the influence of the testicular or ovarian hormones on gastric secretion and ulceration. Pregnancy constitutes a rather unusual physiological state in which most of the endocrine glands are functioning in a manner different from that found in the nonpregnant state. Orchidectomy in the rat has no obvious effect on the structure of the gastric mucosa or on the number of zymogen cells

(z) .Ovariectomy in the same species does not affect secretion. Pituitary gonadotrophins do prevent the development or hasten the healing of experimental ulcers in dogs (46), but the effects of estrogens or testosterone in animals is not definitely known. In man stilbestrol is believed to have some beneficial effect in the longterm management of duodenal ulcer, similar to that found in dogs (47). During pregnancy in women, there is usually a long remission of duodenal ulcer 451

V / ROLE OF THE MAJOR ENDOCRINE GLANDS

symptoms (48). Gastric acid and pepsin secretion are reduced (49). Acid, but not pepsin, production is decreased in the pregnant rat (5o), but in the dog pregnancy has no effect on gastric acidity, although hyperacidity develops during lactation (51). Species differences are well exemplified by these effects of preg-

nancy. This is not surprising in view of the large number of body systems affected by pregnancy. In addition to reducing gastric acidity, pregnancy, as a fundamental biological function, may confer on the organism some currently unmeasurable increase in resistance to stressful and noxious stimuli.

The Pancreatic Islets of Langerhans Insulin augments secretion of gastric juice in both man and animals. This effect occurs at about the same time as hypoglycemia is maximal and is abolished by vagotomy. Thus insulin probably exerts its gastric effect through stimulation of the vagus nerves by hypoglycemia. The action of insulin on gastric secretion was complicated by the report that following the vagal phase of insulin stimulated secretion, there was a quiescent period and then a further rise in secretion of gastric juice (52). This second phase could be abolished by adrenalectomy, and it was postulated that insulin stimulated the posterior hypothalamus and hence the pituitary-adrenal axis, in addition to stimulating vagal pathways in the anterior hypothalamus. This mechanism, which was originally demonstrated in monkeys, is also believed to apply to man (53,54)• This theory is supported by the finding that insulin coma causes an adrenocortical response in man with a rise in plasma 1 7hydroxycorticosteroid levels (55). Insulin secreting tumors of the pancreas cause gastric hypersecretion in man (56) and, occasionally, peptic ulceration (S7). The rarity of the association of f3-celled pancreatic adenomata with peptic ulceration has been stressed, and it has been suggested that if peptic ulceration develops in a patient with hyperinsulinism, it is almost certain there are other lesions in endocrine glands (58). 452

Maximal vagal stimulation is not obtained until blood sugar levels of below 5o mg. per cent are obtained. It may be that insulinomas rarely produce hypoglycemic levels of this order. Another possible explanation of the absence of ulceration in some patients is that in pylorusligated rats, while single doses of insulin produce marked gastric hypersecretion, when the hormone is administered for ten or twenty days gastric secretion is unaffected (59). Change in blood sugar level rather than persistent hypoglycemia may be the effective stimulus. While insulin is formed in the (3-cells of the pancreatic islets it is probable, though not certain, that glucagon is formed in the a-cells. Interest in the gastric effects of glucagon arose from the suggestion that it might be an ulcerogenic hormone secreted from the pancreatic adenomata in the Zollinger-Ellison syndrome (6o). These tumors are usually composed of a-cells (58), and a hyperglycemic substance has been isolated from such a tumor (44). The agent causing hypersecretion in these cases is now known to be gastrin, following the brilliant series of investigations conducted in 196o-1961 by Gregory and Grossman (61,62). Glucagon has also been eliminated from suspicion of causing ulceration by the finding that it does not stimulate gastric secretion, but rather it has a powerful depressant action. In the dog, glucagon

THE EFFECT OF GLUCAGON ON INNERVATED POUCH SECRETION BASAL

VOLUME

6

IN

4

1

PSYCHIC

MEAT EXTR

-57

-69

i

—

MLS

reduces the acid secretory response to insulin and food. Our own studies show that the volume of secretion is reduced in innervated (Fig. za) and denervated pouches (Fig. 2b) under basal and psychic stimulation. The hydrogen-ion concentration was not altered greatly, nor was the response to histamine stimulation. In man also, the volume and acidity of basal secretion are reduced by this substance (63,64). Again there is controversy on the action on histamine-stimulated juice, stimulation being reported by some (65) and depression by others (66,67). It has also been shown that glucagon decreases the secretion of pepsin in man, and lowers the concentration of pepsinogen in the blood of man and the dog (68). It has been suggested that the inhibitory effect is secondary to the concomitant hyperglycemia. Some of the workers referred to observed a similar depression of gastric secretion following intravenous injection of glucose, but others found that the period of inhibition corresponded to that when the difference between the concentration of glucose in the arterial or capillary blood and that in the venous blood was greatest. Our studies in dogs showed that the response was independent of the hyperglycemic effect of glucagon, and that it could be obtained with physiological doses of the hormone (69). The effect of glucagon on the stomach is probably a direct one; the secretory response is still greatly reduced when the hyperglycemic effect is prevented by insulin. Glucagon increases the output of acid

INSULIN

2 % CHANGE

rte

_

NIL

-86

FIG. za. TIE EFFECT CF GLUCAGON ON DENERVATED POUCH SECRETION BASAL

MEAT EXTRACT HISTAMINE

10

8 VOLUME 6

g

4

3

IN ML S.

We CHANGE

1 a

- SO

- 85

-12

FIG 2b. and pepsin from the resting stomach of the pylorus-ligated rat; it abolishes the effect of vagotomy on acid secretion(59). In the rat it is suggested that the inhibitory effect of glucagon on histamine induced secretion might be related to the hypoglycemic phase following administration of this substance. It would therefore appear that, as so frequently found in gastric secretory studies, the response of the rat is different to that in other experimental animals and man. Glucagon affects the stomach in doses similar to those which raise the blood sugar; it is suggested therefore that it may play a part in the physiological regulation of gastric secretion. Glucagon is probably secreted maximally during the fasting state to prevent hypoglycemia. It would perform a useful function in ulcer prevention if at the same time it tended to prevent the secretion of gastric juice by the empty stomach.

Summary The effects on gastric secretion and ulceration produced by the hormones of the six major endocrine glands present a complex and often contradictory picture.

The six glands produce at least fifteen different well-known hormones, some or all of which influence the cells of the gastric mucosa for either good or evil. 453

V / ROLE OF THE MAJOR ENDOCRINE GLANDS

When mucosal resistance to acid pepsin digestion is enhanced, and secretion is normal or low, then the result is good. When, however, nnccosal resistance is impaired, and secretion is proceeding at a rate greater than normal, then peptic ulceration is almost inevitable. The widely differing responses seen after administering different hormones suggest that they affect the mucosal cells in different ways. Most of our knowledge concerns the parietal cells, but the zymogen, mucus and other cells are probably affected in a similar fashion. Some hormones may influence the function of the gastric cells, while others alter the structure of the mucosa. It is probable that insulin changes the stinndi arriving at the parietal cells via the vagus nerve. Cortisone, as well as stimulating the parietal cells to multiply, may render them more susceptible to normal stimuli such as local histamine. There is evidence that in peptic ulceration the number of parietal cells is not the only factor causing hyperacidity (7o). In some patients, there may be an endocrine disturbance not detectable by current methods of hormone assay (71). Adrenal corticoids may have some action on the oxidation-reduction enzyme systems producing hydrochloric acid. When there is an excess of these steroids in the blood reaching the parietal cells, the latter may be enabled to produce unusually large quantities of acid. Conversely acid production may be impeded by a deficiency of adrenal steroids. Such a deficiency could be caused by the local blocking action of some other hormone such as thyroxine. Glucagon may normally tend to inhibit the function of the parietal cells in the same way as cortisone may interfere with the production of rmlcus. Cortisone has been clearly shown to alter mucosal structure, its administration for some days causing a considerable in454

crease in the number of parietal cells. Other hormones are probably involved as well. After hypophysectomy in man, maintenance doses of thyroxine as well as of cortisone are needed to restore secretion to normal (72). It would be surprising if somatotrophin did not play some role in the regulation of gastric structure, but this point has not been fully investigated. While the majority of hormones act directly on the cells of the stomach, some exert their influence by indirect means. Parathormone probably alters parietal cell activity by varying the concentration of calcium ions in the blood; insulin hypoglycemia acts by stivnelating the hypothalanncs, while adrenalin (epinephrine) and noradrenalin can likely alter the supply of blood to the gastric mucosa. It seems clear, however, that aldosterone acts directly on the cells rather than by producing changes in electrolyte balance. The mode of action of the sex hormones is at present unknown. They could influence the anterior pituitary or, directly, have some effect on mucosal resistance. In all experimental and clinical studies involving hormones, the dose administered and the amount of hormone reaching the stomach in the blood stream are most important. When only small physiological quantities of the various hormones are circulating then conditions in the gastric nnucosa are ideal for the normal function, and the parietal and other cells are "permitted" to secrete at the optimal rate and to react normally to stimuli from the vagus nerve and local alimentary canal hormones. The igocosa's resistance to digestion is maximal and ulceration does not occur. The doses of corticotrophin and cortisone which produced beneficial results in Mann-Williamson dogs (15) were probably of this order. With larger pharmacological doses of hormones, gastric secretion may be stimulated, the cells

KYLE, CLARKE, WARD, ADESOLA & WELBOURN

may be unduly sensitive to normal stimuli and some may undergo work hyperplasia, resulting in a large outpouring of acid. At the same time mucus production may be impaired, and the outcome may be the development of a duodenal ulcer in a man or a glandular zone ulcer in a rat. With very large toxic doses producing general systemic disturbances, gastric secretion is depressed. Mucosal resistance may, however, be even more severely impaired, and a gastric ulcer could develop in these circumstances. In our own patients with Cushing's Syndrome, and hyperparathyroidisni gastric acid production was depressed only in those patients who had severe forms of the diseases and long histories. Some days must elapse before most hormones can make manifest their effects

on gastric secretion. At first the changes are rapidly reversible by restoring the hormonal environment to normal. After some months (in animals), the changes produced either by an excess or deficiency of hormones become irreversible. The changes may be in a particular type of gastric cell or in the mucosa generally; occasionally the vascular or nerve supply to the stomach may be permanently impaired. Only when much further work has been done to correlate the effects j'ects o f each of the various hormones on gastric structure and function, and when an experimental preparation for measuring mucosal resistance has been evolved, will we be able to fully understand the role of the endocrine glands in peptic ulceration.

References 1. TUERKISCHER, E. and WERTHEIMER, E. J. Endocr. 4: 143-151, 1945. 2. ABRAMS, G. D. and BAKER, B. L. Gastroenterology 27: 462-468, 1954. 3. WELBOURN, R. B. and CODE, C. F. Gastroenterology 23: 356-362, 1953. 4. KYLE, J. and WELBOURN, R. B. Brit. J. Surg., 44. 241-247, 1956. 5. REID, N. C, HACKETT, R. DI. and WELBOURN, R. B. Gut. 2: 119-122, 1961. 6. BAKER, B. L. and BRIDGMAN, R. M. Amer. J. Anat. 94: 363-397, 1954. 7. CLARKE, S. D., NEILL, D. W. and WELBOURN, R. B. Gut. r: 36-43, 1960. 8. WIEDERANDERS, R. E., CLASSEN, K. L., GOBBEL, W. G. JR., and DOYLE, M. M. Ann. Surg. 152: 119-128, 196o. 9. PLAINOS, T. C., and PHILIPPU, A. J. Gastro-. enterology 35: 183-189, 1958. 1o. OKA, M. and AHO, J. Acta. Rheum. Scand. 3: 68-75, 1957. 11. MCINTOSH, J. W., ANDERSON, N., DUTHIE, H. L. and FORREST, A. P. M. Gut 1: 345-350, 1960. 12. GRAY, S. J. Gastroenterology 37: 412-422o, 1959. 13. SKORYNA, S. C., WEBSTER, D. R. and KAHN, D. S. Gastroenterology 34: 1-l0, 1958. 14. RODRIGUEZ-OLLEROS, A. and GALINDO, L. Gastroenterology 32: 675-688, 1957. 15. SANDWEISS, D. J., SCHEINBERG, S. R. and SALTZ-

STEN, H. C. Gastroenterology 27: 617-624, 1954. 16. GUTTING, W. C., DODDS, E. C., NOBLE, R. L. and WILLIAMs, P. c. Proc. Roy. Soc. Lond. (B) 123: 2 7-38, 1937. 17. ATKINSON, A. J. and IVY, A. C. Amer. J. Dig. Dis. S: 30-35, 1938. t8. JOHNSON, T. A., SKORYNA, S. C., ROTHBALLER, A. B. and WEBSTER, D. R. Surg. Forum 6: 312, 16 1955. 19. MET 19. H. and LACKEY, It. W. Amer. J. Dig. Dis. 7: 27-32, 1940. 20. BAKER, B. L. and ABRAMS, G. D. Amer. J. Physiol. 177: 409-412, 1 954. 21. KYLE, J. Lancet 2: 72 4-72 5, 1955. 22. SMITH, A. W. M., DELAMORE, I. W. and WILLIAMS, A. W. Gut 2: 163-167, 1961. 23. JANOWITZ, H. D., WEINSTEIN, V. A., SHAER, R. G., CEREGHINI, J. F. and HOLLANDER, F. Gastroenterology 34: 11-20, 1958. 24. SALAMANCA, E. F., CASTANO, G. M., PORRUA, L. J. M. and CASTRO-RIAL, M. Arch. Med. Exp. (Madr). 16: 379-392, 1953. 25. ESCAMILLA, R. F. and LISSER, H. J. Clin. Endocr. 2: 65-96, 1942. 26. ZUBIRAN, J. M., KARK, A. E. and DRAGSTEDT, L. R. Gastroenterology 21: 276-279, 1952. 27. FRENCH, J. D., LONGMIRE, R. L., PORTER, R. W. and MovIUS, H. J. Surgery 34: 621-627, 1933. 28. HIRSCHOWITZ, B. fy STREETEN, D. H. P., POLLARD,

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H. M.

and

BOLDT, H. A.

J.A.M.A. 158: 27-32, 51.

1955•

and SPIRO, H. M. Gastroenterology 34: 196-209, 1958. SPIRO, H. M., REIFENSTEIN, R. W. and GRAY, S. J. J. Lab. Clin. Med. 35: 899-910, 1950. INGLE, D. J., PRESTRUD, M. C. and LI, C. H. Amer. J. Physiol. 166: 165-170, 1951. INGLE, D. J., PRESTRUD, M. C. and NEZAMIS, J. E. Amer. J. Physiol. 166: 171-175, 1951. ROGERS, J., RAHE, J. M., FAWCETT, G. G. and HACKETT, G. S. Amer. J. Physiol. 39: 345-353, 1915-16. HANGER, F. M. The Thyroid. Werner, S. C. ed. 516, London, Cassell, 1 955. BLAIR, D. w., and WILLIAMS, M., Brit. Med. J. (in press), 1963.

29. POLINER, I. J. 30. 31. 32. 33.

34. 35.

36. GRAY, S. J., RAMSAY, C., REIFENSTEIN, R. W. and BENSON, J. A. JR., Gastroenterology 25:

37. NASSET,, E. s. and GOLDSMITH, D. P. J. Amer.

J. Physiol. zol: 567-57o, 1961. J. and NASSET, E. S. Amcr. J. Physiol. 197: 1-4, 1959.

38. GOLDSMITH, D. P.

39. ADESOLA, A. 0., WARD, J. T., MCGEO\VN, M. G. and WELBOURN, R. B. Brit. J. Surg. 49: 112,

1961. and MAJNO, G. Presse Med. 6r: 286-288, 1953. SCHIFFRIN, M. J. Amer. J. Physiol. 135: 66o669, 1941-42. DONEGAN, W. L. and SPIRO, H. M. Gastroenterology 38: 750-759, 1960. UNDERDAHL, L. 0., WOOLNER, L. B. and BLACK, B. M. J. Clin. Endocr. 13: 20-47, 1953. FISHER, E. R. and FLANDREAU, R. H. Gastroenterology 32: 1075-1094, 1957. KIRSNER, J. B. Gastroenterology 34: 145-149, 1958. SANDWEISS, D. J., SALTZSTEIN, H. C. and FARBMAN, A. Amer. J. Dig. Dis. 5: 24-30, 1938. TRUELOVE, S. C. Brit. Med. J. 2: 559-566, 1960. CLARKE, D. H. Brit. Med. J. r: 1254-1257, 1953. HUNT, J. N. and MURRAY, F. A. J. Obstet. Gynaec. Brit. Emp. 65: 78-83, 1958. LOGGIO, B. B., GAGLIARDI, 0. P., BIEMPICA, L. and ROYER, M. Gastroenterology 4:: 126-128, 1961.

40. RuTISHAUSER, E. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

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MCCARTHY, J. D., EVANS, S. O. and DRAGSTEDT, L. R. Gastroenterology 27: 275-280, 1 954. 52. PORTER, R. W., MOVIUS, H. 3. and FRENCH, J. D. Surgery 33: 875-880, 1953• 53• SHAY, H., SUN, D. C., DLIN, B. and WEISS, E. J. Appl. Physiol, 12: 461-467, 1958. 54• STEMPIEN, S. J., FRENCH, J. D., DAGRADI, A. MovIOus, J. J., and PORTER, R. W. Gastroenterology 34: 104-116, 1958. 55• CHRISTY, N. P., LONGSON, D. HORWITS, W. A. and KNIGHT, M. M. J. Clin. Invest. 36: 54;-

56.

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SMITH, A. N. and COCHRAN, J. B. Lancet r: 289-293, 195 2 . JANOWITZ, H. C. and CRHHN, B B. Gastro-

58.

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enterology r7: 578-580, 1951. and WELBOURN, R. B. Postgrad. Med. J. 36: 154-159, 1960. 59. JOW, E., WEBSTER, D. R. and SKORYNA, S. C. Gastroenterology 38: 732-739, 1960. 6o. ZOLLINGER, R. M. and ELLISON, E. H. Ann. Surg. 142: 709-728, 1955. 6,. GREGORY, R. A., TRACY, H. J., FRENCH, J. M. and SIRCUS, W. Lancet r: 1045-1048, 1960. 62. GROSSMAN, M. I., TRACY, H. J., and GREGORY, R. A. Gastroenterology, 41: 87-91, 1961. 63. ROBINSON, R. M., HARRIS, K., HLAD, C. J. and EISEMAN, B. Proc. Soc. Exp. Biol. Med. 96: 518-520, 1957. 64. DREILING, D. A. and JANOWITZ, H. D. Gastroenterology, 36: 580-581, 1959. 65. COHEN, N., MASURE, P., DREILING, D. A. and JANOWITZ, H. D. Fed. Proc. r8: 28, 1 959. 66. DOTEVALL, G. and WESrLING, H. Scand. J. Clin. Lab. Invest. 12: 489-492, 1960. 67. MELROSE, A. G. GUT r: 142-145, 1960. 68. EARLE, A. S., CAHILL, G. F. JR., and HOAR, C. S. JR., Ann. Surg. 146: 124-130, 1 957. 69. CLARKE, S. D., NEILL, D. W. and WELBOURN, R. B. Gut r: 146-148, 196o. 70. SIRCUS, W. Peptic Ulceration. Wells, C. and Kyle, J. eds. 29 Edinburgh, Livingstone, 1960. 71. KYLE, J., LOGAN, J. S., NEILL, D. W. and WELBOURN, R. B. Lancet r: 664-666, 1956. 72. DOTEVALL, G. Acta Med. Scand. r7o: Suppl. 368, 1961.

Effects of Insulin and Glycemic Factors on Gastric Secretion*

THE discovery of insulin action by Bant-

ing and Best (1), in MacLeod's Laboratory, has stimulated a great deal of experimental and clinical investigations on the effects of the hormonal secretion of the pancreatic islet cells on other organs. Already by 1924, Bulatao and Carlson (2) were able to show that administration of insulin to normal humans and to dogs increased significantly the motor activity of the duodenum and the colon. However the effect of insulin on gastric secretion remained controversial for a long period. A number of authors (3,4,5,6) observed an augmentation of gastric secretion as well as an increase in acidity, following insulin administration; others (7) were unable to demonstrate such an effect; still others (8,9) have even claimed that insulin has an inhibitory effect on gastric secretion. There is little doubt that the variability of these and other findings is probably due to the lack of uniformity of insulin preparations at that time. Bustamante (1o) was the first to observe an increase of gastric secretion in human subjects receiving insulin, although no blood sugar levels had been recorded in his studies. Meyer (11), who

D. R. Webster G. N. Prohaska*

was one of the first authors to carry out blood sugar determinations in his experimental subjects, found that no stimulation of gastric secretion has occurred in diabetics unless hypoglycemia was present. The lack of pure insulin preparations has prompted several authors to suggest that the gastric secretory effect of insulin was due to impurity of the material rather than to insulin itself. Boldyreff and Stewart (12) were the first to compare the effects of noncrystalline and crystalline insulin preparations, and found a similarity of action. La Barre and de Cespedes (14) have reported detailed experiments on fistula dogs, and established firmly the stimulatory effect of insulin hypoglycemia. They have shown that intravenous administration of glucose at the peak of insulin stimulated secretion, caused an immediate fall in secretory response; it was also observed that intact vagi are necessary for the effect to, take place. These findings were confirmed by further experiments of Boldyreff and Stewart (12). We also have studied this problem in Babkin's laboratory (15) in dogs prepared with esophagotomy and gastric fistula, and have demonstrated

•The experimental work of the authors was supported by a grant from the Medical Research Council of Canada (MBT 77T) ••From the Department of Experimental Surgery, McGill University, Montreal, Canada.

457

V / EFFECTS OF INSULIN AND GLYCEMIC FACTORS

that even severe hypoglycemia (blood sugar levels below 5o mg. per cent) has no effect on gastric secretion when both vagi were transected transthoracically. Advances which have been made in elucidation of the effects of insulin and glycemic factors on gastric secretion since those early days can be listed as follows: a) Insulin preparations have been standardized, and synthetic insulin has been prepared. b) The methods of study of gastric secretion and gastric motility have been improved. c) The biphasic re-

sponse to insulin has been discovered(r6). d) Insulin producing tumors of the pancreas have been described. e) The interrelationship of insulin secretion, glucagon and other hormones has been established. f) A number of pharmacological preparations has been demonstrated to affect the action of insulin. g) Finally oral hypoglycemic agents have been introduced. It is the purpose of this article to review the pertinent data in the light of recent findings as well as to point out some of the problems which remain unsolved.

General Considerations It is now firmly established that insulin has a biphasic action on gastric secretion and motility, as confirmed by studies of Olson and Necheles (17). The first phase, lasting in man 30-45 minutes following insulin administration, represents a decrease of secretory rate and peristalsis corresponding to a phase of profound hypoglycemia, until the blood sugar falls to 27-25 mg. per cent. During the second phase, when blood sugar values are still below normal limits (60-70 mg. per cent), a stimulation of secretion, with a fall in pH and increase in motility, can be observed. In approximately seventy-five minutes, a peak of secretory volume and acidity is reached, and this is followed by a gradual decrease of secretion. It has also been demonstrated that a decrease of blood sugar level has a stimulatory effect on both gastric secretion and motility. The stimulating effect of insulin is observable in human subjects following administration of 25 units of insulin, causing a significant increase in gastric secretion (17). For administration of higher doses, such as a given to schizophrenic patients in the course of shock therapy (18), where the blood sugar falls to very low levels (16-4o mg. per cent), 458

the gastric secretory rate was found to be markedly decreased, and acid values ranged from 6o to 68 units. No exhaustion comparable to secretory achlorhydria of parietal cells has been observed after prolonged insulin stimulation. According to Jögi and Uvnäs (19), in the case of intravenous administration of insulin to dogs starved for twenty hours, with blood sugar levels of 6o-8o mg. per cent, the peak effect is reached after 20-45 minutes, and lasts for one to two hours. The gastric secretory rate was increased when compared to normal. Other species studied include cats, monkeys and rats. The increase in blood sugar levels, such as observed in untreated diabetics, and in dogs on glucose administration, appears to have an inhibitory effect on gastric secretion and motility (20). Winkelstein (21) has postulated that hyperglycemia in diabetics exerts a sedative effect on the vagal nucleus; however, the administration of glucose to dogs with gastric pouches has shown to have inhibited only slightly gastric secretory response, while it had a definite inhibitory effect on gastric motility and emptying time. High concentrations of glucose administered intravenously have

WEBSTER & PROHASKA

been demonstrated to cause a stimulating effect on gastric secretion, probably due to pyrogenlike action. The stimulating effect of hypoglycemia appears to last in man for two to

three hours following insulin administration (19). In dogs, the effect was observed to last one to two hours. In rats the effect subsided gradually in the course of four to six hours ( 22).

Factors Affecting Gastric Response to Insulin Hypoglycemia In dogs histamine has been used gener- ments that relatively slight degrees of inally as a secretory stimulant, and the ef- sulin hypoglycemia cause hypersecretion fect of insulin has been studied in both and hypermotility of the stomach in norinnervated and denervated gastric mal dogs and in man. An interesting obpouches. Karvinen and Karvonen (23) servation was made, namely that during found that intravenous insulin adminis- the time of onset of action of insulin, tration inhibits the secretory response of mostly for 2 5-40 minutes, there was a separated gastric pouches in the absence marked decrease of acidity and motility of vagal nerve supply to the stomach. A of the stomach. The peak of insulin acparallelism between the degree of inhibi- tivity was observed only after forty tion and the decrease in blood sugar was minutes, indicating a biphasic action: first, found. Karvinen and Karvonen suggested an immediate depression of secretion and that a reduction of the carbohydrate sub- motility, then continuous stimulation of strate available to the gastric mucosa may both, for one to two hours. Jögi and Uvbe the cause of inhibition. Forrest and näs (19) demonstrated high acid and Code (24) have further studied this effect pepsin secretion in fasted fistula dogs rein normal dogs, as well as in those with ceiving o. i to 0.2 units of insulin intrasympathectomy and vagotomy. Insulin venously per kilogram of body weight. was found to inhibit gastric secretion In these experiments, the time of onset of from both innervated and denervated gas- gastric secretory response to insulin was tric pouches. Forrest and Code (24) have found to be ten to fifteen minutes followconcluded that the inhibitory effect of in- ing its administration, and lasting one to sulin hypoglycemia on histamine-induced two hours; the peak was reached after 3o gastric secretion was independent of ex- to 45 minutes. The hypoglycemia induced trinsic nerve supply to the gastric mucosa, in this way did not exceed values of 6o to and was directly related to the blood 8o mg. per cent. sugar concentration. When these findings Quigley and Templeton (26) studied are compared with the stimulatory effects gastric motility in dogs following inducof insulin administration on gastric secre- tion of insulin hypoglycemia, and showed tion, the conclusion must be reached that that insulin hypoglycemia inhibited gashistamine stimulation represents a dif- tric motility following total vagotomy. ferent process not affected by the inner- These authors suggested that this was vation of the stomach, but not necessarily caused by a stimulation of the unopposed comparable with other forms of stimula- sympathetic nervous system. Necheles and Olson (27) found that the motility tion through the vagal route. Geziri et al. (2o) show in their experi- of the stomach varied with the depth of 459

V / EFFECTS OF INSULIN AND GLYCEMIC FACTORS

hypoglycemia, and that low blood sugar levels (1 o to 23 mg. per cent) caused a complete inhibition of gastric motility and acidity. It is believed that gastric motility and the psychic phase of gastric secretion are governed by the activity of the vagus nerves, which are stimulated by insulin hypoglycemia. Cannon, McIver and Bliss (28) could prove that a discharge of adrenals takes place when the blood sugar falls below 70 mg. per cent, as in insulin hypoglycemia. Quigley, Johnson and Solomon (29) showed that gastric motility stimulated by insulin hypoglycemia was inhibited by intravenous administration of adrenalin. The effects of insulin on gastrointestinal motility has been studied by Goldberg, Stein and Meyer (3o) by the introduction into the stomach of a tube with a balloon and manometer. Eighty-two per cent of the test patients showed an increase in gastric motility following 15 to 20 units of insulin, lasting for 3o to 240 minutes. Hypermotility usually was observed to start 3o to 5o minutes after insulin administration. The increased colonic motility was shown to be ensured in sixty minutes after insulin injection and to last for about the same period of time as gastric hypermotility. However the colonic hypermotility produced by insulin hypoglycemia was present in only 53 out of 65 patients (approximately 8o per cent). In those experiments a marked biphasicity of insulin action was also observed, namely, an initial inhibition of motility followed by a stimulatory phase

during or immediately after the hypogycemic reaction. Adler, Jacobson, Heitman and Watson (31) have carried out studies of motility during insulin hyperglycemia in normal subjects and in patients with peptic ulcers and gastric neoplasms. These authors have demonstrated that increased peristalsis was visualized in two to five minutes after the administration of insulin in most patients. It was found that the increased motility was of value in aiding the diagnosis of gastric lesions of any kind, since ulcerated or carcinomatous regions of the stomach would not participate in the peristaltic response caused by insulin. The insulin-induced hyperacidity, as well as pepsin, was found to be depressed by chloralose injection (32). A similar effect has been found to follow administration of other anesthetic and analgesic agents, such as ether and nembutal. Jögi and Uvnäs (3 2) have suggested that these drugs produce hyperglycemia, thus counteracting insulin hypoglycemia. Dibutolin, a surface-active derivative of carbominoylcholine with atropine-like properties, was found to inhibit gastrointestinal hypermotility produced by insulin (33) in human subjects. Dibutolin is known to block the action of acetycholine at the level of the effector cells with resulting relaxation of the smooth musculature of the gastrointestinal tract. Thus one may conclude that the stimulating effect of insulin is exerted on the muscle component of the gastric and intestinal wall.

Mechanism of Action Although several mechanisms of action of insulin on gastric secretion have been postulated, there seems to be little doubt that the general consensus of opinion, 460

even in the early phase of studies, favored a mediation of the response to changes in blood sugar concentration through the vagus center. The vagus trunks in the ab-

WEBSTER & PROHASKA

domen give off branches to the celiac plexus; vagal fibres could therefore, at this point, enter the sympathetic distribution. Babkin (34.) suggested that some parasympathetic fibres are present in the Heidenhain pouch preparations, reaching it with the vascular supply. Volborth and Kudriavzeff (35) postulated, in 1927, the existence of cholinergic gastric secretory fibres in the thoracicolumbar outflow which reach the stomach with the sympathetic nerve fibres. However, recent studies of Necheles and Olson (36) definitely reject the possibility of stimulation of secretion by insulin in Heidenhain pouches. The action of insulin on the gastric acid secretion involves three components: a) an initial inhibitory effect, b) an early excitatory effect, and c) a late excitatory effect. The mechanism of the initial inhibitory effect of insulin on gastric acid secretion has not been elucidated. The inhibition is not mediated over the vagal or sympathetic nerve supply to the stomach (24). Several explanations of the inhibitory effect have been offered (36,41), e.g. a direct action of insulin on the acid-secreting glands, and mediation over vasopressin, adrenalin and glucagon. Glucagon causes inhibition of gastric acid secretion in man (37) and in the dog (38), but stimulation has been observed in the rat by high dosage (22). The mechanism of the early excitatory effect of insulin on gastric acid secretion involves a hypoglycemic stimulation of structures of the central nervous system, probably the vagal centers, as was shown by cross-circulation experiments in dogs (40). The vagus nerves constitute the peripherial pathway, and bilateral vagotomy abolishes the early secretory response to insulin hypoglycemia (15). According to Pevsner and Grossman (40), the vagal impulses induced by the hypo-

glycemia evoke secretion by a direct cholinergic action on the acid-secreting glands. In their experiments, insulin hypoglycemia elicited gastric acid secretion in the dog after extirpation of the antrum and the small intestine. However, the vagal impulses also liberate gastrin from the antrum, which contributes to the acid secretory response of insulin hypoglycemia. Acid secretion was thus obtained in Heidenhain pouch dogs by insulin hypoglycemia, and was subsequently abolished by antrectomy (42) or separation of the antral mucosa from the muscle layer (antroneurolysis) (43). In his investigation Nyhus et al. also presented evidence that gastrin liberation during insulin hypoglycemia is mediated over vagal connections with the antral mucosa, and is not secondary to the increased motor activity of the antrum, therefore not a result of another effect of the hypoglycemia. The Seattle group has demonstrated that antroneurolysis removed the acid secretory response to insulin hypoglycemia in Heidenhain pouch dogs, despite the remaining increased motor activity of the antrum. The mechanism of the late excitatory effect of insulin on the gastric acid secretion remains controversial. Insulin-induced acid secretion lasts for many hours in man, monkey and dog. The existence of a late secretory response to insulin hypoglycemia, independent of the vagal innervation of the stomach, was demonstrated by French et al. (44). In vagotomized monkeys, a decrease of the pH values of the gastric secretion appeared during the third to fifth hour after insulin administration. In adrenalectomized monkeys, a pH decrease was obtained only during the first to second hour. It was concluded that the late secretory response was due to adrenal activation, since it was abolished by adrenalectomy (44). A late acid secretory response to insulin hypo461

V / EFFECTS OF INSULIN AND GLYCEMIC FACTORS

glycemia, independent of the vagal innervation of the stomach, has also been demonstrated in man (45). The late secretory response, following vagotomy or vagotomy and antrectomy in man, was of a very low magnitude. In the same study by Sun and Shay, adrenalectomy in two patients markedly reduced the late secretory response to insulin hypoglycemia; also, as judged from the presented curves it reduced the early secretory response. Adrenal corticoids appear to be weak stimuli of the acid secretion in Heidenhain pouch dogs but potentiate the secretory effect of cholinergic drugs (46). Sun and Shay (45) suggested that a synergistic action of the adrenal corticoids and the vagal secretory mechanism determine the late secretory response to insulin hypoglycemia. However, in a preliminary re-

port, Huston et al. (47) stated that the biphasic pattern of the acid secretory response to insulin hypoglycemia in dogs is not influenced by adrenalectomy, and that antrectomy removes the late secretory phase. Huston et al. (47) also claimed that in response to hypoglycemia, the early secretory phase is associated with outpouring of endogenous cortisols but not so the late phase. Estimation of the quantitative significance of the adrenals and the antrum in the late response of gastric acid secretion to insulin hypoglycemia appears to be premature at the present moment. Different species and the different test techniques of the secretory function used in the above-mentioned studies present considerable difficulties for quantitative and qualitative comparison.

Summary It is now firmly established that insulin has a biphasic action on gastric secretion and motility. During the first phase, a decrease of secretory rate and peristalsis occurs corresponding to profound hypoglycemia. During the second phase when blood sugar values are still below normal limits, a stimulation of secretion with a fall in pH and an increase in motility can be observed. A peak of secretory volume and acidity is reached approximately seventy-five minutes after insulin administration. The mechanism of the initial inhibitory effect of insulin on gastric acid secretion has not been elucidated. This inhibition is not mediated over the vagal or sympathetic nerve supply. Several explanations

462

have been offered, including a direct action of insulin on the acid-secreting glands and mediation by vasopressin, adrenalin and glucagon. The mechanism of the early excitatory effect on gastric acid secretion involves a hypoglycemic stimulation of the vagal centres as shown by cross-circulation experiments in dogs. Evidence has been presented that gastrin liberation during insulin hypoglycemia is mediated over vagal connections with the antral mucosa, and is not secondary to the increased motor activity of the antrum. The late excitatory effect of insulin on gastric acid secretion is independent of the vagal innervation. Its mechanism remains controversial.

AVEBSTER & PROHASKA

References I . RANTING, F. B., BEST, C. H. and MACLEOD, J. J. R.,

2. 3. 4•

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Amer. J. Physiol. 59: 479, 1922. BULATAO, E., and CARLSON, A. J. Amer. J. Physiol. 69: 107-115, 1924. DETRE, L., and slvo, R., Z. Ges. Exp. Med. 46: 594-599, 1 925. WIECHMANN, E. and GATZWEILER, W. Deutsch. Arch. Klin. Med. /S7: 208-215, 1927. SIMILI, D., POPESCO, G., and DICULESCO, C.H. Arch. Mal. Appar. Dig. 17: 28-43, 1 927. OKADA, S., KURAMOCHI, K., TSUKAHARA, T. and OOINOUE, T. Arch. Int. Med. 45: 783-813, 1930. IVY A. c., and FISHER, N. F. Amer. J. Physiol. 67: 445-450, 1923-24. SIMNITSKY, s. s. Klin. Wschr. 5: 1 545-1549, 1926. FEISSLY, M. R. Arch. Mal. Appar. Dig. 16: 325-328, 1926.

10. RUsCAmANTE, A. Arch. D. Endocrin y Nutr. Madrid. 6: 295, 1928. 11. KALK, H. and MEYER, P. F. Z. Klin. Med. 12o: 692-7 14, 193 2 . 12. BOLDYREFF, E. B.,

and STEWART, J. F. J. Pharmacol. Exp. Ther. 46: 419-429, 1932. 13. DOBREFF, Al. Arch. Verdauungskr . 50: 157170, 1931. 14. LA BARRE, J. and CESPEDES, C. DE C. R. Soc. Biol (Par) 1931. 15. BABKIN, B. P. Amer. J. Dig. Dis. Editorial 5: 753, 1939. i6. KOMAROV, S. A. Secretory mechanisms of the digestive glands, Babkin, B. P. 2nd. ed., New York, Hoeber, 1950. 17. NECHELES, H., OLSON, W. H., and SCRUGGS, W. Fed. Proc. 1: 62-63, 1942. 18. LEVY, E. M., CORT, A. P. DELL, and LOGAN, V. W. Gastroenterology, 21: 547-550, 1952. 19. JÖGI, P. and uvxns, B. Acta Physiol. Scand. 17: 206-211, 1949. 20. GEZIRI, M. F., ROBERTSON, C .,PLZAK, 1.. F. and WOODWARD, E. R. Surgery 43: 606-609, 1958. 21. WINKELSTEIN, A. and HESS, Al. Gastroenterology, 1 1: 326-336, 1 948. 22. JOW, E., WEBSTER, D. R. and SKORYNA, S. C. Gastroenterology 38: 732-739, 1960. 23. KARVINEN, E., and KARVONEN, M. J. Acta Physiol. Scand. 27: 350-370, 1952-53. 24. FORREST, A. P. M., and CODE, C. F. Amer. J. Physiol. /77: 430-432, 1954.

25. SHARICK, P. R. and CAMPBELL, D. A. Amer. J. Med. Sd. 221: 364-368, 1951. 26. QUIGLEY, J. P. and TEMPLETON, R. D. Amer. J.

Physiol. 91: 482-487, 1929-30. 27. NECHELES, H., OLSON, W. H. and MORRIS, R. Amer. J. Dig. Dis. 8: 270-273, 1941. 28. GANNON, W. B., MCIVER, M. A., and BLISS, S. W. Amer J. Physiol, 69: 46-66, 1924. 29. QUIGLEY, J. P., JOHNSON, V. and SOLOMON, E. T.

Amer. J. Physiol. 90: 89-98, 1929.

30. 31. 32. 33. 34•

35. 36. 37. 38. 39. 40. 41. 42. 4;. 44. 45. 46. 47.

GOLDBERG, E. M., STEIN, I. F. JR., and MEYER, K. A. Gastroenterology. 28: 656-668, 1 955. ADLER, D. C., JACOBSON, G., HEITMANN, K. A. and WATSON, D. D. Radiology 65: 530-S37, 1955. JÖGI, P., STROM, G., and UVNÄS, B. Acta Physiol. Scand. 17: 212-22 I , 1949. PETERSON, C. G., and PETERSON, D. R. Gastro-

enterology, 5: 169-174, 1945. BABKIN, P. B. Secretory Mechanism of the digestive glands. New York, Hoeber, 1944. VOLBORTH, G. W., and KUDRIAVZEFF, N. N. Amer. J. Physiol. 81: 1 54-159, 1 927. OLSON, W. H. and NECHELES, H., J.A.IMA• 159: 1013-1014, 1955. ROBINSON, R. M., HARRIS, K. HLAD, C. J. and EISEMAN, B. Proc. Soc. Exp. Biol. Med. 96: 518-520, 1 957. CLARKE, S. D., NEILL, D. W., and WELBOURN, R. B. Gut. 1: 146-148, 1960. LA BARRE, J., and CESPEDES, C. DE. C. R. Soc. Biol. (Par). to6: 1249-1251, 1931. PEVSNER, L., and GROSSMAN, M. 1. Gastroenterology, 28: 493-499, 19,5 5• BACHRACH, W. H. hysiol. Rev., 33: 566-592, 1953. SURSTALL, P. A. and SCHOFIELD, B. J. Physiol., 123: 168-186, 1954.

NYHUS, L. M., CHAPMAN, N. D., DEVITO, R. V. HARKINS, H. N. Gastroenterology, 39: 5$2-589, 1960. FRENCH, J. D., LANG MIRE, R. L., PORTER, R. W. and MOVIUS, H. J. Surgery, 34: 621-6;2, 1933. SUN, D. C. H. and SHAY, H. J. Appl. Physiol., 15: 697-70 3, 1960. SUN, D. C. H. and SHAY, H. Physiologist. 1: 77, 1 958. HUSTON, C.J.W.,PRESIIAW, R. M. and SIRCUS, W. J. Physiol. 162: 25, 1962.

and

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Röle of Nutritional Factors in Peptic Ulcer*

On the basis of available experimental and clinical data, several systemic factors have been considered as contributing to the genesis of peptic ulcer. The role of nutrition has received its due share of attention, and has been investigated periodically. The literature on nutrition as related to experimental peptic ulcer has been reviewed by Berg (1) and Ivy, Grossman and Bachrach (2). Under experimental conditions of dietary deficiency, it is possible to produce gastroduodenal ulcers in rats, guinea pigs and dogs. In certain areas of India (34,5) and Abyssinia (6), peptic ulcers occur in individuals whose diet is inadequate, and

P. Raghavan**

which contains quantities of irritants such as capsicum and other spices. There is evidence that in India (34,5), Africa (7) and Indonesia (8), ulcers occur in the poorer sections of the community. Increased incidence of peptic ulceration was reported during World War II (9) from Russia, France and Belgium where there was considerable dietary deficiency. These reports suggested the possible role of nutritional factors in the etiology of gastric and duodenal ulcerations. We propose to review the literature on this topic, and to present data on the nutritional status of a group of patients with duodenal ulcer who have come under our care.

General Nutritional Deficiency Bradfield (3) from Madras and Somervell (4,10,11) from Travancore in India found that peptic ulcers were more common in the south of India than in the north. Their figures show that duodenal ulcers are encountered more commonly (85.1 per cent of Bradfield's and 95.1 per cent of Somervell's cases). Somervell (10,

II) felt that these were caused by dietary deficiency and in particular by deficiency of vitamin A. McCarrison (1 z) reported production of ulcer in rats with diets similar to that used by people in Madras and Travancore. He also reported production of gastric ulcers, in addition to degeneration of the myenteron, in guinea

The work carried out by the author on this subject was supported in part by a grant from the Indian Council of Medical Research. "From the Department of Medicine, Seth G. S. Medical College, Bombay, India.

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V / ROLE OF NUTRITIONAL FACTORS

pigs and in monkeys fed on autoclaved rice diets, and stated that deficient and ill-balanced foods may be factors in the causation of acute ulcers of the stomach and duodenum. Dogra (13), however, was not able to produce lesions in monkeys by feeding them on diets similar to those used by McCarrison, although the animals died of starvation. Rao (14) fed a group of monkeys on a poor rice diet comparable to the diet eaten in the southern areas of India. He kept them alive and observed them for a period of three years. No gastroduodenal ulcerations were found in these animals when they were sacrificed, although the gastrointestinal tract showed trophic changes and degeneration of the mucosa. An increase in the incidence of peptic ulcer during World War II was reported by Moutier from France and Van der Hoeden from Belgium (g). A similar increase was noted also in Russia (15). During war time, gastric ulcers were found more frequently than in peace time. An increased frequency was observed in women, and peptic ulcer disease showed an increase in incidence before any dras-

tic changes in food distribution and consumption had occurred. Both Moutier and Van der Hoeden felt that the ulcers were caused by emotional stress and dislocation rather than by dietary deficiency. It is supported by the fact that in the Warsaw ghetto as well as in the concentration camps of Dutch East Indies, ulcers were encountered very infrequently (q). The low incidence in these areas is not surprising, because the fasting gastric acidity and response to histamine of subjects from Warsaw ghetto were found to be low (q). Furthermore, atrophy of the gastric mucosa was seen gastroscopically by Debray and co-workers (16) in people who had symptoms of nutritional deficiency. It is interesting to recall Hoelzel's (17 ) finding that restriction of protein led to increase in gastric acidity, and excess of proteins led to decreased acidity, possibly as a result of fatigue of the secretory mechanism. During prolonged fasting, however, the fasting acidity was less than normal, if control values were above normal, and higher than normal, if control values were lower than normal.

Protein and Amino Acid Deficiency Hoelzel and Da Costa (18) were able to produce ulcers in the forestomach of rats kept on a diet deficient in proteins. They found that the animals developed gastric retention with protein restriction, and the incidence of prepyloric ulcers was high in these animals (19). Weech and Paige (zo) reported peptic ulcer in dogs maintained on hypoproteinemic diets for a period of ninety days. No gross differences in weight loss or hemoglobin levels were noticed between the animals that developed peptic ulcer and those that did not. Li and Freeman (z I) found 47 per cent of thirty-six dogs kept on a pro466

tein-deficient diet developed peptic ulcers after they had been on the restricted diet for a minimum period of twelve weeks. The incidence of ulcer was not related to the survival period or to the hepatic status of these animals. Hahn, Baugh and Foster (z z) found dogs which were bled repeatedly in order to develop an iron deficiency anemia and maintained on a low protein diet developed perforating ulcers of the stomach and duodenum. Mann-Williamson dogs showed a consistent weight loss as a result of deficient absorption, and it was possible to prolong the survival period of these dogs and to

RAGHAVAN

delay the development of peptic ulcer, if they were fed on a highly nutritious diet containing raw ground pancreas and liver (z) . Slive, Bachrach and Fogelson (23) found that the incidence of ulcers in Mann-Williamson dogs was lower if the point of duodenal drainage was closer to the gastrojejunostomy, ensuring better digestion and absorption of nutrients. They felt that the development of ulcer was dependant upon malnutrition rather than gastric acidity. Ivy, Grossman and Bachrach (2) however state that the role of nutrition in Mann-Williamson dogs was not important as one can only delay, but not prevent completely, the development of ulcer in these animals by maintaining them on a high-caloric diet. Sharpless (24,25) found cystine and choline useful in preventing gastric lesions in rats on a deficient diet. They found however that riboflavin and pyridoxine were also necessary. Deficiency of any one of these may prevent the protective action of the other factors. Pantothenate, in their experience, did not prevent or improve the lesions. The story of histidine in relation to peptic ulcer is too fresh in our minds to need repetition

(26,27,28). It is justifiable to state that deficiency of any particular amino acid or the curative action of any one of the amino acids has not been conclusively demonstrated either in the experimental animal or in the human being (q). Marked improvement in peptic ulcer cases, with relief of symptoms and rapid healing, was reported by CoTui and coworkers (29) when patients with peptic ulcers were treated with protein hydrolysate and dextro-maltose. They observed plasma protein deficiency in these subjects before treatment was instituted. Riggs and coworkers (3o) also found a similar deficiency of plasma protein in peptic ulcer patients. Negative nitrogen balance was reported in uncomplicated peptic ulcer disease by Kenamore (31). Those who were put on protein hydrolysate and dextro-maltose supplements showed a positive nitrogen balance. Sappington and Bockus (32) found a negative nitrogen balance in patients with a recent history of hemorrhage or vomiting. It is possible that the beneficial effect was due to the improvement of protein nutrition as well as to the acid-buffering property of protein hydrolvsate.

Vitamins and Calcizu711 Deficiency Vitamin A Deficiency of vitamin A was considered as a cause of peptic ulcer in his patients by Somervel (i i ). The experiments on the role of vitamin A in rats will be discussed later. There is no direct evidence indicating that this vitamin plays an important role in the etiology of human peptic ulcer. Vitamin B and Vitamin B-complex The protective action of vitamin B, and

brewer's yeast in the experimental gastric erosions and ulcers in rats will be described under the heading of experimental ulcer in the rat (33,34,35)• Rao (36) found that five of twelve pups on a thiamin-deficient diet developed chronic gastric and duodenal ulcers. He also studied patients with peptic ulcer, and found an increase of the bisulfite-binding substances in the blood of these patients indicating thiamin deficiency (37). Findlay (38) observed erosions, superficial ulcerations and hemorrhages in the stomach and duodenum of rats on a diet deficient in 467

V / ROLE OF NUTRITIONAL FACTORS

vitamin B,. Zucker (39) was able to produce duodenal ulcers (2.5 cms. below the polyrus) in susceptible strains of rats by pantothenic acid deficiency. These ulcers developed after eleven weeks deficiency; the more chronic cases perforated. Adrenalectomy or hypophysectomy prevented the development of ulcers. In ulcer susceptible rats, the deficiency resulted in an increase in acid secretion and a lowering of pH of the gastric contents. The increased secretion began about eight weeks after the animals started the deficient diets, and reached a peak at about the eleventh or twelfth week, a period which coincided with ulcer development.

within normal limits. He felt that vitamin C deficiency was not an etiological factor in peptic ulcer. Ebbsen and Rasmussen (45) found the serum ascorbic acid levels of sixty-three patients admitted for peptic ulcer to be normal, but the serum levels fell markedly when thirty-five patients were on ulcer diets. Layer and Cody (46) found no evidence of vitamin A, riboflavin, niacin or ascorbic acid deficiency in patients with peptic ulcer. There was nothing to suggest that deficiency of these substances preceded the development of peptic ulcer.

Vitamin C Holst and Frölich (4o) found gastric erosions and ulcers, some of which were perforated, in scorbutic guinea pigs. McCarrison (41) in investigating the effects of deficiency of vitamin C in guinea pigs found that three of nine guinea pigs fed on autoclaved oats and autoclaved milk had punched-out necrotic ulcers in the stomach and marked congestion of the duodenal mucosa. One of these guinea pigs had a duodenal ulcer. The lesions were in various stages of development, from hemorrhages to erosions and ulcers. By feeding cavies on vitamin C deficient diet, Magee, Anderson and McCallum (4=) produced peptic ulcers in seven out of thirty-two guinea pigs. The diet was also deficient in minerals. Smith and McConkey (43) were able to produce duodenal ulcer in guinea pigs on a diet deficient in vitamin C. Addition of vitamin A and D did not protect these animals. They suggested that patients on peptic ulcer diets (Sippy and Lenhartz diet) should be given orange juice in addition, as these diets were grossly deficient in vitamin C. Rao (44) estimated the vitamin C levels in blood and urine of patients with peptic ulcer, and found them to be

In carefully controlled experiments, Zucker and Berg (47,48) found lesions consisting of necrosis, hemorrhage and epithelial hyperplasia in the antrum of rats maintained on a calcium deficient diet. The number of these lesions was reduced by administration of vitamin D and increased by phosphate. Deficiency of thiamine or whole B-complex also produced lesions, but fewer in number. Increased calcium administration to thiamin-deficient rats abolished the incidence of lesions, but thiamin by itself had no protective effect on the lesions produced by calcium deficiency. These lesions differed markedly from the rumenal lesions produced by deficiencies of other nutritional factors in rats.

468

Calcium

Anti-gizzard erosion factor in chicks In the course of an investigation on vitamin K, Dam and associates (49) found that chicks maintained on a vitamin K free diet developed erosions and hemorrhages in the gizzard, which were not prevented by addition of vitamin K. The factor responsible for this was identified later as fat soluble and thermolabile. Cheney (5o) found that the gastric acid-

RAGHAVAN

ity and the response to histamine of chicks were comparable to that in the human being. In chicks with ulcers reared on a diet deficient in the anti-gizzard erosion factor, there was an increase in gastric acidity. He felt that the increase in acidity in chicks with ulcers was caused by the dietary deficiency, and that the acidity by itself was not the cause of lesions. Erosions and ulcers could be produced in chicks by cincophen, and the severity of the lesions was in proportion to the quantity of cincophen in the feeds. These lesions were considerably reduced in

chicks fed on dietary supplements containing the anti-gizzard erosion factor (51,5 2 ). In army personnel, he noticed gastroduodenal ulcers developed when the men had been on canned rations for some time. He was able to demonstrate healing in thirty-one intractable cases who were fed on a high calorie diet containing an anti-ulcer factor, designated by him as vitamin U. The healing was remarkable because no other measures, such as antacids or sedatives, were used (f3). These results have not been confirmed by others.

Nutritional Factors in Experimental Ulcer in the Rat In 1913, Singer (54) was able to produce ulcers and ulcero-papillomas in the forestomach of rats fed on moist bread and wood-shavings which were allowed to be contaminated by rat feces and molds. He felt that these were due to infection and toxins, and considered them similar to the lesions reported by Fibiger (55)• Fibiger produced similar lesions by feeding rats with cockroaches infected with a nematode, Gongylonema neoplasticum; he believed these to be neoplastic. Since that time ulcers and ulcero-papillomas have been produced in the rumen of the rat by a variety of deficient diets. The conclusions drawn from these experiments have been contradictory. Pappenheimer and Larimore (56) were able to produce lesions in rats with deficient diets used for producing rickets. They felt that these were due to some unknown dietary factor, in addition to mechanical irritation as a result of the ingestion of hair. They also thought that the lesions were not due to vitamin A deficiency. Wolbach and Howe(57) did not observe lesions in rats with induced vita-

min A deficiency. Cramer (58) felt that though different strains of rats differ in their resistence to vitamin A deficiency, the ulcerative lesions produced by deficient diets were not due to lack of vitamin A. Harris (S9) noted that animals on diets deficient in vitamin A developed gastroduodenal ulcers only during the period of recovery and treatment with vitamin A. They found that alpha-tocopherol administered along with vitamin A during this period prevented the development of ulcerative lesions. Jensen (6o) also found that lesions in vitamin A deficient rats could be prevented by tocopherol. Alcohol had the opposite effect. Dalldorf and Kellogg (33) found chronic ulcers in the stomach androximal duodenum in rats fed diets deficient in vitamin B, (antineuritic factor) and vitamin C. Since no structural defects had been demonstrated in vitamin C deficiency in rats, these were considered to be due to a deficiency of vitamin B,. Sure and Thatcher (34) were able to produce ulcers of the glandular portion of the 469

V / ROLE OF NUTRITIONAL FACTORS

stomach in rats with a deficiency of vitamin B,, uncomplicated by inanition. Howe and Vivier (61) found that the whole vitamin B-complex was necessary for the prevention of these lesions, and noted the protective action of brewer's yeast, in spite of a protein deficiency. Brunschwig and Rasmussen (35) could demonstrate ulcers in rats by limitation or marked reduction of stock diets. These lesions could be prevented by casein or brewers yeast but not by vitamin K. Berg (6z) found that deficient diets produced gastritis involving mainly the showed hemorrhagic erosions. Ulceration, necrosis and epithelial hyperplasia were seen, more or less as a continuous process, in the rumen and antrum. Ulcers in the rumen of the stomach were produced after starvation for a short period, followed by ligation of the pylorus (63). Ulcers failed to develop if the period of starvation was not long enough, and could not be produced if the animals swallowed blood by self-mutilation or by fighting with other animals. Similar starvation ulcers were produced in the glandular portion of the stomach of mice by Ogawa, Chiles and Necheles (64). Pregnancy had no protective effect on the development of these lesions in older mice. Robert and Nezamis (65,66) found that rats starved for four days developed ulcers in the pro-stomach, some of which showed perforation. Administration of cortisone protected against ulcer formation in the rumen, but induced ulcers in the glandular portion of the stomach even if the pylorus was not ligated. Chen (67) found that starvation ulcers could be prevented by a variety of diets deficient in nutritional factors and by mixtures such as agar and dextrose. Granulomatous lesions developed in rats put on a stock diet after a period of starvation. He felt that the importance of nutritional deficiency, as a cause of these ulcers, was 470

questionable, and that infection may have been responsible. Hoelzel and Da Costa (18) noticed that ulceration of the rumen could be produced by simple protein restriction, prolonged starvation or starvation complicated by pregnancy. A bran diet prevented ulceration, although the animals eventually died of inanition. Feeding high fat, low protein diets induced ulcers more frequently. The same authors (19) found protein restriction produced pylorospasm and prepyloric ulcers in rats. Morris et al. (68) noticed gastric ulcers and fatty and cirrhotic changes in the liver of rats fed on an adequate diet but containing 5o per cent of heated lard. The cause of these ulcers in the rat, whether they were due primarily to deficiency or as secondary to liver disease, is not clear (z) McCarrison (1 2) was able to produce gastric ulcers in albino rats kept on diets comparable to that of people in Travancore and Madras in India. He noticed that rats on a tapioca diet, similar to that used by people in Travancore, developed ulcers in the distal portion of the stomach while those on the rice diet, similar to that consumed by poorer people in Madras, led to ulcers in the proximal portion of the stomach. The reports on the production of experimental gastric ulcers in rats have been reviewed in detail as sonic authors (33) believe that they are related in pathogenesis to human peptic ulcers. But other authors (2,61) have pointed out the nonapplicability of findings on the squamous epithelium of the forestomach of the rat to the human peptic ulcer. The anatomy and histology of the rat's stomach are not similar to that of the human stomach, and ulcers occur most often in the rumen, an area which resembles the lower end of the esophagus, rather than the stomach of the human being(2). Under conditions of acute star-

RAGHAVAN

vation, these are superficial erosions and hemorrhages. In conditions of chronic deficiency, the animals, if they live long enough, develop lesions which are papillomatous and ulcero-papillomatous. Both the acute and chronic lesions can be produced by infection, irritation, starvation, starvation with alternate feeding, protein restriction, restriction of amino acids, choline and cystine restriction of vitamins A or B-complex factors or diets high in heated fats. These lesions heal if the animals are put on an adequate stock diet

after the lesions have been produced. Apparently the rat's stomach responds in the same manner to a variety of deficiencies and irritation. The same type of response of the rat's stomach to various factors mentioned, makes it difficult, however, to assess its significance in relation to the human peptic ulcer. The pyloric lesions in the mouse and the prepyloric lesions in the rat induced by cortisol may not be comparable in pathogenesis, to human peptic ulcer.

Clinical Observations No gross deficiency has been demon- interesting in view of the behavior of gasstrated in human subjects to precede the tric secretion in human peptic ulcer didevelopment of peptic ulcer or in patients sease. In human subjects, no demonstrable with peptic ulcer (44,45,45) • However, increase of gastric acidity was demonnegative nitrogen balance and deficiency strated in the Minnesota experiments on of plasma proteins in patients with peptic human starvation. There is some evidence, ulcer has been noticed by some observers however, that severe and prolonged star(29,30,31,32). Ray Chauduri (69) found vation may depress gastric acidity (9). no deficiency of plasma proteins or hemoglobin in 158 peptic ulcer patients of a Peptic ulcer in undernourished areas of low income group in Calcutta. However, the world a deficiency in the intake of vitamin BHigh incidence of peptic ulcers, parComplex factors was found in those whose income were very low. Increase in ticularly of duodenal ulcers, has been rebisulfite-binding substances in the blood ported by Raper and others from East of patients with peptic ulcer was noted Africa (8,71,72). The high incidence reby Rao (37), indicative of thiamin de- ported by Raper is in contrast to that of ficiency. No significant deficiency of Eagle and Gillman (72) and others (7), vitamin A, riboflavin or ascorbic acid was reported previously. In a university found by Cayer and Cody (46) in the settlement in Kampala, peptic ulcers were cases of peptic ulcer investigated by them. seen mainly in Kikuyu students, who The influence of deficiency on gastric were worried about their homes during secretion is not clear either. Shay et al. Mau Mau activity (73). The food habits (7o) reported an increase in volume of of the African in eating large meals at gastric secretion in thiamin deficient ani- infrequent intervals have been considered mals, although acidity remained normal. (7), and may be one of the causes of The increased acid secretion in pantothe- gastric hypersecretion. Such a hypothesis nate-deficient rats noted by Zucker has is supported by the findings of Bogoras been referred to already (39). This fact is (N) in dogs submitted to intermittent 471

V / ROLE OF NUTRITIONAL FACTORS

starvation. Like Dogra (75) from India, Raper also felt that ulcers occurred frequently in the poorer and uncared-for section of the people. Kouwenaar's (8) figures from Indonesia support the same conclusion. Cheney's (5 3) observations in a military establishment in the U.S.A. showed that ulcers developed when the men had been on canned rations for some time. In the more intractable cases, ulcers showed healing when patients were transferred to a ration of 4200 calories, which included fresh greens. Riggs et al. (76) found in an analysis of the dietary records of sixteen

patients with chronic duodenal ulcer, that the diet was low in calories and deficient in vitamin B and C. They also found lower levels of total serum proteins and plasma ascorbic acid. Whether this finding in peptic ulcer patients is a cause or an effect is open to question. The dietary intake was evaluated, and the quantity of proximate principles and vitamins ingested were assessed carefully in a group of thirty-one patients with duodenal ulcer who had come under our observation. The results are shown in Fig. i and Fig. 2.

FIG. I. Table showing nutritional status in 31 cases of duodenal ulcer. Nutritional Factor Protein in gm. Animal protein in gms. Fat in gms. Carbohydrate in gms. CALORIES

Average intake

Recommended allowances

80 10 54 486 2742

55-65* 16-27 85® 350@ 2400-3000f

No. of persons with intakes less than twothirds recommended allowance 1

16 19

*1 gram/kg. Recommended by the Nutrition Advisory Committee of the Indian Council of Medical Research (1960). ®Approximate requirements for balanced diets. +Varies with type and amount of work done.

FIG. II. Table showing minerals and vitamins in 31 cases of duodenal ulcer.

Nutrient Calcium in gms. Phosphorous in mgs. Iron in mgs. Vitamin A in I.U. Vitamin B1 in I.U. Vitamin C in mgs.

Average intake 0.73 1.86 320 2885 783 55

No. of persons with intake below twoRecommended thirds recommended allowance. allowance* 1.00

8

20.30 3000-4000 333-666 50

1

7 5

*Recommended by the Nutrition Committee of the Indian Council of Medical Research. t.u. = International Units.

472

RAGHAVAN

The intake of proteins and carbohydrate was within the limits recommended by the Nutrition Advisory Committee of the Indian Council of Medical Research. The quantity of protein taken by these patients on the basis of i gram per kilogram body weight was adequate except in one subject. However, the quantity of animal protein consumed by fifteen patients was low. The fat intake was lower than that recommended, and was replaced by an increase in the quantity of carbo-

hydrate. The total caloric intake was deficient in eleven subjects. The quantity of all vitamins was adequate except that of vitamin A. None of these, however, showed clinical evidence of vitamin A deficiency. The hemoglobin level, red cell count and serum proteins were also studied in t 34 cases of duodenal ulcer, and an attempt was made to correlate the basal and maximum parietal secretion with these values.

FIG. 3: Table showing nutrition in relation to maximum parietal secretion of HCI in mgs. No. of patients studied Maximum parietal secretion of HCI in mgs.

134

Hb. in gms

134

RBC in millions

134

Serum proteins in gms.

130

Weight in lbs.

130

Average

Standard Deviation

559

304

14.4 4.93 6.25

—

1.73

.039

0.63

.021

0.57

.004

26.8

107.7

Correlation with M.P.S. of HCl's in mgs.

.008

FIG. 4: Table showing haemoglobin in gms. in relation to maximum parietal secretion of HCI. Correlation coefficient 0.039 (not significant) Hb. in gnu. Maximum parietal secretion of HCl in mgs. 0-300 301-600 601-900 901-1200 1201-1500 1501-1800 Total:

Less than 12.0 gm. 5

6 1

13

12.1 to 14.0 gm.

14.1 to 16.0 gm.

Above 16.1 gm.

6 18 5 4

9 37 14 6 3

6 6 3 3

26 67 22 14 4

1

1

19

134

33

69

Total

473

V / ROLF. OF NUTRITIONAL FACTORS

FIG. 5: Table showing R.B.C. in millions in relation to parietal secretion of HCI in mgs. Correlation coefficient 0.021 (not significant)

RBC in millions Maximum parietal secretion of HCI in mgs.

2.51 to 3.50

3.51 to 4.50

4.51 to 5.50

5.51 to 6.50

1 1 — — —

4 14 3 2 — —

16 45 14 9 4 1

5 7 5 3 —

0-300 301-600 601-900 901-1200 1201-1500 1501-1800

Total 26 67 22 14 4 I

FIG. 6: Table showing serum proteins in mgs. in relation to maximum perietal secretion of HCI in mgs. Correlation coefficient 0.004 (not significant) Serum protein in gm. Maximum Parietal secretion of HC1 in gms.

5.01 to 6.00

4.01 to 5.00 1

0-300 301Ø0

4

2 2

601-900

901-1200 1201-1500 1501-1800

— — —

Total:

5

6.01 to 7.00

Above 7.00 Total

17

16

1

22

47

1

67

0 5 —

14 8 4 1

— 1 — —

22 14 4 1

32

90

3

130

FIG. 7: Table showing weight in pounds in relation to maximum parietal secretion. Correlation coefficient 0.008 (not significant)

Maximum Parietal secretion of HCl in tugs.

61 to 80 lbs.

81 to 100 lbs.

101 to 120 lbs.

0-300 301-600 601-900 901-1200 1201-1500 1501-4800

1 1 1 — — —

15 29 5 4 — —

7 24 11 4 1 —

474

Weight in lbs. 121 141 to to 140 160 lbs. lbs. 3 4 4 3 1 1

— 4 1 — — —

Above 160 lbs. — — — 2 1 —

Total 26 62 22 13 3 1

NUMBER OF PATIENTS

HUMBER OF PATENTS o

0

0

0

Nll

NIL 1-50 SI-100 101 -150