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Memorial Lecture: The Isolation and Chemistry of Gastrin
GASTROENTEROLOGY Official Publication of the American Gastromterological Association ©CoPYRIGHT
VoLUME
51
1966
THE WILLIAMs
&
WILKINs Co.
December 1966
NUMBER
G
MEMORIAL LECTURE: THE ISOLATION AND CHEMISTRY OF GASTRIN R. A. GREGORY, PH.D., D.Sc., M.R.C.S., L.R.C.P., F.R.S. The Physiological Laboratory, The University of Liverpool, Liverpool',' England
Mr. President and members of the Association, I am deeply sensible of the honor you have done me by asking me to give this lecture, and I thank you for your most warm and gracious welcome. This is a memorable occasion for me in another respect; nearly 30 years ago, I began my career in gastroenterology just around the corner from here, in Northwestern University Medical School, and my teacher was Dr. Andrew Ivy. I am proud to feel that I can call myself one of his pupils, and I remember with gratitude the many kindnesses which he showed to me while I worked with him. I came to Northwestern from University College London, where, in 1902, the discovery of secretin by Bayliss and Starling opened a new era in physiological thought by its revelation that there existed beside the nervous system a hormonal system for the integration of the normal bodily functions. This work forms the prelude to my story; for it undoubtedly gave to John Sydney Edkins the germ of his idea that there existed in the gastric antral mucosa a preformed substance which was absorbed into the portal blood stream during gastric digestion and, returning in the circulation to the fundic glands, maintained their acPresented at the Annual Meeting of the American Gastroenterological Association, Chicago, Illinois, May 27, 1966. Address requests for reprints to: Dr. R. A. Gregory, Physiology Laboratory, University of Liverpool, Brownlow Hill, Liverpool 3, England.
953
tivity after the initial vagal reflex excitation associated with the eating of a meal had come to an end. Sixty-one years ago almost to this day (May 18, 1905), Edkins read before th"e Royal Society of London a modest paper entitled "On the chemical mechanism of gastric secretion." In this he desc ribed how he had prepared aqueous extracts from antral and fundic mucosa and had tested them for their power to stimulate gastric secretion by intravenous inj ection into anesthetized cats. The fundic extracts had no action, but the antral extracts excited ac id and pepsin secretion. The active principle he named "gastrin," a term evidently derived from "gastric secretin." It is interesting now to note that in this first paper he did not call the substance a "hormone," for the simple reason that the term was unknown to him at that time. It was in fact first used publicly by Starling a month later when he delivered his famous Croonian Lectures to the Royal College of Physicians of London on "The chemical correlation of the functions of the body"; and in the first lecture, Starling instanced the very recently discovered gastrin as the second example after secretin of a hormone. History, it is said, is the tragedy of events before their time; and certainly t he history of science shows us many examples of men and discoveries before their time. So it was with Edkins; for very soon it seemed that what my friend, Professor W. M. Paton, once described as "the tide of
954
GREGORY
undiscovery" had set in. Those who sought to confirm and extend Edkins' discovery soon found that a stimulant of gastric acid secretion was indeed present in antral mucosa, but an apparently identical stimulant could be extracted by the same means from virtually any other tissue in the body. Edkins had, in fact, fallen first victim to that ubiquitous enigma histamine-years before even its existence in the body was recognized, much less its power to stimulate gastric acid secretion. When these facts were eventually established by the classical studies of Dale and his colleagues and of Popielski, it seemed clear enough that Edkins' antral extracts owed their activity to nothing more specific than the presence in them of histamine. Looking back now, we can appreciate how cruelly unlucky he was, for the oxyntic cells are the only exocrine glands on which histamine has a powerful secretagogue effect; and I, at any rate, am uncertain whether we can attribute to this action any physiological significance despite all that has been written and said upon the subject. If it had chanced to be the pancreatic centroacinar cells which were so powerfully excited by histamine, then there would have been no difficulty in confirming Edkins' claim that there existed in antral mucosa a specific stimulant of gastric secretion-while it might have been Bayliss and Starling who were in trouble! Their joint genius would certainly have coped with such a problem, but history would have been differently written. It was Simon Komarov who, in 1938, realized the trick that Nature-an old hand at the game !-was playing. In all tissues, including antral mucosa, there is histamine; but in antral mucosa-and virtually confined to it-there is present in addition a protein-like substance which can closely mimic the action of histamine on gastric acid secretion, so that the two may not be distinguishable by physiological tests of simple extracts containing both, as did Edkins'. Komarov precipitated with trichloroacetic acid a dilute acid extract of antral mucosa and obtained a protein fraction which was histamine-free but strongly stimulated gastric acid secretion when in-
Vol. 51 , No. 6
jected intravenously into anesthetized cats; he had rediscovered the antral hormone (fig. 1). This classical observation, made 2 years before Edkins' death-and of which, I believe, he learned in time-was the first definite indication that gastrin existed; for not until 10 years later did the work of Grossman, Robertson, and I vy and of Dragstedt and his pupils establish by physiological experiment the existence of an antral hormonal mechanism for the stimulation of gastric acid secretion. Their subsequent studies confirmed and clarified the hypothesis put forward in 1942 by Uvnas that vagal excitation is an important factor in the release and action of the antral hormone; and over the years, they and others have built up a picture with which we are all quite familiar. When a meal is eaten, vagal excitation to the antrum increases the release of gastrin occasioned by local stimulation; at the same time, vagal excitation to the fundic glands increases their response to the circulating hormone. The accumulation of acid in the antral region inhibits the release of hormone by the "gastrin cells," wherever these may be-most probably, I think, in the deeper regions of the pyloric glands. This is so clear and satisfactory a picture that one can only suspect that there must be a great deal in it that we have not yet perceived; I sometimes wonder how it will look in about 20 years' time! In Komarov's work there was one striking observation which seemed to deny its physiological significance. The inhibitory effect of atropine on the response to a meal in conscious animals was well known, but the action of his extract in the anesthetized cat-and he used this preparation almost entirely for his experiments-was completely resistant to repeated large doses of this drug (fig. 1). However, this finding was later confirmed not only with crude antral extracts, but also with the pure gastrin peptides; and it has also been established that the action of both in the conscious dog and human subject is strongly inhibited by atropine. The anomaly turns out to be only one of many examples of remarkable differences in the action of gas-
955
ISOLATION AND CHEMISTRY OF GASTRIN
D ecember 1966
TOTAL Cl TOTAL&rREE ACIOIT1E5 m .eq/1. ISO
.....__ _ _ _ _ TOTAL Cl
140 130 120 /"
80
/
/
/
70 I
VOLUME 60
c .c.
40
-l(
ACIDITY
I
I
I
I
I
/
.),
I
I
I
I
I
f.
/
/
/
.J. I / I
x'
3·0
TOTAL
I I I' I I
90
50
--,.___
/r
100
3·5
)(---)C....
----,. F'I~E.E. ACIDITY / ,./ ><---><-- -><----]<'"
110
/
I
ATROPINE. SULPHATE __,..___
.J.
2 2 2. mg
H J..
I I
,f PEPSIN Mett unit!
2·5 20
I~
1·5
10
10
5
0·5
0
0 TIM[,hrs .
VOLUME I~
J.-2
2.
1 1 1 1 t
(A5TRIN INJECTED 40
20
20
20
20
2~
3
3~
t
1
t
20
20
4
4~
5
20 mg
Fw. I. The effect of "purified" gastrin, extracted from 130 g of canine pyloric mucosa (by method I), and injected intravenously every Yz hr for 3\/z hr, in a cat under urethane-chloralose anesthesia. Total amount of gastrin injected was 180 mg. Intravenous administration of atropine sulfate did not depress the secretagogue effect of gastrin. (Komarov, 1942. Courtesy of Rev. Canad. Biol.)
trin upon gastric secretory responses in different species and different experimental conditions; I shall refer to others later on. Over the years following Komarov's work-first confirmed by Uvnas and his pupils-several efforts were made towards isolation of the active principle in antral extracts. These studies gradually led to a general realization that crude antral extracts not only stimul ated gastric acid secretion, but also pepsin secretion, pancreatic volume flow and enzyme secretion, and gastrointestina l tone and motility, though they had little or no effect on the gall bladder or bile flow ; there was too an indi-
cation that an inhibitory effect against a "background" response stimulated by histamine could be obtained. It looked as though the antral mucosa might be a veritable storehouse of biologically active .~ub stances, some of them at least of known hormonal status; but efforts to separate them were unsuccessful. There is now seen to have been implicit in the interpretation of these findings at the time the traditional assumption that with each of the gastrointestinal hormones was associated a single physiological function, on the basis of which its existence had originally been recognized. No one supposed-certainly not I-
956
GREGORY
that all these actions of crude antral extracts would eventually be shown to be properties of a single antral peptide. This has led, I think, to a significant change in our way of thinking about the other gastrointestinal hormones; we are now prepared to find that when they are studied in a pure state, they will be shown to have more than one effect of physiological significance, as has gastrin. My partner, Hilda Tracy, and I began our work almost by accident in the summer of 1959, when we sought to prepare for use in other experiments a histaminefree gastrin extract which would stimulate effectively when injected subcutaneously or intramuscularly in the conscious dog. Komarov and others after him had used the anesthetized cat and intravenous injection, almost exclusively; and there was some doubt whether gastrin preparations had more than a slight effect in conscious dogs. I shall not weary you with the details of the path by which we eventually arrived some 5 years later-and I might add, some 50,000 hog antrums later!-at the isolation, structure, and synthesis of two almost identical peptides from antral mucosa, which we named gastrins I and II. Early in 1962, we had isolated gastrin I; and at that time we had the immense good fortune not only to secure financial support for the furtherance of our work from your National Institutes of Healthfor which we shall always be grateful-but also to enlist the collaboration of a distinguished group of peptide chemists in the Department of Organic Chemistry of our own University, headed by Professor G. W. Kenner, an authority in the field of peptide chemistry. The second gastrin-gastrin II-turned up on Christmas morning of the same year, when we completed our first large-scale production after scalingup and improving the method in order to provide our chemist colleagues with material for their structural studies. By September 1964, they had achieved complete success; the structures of both gastrins were elucidated, and their total synthesis was accomplished. In the meantime, using the pure natural products-and later, of course, syn-
Vol. 51, No.6
thetic material-we were enabled to explore in meaningful terms their range of physiological properties. An added impetus to this study-long contemplated but postponed until material of attested homogeneity should be available-was the private communication from Grossman in February 1962, that a gastrin extract made by our earlier method would powerfully inhibit a "background" response maintained by histamine, if injected as a single rapid intravenous dose. This we confirmed using pure gastrin I which we had just isolated; and our subsequent studies showed that all of the actions previously described for crude antral extracts were displayed by the pure gastrin peptides, whether natural or synthetic. I can only refer in passing to some of these effects, in particular those on gastric and pancreatic secretion; the former because our studies and those of others have shown the remarkable diversity of action which is revealed in different species and types of animal preparation, the latter because it has led to recognition of a second physiological role for gastrin besides the stimulation of gastric acid secretion. The inhibitory effect of single intravenous injections in the conscious dog, first described in detail by Gillespie and Grossman using a gastrin extract, is seen also in the anesthetized rat; but it does not occur in man or the anesthetized cat. The associated stimulation of pepsin secretion, which we first noted in the conscious dog, occurs also in the anesthetized cat, and there is a small effect in man; information for the conscious cat and anesthetized or conscious rat is not yet available. In manas might be anticipated from the absence of the inhibitory effect-single intravenous injections (fig. 2), as well as subcutaneous or intramuscular administration, are highly effective as a means of exciting acid secretion; in fact, gastrin II has been found to be about 500 times more potent than histamine on a molar basis of comparison. The effect of the gastrin peptides on pancreatic volume flow in the conscious dog is small but definite, while the effect on pancreatic enzyme secretion is quite pronounced; and the elegant work of Preshaw, Cooke, and Grossman seems to establish
ISOLATION AND CHEMISTRY OF GASTRIN
December 1966
957 140
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40
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BO 5 10 15 20 25 30 35 .. o 45 50 55 MINUTES
Fw. 2. Output in milliequivalents per 5 min, secretory rate in milliliters per 5 min, and H• ion concentrations following an intravenous injection of gastrin (4 JLg) in subject G. M. The secretory rate is divided into its parietal and nonparietal (blacked-out portion) components. (From Makhlouf, McManus, and Card, 1966.)
that these actions of gastrin are of physiological significance. They prepared conscious dogs with an antral transplant, a gastric fistula, and a pancreatic fistula. The antral pouch was irrigated with acetylcholine solution at pH 7 to cause a release of endogenous gastrin; the expected acid secretion occurred, and the acid was drained from the open gastric fistula so that none of it entered the duodenum. Nevertheless, there occurred a quite obvious pancreatic response involving volume flow and enzyme secretion which, like the gastric response, disappeared when the acetylcholine solution irrigating the antral pouch was acidified (fig. 3). We may have little doubt, I think, that there exists an antral hormonal phase in the pancreatic secretory response to a meal and that gastrin is the mediator. In continuance of our studies on the gastrin peptides, Tracy and I have isolated a similar peptide from the antral region of the sheep abomasum, and there is evidence of a second in much smaller amount. More recently, with Grossman, we have isolated two peptides from human
antral mucosa and, during the last few weeks, two such peptides from dog antral mucosa. The established or probable constitutions of this family of gastrin peptides are shown in scheme 1. They are all heptadecapeptide amides of closely similar amino acid composition, occurring in unsulfated and sulfated forms. The significance of the sulfate group attached in an ester linkage to the single tyrosy I residue in each case is not known; it has no effect upon the physiological activities so far as we can tell at present, but clearly plays some metabolic role. The differences between the peptides of the various species are seen to lie in the body of the molecule, in close relation to the sequence of glutamyl residues; but as will be seen, we cannot associate these differences in structure with any differences in physiological properties. During the work leading to the successful synthesis of the hog gastrins in 1964 by our chemist colleagues, we were greatly fortunate in that they made available to us, for physiological study, a series of synthetic peptides which corresponded to vari-
ANTRAL TRANSPLANT
~
IRRIGATION OF ANTRAL TRANSPLANT WITH 0
5"/o ACETYLCHOLINE SOLUTION
B VI
GASTRIC FISTULA
! 6 :::£
.,.,
.,.
::: 4 w
E - 2 u
:I:
0 200
z
PANCREATIC FISTULA
~6
150 .,., ~
:::4 i
100
.,.,i
:.'? z :::>
~ 2 ,_ _ _,.____, .....
50~
>-
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N
z
2
5
4
3
6
HOURS
FIG. 3. Redrawn from Preshaw, Cook, and Grossman, 1965. For description see text. Hog I - Pyro·Gly ·Pro · Try·Met•(Giu) 5 ·Ala · Tyr ·Gly · Try·Met·Asp·Phe · NH 2
r
Il (SO,) Human I - Pyro · Gly · Pro ·Tyr · Leu•(Giu) 5 ·Ala·Tyr·Gly·Try·Met·Asp ·Phe · NH 2
r
II (SO,) Dog J - Pyro ·Gly· Pro·Try ·Met• (Aia,Glu,) ·Ala·Tyr ·Gly·Try·Met·Asp·Phe·NH2
r
II (SO,) Sheep I - Pyro · Gly ·Pro · Try· Val• (Aia,Glu,) ·Ala · Tyr · Gly·Try·Met·Asp·Phe·NH 2
r
II (SO,) Scheme 1. The constitutions of the hog , human, dog, and sheep gastrins. -Pyro, pyroglutamyl. The bracketed sequences in dog I and sheep I are not yet fin ally resolved. 958
December 1966
ISOLATION AND CHEMISTRY OF GASTRIN
ous portions of the total molecule of gastrin I; our earlier recognition of the range of actions displayed by the total molecule had naturally raised the question of structure-function relationships. We were disappointed in our expectation that some segregation of the different actions in different portions of the total molecule might be revealed; but as a consolation prize we were given the remarkable discovery that all of the effects of the total structure were displayed by no more than the C-terminal tetrapeptide and that when the amide group masking the C-terminus of this simple sequence was removed, almost all of its actions disappeared; only a slight effect on pancreatic secretion remained. These observations were confirmed and extended in a subsequent study with Morley of a large series of analogues of the active C-terminal tetra- and pentapeptides ; and the results showed that most substitutions led to a considerable decline, and sometimes a virtually total disappearance of the actions of the parent structure. There was no strikingly selective preservation or destruction of the various activities as between the different analogues; minor quantitative variations were of course detected, but in general the picture was that of a fairly uniform change in activity. The results seemed to indicate that in the tetrapeptide sequence, and perhaps also in the total gastrin molecule, the tryptophan and methionine residues were in the nature of binding, rather than functionally active sites, and that the most important regions of the molecule for physiological activity were the aspartic carboxylic and phenylalanine-amide residues. The results of these studies would perhaps lend some support to the speculation t hat the mode of action of the gastrin peptides on their various target sites may be of the same fundamental nature; and indeed Bennett has suggested, on the basis of a pharmacological analysis of the action of pure gastrin II on the isolated guinea pig ileum, that it may cause a release of
959
acetylcholine from nerve terminals in the tissue and that this might possibly be the case elsewhere. This stimulating suggestion certainly merits further exploration , for it would indeed be remarkable if gastrin turned out to be an acetylcholine liberator; but H eraclitus has warned us that if we do not expect the unexpected, we will never find it! It seems to me likely that vve shall not solve the problem of the mode of action of gastrin on the target cell until we can bring to bear on it the same techniques as have reaped such great harvest in the study of the nervous system-the investigation with microelectrodes and micropipettes of single cells. In the mammalian, or even the frog, gastric mucosa, there are formidable difficulties still to be surmounted; but if we go further afield, into the realm of marine biology, we can find a simpler situation, in which acetylcholine or histamine do not seem to be involved in the action of gastrin. From time to time, on the seacoast near Liverpool, it is possible to find a small marine animal named Pleurobranchus membranaceus; it is one of a group of such creatures which protect themselves from inquisitive fish by secreting HCl from cells on their external surface when touched. My colleague, Dr. D . V. Roberts, has found that when acetylcholine or histamine (10 p.g per ml) is added to the surrounding seawater, they show no response, although they remain sensitive to touch; but if gastrin (pure gastrin II) is added (1 p.g per ml), they give a very large and prolonged secretion of acid. How specific this response is for gastrin we do not yet know, but the preparation might obviously be useful for studying its action at a rather primitive level. My tale is done; I thank you for your kind attention and for the privilege of addressing you. I hope that in these few words I have said enough to show how much there is that we still do not understand about gastrin and that this fascinating story is far from concluded.
VoLUME
51
1966
THE WILLIAMs
&
WILKINs Co.
December 1966
NUMBER
G
MEMORIAL LECTURE: THE ISOLATION AND CHEMISTRY OF GASTRIN R. A. GREGORY, PH.D., D.Sc., M.R.C.S., L.R.C.P., F.R.S. The Physiological Laboratory, The University of Liverpool, Liverpool',' England
Mr. President and members of the Association, I am deeply sensible of the honor you have done me by asking me to give this lecture, and I thank you for your most warm and gracious welcome. This is a memorable occasion for me in another respect; nearly 30 years ago, I began my career in gastroenterology just around the corner from here, in Northwestern University Medical School, and my teacher was Dr. Andrew Ivy. I am proud to feel that I can call myself one of his pupils, and I remember with gratitude the many kindnesses which he showed to me while I worked with him. I came to Northwestern from University College London, where, in 1902, the discovery of secretin by Bayliss and Starling opened a new era in physiological thought by its revelation that there existed beside the nervous system a hormonal system for the integration of the normal bodily functions. This work forms the prelude to my story; for it undoubtedly gave to John Sydney Edkins the germ of his idea that there existed in the gastric antral mucosa a preformed substance which was absorbed into the portal blood stream during gastric digestion and, returning in the circulation to the fundic glands, maintained their acPresented at the Annual Meeting of the American Gastroenterological Association, Chicago, Illinois, May 27, 1966. Address requests for reprints to: Dr. R. A. Gregory, Physiology Laboratory, University of Liverpool, Brownlow Hill, Liverpool 3, England.
953
tivity after the initial vagal reflex excitation associated with the eating of a meal had come to an end. Sixty-one years ago almost to this day (May 18, 1905), Edkins read before th"e Royal Society of London a modest paper entitled "On the chemical mechanism of gastric secretion." In this he desc ribed how he had prepared aqueous extracts from antral and fundic mucosa and had tested them for their power to stimulate gastric secretion by intravenous inj ection into anesthetized cats. The fundic extracts had no action, but the antral extracts excited ac id and pepsin secretion. The active principle he named "gastrin," a term evidently derived from "gastric secretin." It is interesting now to note that in this first paper he did not call the substance a "hormone," for the simple reason that the term was unknown to him at that time. It was in fact first used publicly by Starling a month later when he delivered his famous Croonian Lectures to the Royal College of Physicians of London on "The chemical correlation of the functions of the body"; and in the first lecture, Starling instanced the very recently discovered gastrin as the second example after secretin of a hormone. History, it is said, is the tragedy of events before their time; and certainly t he history of science shows us many examples of men and discoveries before their time. So it was with Edkins; for very soon it seemed that what my friend, Professor W. M. Paton, once described as "the tide of
954
GREGORY
undiscovery" had set in. Those who sought to confirm and extend Edkins' discovery soon found that a stimulant of gastric acid secretion was indeed present in antral mucosa, but an apparently identical stimulant could be extracted by the same means from virtually any other tissue in the body. Edkins had, in fact, fallen first victim to that ubiquitous enigma histamine-years before even its existence in the body was recognized, much less its power to stimulate gastric acid secretion. When these facts were eventually established by the classical studies of Dale and his colleagues and of Popielski, it seemed clear enough that Edkins' antral extracts owed their activity to nothing more specific than the presence in them of histamine. Looking back now, we can appreciate how cruelly unlucky he was, for the oxyntic cells are the only exocrine glands on which histamine has a powerful secretagogue effect; and I, at any rate, am uncertain whether we can attribute to this action any physiological significance despite all that has been written and said upon the subject. If it had chanced to be the pancreatic centroacinar cells which were so powerfully excited by histamine, then there would have been no difficulty in confirming Edkins' claim that there existed in antral mucosa a specific stimulant of gastric secretion-while it might have been Bayliss and Starling who were in trouble! Their joint genius would certainly have coped with such a problem, but history would have been differently written. It was Simon Komarov who, in 1938, realized the trick that Nature-an old hand at the game !-was playing. In all tissues, including antral mucosa, there is histamine; but in antral mucosa-and virtually confined to it-there is present in addition a protein-like substance which can closely mimic the action of histamine on gastric acid secretion, so that the two may not be distinguishable by physiological tests of simple extracts containing both, as did Edkins'. Komarov precipitated with trichloroacetic acid a dilute acid extract of antral mucosa and obtained a protein fraction which was histamine-free but strongly stimulated gastric acid secretion when in-
Vol. 51 , No. 6
jected intravenously into anesthetized cats; he had rediscovered the antral hormone (fig. 1). This classical observation, made 2 years before Edkins' death-and of which, I believe, he learned in time-was the first definite indication that gastrin existed; for not until 10 years later did the work of Grossman, Robertson, and I vy and of Dragstedt and his pupils establish by physiological experiment the existence of an antral hormonal mechanism for the stimulation of gastric acid secretion. Their subsequent studies confirmed and clarified the hypothesis put forward in 1942 by Uvnas that vagal excitation is an important factor in the release and action of the antral hormone; and over the years, they and others have built up a picture with which we are all quite familiar. When a meal is eaten, vagal excitation to the antrum increases the release of gastrin occasioned by local stimulation; at the same time, vagal excitation to the fundic glands increases their response to the circulating hormone. The accumulation of acid in the antral region inhibits the release of hormone by the "gastrin cells," wherever these may be-most probably, I think, in the deeper regions of the pyloric glands. This is so clear and satisfactory a picture that one can only suspect that there must be a great deal in it that we have not yet perceived; I sometimes wonder how it will look in about 20 years' time! In Komarov's work there was one striking observation which seemed to deny its physiological significance. The inhibitory effect of atropine on the response to a meal in conscious animals was well known, but the action of his extract in the anesthetized cat-and he used this preparation almost entirely for his experiments-was completely resistant to repeated large doses of this drug (fig. 1). However, this finding was later confirmed not only with crude antral extracts, but also with the pure gastrin peptides; and it has also been established that the action of both in the conscious dog and human subject is strongly inhibited by atropine. The anomaly turns out to be only one of many examples of remarkable differences in the action of gas-
955
ISOLATION AND CHEMISTRY OF GASTRIN
D ecember 1966
TOTAL Cl TOTAL&rREE ACIOIT1E5 m .eq/1. ISO
.....__ _ _ _ _ TOTAL Cl
140 130 120 /"
80
/
/
/
70 I
VOLUME 60
c .c.
40
-l(
ACIDITY
I
I
I
I
I
/
.),
I
I
I
I
I
f.
/
/
/
.J. I / I
x'
3·0
TOTAL
I I I' I I
90
50
--,.___
/r
100
3·5
)(---)C....
----,. F'I~E.E. ACIDITY / ,./ ><---><-- -><----]<'"
110
/
I
ATROPINE. SULPHATE __,..___
.J.
2 2 2. mg
H J..
I I
,f PEPSIN Mett unit!
2·5 20
I~
1·5
10
10
5
0·5
0
0 TIM[,hrs .
VOLUME I~
J.-2
2.
1 1 1 1 t
(A5TRIN INJECTED 40
20
20
20
20
2~
3
3~
t
1
t
20
20
4
4~
5
20 mg
Fw. I. The effect of "purified" gastrin, extracted from 130 g of canine pyloric mucosa (by method I), and injected intravenously every Yz hr for 3\/z hr, in a cat under urethane-chloralose anesthesia. Total amount of gastrin injected was 180 mg. Intravenous administration of atropine sulfate did not depress the secretagogue effect of gastrin. (Komarov, 1942. Courtesy of Rev. Canad. Biol.)
trin upon gastric secretory responses in different species and different experimental conditions; I shall refer to others later on. Over the years following Komarov's work-first confirmed by Uvnas and his pupils-several efforts were made towards isolation of the active principle in antral extracts. These studies gradually led to a general realization that crude antral extracts not only stimul ated gastric acid secretion, but also pepsin secretion, pancreatic volume flow and enzyme secretion, and gastrointestina l tone and motility, though they had little or no effect on the gall bladder or bile flow ; there was too an indi-
cation that an inhibitory effect against a "background" response stimulated by histamine could be obtained. It looked as though the antral mucosa might be a veritable storehouse of biologically active .~ub stances, some of them at least of known hormonal status; but efforts to separate them were unsuccessful. There is now seen to have been implicit in the interpretation of these findings at the time the traditional assumption that with each of the gastrointestinal hormones was associated a single physiological function, on the basis of which its existence had originally been recognized. No one supposed-certainly not I-
956
GREGORY
that all these actions of crude antral extracts would eventually be shown to be properties of a single antral peptide. This has led, I think, to a significant change in our way of thinking about the other gastrointestinal hormones; we are now prepared to find that when they are studied in a pure state, they will be shown to have more than one effect of physiological significance, as has gastrin. My partner, Hilda Tracy, and I began our work almost by accident in the summer of 1959, when we sought to prepare for use in other experiments a histaminefree gastrin extract which would stimulate effectively when injected subcutaneously or intramuscularly in the conscious dog. Komarov and others after him had used the anesthetized cat and intravenous injection, almost exclusively; and there was some doubt whether gastrin preparations had more than a slight effect in conscious dogs. I shall not weary you with the details of the path by which we eventually arrived some 5 years later-and I might add, some 50,000 hog antrums later!-at the isolation, structure, and synthesis of two almost identical peptides from antral mucosa, which we named gastrins I and II. Early in 1962, we had isolated gastrin I; and at that time we had the immense good fortune not only to secure financial support for the furtherance of our work from your National Institutes of Healthfor which we shall always be grateful-but also to enlist the collaboration of a distinguished group of peptide chemists in the Department of Organic Chemistry of our own University, headed by Professor G. W. Kenner, an authority in the field of peptide chemistry. The second gastrin-gastrin II-turned up on Christmas morning of the same year, when we completed our first large-scale production after scalingup and improving the method in order to provide our chemist colleagues with material for their structural studies. By September 1964, they had achieved complete success; the structures of both gastrins were elucidated, and their total synthesis was accomplished. In the meantime, using the pure natural products-and later, of course, syn-
Vol. 51, No.6
thetic material-we were enabled to explore in meaningful terms their range of physiological properties. An added impetus to this study-long contemplated but postponed until material of attested homogeneity should be available-was the private communication from Grossman in February 1962, that a gastrin extract made by our earlier method would powerfully inhibit a "background" response maintained by histamine, if injected as a single rapid intravenous dose. This we confirmed using pure gastrin I which we had just isolated; and our subsequent studies showed that all of the actions previously described for crude antral extracts were displayed by the pure gastrin peptides, whether natural or synthetic. I can only refer in passing to some of these effects, in particular those on gastric and pancreatic secretion; the former because our studies and those of others have shown the remarkable diversity of action which is revealed in different species and types of animal preparation, the latter because it has led to recognition of a second physiological role for gastrin besides the stimulation of gastric acid secretion. The inhibitory effect of single intravenous injections in the conscious dog, first described in detail by Gillespie and Grossman using a gastrin extract, is seen also in the anesthetized rat; but it does not occur in man or the anesthetized cat. The associated stimulation of pepsin secretion, which we first noted in the conscious dog, occurs also in the anesthetized cat, and there is a small effect in man; information for the conscious cat and anesthetized or conscious rat is not yet available. In manas might be anticipated from the absence of the inhibitory effect-single intravenous injections (fig. 2), as well as subcutaneous or intramuscular administration, are highly effective as a means of exciting acid secretion; in fact, gastrin II has been found to be about 500 times more potent than histamine on a molar basis of comparison. The effect of the gastrin peptides on pancreatic volume flow in the conscious dog is small but definite, while the effect on pancreatic enzyme secretion is quite pronounced; and the elegant work of Preshaw, Cooke, and Grossman seems to establish
ISOLATION AND CHEMISTRY OF GASTRIN
December 1966
957 140
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that these actions of gastrin are of physiological significance. They prepared conscious dogs with an antral transplant, a gastric fistula, and a pancreatic fistula. The antral pouch was irrigated with acetylcholine solution at pH 7 to cause a release of endogenous gastrin; the expected acid secretion occurred, and the acid was drained from the open gastric fistula so that none of it entered the duodenum. Nevertheless, there occurred a quite obvious pancreatic response involving volume flow and enzyme secretion which, like the gastric response, disappeared when the acetylcholine solution irrigating the antral pouch was acidified (fig. 3). We may have little doubt, I think, that there exists an antral hormonal phase in the pancreatic secretory response to a meal and that gastrin is the mediator. In continuance of our studies on the gastrin peptides, Tracy and I have isolated a similar peptide from the antral region of the sheep abomasum, and there is evidence of a second in much smaller amount. More recently, with Grossman, we have isolated two peptides from human
antral mucosa and, during the last few weeks, two such peptides from dog antral mucosa. The established or probable constitutions of this family of gastrin peptides are shown in scheme 1. They are all heptadecapeptide amides of closely similar amino acid composition, occurring in unsulfated and sulfated forms. The significance of the sulfate group attached in an ester linkage to the single tyrosy I residue in each case is not known; it has no effect upon the physiological activities so far as we can tell at present, but clearly plays some metabolic role. The differences between the peptides of the various species are seen to lie in the body of the molecule, in close relation to the sequence of glutamyl residues; but as will be seen, we cannot associate these differences in structure with any differences in physiological properties. During the work leading to the successful synthesis of the hog gastrins in 1964 by our chemist colleagues, we were greatly fortunate in that they made available to us, for physiological study, a series of synthetic peptides which corresponded to vari-
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FIG. 3. Redrawn from Preshaw, Cook, and Grossman, 1965. For description see text. Hog I - Pyro·Gly ·Pro · Try·Met•(Giu) 5 ·Ala · Tyr ·Gly · Try·Met·Asp·Phe · NH 2
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II (SO,) Scheme 1. The constitutions of the hog , human, dog, and sheep gastrins. -Pyro, pyroglutamyl. The bracketed sequences in dog I and sheep I are not yet fin ally resolved. 958
December 1966
ISOLATION AND CHEMISTRY OF GASTRIN
ous portions of the total molecule of gastrin I; our earlier recognition of the range of actions displayed by the total molecule had naturally raised the question of structure-function relationships. We were disappointed in our expectation that some segregation of the different actions in different portions of the total molecule might be revealed; but as a consolation prize we were given the remarkable discovery that all of the effects of the total structure were displayed by no more than the C-terminal tetrapeptide and that when the amide group masking the C-terminus of this simple sequence was removed, almost all of its actions disappeared; only a slight effect on pancreatic secretion remained. These observations were confirmed and extended in a subsequent study with Morley of a large series of analogues of the active C-terminal tetra- and pentapeptides ; and the results showed that most substitutions led to a considerable decline, and sometimes a virtually total disappearance of the actions of the parent structure. There was no strikingly selective preservation or destruction of the various activities as between the different analogues; minor quantitative variations were of course detected, but in general the picture was that of a fairly uniform change in activity. The results seemed to indicate that in the tetrapeptide sequence, and perhaps also in the total gastrin molecule, the tryptophan and methionine residues were in the nature of binding, rather than functionally active sites, and that the most important regions of the molecule for physiological activity were the aspartic carboxylic and phenylalanine-amide residues. The results of these studies would perhaps lend some support to the speculation t hat the mode of action of the gastrin peptides on their various target sites may be of the same fundamental nature; and indeed Bennett has suggested, on the basis of a pharmacological analysis of the action of pure gastrin II on the isolated guinea pig ileum, that it may cause a release of
959
acetylcholine from nerve terminals in the tissue and that this might possibly be the case elsewhere. This stimulating suggestion certainly merits further exploration , for it would indeed be remarkable if gastrin turned out to be an acetylcholine liberator; but H eraclitus has warned us that if we do not expect the unexpected, we will never find it! It seems to me likely that vve shall not solve the problem of the mode of action of gastrin on the target cell until we can bring to bear on it the same techniques as have reaped such great harvest in the study of the nervous system-the investigation with microelectrodes and micropipettes of single cells. In the mammalian, or even the frog, gastric mucosa, there are formidable difficulties still to be surmounted; but if we go further afield, into the realm of marine biology, we can find a simpler situation, in which acetylcholine or histamine do not seem to be involved in the action of gastrin. From time to time, on the seacoast near Liverpool, it is possible to find a small marine animal named Pleurobranchus membranaceus; it is one of a group of such creatures which protect themselves from inquisitive fish by secreting HCl from cells on their external surface when touched. My colleague, Dr. D . V. Roberts, has found that when acetylcholine or histamine (10 p.g per ml) is added to the surrounding seawater, they show no response, although they remain sensitive to touch; but if gastrin (pure gastrin II) is added (1 p.g per ml), they give a very large and prolonged secretion of acid. How specific this response is for gastrin we do not yet know, but the preparation might obviously be useful for studying its action at a rather primitive level. My tale is done; I thank you for your kind attention and for the privilege of addressing you. I hope that in these few words I have said enough to show how much there is that we still do not understand about gastrin and that this fascinating story is far from concluded.