Gastrin: UCLA Forum in Medical Sciences, Number 5

UCLA FORUM IN MEDICAL SCIENCES

VICTOR E. HALL, Editor

MARTHA BASCOPÉ-VARGAS, Assistant Editor

EDITORIAL BOARD

UNIVERSITY OF CALIFORNIA, LOS ANGELES

GASTRIN

UCLA FORUM IN MEDICAL SCIENCES

NUMBER 5

GASTRIN

Proceedings of a Conference held in September, 1964

Sponsored by the School of Medicine, University of California, Los Angeles

EDITOR

MORTON I. GROSSMAN

UNIVERSITY OF CALIFORNIA PRESS

BERKELEY AND LOS ANGELES

1966

CITATION FORM

Grossman, M. I. (Ed.), Gastrin. UCLA Forum Med. Sci. No. 5, University of

California Press, Los Angeles, 1966.

University of California Press

Berkeley and Los Angeles, California

© 1966 by The Regents of the University of California

Library of Congress Catalog Card Number: 66-25614

Printed in the United States of America

PARTICIPANTS IN THE CONFERENCE

MORTON I. GROSSMAN, Chairman and Editor

Division of Gastroenterology, Veterans Administration Center

Los Angeles, California

SVEN ANDERSSON

Department of Pharmacology, Karolinska Institutet

Stockholm, Sweden

E. L. BLAIR

Department of Physiology

The Medical School, The University of Newcastle upon Tyne

Newcastle upon Tyne, England

W. I. CARD

Gastro-Intestinal Unit, Western General Hospital

and University of Edinburgh

Edinburgh, Scotland

CHARLES F. CODE

Section of Physiology, Mayo Foundation

Rochester, Minnesota

LESTER DRAGSTEDT

Department of Surgery, University of Florida

Gainesville, Florida

IAIN E. GILLESPIE

Department of Surgery, Western Infirmary

University of Glasgow

Glasgow, Scotland

R. A. GREGORY

The Physiological Laboratory, The University of Liverpool

Liverpool, England

GEORGE A. HALLENBECK

Section of Surgical Research, Mayo Clinic and Mayo Foundation

Rochester, Minnesota

BERNARD J. HAVERBACK

Department of Medicine, University of Southern California

Los Angeles, California

GABRIEL M. MAKHLOUF

Gastro-Intestinal Unit, Western General Hospital

Edinburgh, Scotland

LARS OLBE®

Department of Pharmacology, Karolinska Institutet

Stockholm, Sweden

ROY M. PRESHAw$

Gastrointestinal Research Laboratory, Veterans Administration Center

Los Angeles, California

BRIAN SCHOFIELD

Department of Physiology

The Medical School, The University of Newcastle upon Tyne

Newcastle upon Tyne, England

EMIL L. SMITH

Department of Biological Chemistry, University of California, Los Angeles

Los Angeles, California

STUART D. TAUBER

Department of Internal Medicine

Southwestern Medical School, The University of Texas

Dallas, Texas

JAMES C. THOMPSON

Department of Surgery, University of California, Los Angeles

Los Angeles, California

BÖRJE UVNÄS

Department of Pharmacology, Karolinska Institutet

Stockholm, Sweden

° Present Address: Surgical Clinic II, Sahlgrenska Hospital Göteborg, Sweden

t Present Address: Department of Experimental Surgery, McGill University Montreal, Canada

JOHN SYDNEY EDK1NS

1863-1940

The Discoverer of Gastrin

CONTENTS 1

CONTENTS 1

SOME NOTES ON THE HISTORY OF GASTRIN

STUDIES ON THE CHEMISTRY OF GASTRINS I AND II†

CHARACTERIZATION OF A PURE GASTRIN

ASSAY OF GASTRIN§

DISTRIBUTION AND LOCAL RELEASE OF GASTRIN**

VAGAL RELEASE OF GASTRIN

VAGAL AND ANTRAL INFLUENCES ON THE ACTION OF GASTRIN

ACTION OF GASTRIN II ON GASTRIC SECRETION IN MAN†††

INHIBITION BY ACID OF GASTRIN RELEASE

THE QUESTION OF AN ANTRAL CHALONE

INHIBITION OF ACID SECRETION BY GASTRIN EXTRACTS

THE QUESTION OF RELEASE OF HISTAMINE BY GASTRIN

GASTRIN-LIKE ACTIVITY OF TUMORS: A REVIEW

STIMULATION OF PANCREATIC SECRETION BY GASTRIN EXTRACTS

AFTER THE CONFERENCE: REVIEW AND PERSPECTIVE

ADDITIONAL BIBLIOGRAPHY

NAME INDEX

SUBJECT INDEX

SOME NOTES ON THE HISTORY OF GASTRIN

MORTON I. GROSSMAN

Chairman

In a sense, one might say that Edkins invented gastrin rather than discovered it. Discoveries usually represent the culmination of a search baited by fragmentary and equivocal clues that cannot be rationalized on the basis of current doctrine. In the case of secretin, it was necessary to learn how acid in the intestine could stimulate pancreatic secretion even after nervous connections between the gut and pancreas had been severed. In the case of gastrin, no comparable phenomenon had been observed that demanded to be accounted for.

There was, of course, what we now know to be a powerful piece of evidence which indicated hormonal control of gastric secretion, namely the well-known observation that the vagally-denervated Heidenhain pouch secreted acid in response to feeding. However, there was uncertainty as to whether the Heidenhain pouch was completely deprived of vagal fibers and also as to whether the vagus nerves were the sole secretory nerves to the stomach. Finally, Heidenhain’s own explanation (15), that absorbed foodstuffs were the stimulant, had not been disproved.

The first of the two volumes of E. A. Schäfers Text-Book of Physiology (23) appeared in 1898, and contained a chapter, Mechanism of Secretion of Gastric, Pancreatic, and Intestinal Juices (6), written by J. S. Edkins, who was then instructor on practical physiology in St. Bartholomew’s Hospital Medical School in London. This scholarly piece of writing reveals that Edkins was thoroughly conversant with the entire field of gastric physiology a full seven years before he announced his discovery of gastrin. In this chapter are several observations and interpretations that may be considered to have special significance as possibly revealing the framework of his great accomplishment.

Edkins cites the studies of Heidenhain (15) showing that food placed in the main stomach caused the separated pouch to secrete, and comments: The conclusion which Heidenhain arrived at was that certain products of digestion when absorbed stimulate the flow of gastric juice. The question then arises, What are these products of digestion, and by what paths are they absorbed? Are the completely digested foodstuffs (that is to say, completely digested so far as gastric digestion is concerned) passed on to the intestine and there absorbed, or are they directly absorbed in the stomach?

Edkins then goes on to cite the work of von Mering (27), who claimed to have demonstrated the absorption of sugars from the stomach. Accepting this as evidence that absorption of foodstuffs can occur in the stomach, and combining it with the observations of Chischin (4), from Pavlov’s laboratory, that peptones were particularly effective in stimulating pouches to secrete when placed in the main stomach, Edkins (6) theorizes as follows: According to these experiments, then, we may assume that small quantities of peptone may be normally formed in the stomach, and, becoming absorbed there, in some way influence the epithelium so that secretion results.

Two additional elements were required to complete the gastrin hypothesis. First, there was the revolutionary concept introduced in 1902 by Bayliss & Starling (3) that in the case of the pancreas it was not the absorbed materials themselves that excited pancreatic secretion, but the release of a chemical stimulant, the hormone secretin, from the intestinal mucosa by the materials being absorbed. Second, there was the assumption made by Edkins that the pyloric gland area of the stomach would be a more likely site of absorption of foodstuffs than the fundic gland area.

This is the way Edkins expressed his idea in his preliminary communication to the Royal Society in May, 1905 (7): On the analogy of what has been held to be the mechanism at work in the secretion of pancreatic juice by Bayliss and Starling, it is probable that, in the process of absorption of digested food in the stomach, a substance may be separated from the cells of the mucous membrane which, passing into the blood or lymph, later stimulates the secretory cells of the stomach to functional activity.

Today the gastric absorption of foodstuffs is known to be negligible. It was on the basis of the supposition that the release of gastrin is brought about by the absorption of foodstuffs that Edkins decided the pyloric gland area was a more likely site of formation of his hypothetical hormone than the fundic gland area—because, he supposed, the former, on the basis of its histologic appearance, was more likely to have an absorptive function.

The direct demonstration that the pyloric gland area of the stomach plays a special role as a receptor site for substances that stimulate gastric secretion came after Edkins had demonstrated that extracts of the pyloric gland area contain a gastric secretory stimulant not found in extracts of the oxyntic gland area. The first such demonstration came from Walter Gross (12), who worked in Pavlov’s laboratory and performed studies on a dog that had been surgically prepared by Pavlov. He found that introduction of meat extract into the main stomach caused secretion from the Pavlov pouch, but this effect no longer occurred when the pyloric gland area of the stomach was separated from the main stomach so that the meat extract could bathe only the oxyntic gland area. Gross was aware of the work of Edkins and interpreted his results as being consistent with Edkins’ hypothesis. Although his paper appeared in 1906, very shortly after Edkins’ first paper, Gross gives no indication of whether his work was started before or after he became aware of Edkins’ work. In any case, there is no documentary support for the impression given in some historical accounts (2, 8) that the discovery of the special role of the pyloric gland area by the Pavlov school long preceded Edkins’ work.

As cited by Babkin (1), Pavlov gave in his M.D. thesis, written in 1883 (18), this definition of nervism, a doctrine he had evolved under the influence of Botkin and which he was to espouse all his scientific life: I understand by nervism a physiological theory which tries to prove that the nervous system controls the greatest possible number of bodily activities. The temporary lapse from this doctrine of nervism manifested in Gross’ paper was the sole departure workers in Pavlov’s laboratory were to make. Later workers in Pavlov’s laboratory, particularly Sawitsch & Zeliony (22), showed that the stimulation of gastric secretion brought about by agents acting in the pyloric gland area could be counteracted by atropine, and concluded that it represented a vagal reflex. It was to be many years before these two apparently irreconcilable views were brought into harmony by the work of Uvnäs (25), who showed that vagal impulses to the pyloric gland area could release gastrin. Thus the regulation of gastric secretion by the pyloric gland area is neurohormonal.

Of the nearly sixty years that have passed since the discovery of gastrin, all but the last dozen have been occupied by the controversy over whether the hormone did in fact exist. Once that hurdle was passed, the pace was indeed swift, and in the past few years it has been truly meteoric.

The middle years of the gastrin story, when its existence was being questioned, were marked by desultory and indecisive studies. Two obstacles blocked the path of progress. One led down the blind alley of histamine as the pretender to the throne of gastrin. The other was concerned with the great difficulty of demonstrating the endogenous release of gastrin under conditions in which contributions from nerves or absorbed secretagogues could be ruled out.

Soon after Edkins announced the discovery of gastrin, many workers showed that crude extracts prepared from a variety of organs stimulated gastric acid secretion when injected subcutaneously in conscious dogs with gastric pouches. Popielski (19) ascribed the action of these extracts to a substance he called vasodilatin that was eventually identified by Dale & Laidlaw (5) as histamine.

Once it had been shown that histamine was a powerful stimulant of gastric secretion (20) and that it was widely distributed in the body (5), including the pyloric gland area of the gastric mucosa (21), the whole gastrin hypothesis fell under a pall from which it did not soon emerge. It was

Figure 1. Simon Andrew Komarov (1892-1964), the first to demonstrate that histamine- free extracts of pyloric mucosa could stimulate gastric secretion.

Komarov1 (16) who in 1938 put our thinking back on the right track when he correctly assumed that gastrin was a polypeptide like secretin and could therefore be separated from histamine by precipitation with trichloracetic acid. Unfortunately, the methods he devised for extraction of gastrin did not always give active preparations, and several modifications based on them gave only indifferent success. It was not until Gregory & Tracy (9) took up the problem again in 1961 that a fully reliable method for preparing the hormone became available. It was only a few years after that, as is recorded in the proceedings of this conference, that these investigators and their coworkers succeeded in isolating two gastrins in pure form, characterizing their chemical composition completely, and proving their structure by total synthesis.

To demonstrate that there need be no nervous pathways between the pyloric gland area and the fundic gland area for the gastrin mechanism to operate, it is necessary to deprive one or both of these structures of its nerve supply, and this can best be done by transplantation. When the fundic gland area is vagally denervated, it becomes much less sensitive to the stimulating action of gastrin. To compensate for this decreased sensitivity, one can give a background dose of a stable choline ester. Using such conditions and distention of the pyloric gland area as the stimulant, thus avoiding the question of blood-borne absorbed secretagogues, we (14) were able to provide what we considered to be crucial proof for the physiological existence of the gastrin mechanism.

As so often happens in science, secondary discoveries had to be made before much of th information on gastrin could fall into place. Thus many workers gave gastrin extracts as single rapid intravenous injections, not realizing that supramaximal doses are highly inhibitory and that therefore the best way to demonstrate the stimulatory action is to give the material by subcutaneous or intramuscular injection or by slow intravenous infusion. This inhibitory action of large doses was first observed by Uvnäs (26), who thought that it was caused by impurities in the extract, but Gregory & Tracy (10) showed that pure gastrin had a similar inhibitory action.

Not until it was recognized that acid bathing the pyloric gland area mucosa can inhibit the release of gastrin (28) was it possible to demonstrate unequivocally that vagal impulses can release gastrin. Straaten (24) had shown that resection of the pyloric part of the stomach reduced the response to sham feeding, and Uvnäs (25) had confirmed this by showing that resection of the pyloric part of the stomach greatly depressed the response of the fundic glands to electrical stimulation of the vagus nerves. Uvnäs was correct in his interpretation that vagal impulses released gastrin. Because it was well known that vagal stimuli, such as sham feeding and insulin hypoglycemia, did not stimulate secretion from Heidenhain pouches, Uvnäs’ hypothesis of vagal release of gastrin was questioned (13). However, when Schofield and coworkers (17) took the pyloric gland area out of the path of the gastric acid by making it into a vagally-innervated pouch, they showed that vagal stimulation released gastrin and that placing acid in the pyloric pouch prevented this.

First assumptions are not always correct. When it was found that extracts of the pyloric gland area of the stomach stimulated flow and enzyme output from the pancreas, it was assumed that this was caused by secretin and pancreozymin in the extracts, but it has now been established that gastrin itself is the pancreatic stimulant (10).

With the discovery that gastric hypersecretion in patients with Zollinger- Ellison syndrome is caused by unregulated production of gastrin by the tumor of the islet cells of the pancreas (11), a whole new dimension was added to research in gastrin.

It does not seem unlikely that the success achieved in determining the structure of two gastrins will serve to topple the entire row of dominoes. Perhaps the next few years will see the isolation, chemical characterization, and synthesis of each of the presently known polypeptide gastrointestinal hormones.

REFERENCES

1. BABKIN, B. P., Pavlov; a Biography. Univ, of Chicago Press, 1949.

2. , Secretory Mechanism of the Digestive Glands (2nd ed.). Hoeber,

New York, 1950.

3. BAYLISS, W. M., and STARLING, E. H., The mechanism of pancreatic secretion. J. Physiol., 1902,28: 325-353.

4. CHISCHIN, P., Die sekretorische Thätigkeit des Hundemagens. Jahresb. Fortschr. Thier.-Chem., 1894, 24: 347-351.

5. DALE, H. H., and LAIDLAW, P. P., The physiological action of s-iminazolyl- ethylamine. J. Physiol., 1910-11, 41: 318-344.

6. EDKINS, J. S. Mechanism of secretion of gastric, pancreatic, and intestinal juices. In: Text-Book of Physiology, Vol. I (E. A. Schäfer, Ed.). Pentland, Edinburgh, 1898: 531-558.

7. , On the chemical mechanism of gastric secretion. Proc. Roy. Soc. Lon

don B, 1905, 76: 376.

8. GREGORY, R. A., Secretory Mechanisms of the Gastro-Intestinal Tract. Arnold, London, 1962.

9. GREGORY, R. A., and TRACY, H. J., The preparation and properties of gastrin. J. Physiol., 1961,156: 523-543.

10. , The constitution and properties of two gastrins extracted from hog

antral mucosa. Gut, 1964,5: 103-117.

11. GREGORY, R. A., TRACY, H. J., FRENCH, J. M., and SIRCUS, W., Extraction of a gastrin-like substance from a pancreatic tumour in a case of Zollinger- Ellison syndrome. Lancet, 1960, 1: 1045-1048.

12. GROSS, W., Beitrag zur Kenntnis der Sekretionsbedingungen des Magens nach Versuchen am Hund. Arch. Verdauungs-Krankh., 1906, 12: 507-516.

13. GROSSMAN, M. L, Gastrointestinal hormones. Physiol. Rev., 1950, 30: 33-90.

14. GROSSMAN, M. L, ROBERTSON, C. R., and IVY, A. C., Proof of a hormonal mechanism for gastric secretion—the humoral transmission of the distention stimulus. Am. J. Physiol., 1948,153:1-9.

15. HEIDENHAIN, R., Ueber der Absonderung der Fundusdrüsen des Magens. Pflüg, Arch. Physiol., 1879, 19: 148-166.

16. KOMAROV, S. A., Gastrin. Proc. Soc. Exp. Biol. Med., 1938, 38: 514-516.

17. MAUNG PE THEIN, and SCHOFIELD, B., Release of gastrin from the pyloric antrum following vagal stimulation by sham feeding in dogs. J. Physiol., 1959, 148: 291-305.

18. PAVLOV, I. P., The Centrifugal Nerves of the Heart. Thesis, Kotomina, St. Petersburg, 1883 (in Russian).

19. POPIELSKI, L., Die Wirkung der Organextrakte und die Theorie der Hormone. Münch. Med. Wschr., 1912,59: 534-535.

20. , ß-imidazolyläthylamin und die Organextrakte; ß-imidazolyläthylamin

als mächtiger Erreger der Magendrüsen. Pflüg. Arch. Physiol., 1920, 178: 214-236.

21. SACKS, J., IVY, A. C., BURGESS, J. P., and VANDOLAH, J. E., Histamine as the hormone for gastric secretion. Am. J. Physiol., 1932, 101: 331-338.

22. SAWITSCH, W., and ZELIONY, G., Zur Physiologie des Pylorus, Pflüg. Arch. Physiol., 1913, 150: 128-138.

23. SCHÄFER, E. A. (Ed.), Text-Book of Physiology. Pentland, Edinburgh, 1898.

24. STRAATEN, T., Die Bedeutung der Pylorusdrüsenzone für die Magensaftsekretion. Ein experimenteller Beitrag zur Resektionsbehandlung des Geschwürsleidens. Arch. klin. Chir., 1933, 176: 236-251.

25. UVNÄS, B., The part played by the pyloric region in the cephalic phase of gastric secretion. Acta Physiol. Scand., 1942, 4, Supp. 13: 1-86.

26. , The gastric secretory excitant from the pyloric mucosa. Acta ‘Physiol.

Scand., 1943,6: 97-107.

27. VON MERING, J., Ueber die Function des Magens. Verhandl. deut. Ges. inn. Med., 1893, 12: 471-487.

28. WOODWARD, E. R., LYON, E. S., LANDOR, J., and DRAGSTEDT, L. R., The physiology of the gastric antrum; experimental studies on isolated antrum pouches in dogs. Gastroenterology, 1954, 27: 766-785.

1 Dr. Simon Komarov died on March 29, 1964. The portrait in Figure 1 was taken shortly before his death.

STUDIES ON THE CHEMISTRY OF GASTRINS I AND II1

R. A. GREGORY and HILDA J. TRACYt

The University of Liverpool

England

Our work on gastrin was begun almost by accident, and certainly without any particular enthusiasm, in August 1959. For some time we had been interested in the mechanism by which enterogastrone inhibits gastric secretion; and a need had arisen in our experiments for a gastrin preparation which would stimulate secretion effectively in the conscious dog, preferably when given by subcutaneous injection. We soon learned from the literature that only Komarov (17) and Linde (18) had tested a gastrin preparation on conscious dogs, obtaining rather small responses to intravenous injections. All other investigators had followed Komarov in testing their preparations by intravenous injection in anesthetized cats; he had stated that subcutaneous injections into conscious dogs were ineffective (17).

Professor A. A. Harper was at the time a not infrequent visitor to our laboratory. Knowing of his interest in the extraction of gastrin (14), we begged from him a sample of crude gastrin extract, which he had found to be an effective stimulant of gastric acid secretion when injected intravenously into anesthetized cats. This extract we tested on conscious dogs; to his and our disappointment, it did not stimulate secretion. If we had obtained a response, we would probably not have taken any further interest in the problem of extracting gastrin! An unexplained point is that when the method used for making this extract was eventually published (4) and we tried it for ourselves, we found the material obtained to be an excellent stimulant of gastric acid secretion when given subcutaneously to conscious dogs.

After Grossman (7) had shown that gastrin extracts strongly inhibit gastric acid secretion when given intravenously, we realized that when we had tried out Harper’s extract in 1959, we gave it first of all intravenously and, after obtaining no response, gave further doses subcutaneously and intramuscularly. Harper’s extract, like other gastrin preparations, strongly inhibits secretion when given as a rapid intravenous injection in conscious dogs.

There must have been many investigators during the years following Komarov’s classic papers (15, 16, 17) who concluded that gastrin extracts made by his methods were inactive because they obtained no response upon intravenous injection into conscious dogs.

We decided to try to make some gastrin for ourselves, and soon evolved a new method of extraction which provided a crude but histamine-free preparation, highly effective in stimulating gastric acid secretion when injected subcutaneously into conscious dogs (9). Two further stages of purification were then incorporated into the procedure, thereby providing material sufficiently pure to stimulate gastric acid secretion without producing significant side effects in a human subject when given subcutaneously, intramuscularly and intravenously (10, 11). The product was far from homogenous, showing several components on paper electrophoresis, but it was more potent than histamine on a simple weight basis. The properties of the material suggested that the active principle was of protein or polypeptide nature. The first stage of this method has been widely used since then, not only for extracting gastrin from hog antral mucosa, but also for extracting Zollinger-Ellison tumors.

In September 1960, Dr. Grossman came to work with us in Liverpool, and we enjoyed a most happy and profitable period of close association. Using our gastrin extract in its most highly purified form (the Stage III product) he made several brilliant observations on its physiological actions. In January 1961, Harper (4) published his method for preparing a crude gastrin extract from antral mucosa. This was virtually the same as the method originally used by Edkins (6), with the addition of a stage of acetone precipitation which removed all significant amounts of histamine. Incidentally, this work provided the first decisive evidence that Edkins’ extracts had in fact contained gastrin, as well as histamine. Harper’s method was suitable for use on a very small scale only because the stages involving filtration through paper and acetone precipitation were practical only for small batches, but it was a highly effective means of extracting gastrin from antral mucosa, and Dr. Grossman joined with us in adapting it to large- scale use as a starting point for the purification of gastrin by our 1961 method. This modification was later described by Gillespie & Grossman (7).,

After Dr. Grossman’s return to Los Angeles in the spring of 1961, we made an observation that led us for the first time seriously to contemplate attempting to isolate gastrin on a scale adequate for chemical, as well as physiological studies. This significant observation was that certain cellulose derivatives (DEAE-, AE-, and ECTEOLA-cellulose) would take up gastrin activity from dilute solutions at appropriate pH and low salt content, and release it in solutions of higher pH and salt content. We were, as a matter of fact, seeking a means of extracting from the urine of patients with the Zollinger-Ellison syndrome any gastrin which might be present there. When we found that these cellulose derivatives would also remove gastrin from a crude aqueous extract of antral mucosa made by Harper’s method and that, if the floc form of the material was used, large-scale batch operations were easily carried out, we realized that here was an Open Sesame to the large- scale extraction of gastrin from hog antral mucosa.

Soon we were studying the behavior of gastrin activity by using Sephadex columns, counter-current distribution systems, and chromatographic columns of various types. Gradually a method evolved by which in early April 1962 we obtained what we soon became reasonably certain was gastrin in a pure, i.e., homogenous state. Our preliminary data on its amino acid composition and molecular weight convinced us that it was a fairly small peptide, and, as expected, it was many times more potent than histamine in stimulating gastric acid secretion. These results we reported at the Gastrin Symposium held in New York on April 26, 1962, and organized by the Gastroenterology Research Group. Simon Komarov was to be chairman of this symposium. We wrote to him a few days beforehand and sent him a photograph of our preparation in the ultracentrifuge (Figure 2). We remember with what joy he greeted us when we arrived in New York—he could not have been more pleased if he had done it himself!

During the summer of 1962 we obtained the first accurate quantitative amino acid analysis of our product; and this, together with the results of high-voltage paper electrophoresis and end-group determination, supported our previously tentative conclusions that gastrin—or rather a gastrin—had been isolated. We also worked out improvements in the method so that we could deal with 600 antrums weekly—an amount which we felt sure would provide enough gastrin for all our needs—and began active collaboration with a group of colleagues in the department of organic chemistry at the University of Liverpool, headed by a distinguished peptide chemist, Professor G. W. Kenner. Difficulties in obtaining antrums soon arose, however, for in our part of England there is a consumer market for them, and it was not, in fact, until the morning of December 25, 1962, that we read the samples coming off the AE column—the final stage of isolation—from our first batch of 600 antrums. An unexpected and most exciting Christmas present awaited us—a second gastrin, almost completely separated from the first and evidently slightly more acidic in behavior (Figure 3). Henceforth, we called these two substances Gastrin I and Gastrin II. It was eventually confirmed that what we had isolated the previous April was, appropriately enough, Gastrin I.

By a second stage of chromatography on AE columns, both gastrins could be obtained in a virtually pure state. It was soon found that the two substances had an identical quantitative amino acid composition, even to the single amide group present in each. For some time, a peak in the quantitative amino acid analysis pattern remained unidentified; it was eventually shown that this was tryptophan, giving the following composition for each gastrin: aspartic acid, 1; glutamic acid, 6; glycine, 2; alanine, 1; methionine, 2; proline, 1; phenylalanine, 1; tryosine, 1; tryptophan, 2; and ammonia, 1. The calculated molecular weight was 2114.

Our chemist colleagues decided to concentrate their studies of the amino acid sequence upon Gastrin II, as this was available in larger amounts than Gastrin I. In view of the identical amino acid composition of the two substances, it was anticipated that it would probably not be a difficult task to ascertain in what respect Gastrin I differed from Gastrin II. As it turned out, the nature of this difference was revealed in a rather novel, and indeed dramatic, fashion.

By the summer of 1963 the greater part of the amino acid sequence in

Figure 2. Ultracentrifugal pattern of a one per cent solution of Gastrin II after 90 minutes at approximately 59,000 rpm (250,000 G). Synthetic boundary cell. Spinco Model E ultracentrifuge.

Gastrin II had been elucidated, and we felt able to present a preliminary account of the isolation and properties of the two gastrins (12). By that time, incidentally, we had become well aware that both substances produced wide and varied effects on alimentary tract structures besides stimulating gastric acid secretions. The initial impetus for these studies had come

Figure 3. The separation of Gastrins I and II by gradient elution chromatography on aminoethylcellulose in ammonium bicarbonate buffer.

from Dr. Grossman’s private communication to us (January 8, 1962) reporting that his gastrin extract strongly inhibited a background secretory response to histamine when given intravenously to a conscious pouch dog (7), an observation which we had confirmed, using pure Gastrin I, just before the meeting in New York.

Early in 1964, what was believed to be the complete amino acid sequence for Gastrin II was arrived at by our colleagues (8) who were using the classical methods of enzymic and acid hydrolysis, stepwise degradation of peptides, and end-group determination. The program of synthesis commenced forthwith, and when success seemed reasonably certain, we published a full account of the method of isolation and the properties of the two peptides (13). The structure, which was believed to be that of Gastrin II, and upon which the schemes of the synthesis were based, is shown in Figure 4. It is, however, labeled Gastrin I.

M. W. = 2114

Figure 4. The amino acid sequence of Gastrin I, as worked

out by Kenner et al. in 1964 (cf. 1, 8). See text.

The realization that this was in fact the structure not of Gastrin II but of Gastrin I came about in the following way. Figure 4 illustrates the scheme of synthesis which was first successfully used. The three portions in which the sequence is written correspond to three peptides which were first prepared separately and then coupled together. The first step gave the 13-residue peptide Glu … Gly; this was then coupled with the C-terminal tetrapeptide Try.Met.Asp.Phe-NH2. Excess tetrapeptide was easily removed from the reaction mixture leaving (it was hoped) a mixture of the 13-residue peptide and gastrin. A run of the mixture on a thin-layer chromatogram with pure natural Gastrin II as a reference substance (staining with Ehrlich’s reagent, dimethylaminobenzaldehyde, which reacts well with the tryptophan residues) showed the presence of a prominent component almost exactly corresponding in position to Gastrin II. The two compounds were separated by running the mixture on a column of Sephadex G-25 in a volatile buffer (ammonium bicarbonate). The column had previously been calibrated with a mixture of natural Gastrins I and II and the 13-residue peptide, so that their relative positions in the effluent fractions were known.

To the great surprise of all concerned, a peak corresponding to gastrin was indeed obtained—but it was clearly in the position which indicated Gastrin I and not Gastrin II. This fraction was then run on aminoethylcellulose, as is done when isolating natural gastrin; and again the product emerged in a position indicating not Gastrin II, but Gastrin I. Electrophoretic and thin-layer chromatographic studies, quantitative amino acid analysis, and determination of biological activity all substantiated the conclusion that what had been synthesized was Gastrin I (1). Obviously, the amino acid sequence previously thought to be that of Gastrin II was in fact that of Gastrin I.

It was Kenner who in a few hours found the solution to this puzzle. Both gastrins had an identical amino acid composition, including even the single amide residue present in each, but Gastrin II was known to be slightly more acidic than Gastrin I. Therefore there must have been present in Gastrin II an acidic group, absent from Gastrin I, which was not an amino acid residue and which therefore had not been detected in arriving at the amino acid sequence for Gastrin II. An intensive reappraisal of the data from the structural analysis revealed that during degradation studies on Gastrin II the dipeptide alanyl-tyrosine had behaved anomalously on paper electrophoresis; this provided the clue. Perhaps the tyrosine had been sulfated (tyrosine-Q-sulfate); this compound had been found in normal human urine (20) and was also known to be present in one of the peptides split off from rabbit fibrinogen by the action of thrombin (2). Within a day or two, the presence of tyrosine-O-sulfate in Gastrin II was proved by its identification in the products of alkaline hydrolysis; it has since been shown that each gastrin can be converted into the other by the addition or removal of the sulfate group (8).

What the significance may be of this slight but remarkable difference between the two gastrins we have as yet no idea. Whether one is a product of the other, formed postmortem or during extraction, we are not sure; the latter seems unlikely. However, since the antrums we used for extraction had of necessity to be collected the day before and chilled overnight before processing, and since both gastrins have an apparently identical power to stimulate gastric secretion, the change of one into the other could have occurred postmortem without our noticing it. But we have very recently found that antrums collected in smaller numbers immediately after death also show the presence of both gastrins in about the same amounts as do antrums kept overnight; so either the post-mortem change is rapid but limited in extent, or this is not the answer to the problem at all. In due course, we hope to prepare gastrin from other species by this method. It will be interesting to see whether or not a similar pair of peptides is obtained.

Finally, we should like to review briefly the physiological actions of some of a large series of peptide derivatives which, through the kindness of our chemist colleagues, we were able to study toward the end of the work just described (21). These peptides mostly resulted from the various steps in the synthesis, and comprise various portions of the molecule of Gastrin I, but some of them were produced specifically at our request, and we are especially grateful to our colleagues in this regard.

Each of the natural gastrins (I and II) produces, in apparently identical form and degree, the remarkable effects on alimentary tract function which we described in detail in our account of the isolation and properties of Gastrins I and II (13). These effects had all been recognized by previous workers who used more or less crude extracts of antral mucosa. It had come to be generally accepted that they were attributable to the presence in such extracts of a variety of substances besides gastrin, which was considered to stimulate only gastric acid secretion. These actions may be summarized as follows:

a. Stimulation of gastric acid secretion. In conscious dogs, subcutaneous injections cause a protracted secretion of highly acid gastric juice which contains little pepsin unless the dosage is high.

b. Inhibition of gastric acid secretion. In conscious pouch dogs intravenous injections inhibit a background secretion established by histamine (or by Gastrin I or II).

c. Stimulation of pepsin output. In conscious dogs provided with denervated fundic pouches, a large output of pepsin occurs as the inhibition following such an intravenous dose wears off.

d. Stimulation of pancreatic secretion. In either conscious or anesthetized dogs, intravenous injections cause a prompt increase in volume-flow and enzyme output.

e. Stimulation of gastric tone and motility. Small rapid intravenous injections cause a prompt and powerful spasm of the gastric musculature followed by some increase of motility at a lower tone level as the spasm wears off.

f. Effects on small intestinal tone and motility. Small rapid intravenous injections cause a prompt spasm, or series of contractions, which is quickly converted into more prolonged inhibition of tone and motility.

g. Biliary tract and blood pressure. Neither of the gastrins has a significant effect on gallbladder tone or hepatic bile flow. A small and inconstant depressant effect upon the arterial blood pressure may be observed in lightly anesthetized animals following rapid intravenous injections.

The peptides available to us were examined for the presence of these effects, with the exception of possible effects on the biliary tract, by using the same methods described previously (13); the dose of each peptide routinely employed was such as would give