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Some Aspects of Gastric Secretion
Vol. 52, No.5 Printed in U.S.A .
GASTROENTEROLOGY
Copyright © 1961 by The Williams & Wilkins Co.
PROGRESS IN GASTROENTEROLOGY
SOME ASPECTS OF GASTRIC SECRETION MORTON
1.
GROSSMAN, M.
D.,
PH.D.
Research, Veterans Administration Center, Los Angeles, California, and Departments of Medicine and Physiology UCLA School of Medicine , Los Angeles, California
The plan (or lack thereof) of this review has been to identify an area of interest in a recent paper, to analyze the topic critically with the aid of citations to appropriate literature, ancient or current, and then to pass on to another, not necessarily related, subject that happened to capture my fancy. Those who might be displeased because this review does not provide a comprehensive bibliography of all recent papers on gastric secretion (or even on the narrow topics selected for discussion) should find some reassurance in learning that a source now exists that should satisfy even the most ardent seekers after completeness of references to literature. I refer, of course, to the new publication, Gastroenterology Abstracts and Citations, published by the National Institute of Arthritis and Metabolic Diseases. This excellent bibliographic citation and abstracting service now makes it possible for writers of reviews such as this to devote themselves to a critical appraisal of a few topics without feeling guilty for not having mentioned all papers dealing with the topic. There is presently an abundance of writing on gastric secretion. The first volume of Section 6 of the Handbook oj Physiology, devoted to "Physiology of the Alimentary Tract," published by the American Physiological Society, contains many chapters on gastric secretion and it should be off the press at about the same time as this review. The proceedings of an international conference on gastrin held in September 1964 have been published reAddress requests for reprints to: Dr. M. 1. Grossman, Room 231, Building 114, Veterans Administration Center, Los Angeles, California 90073.
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centlyl and the proceedings of another conference on gastric secretion held in Edmonton, Albert a, Canada, in September 1965 are in press. The book edited by Thomson and Gillespie 1a has a number of chapters devoted to gastric secretion. Gastrin
Chem1'cal structure. In his brilliant Memorial Lecture delivered at the Annual Meeting of the American Gastroenterological Association in May 1966, Professor R. A. Gregory2 presented further information on the structure of gastrin. The original studies on the isolation, structure, and synthesis of gastrin had been on material derived from hog stomachs. 3 - 5 Three additional species have now been studied: man, dog, and sheep.2 Human gastrin has been isolated, chemically characterized, and synthesized. 6 - s Dog and sheep gastrins have been isolated and their amino acid compositions have been determined but there is still some uncertainty about the sequence of a few of the amino acids. 2 Diagram 1 shows the structure of gastrin in these four species. A number of properties are common to the gastrins from all four species (diagram 1). The N-terminal residue is pyroglutamyl (pyrrolidone carboxyl). The C-terminal residue is phenylalanyl amide. Thus both the amino and the carboxyl terminal groups are blocked, so these molecules do not give the common chemical reactions that depend on these groups being reactive. All of the gastrins occur in two forms: without sulfate, designated I, and with an ethereal sulfate on the phenolic hydroxyl radical of tyrosine, designated II. All of the gastrins have a number of dicarboxylic
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883
,r-------------------,,
Hog
1 2 3 4 5 6 7 8 9 Ie} 11 12 13 : 14 15 16 17 ' ,-/\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1 \ 1 \ ' 1\ 1\ 1\ 1\ : LGlu-Gly-Pro-Try-Met-Glu-Glu-Glu-Glu-Glu-Ala- Tyr- Gly+Try-Met-Asp-Phe-NH 2 :
,
L- _________________ 1
Man Dog Sheep
Leu (Ala) Val-(Ala)
£Glu = pyroglutamyl (pyrrolidone carboxyl)
H 2C--CH 2 I I O==C", / CH-CoN H DIAGRAM 1. Structure of gastrins from hog, man, dog, and shee p. In dog and sheep gastrins the Ala is in parentheses because it is not yet known which glutamyl residue (6--10) is replaced by alanyl. In each species, gastrin occurs in two forms : I, without sulfate, and II, wi th sulfate as tYl'osyl-O-sulfate in position 12. The carboxyl terminal tetrapeptide amide surrounded by t he dotted line is the "active center" of the molecule. (Data of Gregory: )
amino acids and no dibasic ones, so they behave e lectrophoretic ally and chromatographically as acids. The acidic polarity is increased by the sulfate group. Most importantly, all of the gastrins have the same carboxyl terminal tetrapeptide amide sequence: -Try-Met-Asp-PheNH2 . This portion of the molecule may be regard ed as the "active center" because all of the biological actions of gastrin can be produced by this fragment but not by smaller portions of the molecule. 5 To date, no differences in potency or in spectrum of biological actions have been found between gastrins from various species or between the sulfated and un sulfated forms. The differences between the species so far studied (diagram 1) involve substitution of one or two of the 17 amino acid residues. This is in keeping with other observations on evolutionary changes in protein structure which show that the active site is usually not involved, that changes usually are substitutions of amino acids rather than alterations of chain length, and that the number of variant amino acid residues between any two species i s afunction of the time since their evolutionary divergence from a common ancestor. It is estimated that mutations involving one
amino acid may b e expected to occur and to be ret ained at intervals that have a mean value of about 12 million years. 9 Other gastrins. Are the gastrins of Gregory and Tracy t he unaltered molecules as they exist n aturally in the mucosa 0 1' have they been modified by the extraction process? Gregory lO has shown that modifications of the extraction procedure designed to lessen t he likelihood of chemical alterations during extraction did not change the products e xtracted. While the question must b e regarded as .still open, there is presently no evidence that the GregoryTracy gastrins have be en modified by extraction. Are there gastrins other than the two isolated by Gregory and Tracy? The strongest indication that there might be another form o f gastrin comes from the work of T auber and Madison .1l - 13 They isolated from hog antral mucosa a polypeptide containing 107 amino acid residues with a minimal molecular weight of 12,154 (compared wit h 2114 for Gregory-Tracy gastrin I). The material stimulated gastric secretion of ac id when injected intravenously during a 30-min period in dogs with gastric fistulas ; other physiological effect s known to be produced by the GregoryTracy peptides were not sought. The work
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of Tauber and Madison suggested that they had isolated a substance that might represent gastrin in a form from which the Gregory-Tracy peptides had been derived by scission during the extraction process. However, Tauber and Madison found no tryptophan in their gastrin molecule, and there are two tryptophan residues in the Gregory-Tracy peptides, one in the active site. From the dose-response data presented by TauberI3 it is clear that his final product has a potency far lower than that of the Gregory-Tracy peptides. For example, 0.1 p.g per kg per hr of Gregory-Tracy gastrin II will produce a secretory response in dogs with gastric fistulas that is about onefourth of maximal rate, whereas 20 p.g per kg per hr of Tauber's material were needed for a threshold response. 13 In view of this marked discrepancy in potency (much greater than the relative molecular weights of the two materials), it is possible that the activity of the Tauber-Madison material was attributable to the presence of a small percentage of Gregory-Tracy peptides and that the amount of tryptophan thus introduced was too small to be detected by the methods used. The methods of staining used by Tauber and Madison to demonstrate apparent homogeneity of their final product would not have revealed the presence of traces of the smaller peptides and it was not shown that the single component stained actually contained the gastrin activity. Until this possibility of contamination by traces of Gregory-Tracy peptides has been excluded, the advocates of the existence of high molecular weight gastrins 14 • 15 will have difficulty in presenting a convincing case. The simplest way to determine whether the purported high molecular weight gastrins contain GregoryTracy peptides would be to subject them to the Gregory-Tracy purification procedure and determine whether the smaller pep tides can be found. Relation between chemical structure and biological function. The entire range of physiological activities displayed by natural gastrin can also be produced by the Cterminal tetrapeptide amide. 5 Fragments of the molecule smaller than this tetrapeptide had little or no biological action, and no
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other portion of the molecule, including the entire 13 amino acid sequence from the N-terminus to the active tetrapeptide, had any significant biological action. As the peptide chain was progressively lengthened toward the N-terminus, the potency of the molecule increased. The synthetic peptide that differs from gastrin only in having two instead of five glutamyl residues in the body of the molecule had the same potency as gastrin itself. Most of the studies to date on the various chemical forms of gastrin and on the synthetic peptides related to gastrin have been rough screening tests. In only a few instances have truly quantitative biological assays been carried out. The generalizations about kinds and amounts of activity of the various substances are subject to this qualification. More careful study may make it necessary to revise some of the statements made here. A large number of pep tides related to the C-terminal tetrapeptide sequence of gastrin have been synthesized and examined for biological activity by Tracy and Gregory5 and by Morley, Tracy, and Gregory.16 In general, peptide derivatives that had the power to stimulate gastric acid secretion also had all of the other biological actions of the whole gastrin molecule, but some exceptions were encountered. Thus, several peptides stimulated gastric acid secretion but did not have an effect on gastrointestinal motility. Some peptides that stimulated gastric acid secretion at high (500 p.g) but not at low (50 p.g) doses subcutaneously did not inhibit gastric acid secretion with the high dose given intravenously, but this might only mean that the threshold for inhibition was still higher. The most interesting dissociation, namely inhibition of gastric acid secretion by large doses given intravenously without stimulation by small or large doses given subcutaneously, was encountered only once, in a tetrapeptide in which D-aspartic acid had replaced the Lmethionine of the natural tetrapeptide. No studies of this compound beyond the preliminary report of Morley et aJ.16 have appeared. It seems safe to guess that the search for an analogue of gastrin that acts
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as a competitive inhibitor of the hormone will be vigorously pursued. Since nonspecific inhibition of gastric acid secretion by large doses of gastrin given intravenously occurs in dogs but is not seen in cats,17 the latter species may be more suitable for screening synthetic analogues of gastrin for specific anti-gastrin activity. The further discussion of structure-function relationships will be restricted to stimulation of gastric acid secretion. Table 1 summarizes the effect of substitutions in various positions of the C-terminal tetrapeptide amide. Lengthening the chain by acylation of the N-terminal position often enhances activity. Only very minor changes in the active tetrapeptide itself can be made without complete loss of activity; the methionine position appears to tolerate the greatTABLE
est amount of change. Whereas the amino group at the N-terminal position can be either removed or blocked without significant change in activity, the amide group at the C-terminal position is apparently of crucial importance, for it can neither be removed nor substituted without almost total loss of activity. Pentapeptide. Of the various derivatives of the C-terminal tetrapeptide amide that have been examined, the one that has been widely available and extensively studied is the pentapeptide analogue in which a (3alanyl residue is attached to the tryptophan residue of the C-terminal tetrapeptide amide. This synthetic peptide has been manufactured and distributed by Imperial Chemical Industries. It is most commonly referred to by its code number, ICI 50,123.
1. Effect of substitutions on the activity of the C-terminal tetrapeptide amide in stimulation of gastric acid secretion" 1 2 3 4 5 6 R-Try-Met-Asp--Phe-NR 2 Substitutions tbat cause:
Position number No effect on activity
Enhanced activity
Depressed activity
1
t-Butyloxycarbonyl Benzyloxycarbonyl (blocking groups on amino of tryptophan)
2
4-CHatryptophan 5-CHatryptophan 6-CH atryptophan 2-Indolyl (CR 2 ) 2-CO-
Phenylalanine Histidine D-Tryptophan
3
Norleucine Ethionine
Norvaline a-Amino butyric Alanine Methionine sulfone Methionine sulfoxide
{3-Alanyl Lysine Carbamoyl Glutamic acid
4
{3-Aspartic Glutamic
5
Tyrosine O-Methyl tyrosine D-Phenylalanine
6
Cyclohexyl
" Data of Morley et al. 16
Abolition of activity
D-Methionine D-Aspartic
OH (free acid) Methyl ester
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In the remainder of this review it will be referred to simply as pentapeptide. M orley and co-workers16 st at ed that this pentapeptide was considerably more potent than the tetrapeptide but in the one e xample they give the response of a H eidenhain pouch to subcutaneous injection of 10 p.g was 1.2 mEq for the pentapeptide (their compound no. 29) and 1.5 mEq for the tetrapept ide (their compound no. 33). In studies on dogs with gastric fistul as we 1S found that the dose required for half-maximal response was about the same for tetrapeptide and pent apeptide. In these same studies we 1S found that gastrin was about 4 times as potent as the pentapeptide. In human subjects M akhlouf and co-work ers19 found that gastrin was a bout 10 times as potent as pentapetide. Although t here isstill some uncert ainty about exact potency it is clear that on a weight basis pentapeptide st ands between gastrin a nd hista mine. When given in dosefl required for maximal stimulation of acid secretion eit her by the subcutaneous route (2 p.g per k g) 20 or by continuous intravenous injection (1 p.g per kg per hI') 21 in man, gastrin produces no side effects. Rapid intravenous inj ection of large doses of gastrin may produce severe s ide effects including collapse.21, 22 With p entapeptide as the stimulant it was not possible to give human subj ect s doses t hat produced maximal response eithcr b y continuous intravenous injection 23 or by single subcutaneous injection 19 wit hout a high incidence of side effects, in cluding abdominal cramps, nausea, sinking sensation, and ret ching. However, the highest doses of pent apeptide that could be given without side effect (6 p.g per k g subcutaneously19 or 0.01 p.g per kg per min intravenously23 ) produced very high and reproducible rates of acid secretion, amounting t o 80% or more of the maximal r at e that can b e achieved wit h other stimulants such as porcine gastrin , hist amine, 01' betazole. The mechanism of the production of side effects by pentapeptide is not clear. Although there is a tendency for increased motor activity to occur throughout t he alimentary tract, particula ry in the colon, t his has not been correlated with
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side effect s.24 The s mall increases in arterial blood pressure and heart rate seen during sympt oms induced by pentapeptide a re probably the result rather than the cause of the symptoms.ll5 Pentapeptide has been extensively studied in human subj ects in Great Britain but has thus far been used only in animals in the United States. There is at present n o indication t hat pentapeptide or any ot her synthetic peptid e related to gastrin, or even gastrin itself, will have any theoretical advantage over hist a mine or betazole for clinical diagnostic testing of gastric secretion. The responses to all of these agents arc highly correlat ed so that anyone of them will serve to give a reliable index of the secretory responsiveness of an individ ual. It is possible that differences in responsiveness t o various stimulants, for example, histamine versus gastrin 01' one of the gastrin-related peptides, may b e foun d in certain diseases but th e experience to date does not favor this possibility. Strong stimulation of gastric secretion is desirable in diagnost ic t est ing because it is required when achlorhydria must be ruled out and because the reproducibility on replicate testing is greatly improved. The selection of an agent for stimula tion of gast ric acid secretion for clinical diagnostic testing resolves itself then into practical considerations of availa bility, sta bility, cost, and occurrence of side effects. Natural or synt het ic gastrin would be ideal but there is no current pr ospect t hat it will become widely avail able a t low cost. The sy nthetic partial peptides such as the penta peptide offer much p romise, but the search will probably not end with the pentapeptide because it cannot be given in doses t hat produce maximal response without in cu rring side effect s. Nevertheless, the pentapeptide deserves attention because when gi ven in the largest doses that do not provoke sid e effects (6 p.g per kg subcutaneously ) it produces ra tes of secretion only slightly lower than those obtained with the socalled " augmented histamine test" (40 p.g of histamine phosphate per kg subcutaneously). Of the readily available stimula nts, histamine, betazole, and pentapeptide, the pentapeptide is c le arly the "best" in t erms
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of being the one that can produce the highest rate of secretion without side effects. Extensive experience in the use of pentapeptide in human subjects will be required to establish its safety. Is maximal the utmost? The word "maximal" has been used in various senses in regard to gastric secretion of acid, and since authors do not always define the term confusion has arisen. There are at least three meanings that have been ascribed to the word "maximal": 1. The highest rate observed during the course of an individual study. The word "peak" would seem more appropriate than "maximal" for this meaning. 2. The "observed maximal" rate, that is, the highest rate attainable with a given stimulant. This is the commonest sense in which the term is used and is the meaning intended elsewhere in this review when the term is not qualified. The "maximal" rate, used in this sense, is determined by giving progressively larger doses of the stimulant and observing that portion of the dose-response curve in which increasing the dose does not increase the response. The word "maximal" used in this sense must be regarded as an operational term because the maximal rate observed will depend on a number of factors such as the stimulant used, the route of its administration, the sequence of doses, the frequency of sampling, and other such factors. Thus, defined in this way, the maximal response to subcutaneous gastrin is higher than to subcutaneous histamine,20 the maximal response to intravenous histamine is higher than to subcutaneous histamine,26 and the maximal response to subcutaneous betazole is higher than to subcutaneous histamine.27 3. The "calculated maximal" rate. Makhlouf and co-workers19 have proposed that maximal rate of secretion should be determined by extrapolation of the dose-response line. When the reciprocal of dose is plotted against the reciprocal of response the points fall on a straight line and the calculated intercept on the response axis represents the reciprocal of the theoretical response at infinitely large dose. In support of this proposed method for calculating maximal rate is the finding of Makhlouf
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and co-workers19 that the maximal rate so calculated was the same for various stimulants, including gastrin, pentapeptide, histamine, and betazole, and for various routes, intravenous and subcutaneous. It remains to be seen whether this calculated maximal rate does in fact represent the "true" maximal rate or the "utmost" of which the glands are capable, as Makhlouf and coworkers 19 have suggested, or whether situations will be found, perhaps with two stimulants acting together to potentiate each other,28.29 in which the calculated maximal rate for a single stimulant is exceeded. It would be helpful if the terms "observed maximal rate" and "calculated maximal rate" would be used where appropriate to avoid ambiguity. The many actions of gastrin. Before gastrin was isolated, it was assumed to have only one action, stimulation of gastric secretion of acid. Once pure gastrin was available it became apparent that it had a surprisingly wide spectrum of actions, including some that had been thought to be due to other constituents in crude antral mucosal extracts and some completely unexpected actions. The actions of gastrin can be classified into two broad categories 30 : (a) those that require only small doses of exogenous gastrin and can be mimicked by endogenously released gastrin (to use a much-maligned expression, these might be regarded as "physiological")31; and (b) those that occur only with large doses given intravenously and cannot be mimicked by endogenously released gastrin. Under category (a) are included (1) stimulation of gastric acid secretion, (2) stimulation of gastric pepsin secretion, (3) stimulation of pancreatic flow and bicarbonate secretion, (4) stimulation of pancreatic enzyme secretion, and (5) stimulation of hepatic biliary flow and bicarbonate secretion. Under category (b) are included (1) inhibition of gastric secretion of acid, (2) very strong stimulation of gastric pepsin secretion, (3) stimulation of gastric motility, (4) stimulation followed by inhibition of small intestinal motility, and (5) variable effects on blood pressure.
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For all five of the actions listed under category (a) it has been shown that endogenuus gastrin can produce an effect like that of small doses of exogenous gastrin. Cooke and co-workers32 found that the maximal rate of acid secretion from Heidenhain pouches in dogs W I'.8 higher with exogenous porcine gastrin than with release of the dog's own gastrin from an antral pouch by irrigation with acetylcholine. In unpublished studies Cooke and I have found that in dogs with gastric fistulas maximal acid response w as the same to exogenous porcine gastrin and to endogenous gastrin released from an antral pouch by bathing it with acetylcholine. In these same studies the pepsin responses to both exogenous and endogenous gastrin were higher than to histamine during maximal rates of acid secretion. With porcine gastrin as the stimulant pepsin secretion increased parallel with acid secretion over the entire range of acid responses in both dogs and cats.33 With histamine as the stimulant, the highest output of pepsin occurred with low doses and with doses that produced maximal rates of acid secretion pepsin output was markedly depressed. 33 In man, doses of histamine that produced maximal r ates of acid secretion did not depress pepsin secretion , and with such doses pepsin secretion in response to histamine and pentapeptide were equal. 19 Although gastrin is a stimulant of pepsin secretion, it does not appear to be capable of producing rates of pepsin output comparable to t hose achieved with cholinomimetic drugs.34 Both exogenous 35 and endogenous86 gastrin produced weak stimulation of pancreatic flow and bicarbonate output and strong stimulation of pancreatic enzyme secretion in dogs. The highest r ate of flow attainable with gastrin was about one-third of t hat seen with secretin, whereas the highest rat e of enzyme secretion with gastrin was about three-fourths of that seen with pancreozymin. In man pentapeptide was foun d to stimulate pancreatic secretion under cert ain conditions. 23 The effect of gastrin on secretion of bile in dogs is like that of secretin but is considerably weaker37 ; both are characterized
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by an increase in bicarbonate concentration accompanying the increase in flow rate. Endogenously released gastrin produced stimulation of bile flow with a time course similar to the stimulation of gastric acid secretion. 3s It is clearly established that gastrin is a major factor in regulation of gastric secretion ; its relative importance in stimulation of pancreatic and biliary secretion is not yet clear. Of the actions of gastrin t hat r equire large doses given r apidly intravenously, category (b ) , above, only inhibition of gastric acid secretion in dogs has been extensively studied. 39 ,4.0 The mechanism of this effect is not known but now that it has been shown that the phenomenon does not occur in man 22 or in cat s17 it is of interest mainly as a species peculiarity rather than as a general property of gastrin. Large doses of gastrin given rapidly intravenously caused very strong stimulation of pepsin secretion in both dogs and cats17 ; thus, t he effect on pepsin secretion appears to be independent of the effect on acid secretion. It is not yet clear whether higher rates of pepsin secretion occur when gastrin is given as a single large dose rapidly intravenously than when gastrin is given subcutaneously or continuously intravenously in doses producing maximal acid secretion. Gastrin causes contraction of smooth muscle under a variety of circumstances, including gastric and small intestinal smooth muscle in conscious dogs,5 isolated stomach, duodenum, and colon of the rat,41 and isolated ileum of the guinea pig.41 The amount of gastrin that must be added to the bath to cause the guinea pig ileum to contract (0.1 to 4 fLg per ml) would be enough to evoke a gastric secretory response in a dog. The concent ration of gastrin in a bath required to stimulate gastric secretion in isolated gastric m ucosa of frog 4 2 was a bou t one-thousandth of that needed for contraction of guinea pig ileum.41 The molar concentration of gastrin required to produce an equivalent degree of contraction of isolated guinea pig ileum was from 10 to 30 times that of acetylcholine or histamine.41 Pharmacological analy sis of the mechanism
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by which gastrin causes contraction of guinea pig ileum led to the conclusion that it acted on the postganglionic parasympathetic nerves to cause the release of acetylcholine which was the actual mediator of the effect.41 From these studies on guinea pig ileum Bennett41 suggested that stimulation of gastric secretion by gastrin might also be effected by the hormone acting on nerves to release acetylcholine. While it is true that atropine is a more effective inhibitor of gastrin stimulated than of histamine stimulated gastric acid secretion in dogs,43 there are circumstances, for example, in the anesthetized cat,44 under which atropine causes no inhibition. Since gastrin stimulates acid secretion in the isolated gastric mucosa of frogs,42 the possi bility exists of doing a pharmacological analysis like that on the isolated guinea pig ileum. In any case the hypothesis that gastrin acts by releasing ,acetylcholine deserves at least as much attention as the more familiar hypothesis that gastrin acts by releasing histamine. Tracy and Gregory5 stated that in anesthetized dogs gastrin had a small and inconstant depressant effect on the blood pressure. In unpublished studies in my laboratory, Jacobson and Swan found that both gastrin and penta peptide produced a small and inconstant depression of blood pressure · in conscious dogs. In man Logan and co-workers 25 observed an increase in blood pressure, heart rate , and blood flow in the forearm and hand of subjects receiving penta peptide intravenously. Since these changes did not occur until unpleasant symptoms had appeared, the cardiovascular effects may be indirect. Is Histamine Involved
If scientific theories could be proven by proclamation of faith in them, the theory that histamine is the final common mediator for gastric secretion of acid would be assured acceptance. There is no lack of ringing declarations of the validity of the theory.45-47a What seems to be lacking, in my view, is carefully reasoned evidence supporting the critical assumptions of the theory. In a sense, the question of the role of
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histamine in gastric secretion has about the same status as the question of other physiological roles for histamine, such as vasodilation, neuronal activation or inhibition, or contraction of visceral smooth muscle. In each instance it is clear that histamine is abundantly present in or near a responsive tissue and that exogenous histamine can activate or inhibit the tissue. The question has been and is: does endogenous histamine participate in the regulation of the tissue in question? In no instance has it been possible to devise crucial experiments that unequivocally implicate histamine in any normal process, and the possible role of histamine in pathological processes such as anaphylaxis and injury would appear to be partial at best. On the other hand, in no instance has a role for histamine in any of these processes been clearly disproved. The broad question of a possible role for histamine in regulation of gastric secretion can be resolved into three more restricted quesions. 1. Is gastrin histamine? Gastrin quite clear ly is not histamine but is a fully identified polypeptide amide that does not contain histamine or its precursor histidine. Since gastrin is several hundred times more potent than histamine on a molar basis, the possibility that the action of gastrin is due to bound histamine is ruled out. Much of the early controversy about a physiological role of histamine in gastric secretion concerned the possibility that gastrin was histamine; that question can now be regarded as definitely answered in the negative. 2. Is activation of gastric secretion associated with an increase in levels of histamine in plasma, or in urinary excretion of free histamine as a reflection of plasma levels? Although feeding caused an increase both in urinary histamine and in gastric secretion of acid in dogs, the two events could be made to occur independently, indicating that they probably were not causally related. For example, Irvine and Code 48 showed that meat given into the jejunum caused a transient increase in urinary histamine output and that the gastric secretory response occurred after the
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urinary histamine excretion had returned to cont rol levels. When meat was confined to the stomach it caused an increase in gastric secretion without a change in urinary histamine output. While these findings show that stimulation of gastric secretion produced by feeding meat cannot be ascribed to elevation of plasma histamine (on t he reasonable assumption that urinary e xcretion of histamine is a as tisfactory index of plasma levels) , they leave unexplained the failure of gastric secretion to occur during certain periods of elevated urinary excretion of histamine. Irvine and Code48 showed that infusion of histamine at ra tes too low to stimulate gastric secretion caused detectable increases in urinary excretion of histamine, and the rate of urinary excretion of hist amine increased by less than 2-fold when the rate of injection of histamine was increased 4-fold. Thus it is possible that feeding may produce elevations of circulating histamine that are too small to stimulate secretion but are still detectable on the basis of increase in urinary excretion. Since histamine can potentiate other stimuli for gastric secretion 28 , 29 these subthreshold amounts of histamine may be effective when acting with other stimuli. A systematic study is needed of the relation between r ate of infusion of histamine and (a) rate of urinary excretion and (b) degree of direct stimulation and potentiation of gastric secretion. It might t hen be possible to assess whether the levels of urinary histamine excretion produced by feeding are significant in terms of potentiation of other stimuli for gastric secretion. Another role that has been suggested for hist amine in gastric secretion is t hat it may be released widely in the body by gastrin. The careful studies of Bla ir,49 showing that plasma and urinary levels of histamine failed to rise when doses of gastrin t hat stimulated acid secretion were given to cats, would seem to dispose of this notion. 3. Do stimulants of gastric secretion such as gastrin and acetylcholine release hist amine in or near the parietal c ell and t hereby cause stimulation of secretion of acid? This is the question that continues
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to give v it ality to the hypothesis of a physiological role for histamine in gastric secretion. This aspect of the histamine-gastric secretion hypothesis has received fresh impetus from three kinds of recent studies. First, several groups50-52 have shown t hat the hi stamine content of the gastric mucosa of rats is decreased by gastrin and a variety of ot her gastric secretory stimulants. Second, the enzyme that synthesizes histamine, histidine decarboxylase, has been shown to be present in gastric mucosa of the rat5 3 and to show an increase in activity in response to feeding or inj ection of gastrin .50 Third, drugs that inhibit histidine decarboxylase and thereby deplete hist amine stores in gastri c mucosa in rats have been shown to inhibit gastric secretion in response to gastrin and a variety of other stimulants of acid secretion but did not inhibit the response to hist amine.54 The failure of the drugs to block the action o f histamine is of special significance because it indicat es that they did not cause a nonspecific depression of acid secretion. Do these three groups of findin gs establish wit hout doubt t hat histamine is a mediator of gastric secretion? Unfortunately, no. All three groups of evidence pertain exclusively to the rat and are probably not valid for other species. It has not been possible to show depletion of mucosal histamine on activation of gastric secretion in other species. 42 , 45 The gastric mucosa of the rat has a high concentration of a specific h istidine decarboxylase. This enzyme has been looked for but not found in other species ; the very low rate of hi stamine form ation produced by extracts of tissues from other species is assumed to be caused by nonspecific amino acid decarboxylases.53 D epression of gastric secretion by drugs that inhibi t histidine decarboxylase has not been demonstrated in species other than the rat . From the evidence presently available, one m ust conclude either that histamine is a me diator of gastric secretion in the rat but not in other species or that the evidence for histamine as a m ediator
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of gastric secretion is inconclusive in all species. The studies of Blair49 have shown that during stimulation of gastric secretion by gastrin in the cat there is no increase in the level of histamine in arterial or gastric venous blood plasma or in the rate of excretion of histamine in urine or gastric juice. To accommodate t he theory of mediation by histamine it thus becomes necessary to postulate (a) that the histamine involved in stimulation of gastric secretion is consumed in the process, and (b) that the histamine that appears in gastric juice is not involved in stimulation of secretion of acid. There is no a priori theoretical objection to either of these postulates but they obviously are formidable obstacles in verifying that histamine is a mediator of gastric secretion. REFERENCES 1. Grossman, M . I. [ed.]. 1966. Gastrin. University of California Press, Berkeley. 1a. Thomson, T. J ., and I. E . Gillespie [ed.]. 1966. Postgraduate Gastroenterology. Bailliere, Tindall, and Cassell, London . 2. Gregory, R. A. 1966. The isolation and chemistry of gastrin. Gastroenterology 51: 953959. 3. Gregory, H ., P. M. Hardy, D. S. J ones, G. W. Kenner, and R. C. Sheppard. 1964. Structure of gastrin. Nature (London) 204 : 931-933 . 4. Andersbn, J . C., M. A. Barton, R. A. Gregory, P. M . Hardy, G. W. Kenner, J. K . Macleod, J . Preston, R. C .. Sheppard, and J . S. Morley. 1964. Synthesis of gastrin. Nature (London) 204 : 933-934. 5. Tracy, H. J., and R. A. Gregory. 1964. Physiological properties of a series of synthetic peptides structurally related to gastrin I. N ature (London) 204: 935-938. 6. Gregory, R. A., H. J. Tracy, and M . I. Grossman . 1966. Isolation of two gastrins from human antral mucosa. Nature (London) 209 : 583 . 7. Bentley, P . H., G. W. K enner, and R. C. Sheppard . 1966. Structures of human gastrins I and II. ~ature (London) 209 : 583-585. 8. Beacham, J ., P. H. Bentley, R. A. Gregory, G. W. K enner, J. K. M acLeod, and R. C. Sheppard. 1966. Synthesis of hum an gastrin I. Nature (London) 209: 585-586. 9. White, A., P. Handler, and E. L . Smith . 1964. Principles of biochemistry, Ed. 3, p .611-626. M cGraw-Hill Book Company, Inc., New York.
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10. Gregory, R. A. 1967. I solation and chemistry of gastrin . In C. Code [ed.], Handbook of Physiology, Section 6, The Alimentary Tract. American Physiological Society, Wash ington, D. C., in press. 11. Tauber, S. D., and L. L. Madison. 1964. Isolation, physicochemical and physiological charact erization of gastrin. J. Clin. Invest. 43: 1271-1272. 12. T auber, S. D., and L. L. Madison. 1965. The isolation and characterization of porcine gastrin. J .BioI. Chern. 240: 645-650 . 13. T a uber; S. D. 1966. Characteriza tion of a pure gastrin, p. 27-48. In M. I. Grossman [ed.] , Gastrin. University of California Press, Berkeley . 14. Fletcher, T . L., W. R. Anderson, C. L. Pitts, and H. N. H arkins. 1961. A new preparation of gastrin; preliminary characterization. Nature (London) 190: 448. 15. Wilding, P ., B . J. Dyce, H. Riderknecht, and B . J . H averback. 1966. The most probable gastrin? Gastroenterology 50: 879. 16. Morley, J . S., H . JTracy, . and R. A. Gregory . 1965. Structure-function relationships in the active C-terminal tetrapeptide sequence of gastrin. Nature (London) 207: 1356-1359 . 17. Emits, S., and M . I. Grossman. 1966. Difference between dogs and cats in effect of large dose of gastrin on gastric secretion. Physiologist 9: 175. 18. Konturek, S., and M. I. Grossman. 1966. Acid response to gastrin and related peptides. Gastroenterology 50 : 650-652. 19. Makh louf, G. M ., J. P . A. McManus, and W. I. Card . 1966. The action of t he pentapeptide (lCI 50, 123) on gastric secretion in man. Gastroenterology 51 : 455-465. 20. Makhlouf, G. M., J . P. A. M cManus, and W. I. Card. 1964. The action of gastrin II on gastric-ac id secretion in man. Lancet 2 : 485-490. 21. Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1964. Dose-response curves for the effect of gastrin II on acid secretion in man. Gut 5: 379-384. 22. Makhlouf, G. M ., J. P . A. M cM anus, and W . I. Card. 1966. Action of gastrin II on gastric secretion in m an, p. 139-169. In M . I. Grossman [ed.], Gastrin. University of California Press, Berkeley. 23. Wormsley, K. G., M . P. M ahoney, and M. Ng. 1966. Effects of a gastrin-like penta peptide (I.C.I. 50,123) on stomach and pancreas. Lancet 1: 993-996. 24. Logan, C. J. H ., and A. M. Connell. 1966. The effect of a synthetic gastrin-like pentapeptide (I .C.I. 50,123) on intestinal motility in man . Lancet 1: 996-999.
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25. Logan, C. J. H., 1. Brick, and I. C. Roddie. 1966. Circulatory effects of a synthetic gastrin-like pentapeptide (I.C.!. 50,123). Lancet 1: 999-1000. 26. Lawrie, J. H., G. M. R Smith, and A. P. M. Forrest. 1964. The histamine-infusion test. Lancet 2: 270-273. 27. Ward, S., I. E. Gillespie, E. P. Passaro, Jr., and M. 1. Grossman. 1963. Comparison of Histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs. Gastroenterology 44: 620-626. 28. Passaro, E. P., Jr., I. E. Gillespie, and M. 1. Grossman. 1963. Potentiation between gastrin and histamine in stimulation of gastric secretion. Proc. Soc. Exp. BioI. Med. 114: 50-52. 29. Gillespie, I. E., and M. I. Grossman. 1964. Potentiation between Urecholine and gastrin extract and between Urecholine and histamine in the stimulation of Heidenhain pouches. Gut 5: 71-76. 30. Grossman, M. I. 1966. Gastrin. Ann. Intern. Med. 64: 212-216. 31. Hogben, C. A. M. 1960. The alimentary tract. Ann. Rev. Physiol. 1212: 381-406 (see footnote to p. 388). 32. Cooke, A. R, R M. Preshaw, D. L. Nahrwold, and M. 1. Grossman. 1966. Comparison of acid secretory responses to endogenous and exogenous gastrin. Fed. Proc. 25: 513. 33. Emas, S., and M. 1. Grossman. 1967. Comparison of gastric secretion in conscious dogs and cats. Gastroenterology 512: 29-34. 34. Andersson, S., and M. 1. Grossman. 1965. Effect of vagal denervation of pouches on gastric secretion in dogs with intact or resected antrums. Gastroenterology 48: 449-462. 35. Preshaw, R M., and M. 1. Grossman. 1965. Stimulation of pancreatic secretion by extracts of the pyloric gland area of the stomach. Gastroenterology 48: 36-44. 36. Preshaw, R M., A. R Cooke, and M. 1. Grossman. 1965. Stimulation of pancreatic secretion by a humoral agent from the pyloric gland area of the stomach. Gastroenterology 49: 617-622. 37. Zaterka, S., and M. 1. Grossman. 1966. The effect of gastrin and histamine on secretion of bile. Gastroenterology 50: 500-505. 38. Nahrwold, D. L., A. R Cooke, and M. 1. Grossman. 1967. Choleresis induced by stimulation of the gastric antrum. Gastroenterology 52: 18-22. 39. Gillespie, 1. E., and M. 1. Grossman. 1963. Inhibition of gastric secretion by extracts containing gastrin. Gastroenterology 44: 301-310.
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40. Gillespie, 1. E. 1966. Inhibition of acid secretion by gastrin extracts. p. 229-253. In: M. I. Grossman [ed.], Gastrin. University of California Press, Berkeley. 41. Bennett, A. 1965. Effect of gastrin on isolated smooth muscle preparations. Nature (London) 1208: 170-173. 42. Davidson, W. D., C. A. E. Lemmi, and J. C. Thompson. 1966. Action of gastrin on the isolated gastric mucosa of the bullfrog. Proc. Soc. Exp. BioI. Med. 121: 545-547. 43. Grossman, M. I. 1961. Cholinergic potentiation of the response to gastrin. J. Physiol. (London) 157: 14-15. 44. Blair, E. L., A. A. Harper, H. J. Lake, and J. D. Reed. 1961. The effect of atropine upon gastrin-stimulated gastric secretion. J. Physiol. (London) 159: 72-73P. 45. Code, C. F. 1965. Histamine and gastric secretion: a later look, 1955-1965. Fed. Proc. 124: 1311-1321. 46. Code, C. F. 1966. Histamine and gastric secretion. Gastroenterology 51: 272. 47. Haverback, B. J. 1966. Role of histamine in gastric secretion. Gastroenterology 50: 898899. 47a. Ivy, A. C., and W. H. Bachrach. 1966. Physiological significance of the effect of histamine on gastric secretion, p. 810-891. In O. Eichler and A. Farah [eds.], Handbook of experimental pharmacology, Vol. XVIII/I. Springer-Verlag, Berlin. 48. Irvine, W. T., and C. F. Code. 1958. Gastric secretion and free histamine III urine. Amer. J. Physiol. 195: 202-208. 49. Blair, E. L. 1966. The question of release of histamine by gastrin, p. 255-284. In M. 1. Grossman [ed.], Gastrin. University of California Press, Berkeley. 50. Kahlson, G., E. Rosengren, D. Svahn, and R. Thunberg. 1964. Mobilization and formation of histamine in the gastric mucosa as related to acid secretion. J. Physiol. (London) 174: 400-416. 51. Haverback, B. J., M. I. Stubrin, and B. J. Dyce. 1965. Relationship of histamine to gastrin and other secretagogues. Fed. Proc. 24: 1326-1330. 52. Shore, P. A. 1965. Release of histamine from the stomach by vagus-stimulating drugs; association with gastric acid secretion. Fed. Proc. 24: 1322-1325. 53. Hakanson, R, and C. Owman. 1966. Distribution and properties of amino acid decarbox~ ylases in gastric mucosa. Biochem. Pharmacol. 15: 489-499. 54. Levine, R J. 1965. Effect of histidine decarboxylase inhibition on gastric acid secretion in the rat. Fed. Proc. 124: 1331-1333.
GASTROENTEROLOGY
Copyright © 1961 by The Williams & Wilkins Co.
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SOME ASPECTS OF GASTRIC SECRETION MORTON
1.
GROSSMAN, M.
D.,
PH.D.
Research, Veterans Administration Center, Los Angeles, California, and Departments of Medicine and Physiology UCLA School of Medicine , Los Angeles, California
The plan (or lack thereof) of this review has been to identify an area of interest in a recent paper, to analyze the topic critically with the aid of citations to appropriate literature, ancient or current, and then to pass on to another, not necessarily related, subject that happened to capture my fancy. Those who might be displeased because this review does not provide a comprehensive bibliography of all recent papers on gastric secretion (or even on the narrow topics selected for discussion) should find some reassurance in learning that a source now exists that should satisfy even the most ardent seekers after completeness of references to literature. I refer, of course, to the new publication, Gastroenterology Abstracts and Citations, published by the National Institute of Arthritis and Metabolic Diseases. This excellent bibliographic citation and abstracting service now makes it possible for writers of reviews such as this to devote themselves to a critical appraisal of a few topics without feeling guilty for not having mentioned all papers dealing with the topic. There is presently an abundance of writing on gastric secretion. The first volume of Section 6 of the Handbook oj Physiology, devoted to "Physiology of the Alimentary Tract," published by the American Physiological Society, contains many chapters on gastric secretion and it should be off the press at about the same time as this review. The proceedings of an international conference on gastrin held in September 1964 have been published reAddress requests for reprints to: Dr. M. 1. Grossman, Room 231, Building 114, Veterans Administration Center, Los Angeles, California 90073.
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centlyl and the proceedings of another conference on gastric secretion held in Edmonton, Albert a, Canada, in September 1965 are in press. The book edited by Thomson and Gillespie 1a has a number of chapters devoted to gastric secretion. Gastrin
Chem1'cal structure. In his brilliant Memorial Lecture delivered at the Annual Meeting of the American Gastroenterological Association in May 1966, Professor R. A. Gregory2 presented further information on the structure of gastrin. The original studies on the isolation, structure, and synthesis of gastrin had been on material derived from hog stomachs. 3 - 5 Three additional species have now been studied: man, dog, and sheep.2 Human gastrin has been isolated, chemically characterized, and synthesized. 6 - s Dog and sheep gastrins have been isolated and their amino acid compositions have been determined but there is still some uncertainty about the sequence of a few of the amino acids. 2 Diagram 1 shows the structure of gastrin in these four species. A number of properties are common to the gastrins from all four species (diagram 1). The N-terminal residue is pyroglutamyl (pyrrolidone carboxyl). The C-terminal residue is phenylalanyl amide. Thus both the amino and the carboxyl terminal groups are blocked, so these molecules do not give the common chemical reactions that depend on these groups being reactive. All of the gastrins occur in two forms: without sulfate, designated I, and with an ethereal sulfate on the phenolic hydroxyl radical of tyrosine, designated II. All of the gastrins have a number of dicarboxylic
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883
,r-------------------,,
Hog
1 2 3 4 5 6 7 8 9 Ie} 11 12 13 : 14 15 16 17 ' ,-/\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1\ 1 \ 1 \ ' 1\ 1\ 1\ 1\ : LGlu-Gly-Pro-Try-Met-Glu-Glu-Glu-Glu-Glu-Ala- Tyr- Gly+Try-Met-Asp-Phe-NH 2 :
,
L- _________________ 1
Man Dog Sheep
Leu (Ala) Val-(Ala)
£Glu = pyroglutamyl (pyrrolidone carboxyl)
H 2C--CH 2 I I O==C", / CH-CoN H DIAGRAM 1. Structure of gastrins from hog, man, dog, and shee p. In dog and sheep gastrins the Ala is in parentheses because it is not yet known which glutamyl residue (6--10) is replaced by alanyl. In each species, gastrin occurs in two forms : I, without sulfate, and II, wi th sulfate as tYl'osyl-O-sulfate in position 12. The carboxyl terminal tetrapeptide amide surrounded by t he dotted line is the "active center" of the molecule. (Data of Gregory: )
amino acids and no dibasic ones, so they behave e lectrophoretic ally and chromatographically as acids. The acidic polarity is increased by the sulfate group. Most importantly, all of the gastrins have the same carboxyl terminal tetrapeptide amide sequence: -Try-Met-Asp-PheNH2 . This portion of the molecule may be regard ed as the "active center" because all of the biological actions of gastrin can be produced by this fragment but not by smaller portions of the molecule. 5 To date, no differences in potency or in spectrum of biological actions have been found between gastrins from various species or between the sulfated and un sulfated forms. The differences between the species so far studied (diagram 1) involve substitution of one or two of the 17 amino acid residues. This is in keeping with other observations on evolutionary changes in protein structure which show that the active site is usually not involved, that changes usually are substitutions of amino acids rather than alterations of chain length, and that the number of variant amino acid residues between any two species i s afunction of the time since their evolutionary divergence from a common ancestor. It is estimated that mutations involving one
amino acid may b e expected to occur and to be ret ained at intervals that have a mean value of about 12 million years. 9 Other gastrins. Are the gastrins of Gregory and Tracy t he unaltered molecules as they exist n aturally in the mucosa 0 1' have they been modified by the extraction process? Gregory lO has shown that modifications of the extraction procedure designed to lessen t he likelihood of chemical alterations during extraction did not change the products e xtracted. While the question must b e regarded as .still open, there is presently no evidence that the GregoryTracy gastrins have be en modified by extraction. Are there gastrins other than the two isolated by Gregory and Tracy? The strongest indication that there might be another form o f gastrin comes from the work of T auber and Madison .1l - 13 They isolated from hog antral mucosa a polypeptide containing 107 amino acid residues with a minimal molecular weight of 12,154 (compared wit h 2114 for Gregory-Tracy gastrin I). The material stimulated gastric secretion of ac id when injected intravenously during a 30-min period in dogs with gastric fistulas ; other physiological effect s known to be produced by the GregoryTracy peptides were not sought. The work
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of Tauber and Madison suggested that they had isolated a substance that might represent gastrin in a form from which the Gregory-Tracy peptides had been derived by scission during the extraction process. However, Tauber and Madison found no tryptophan in their gastrin molecule, and there are two tryptophan residues in the Gregory-Tracy peptides, one in the active site. From the dose-response data presented by TauberI3 it is clear that his final product has a potency far lower than that of the Gregory-Tracy peptides. For example, 0.1 p.g per kg per hr of Gregory-Tracy gastrin II will produce a secretory response in dogs with gastric fistulas that is about onefourth of maximal rate, whereas 20 p.g per kg per hr of Tauber's material were needed for a threshold response. 13 In view of this marked discrepancy in potency (much greater than the relative molecular weights of the two materials), it is possible that the activity of the Tauber-Madison material was attributable to the presence of a small percentage of Gregory-Tracy peptides and that the amount of tryptophan thus introduced was too small to be detected by the methods used. The methods of staining used by Tauber and Madison to demonstrate apparent homogeneity of their final product would not have revealed the presence of traces of the smaller peptides and it was not shown that the single component stained actually contained the gastrin activity. Until this possibility of contamination by traces of Gregory-Tracy peptides has been excluded, the advocates of the existence of high molecular weight gastrins 14 • 15 will have difficulty in presenting a convincing case. The simplest way to determine whether the purported high molecular weight gastrins contain GregoryTracy peptides would be to subject them to the Gregory-Tracy purification procedure and determine whether the smaller pep tides can be found. Relation between chemical structure and biological function. The entire range of physiological activities displayed by natural gastrin can also be produced by the Cterminal tetrapeptide amide. 5 Fragments of the molecule smaller than this tetrapeptide had little or no biological action, and no
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other portion of the molecule, including the entire 13 amino acid sequence from the N-terminus to the active tetrapeptide, had any significant biological action. As the peptide chain was progressively lengthened toward the N-terminus, the potency of the molecule increased. The synthetic peptide that differs from gastrin only in having two instead of five glutamyl residues in the body of the molecule had the same potency as gastrin itself. Most of the studies to date on the various chemical forms of gastrin and on the synthetic peptides related to gastrin have been rough screening tests. In only a few instances have truly quantitative biological assays been carried out. The generalizations about kinds and amounts of activity of the various substances are subject to this qualification. More careful study may make it necessary to revise some of the statements made here. A large number of pep tides related to the C-terminal tetrapeptide sequence of gastrin have been synthesized and examined for biological activity by Tracy and Gregory5 and by Morley, Tracy, and Gregory.16 In general, peptide derivatives that had the power to stimulate gastric acid secretion also had all of the other biological actions of the whole gastrin molecule, but some exceptions were encountered. Thus, several peptides stimulated gastric acid secretion but did not have an effect on gastrointestinal motility. Some peptides that stimulated gastric acid secretion at high (500 p.g) but not at low (50 p.g) doses subcutaneously did not inhibit gastric acid secretion with the high dose given intravenously, but this might only mean that the threshold for inhibition was still higher. The most interesting dissociation, namely inhibition of gastric acid secretion by large doses given intravenously without stimulation by small or large doses given subcutaneously, was encountered only once, in a tetrapeptide in which D-aspartic acid had replaced the Lmethionine of the natural tetrapeptide. No studies of this compound beyond the preliminary report of Morley et aJ.16 have appeared. It seems safe to guess that the search for an analogue of gastrin that acts
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as a competitive inhibitor of the hormone will be vigorously pursued. Since nonspecific inhibition of gastric acid secretion by large doses of gastrin given intravenously occurs in dogs but is not seen in cats,17 the latter species may be more suitable for screening synthetic analogues of gastrin for specific anti-gastrin activity. The further discussion of structure-function relationships will be restricted to stimulation of gastric acid secretion. Table 1 summarizes the effect of substitutions in various positions of the C-terminal tetrapeptide amide. Lengthening the chain by acylation of the N-terminal position often enhances activity. Only very minor changes in the active tetrapeptide itself can be made without complete loss of activity; the methionine position appears to tolerate the greatTABLE
est amount of change. Whereas the amino group at the N-terminal position can be either removed or blocked without significant change in activity, the amide group at the C-terminal position is apparently of crucial importance, for it can neither be removed nor substituted without almost total loss of activity. Pentapeptide. Of the various derivatives of the C-terminal tetrapeptide amide that have been examined, the one that has been widely available and extensively studied is the pentapeptide analogue in which a (3alanyl residue is attached to the tryptophan residue of the C-terminal tetrapeptide amide. This synthetic peptide has been manufactured and distributed by Imperial Chemical Industries. It is most commonly referred to by its code number, ICI 50,123.
1. Effect of substitutions on the activity of the C-terminal tetrapeptide amide in stimulation of gastric acid secretion" 1 2 3 4 5 6 R-Try-Met-Asp--Phe-NR 2 Substitutions tbat cause:
Position number No effect on activity
Enhanced activity
Depressed activity
1
t-Butyloxycarbonyl Benzyloxycarbonyl (blocking groups on amino of tryptophan)
2
4-CHatryptophan 5-CHatryptophan 6-CH atryptophan 2-Indolyl (CR 2 ) 2-CO-
Phenylalanine Histidine D-Tryptophan
3
Norleucine Ethionine
Norvaline a-Amino butyric Alanine Methionine sulfone Methionine sulfoxide
{3-Alanyl Lysine Carbamoyl Glutamic acid
4
{3-Aspartic Glutamic
5
Tyrosine O-Methyl tyrosine D-Phenylalanine
6
Cyclohexyl
" Data of Morley et al. 16
Abolition of activity
D-Methionine D-Aspartic
OH (free acid) Methyl ester
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In the remainder of this review it will be referred to simply as pentapeptide. M orley and co-workers16 st at ed that this pentapeptide was considerably more potent than the tetrapeptide but in the one e xample they give the response of a H eidenhain pouch to subcutaneous injection of 10 p.g was 1.2 mEq for the pentapeptide (their compound no. 29) and 1.5 mEq for the tetrapept ide (their compound no. 33). In studies on dogs with gastric fistul as we 1S found that the dose required for half-maximal response was about the same for tetrapeptide and pent apeptide. In these same studies we 1S found that gastrin was about 4 times as potent as the pentapeptide. In human subjects M akhlouf and co-work ers19 found that gastrin was a bout 10 times as potent as pentapetide. Although t here isstill some uncert ainty about exact potency it is clear that on a weight basis pentapeptide st ands between gastrin a nd hista mine. When given in dosefl required for maximal stimulation of acid secretion eit her by the subcutaneous route (2 p.g per k g) 20 or by continuous intravenous injection (1 p.g per kg per hI') 21 in man, gastrin produces no side effects. Rapid intravenous inj ection of large doses of gastrin may produce severe s ide effects including collapse.21, 22 With p entapeptide as the stimulant it was not possible to give human subj ect s doses t hat produced maximal response eithcr b y continuous intravenous injection 23 or by single subcutaneous injection 19 wit hout a high incidence of side effects, in cluding abdominal cramps, nausea, sinking sensation, and ret ching. However, the highest doses of pent apeptide that could be given without side effect (6 p.g per k g subcutaneously19 or 0.01 p.g per kg per min intravenously23 ) produced very high and reproducible rates of acid secretion, amounting t o 80% or more of the maximal r at e that can b e achieved wit h other stimulants such as porcine gastrin , hist amine, 01' betazole. The mechanism of the production of side effects by pentapeptide is not clear. Although there is a tendency for increased motor activity to occur throughout t he alimentary tract, particula ry in the colon, t his has not been correlated with
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side effect s.24 The s mall increases in arterial blood pressure and heart rate seen during sympt oms induced by pentapeptide a re probably the result rather than the cause of the symptoms.ll5 Pentapeptide has been extensively studied in human subj ects in Great Britain but has thus far been used only in animals in the United States. There is at present n o indication t hat pentapeptide or any ot her synthetic peptid e related to gastrin, or even gastrin itself, will have any theoretical advantage over hist a mine or betazole for clinical diagnostic testing of gastric secretion. The responses to all of these agents arc highly correlat ed so that anyone of them will serve to give a reliable index of the secretory responsiveness of an individ ual. It is possible that differences in responsiveness t o various stimulants, for example, histamine versus gastrin 01' one of the gastrin-related peptides, may b e foun d in certain diseases but th e experience to date does not favor this possibility. Strong stimulation of gastric secretion is desirable in diagnost ic t est ing because it is required when achlorhydria must be ruled out and because the reproducibility on replicate testing is greatly improved. The selection of an agent for stimula tion of gast ric acid secretion for clinical diagnostic testing resolves itself then into practical considerations of availa bility, sta bility, cost, and occurrence of side effects. Natural or synt het ic gastrin would be ideal but there is no current pr ospect t hat it will become widely avail able a t low cost. The sy nthetic partial peptides such as the penta peptide offer much p romise, but the search will probably not end with the pentapeptide because it cannot be given in doses t hat produce maximal response without in cu rring side effect s. Nevertheless, the pentapeptide deserves attention because when gi ven in the largest doses that do not provoke sid e effects (6 p.g per kg subcutaneously ) it produces ra tes of secretion only slightly lower than those obtained with the socalled " augmented histamine test" (40 p.g of histamine phosphate per kg subcutaneously). Of the readily available stimula nts, histamine, betazole, and pentapeptide, the pentapeptide is c le arly the "best" in t erms
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of being the one that can produce the highest rate of secretion without side effects. Extensive experience in the use of pentapeptide in human subjects will be required to establish its safety. Is maximal the utmost? The word "maximal" has been used in various senses in regard to gastric secretion of acid, and since authors do not always define the term confusion has arisen. There are at least three meanings that have been ascribed to the word "maximal": 1. The highest rate observed during the course of an individual study. The word "peak" would seem more appropriate than "maximal" for this meaning. 2. The "observed maximal" rate, that is, the highest rate attainable with a given stimulant. This is the commonest sense in which the term is used and is the meaning intended elsewhere in this review when the term is not qualified. The "maximal" rate, used in this sense, is determined by giving progressively larger doses of the stimulant and observing that portion of the dose-response curve in which increasing the dose does not increase the response. The word "maximal" used in this sense must be regarded as an operational term because the maximal rate observed will depend on a number of factors such as the stimulant used, the route of its administration, the sequence of doses, the frequency of sampling, and other such factors. Thus, defined in this way, the maximal response to subcutaneous gastrin is higher than to subcutaneous histamine,20 the maximal response to intravenous histamine is higher than to subcutaneous histamine,26 and the maximal response to subcutaneous betazole is higher than to subcutaneous histamine.27 3. The "calculated maximal" rate. Makhlouf and co-workers19 have proposed that maximal rate of secretion should be determined by extrapolation of the dose-response line. When the reciprocal of dose is plotted against the reciprocal of response the points fall on a straight line and the calculated intercept on the response axis represents the reciprocal of the theoretical response at infinitely large dose. In support of this proposed method for calculating maximal rate is the finding of Makhlouf
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and co-workers19 that the maximal rate so calculated was the same for various stimulants, including gastrin, pentapeptide, histamine, and betazole, and for various routes, intravenous and subcutaneous. It remains to be seen whether this calculated maximal rate does in fact represent the "true" maximal rate or the "utmost" of which the glands are capable, as Makhlouf and coworkers 19 have suggested, or whether situations will be found, perhaps with two stimulants acting together to potentiate each other,28.29 in which the calculated maximal rate for a single stimulant is exceeded. It would be helpful if the terms "observed maximal rate" and "calculated maximal rate" would be used where appropriate to avoid ambiguity. The many actions of gastrin. Before gastrin was isolated, it was assumed to have only one action, stimulation of gastric secretion of acid. Once pure gastrin was available it became apparent that it had a surprisingly wide spectrum of actions, including some that had been thought to be due to other constituents in crude antral mucosal extracts and some completely unexpected actions. The actions of gastrin can be classified into two broad categories 30 : (a) those that require only small doses of exogenous gastrin and can be mimicked by endogenously released gastrin (to use a much-maligned expression, these might be regarded as "physiological")31; and (b) those that occur only with large doses given intravenously and cannot be mimicked by endogenously released gastrin. Under category (a) are included (1) stimulation of gastric acid secretion, (2) stimulation of gastric pepsin secretion, (3) stimulation of pancreatic flow and bicarbonate secretion, (4) stimulation of pancreatic enzyme secretion, and (5) stimulation of hepatic biliary flow and bicarbonate secretion. Under category (b) are included (1) inhibition of gastric secretion of acid, (2) very strong stimulation of gastric pepsin secretion, (3) stimulation of gastric motility, (4) stimulation followed by inhibition of small intestinal motility, and (5) variable effects on blood pressure.
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For all five of the actions listed under category (a) it has been shown that endogenuus gastrin can produce an effect like that of small doses of exogenous gastrin. Cooke and co-workers32 found that the maximal rate of acid secretion from Heidenhain pouches in dogs W I'.8 higher with exogenous porcine gastrin than with release of the dog's own gastrin from an antral pouch by irrigation with acetylcholine. In unpublished studies Cooke and I have found that in dogs with gastric fistulas maximal acid response w as the same to exogenous porcine gastrin and to endogenous gastrin released from an antral pouch by bathing it with acetylcholine. In these same studies the pepsin responses to both exogenous and endogenous gastrin were higher than to histamine during maximal rates of acid secretion. With porcine gastrin as the stimulant pepsin secretion increased parallel with acid secretion over the entire range of acid responses in both dogs and cats.33 With histamine as the stimulant, the highest output of pepsin occurred with low doses and with doses that produced maximal rates of acid secretion pepsin output was markedly depressed. 33 In man, doses of histamine that produced maximal r ates of acid secretion did not depress pepsin secretion , and with such doses pepsin secretion in response to histamine and pentapeptide were equal. 19 Although gastrin is a stimulant of pepsin secretion, it does not appear to be capable of producing rates of pepsin output comparable to t hose achieved with cholinomimetic drugs.34 Both exogenous 35 and endogenous86 gastrin produced weak stimulation of pancreatic flow and bicarbonate output and strong stimulation of pancreatic enzyme secretion in dogs. The highest r ate of flow attainable with gastrin was about one-third of t hat seen with secretin, whereas the highest rat e of enzyme secretion with gastrin was about three-fourths of that seen with pancreozymin. In man pentapeptide was foun d to stimulate pancreatic secretion under cert ain conditions. 23 The effect of gastrin on secretion of bile in dogs is like that of secretin but is considerably weaker37 ; both are characterized
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by an increase in bicarbonate concentration accompanying the increase in flow rate. Endogenously released gastrin produced stimulation of bile flow with a time course similar to the stimulation of gastric acid secretion. 3s It is clearly established that gastrin is a major factor in regulation of gastric secretion ; its relative importance in stimulation of pancreatic and biliary secretion is not yet clear. Of the actions of gastrin t hat r equire large doses given r apidly intravenously, category (b ) , above, only inhibition of gastric acid secretion in dogs has been extensively studied. 39 ,4.0 The mechanism of this effect is not known but now that it has been shown that the phenomenon does not occur in man 22 or in cat s17 it is of interest mainly as a species peculiarity rather than as a general property of gastrin. Large doses of gastrin given rapidly intravenously caused very strong stimulation of pepsin secretion in both dogs and cats17 ; thus, t he effect on pepsin secretion appears to be independent of the effect on acid secretion. It is not yet clear whether higher rates of pepsin secretion occur when gastrin is given as a single large dose rapidly intravenously than when gastrin is given subcutaneously or continuously intravenously in doses producing maximal acid secretion. Gastrin causes contraction of smooth muscle under a variety of circumstances, including gastric and small intestinal smooth muscle in conscious dogs,5 isolated stomach, duodenum, and colon of the rat,41 and isolated ileum of the guinea pig.41 The amount of gastrin that must be added to the bath to cause the guinea pig ileum to contract (0.1 to 4 fLg per ml) would be enough to evoke a gastric secretory response in a dog. The concent ration of gastrin in a bath required to stimulate gastric secretion in isolated gastric m ucosa of frog 4 2 was a bou t one-thousandth of that needed for contraction of guinea pig ileum.41 The molar concentration of gastrin required to produce an equivalent degree of contraction of isolated guinea pig ileum was from 10 to 30 times that of acetylcholine or histamine.41 Pharmacological analy sis of the mechanism
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by which gastrin causes contraction of guinea pig ileum led to the conclusion that it acted on the postganglionic parasympathetic nerves to cause the release of acetylcholine which was the actual mediator of the effect.41 From these studies on guinea pig ileum Bennett41 suggested that stimulation of gastric secretion by gastrin might also be effected by the hormone acting on nerves to release acetylcholine. While it is true that atropine is a more effective inhibitor of gastrin stimulated than of histamine stimulated gastric acid secretion in dogs,43 there are circumstances, for example, in the anesthetized cat,44 under which atropine causes no inhibition. Since gastrin stimulates acid secretion in the isolated gastric mucosa of frogs,42 the possi bility exists of doing a pharmacological analysis like that on the isolated guinea pig ileum. In any case the hypothesis that gastrin acts by releasing ,acetylcholine deserves at least as much attention as the more familiar hypothesis that gastrin acts by releasing histamine. Tracy and Gregory5 stated that in anesthetized dogs gastrin had a small and inconstant depressant effect on the blood pressure. In unpublished studies in my laboratory, Jacobson and Swan found that both gastrin and penta peptide produced a small and inconstant depression of blood pressure · in conscious dogs. In man Logan and co-workers 25 observed an increase in blood pressure, heart rate , and blood flow in the forearm and hand of subjects receiving penta peptide intravenously. Since these changes did not occur until unpleasant symptoms had appeared, the cardiovascular effects may be indirect. Is Histamine Involved
If scientific theories could be proven by proclamation of faith in them, the theory that histamine is the final common mediator for gastric secretion of acid would be assured acceptance. There is no lack of ringing declarations of the validity of the theory.45-47a What seems to be lacking, in my view, is carefully reasoned evidence supporting the critical assumptions of the theory. In a sense, the question of the role of
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histamine in gastric secretion has about the same status as the question of other physiological roles for histamine, such as vasodilation, neuronal activation or inhibition, or contraction of visceral smooth muscle. In each instance it is clear that histamine is abundantly present in or near a responsive tissue and that exogenous histamine can activate or inhibit the tissue. The question has been and is: does endogenous histamine participate in the regulation of the tissue in question? In no instance has it been possible to devise crucial experiments that unequivocally implicate histamine in any normal process, and the possible role of histamine in pathological processes such as anaphylaxis and injury would appear to be partial at best. On the other hand, in no instance has a role for histamine in any of these processes been clearly disproved. The broad question of a possible role for histamine in regulation of gastric secretion can be resolved into three more restricted quesions. 1. Is gastrin histamine? Gastrin quite clear ly is not histamine but is a fully identified polypeptide amide that does not contain histamine or its precursor histidine. Since gastrin is several hundred times more potent than histamine on a molar basis, the possibility that the action of gastrin is due to bound histamine is ruled out. Much of the early controversy about a physiological role of histamine in gastric secretion concerned the possibility that gastrin was histamine; that question can now be regarded as definitely answered in the negative. 2. Is activation of gastric secretion associated with an increase in levels of histamine in plasma, or in urinary excretion of free histamine as a reflection of plasma levels? Although feeding caused an increase both in urinary histamine and in gastric secretion of acid in dogs, the two events could be made to occur independently, indicating that they probably were not causally related. For example, Irvine and Code 48 showed that meat given into the jejunum caused a transient increase in urinary histamine output and that the gastric secretory response occurred after the
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urinary histamine excretion had returned to cont rol levels. When meat was confined to the stomach it caused an increase in gastric secretion without a change in urinary histamine output. While these findings show that stimulation of gastric secretion produced by feeding meat cannot be ascribed to elevation of plasma histamine (on t he reasonable assumption that urinary e xcretion of histamine is a as tisfactory index of plasma levels) , they leave unexplained the failure of gastric secretion to occur during certain periods of elevated urinary excretion of histamine. Irvine and Code48 showed that infusion of histamine at ra tes too low to stimulate gastric secretion caused detectable increases in urinary excretion of histamine, and the rate of urinary excretion of hist amine increased by less than 2-fold when the rate of injection of histamine was increased 4-fold. Thus it is possible that feeding may produce elevations of circulating histamine that are too small to stimulate secretion but are still detectable on the basis of increase in urinary excretion. Since histamine can potentiate other stimuli for gastric secretion 28 , 29 these subthreshold amounts of histamine may be effective when acting with other stimuli. A systematic study is needed of the relation between r ate of infusion of histamine and (a) rate of urinary excretion and (b) degree of direct stimulation and potentiation of gastric secretion. It might t hen be possible to assess whether the levels of urinary histamine excretion produced by feeding are significant in terms of potentiation of other stimuli for gastric secretion. Another role that has been suggested for hist amine in gastric secretion is t hat it may be released widely in the body by gastrin. The careful studies of Bla ir,49 showing that plasma and urinary levels of histamine failed to rise when doses of gastrin t hat stimulated acid secretion were given to cats, would seem to dispose of this notion. 3. Do stimulants of gastric secretion such as gastrin and acetylcholine release hist amine in or near the parietal c ell and t hereby cause stimulation of secretion of acid? This is the question that continues
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to give v it ality to the hypothesis of a physiological role for histamine in gastric secretion. This aspect of the histamine-gastric secretion hypothesis has received fresh impetus from three kinds of recent studies. First, several groups50-52 have shown t hat the hi stamine content of the gastric mucosa of rats is decreased by gastrin and a variety of ot her gastric secretory stimulants. Second, the enzyme that synthesizes histamine, histidine decarboxylase, has been shown to be present in gastric mucosa of the rat5 3 and to show an increase in activity in response to feeding or inj ection of gastrin .50 Third, drugs that inhibit histidine decarboxylase and thereby deplete hist amine stores in gastri c mucosa in rats have been shown to inhibit gastric secretion in response to gastrin and a variety of other stimulants of acid secretion but did not inhibit the response to hist amine.54 The failure of the drugs to block the action o f histamine is of special significance because it indicat es that they did not cause a nonspecific depression of acid secretion. Do these three groups of findin gs establish wit hout doubt t hat histamine is a mediator of gastric secretion? Unfortunately, no. All three groups of evidence pertain exclusively to the rat and are probably not valid for other species. It has not been possible to show depletion of mucosal histamine on activation of gastric secretion in other species. 42 , 45 The gastric mucosa of the rat has a high concentration of a specific h istidine decarboxylase. This enzyme has been looked for but not found in other species ; the very low rate of hi stamine form ation produced by extracts of tissues from other species is assumed to be caused by nonspecific amino acid decarboxylases.53 D epression of gastric secretion by drugs that inhibi t histidine decarboxylase has not been demonstrated in species other than the rat . From the evidence presently available, one m ust conclude either that histamine is a me diator of gastric secretion in the rat but not in other species or that the evidence for histamine as a m ediator
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of gastric secretion is inconclusive in all species. The studies of Blair49 have shown that during stimulation of gastric secretion by gastrin in the cat there is no increase in the level of histamine in arterial or gastric venous blood plasma or in the rate of excretion of histamine in urine or gastric juice. To accommodate t he theory of mediation by histamine it thus becomes necessary to postulate (a) that the histamine involved in stimulation of gastric secretion is consumed in the process, and (b) that the histamine that appears in gastric juice is not involved in stimulation of secretion of acid. There is no a priori theoretical objection to either of these postulates but they obviously are formidable obstacles in verifying that histamine is a mediator of gastric secretion. REFERENCES 1. Grossman, M . I. [ed.]. 1966. Gastrin. University of California Press, Berkeley. 1a. Thomson, T. J ., and I. E . Gillespie [ed.]. 1966. Postgraduate Gastroenterology. Bailliere, Tindall, and Cassell, London . 2. Gregory, R. A. 1966. The isolation and chemistry of gastrin. Gastroenterology 51: 953959. 3. Gregory, H ., P. M. Hardy, D. S. J ones, G. W. Kenner, and R. C. Sheppard. 1964. Structure of gastrin. Nature (London) 204 : 931-933 . 4. Andersbn, J . C., M. A. Barton, R. A. Gregory, P. M . Hardy, G. W. Kenner, J. K . Macleod, J . Preston, R. C .. Sheppard, and J . S. Morley. 1964. Synthesis of gastrin. Nature (London) 204 : 933-934. 5. Tracy, H. J., and R. A. Gregory. 1964. Physiological properties of a series of synthetic peptides structurally related to gastrin I. N ature (London) 204: 935-938. 6. Gregory, R. A., H. J. Tracy, and M . I. Grossman . 1966. Isolation of two gastrins from human antral mucosa. Nature (London) 209 : 583 . 7. Bentley, P . H., G. W. K enner, and R. C. Sheppard . 1966. Structures of human gastrins I and II. ~ature (London) 209 : 583-585. 8. Beacham, J ., P. H. Bentley, R. A. Gregory, G. W. K enner, J. K. M acLeod, and R. C. Sheppard. 1966. Synthesis of hum an gastrin I. Nature (London) 209: 585-586. 9. White, A., P. Handler, and E. L . Smith . 1964. Principles of biochemistry, Ed. 3, p .611-626. M cGraw-Hill Book Company, Inc., New York.
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10. Gregory, R. A. 1967. I solation and chemistry of gastrin . In C. Code [ed.], Handbook of Physiology, Section 6, The Alimentary Tract. American Physiological Society, Wash ington, D. C., in press. 11. Tauber, S. D., and L. L. Madison. 1964. Isolation, physicochemical and physiological charact erization of gastrin. J. Clin. Invest. 43: 1271-1272. 12. T auber, S. D., and L. L. Madison. 1965. The isolation and characterization of porcine gastrin. J .BioI. Chern. 240: 645-650 . 13. T a uber; S. D. 1966. Characteriza tion of a pure gastrin, p. 27-48. In M. I. Grossman [ed.] , Gastrin. University of California Press, Berkeley . 14. Fletcher, T . L., W. R. Anderson, C. L. Pitts, and H. N. H arkins. 1961. A new preparation of gastrin; preliminary characterization. Nature (London) 190: 448. 15. Wilding, P ., B . J. Dyce, H. Riderknecht, and B . J . H averback. 1966. The most probable gastrin? Gastroenterology 50: 879. 16. Morley, J . S., H . JTracy, . and R. A. Gregory . 1965. Structure-function relationships in the active C-terminal tetrapeptide sequence of gastrin. Nature (London) 207: 1356-1359 . 17. Emits, S., and M . I. Grossman. 1966. Difference between dogs and cats in effect of large dose of gastrin on gastric secretion. Physiologist 9: 175. 18. Konturek, S., and M. I. Grossman. 1966. Acid response to gastrin and related peptides. Gastroenterology 50 : 650-652. 19. Makh louf, G. M ., J. P . A. McManus, and W. I. Card . 1966. The action of t he pentapeptide (lCI 50, 123) on gastric secretion in man. Gastroenterology 51 : 455-465. 20. Makhlouf, G. M., J . P. A. M cManus, and W. I. Card. 1964. The action of gastrin II on gastric-ac id secretion in man. Lancet 2 : 485-490. 21. Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1964. Dose-response curves for the effect of gastrin II on acid secretion in man. Gut 5: 379-384. 22. Makhlouf, G. M ., J. P . A. M cM anus, and W . I. Card. 1966. Action of gastrin II on gastric secretion in m an, p. 139-169. In M . I. Grossman [ed.], Gastrin. University of California Press, Berkeley. 23. Wormsley, K. G., M . P. M ahoney, and M. Ng. 1966. Effects of a gastrin-like penta peptide (I.C.I. 50,123) on stomach and pancreas. Lancet 1: 993-996. 24. Logan, C. J. H ., and A. M. Connell. 1966. The effect of a synthetic gastrin-like pentapeptide (I .C.I. 50,123) on intestinal motility in man . Lancet 1: 996-999.
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25. Logan, C. J. H., 1. Brick, and I. C. Roddie. 1966. Circulatory effects of a synthetic gastrin-like pentapeptide (I.C.!. 50,123). Lancet 1: 999-1000. 26. Lawrie, J. H., G. M. R Smith, and A. P. M. Forrest. 1964. The histamine-infusion test. Lancet 2: 270-273. 27. Ward, S., I. E. Gillespie, E. P. Passaro, Jr., and M. 1. Grossman. 1963. Comparison of Histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs. Gastroenterology 44: 620-626. 28. Passaro, E. P., Jr., I. E. Gillespie, and M. 1. Grossman. 1963. Potentiation between gastrin and histamine in stimulation of gastric secretion. Proc. Soc. Exp. BioI. Med. 114: 50-52. 29. Gillespie, I. E., and M. I. Grossman. 1964. Potentiation between Urecholine and gastrin extract and between Urecholine and histamine in the stimulation of Heidenhain pouches. Gut 5: 71-76. 30. Grossman, M. I. 1966. Gastrin. Ann. Intern. Med. 64: 212-216. 31. Hogben, C. A. M. 1960. The alimentary tract. Ann. Rev. Physiol. 1212: 381-406 (see footnote to p. 388). 32. Cooke, A. R, R M. Preshaw, D. L. Nahrwold, and M. 1. Grossman. 1966. Comparison of acid secretory responses to endogenous and exogenous gastrin. Fed. Proc. 25: 513. 33. Emas, S., and M. 1. Grossman. 1967. Comparison of gastric secretion in conscious dogs and cats. Gastroenterology 512: 29-34. 34. Andersson, S., and M. 1. Grossman. 1965. Effect of vagal denervation of pouches on gastric secretion in dogs with intact or resected antrums. Gastroenterology 48: 449-462. 35. Preshaw, R M., and M. 1. Grossman. 1965. Stimulation of pancreatic secretion by extracts of the pyloric gland area of the stomach. Gastroenterology 48: 36-44. 36. Preshaw, R M., A. R Cooke, and M. 1. Grossman. 1965. Stimulation of pancreatic secretion by a humoral agent from the pyloric gland area of the stomach. Gastroenterology 49: 617-622. 37. Zaterka, S., and M. 1. Grossman. 1966. The effect of gastrin and histamine on secretion of bile. Gastroenterology 50: 500-505. 38. Nahrwold, D. L., A. R Cooke, and M. 1. Grossman. 1967. Choleresis induced by stimulation of the gastric antrum. Gastroenterology 52: 18-22. 39. Gillespie, 1. E., and M. 1. Grossman. 1963. Inhibition of gastric secretion by extracts containing gastrin. Gastroenterology 44: 301-310.
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40. Gillespie, 1. E. 1966. Inhibition of acid secretion by gastrin extracts. p. 229-253. In: M. I. Grossman [ed.], Gastrin. University of California Press, Berkeley. 41. Bennett, A. 1965. Effect of gastrin on isolated smooth muscle preparations. Nature (London) 1208: 170-173. 42. Davidson, W. D., C. A. E. Lemmi, and J. C. Thompson. 1966. Action of gastrin on the isolated gastric mucosa of the bullfrog. Proc. Soc. Exp. BioI. Med. 121: 545-547. 43. Grossman, M. I. 1961. Cholinergic potentiation of the response to gastrin. J. Physiol. (London) 157: 14-15. 44. Blair, E. L., A. A. Harper, H. J. Lake, and J. D. Reed. 1961. The effect of atropine upon gastrin-stimulated gastric secretion. J. Physiol. (London) 159: 72-73P. 45. Code, C. F. 1965. Histamine and gastric secretion: a later look, 1955-1965. Fed. Proc. 124: 1311-1321. 46. Code, C. F. 1966. Histamine and gastric secretion. Gastroenterology 51: 272. 47. Haverback, B. J. 1966. Role of histamine in gastric secretion. Gastroenterology 50: 898899. 47a. Ivy, A. C., and W. H. Bachrach. 1966. Physiological significance of the effect of histamine on gastric secretion, p. 810-891. In O. Eichler and A. Farah [eds.], Handbook of experimental pharmacology, Vol. XVIII/I. Springer-Verlag, Berlin. 48. Irvine, W. T., and C. F. Code. 1958. Gastric secretion and free histamine III urine. Amer. J. Physiol. 195: 202-208. 49. Blair, E. L. 1966. The question of release of histamine by gastrin, p. 255-284. In M. 1. Grossman [ed.], Gastrin. University of California Press, Berkeley. 50. Kahlson, G., E. Rosengren, D. Svahn, and R. Thunberg. 1964. Mobilization and formation of histamine in the gastric mucosa as related to acid secretion. J. Physiol. (London) 174: 400-416. 51. Haverback, B. J., M. I. Stubrin, and B. J. Dyce. 1965. Relationship of histamine to gastrin and other secretagogues. Fed. Proc. 24: 1326-1330. 52. Shore, P. A. 1965. Release of histamine from the stomach by vagus-stimulating drugs; association with gastric acid secretion. Fed. Proc. 24: 1322-1325. 53. Hakanson, R, and C. Owman. 1966. Distribution and properties of amino acid decarbox~ ylases in gastric mucosa. Biochem. Pharmacol. 15: 489-499. 54. Levine, R J. 1965. Effect of histidine decarboxylase inhibition on gastric acid secretion in the rat. Fed. Proc. 124: 1331-1333.