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Studies on the regulation of gastric functionKleibeuker, Jan Hendrik

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STUDIES ON THE REGULATION

OF GASTRIC FUNCTION

J. H. KLEIBEUKER

STUDIES ON THE REGULATION OF GASTRIC FUNCTION

Omslag: Janke de Vries

STELLING EN

I.

De hormonen secretine en cholecystokinine hebben bij de mens een fysiolo­

gische functie bij de regulatie van de ontlediging van de maag.

II.

Secretine is bij de mens niet van fysiologische betckenis voor de regulatie van

de secretie van maagzuur.

III.

Het gevoel van verzadiging, dat wordt teweeggebracht door cholecystokini­

ne, is voor een deel toe te schrijven aan de vertraging van de maagontlediging,

die door dit hormoon wordt veroorzaakt.

IV.

Het gebruik van de "power exponential curve" is een belangrijke aanwinst

voor het onderzoek van de maagontlediging.

V.

Het verschil, dat is aangetoond tussen de mens en de hood ten aanzien van

het effect van histamine op de secretie van gastrine, is toe te schrijven aan on­

derling verschillende, in dit opzicht soortspecifieke eigenschappen van de

mens en de hood.

VI.

De waarde van radioaktief gemerkte leukocyten voor de diagnostiek van in­

flammatoire aandoeningen van de darm is zeer beperkt.

VII.

Een ernstig tekortschietende postprandiale doorbloeding van de mesente­

riele vaten kan warden verbeterd door stimulatie van de collaterale circulatie

met behulp van continue enterale voeding.

VIII.

Voor het optimale herstel van de aktiviteit van de disaccharidasen in het jeju­

numslijmvlies bij volwassen patienten met glutengevoelige spruw na het instel­

len van een glutenvrij dieet is meestal mcer dan een jaar nodig.

IX.

De "arteriespuit" is niet geschikt voor het beoordelen van een verstoring van het zuur-base evenwicht tijdens cardiopulmonale resuscitatie.

X.

Vroegtijdige herkenning van somatisatie als oorzaak van lichamelijke klach­ten en adequate begeleiding van de patienten hiermee, kunnen patient, arts en maatschappij veel leed, tijd en kosten besparen.

XI.

Het drinken van zure vruchtensappen bevordert in het algemeen de alkalisa­tie van de urine.

XII.

De professionele balletdanseres lijdt een breekbaar bestaan.

Stellingen behorende bij het proefschrift van

J. H. Kleibeuker

Studies on the regulation of gastric function

Groningen 1987

RIJKSUNIVERSITEIT TE GRONINGEN

STUDIES ON THE REG ULA TION

OF GASTRIC FUNCTION

PROEFSCHRIFT

ter vcrkrijging van het doctoraat in de Gcnce�kundc

aan de Rijksunivcrsih:it te Groning._':!n

op gczag van de Rector Magnificu� Dr. E. Blcumink

in hct openhaar te vcrdcdigen op

wocnsdag I april 1987

des namiddags tc 4.00 uur

door

JAN HENDRIK KLEIBEUKER

gehoren te Mcppel

1987

DRUKKERIJ VAN DENDEREN B.V.

GRONINGEN

Promotorc�: Prof. Dr. W. C. Vccgcr Prof. Dr. C. B. H. W. Lamer-; Prof. J. H. Walsh. M.D.

CONTENTS

Voorwoord .

Introduction

Chapter 1 Regulation of gastric acid secretion in man: an update Neth. J. Med. 1986; 29: 325-333. . . . . . . . . . .

Chapter 2 Regulation of gastric emptying in man: physiology and effects of medical and surgical therapy. An update.

3

Submitted. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Chapter3 Intravenous histamine reduces bombesin-stimulated gastrin release in dogs. Regul. Pept. 1985; 11: 209-215. . . . . . . . . . . . . . . . . . . 31

Chapter 4 Effect of histamine Hr receptor stimulation on bombesin- and peptone­stimulated gastrin release in man. Dig. Dis. Sci. 1986;31: 1095-1099.

Chapter 5 Effect of selective and nonselective cholinergic blockade on bombesin­and peptone-stimulated gastrin release.

39

Submitted. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Chapter 6 Role of endogenous secretin in acid-induced inhibition of human gastric function. J. Clin. Invest. 1984; 73: 526-532.

Chapter7 Retardation of gastric emptying of solid food by secretin. Submitted. . . . . . . . . . . . . . . . . . . . . . . .

57

73

Chapter8

Cholecystokinin is a physiologic hormonal mediator of fat-induced

inhibition of gastric emptying.

Submitted. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Summary . 93

Samenvatting 97

VOORWOORD

De studies, beschreven in dit procfschrift, wcrdcn vcrricht in hct Center for Ulcer Research and Education (CURE) in Los Angeles en in de afdclingcn Gastrocnterologic en Nucleairc Gcnceskundc van hct Academisch Zickcnhuis in Groningcn, in samenwcrking met de afdcling Gastrocntcrologic van hct Acadcmisch Zickcnhuis in Leiden en de afdcling Klinischc Chemic van hct Bispebjcrg Zickcnhuis in Kopcnhagcn.

lk dank vooral alle vrijwilligers. in Los Angeles en in Groningcn, voor hun bcrcidwillighcid om als procfpersoon aan mijn onderzockingcn dee! tc ncmcn.

Ondcrgenoemde personen hcbbcn ieder op hun cigen wijze bijgcdragen aan de totstandkoming van dit procfschrift. Daarvoor ben ik hen alien zccr crkcn­tclijk.

Professor W. Yccgcr bood mij de gclcgcnhcid de oplciding tot gastrocntcro­loog te vol gen en gaf mij allc vrijhcid voor mij n ondcrzoek. Professor Cock La­mers hccft mij vanaf hct allcrccrstc begin van mij n intcrcssc in de gastrointcsti­nalc hormoncn ondcrstcund met nict aflatcnd cnthousiasmc. Yan Professor John Walsh krccg ik ccn intcnsievc training in de gastrointcstinalc research. Gordon Kauffman. Yiktor Eyssclcin, Vernon Maxwell, Henk Bcckhuis, Bert Piers, Henk Kooi. Ove Schaffalitzky de Muckadcll en Jan Jansen waren als mc­dcondcrzockcr ten nauwstc bij de studies bctrokkcn. Professor E. Mandema en Professor J. H. Scholten hcbbcn er in bclangrij kc mate toe bijgcdragcn dat ik ccn jaar in hct CURE kon wcrkcn.

Verdcr hcbbcn aan de totstandkoming van dit proefschrift meegewcrkt: de mcdcwcrkcrs van de afdeling Nuclcaire Gcnccskundc, Wytske Boersma-van Ek, Annckc Bos-Wijma, Henk Brcukclman, Swanct Brooymans, Jan Brou­wer, Kiki Bugler. Peter Chew. Janet Elashoff, Annemarie Grcftc, Anita Han­sen, Jules Kcyzcr, Minze Luinstra, P. de Noord, Evrard Orcmus, Terry Reedy, Bert Schaalma, Wits Stevenson, Jacques de Vries, John Washington en Wiebe Zcinstra.

De Jan Kornclis de Cock-Stich ting hceft mijn vcrblijf in Los Angeles en de studies, bcschrevcn in de hoofdstukkcn 4 en 5, financiccl ondcrstcund. De fir­ma's Smith, Kline & French B. V. en Glaxo B.Y. hchbcn ook ecn financicle bij­drage gclcvcrd.

INTRODUCTION

Gastric function has been one of the most extensively studied topics in gas­trointestinal physiology for many decades. Due to modern investigational tech­niques our understanding of the physiology and pathophysiology of the gastric functions has greatly increased during the past few decades.

The function of the human stomach can be divided in the gastric motoric and the secretory function. Gastric motility has an important role in the digestive process. After a meal the stomach provides the small intestine little by little with a steady flow of mostly liquificd food, so that optimal digestion and ab­sorption can take place. Gastric secretions have only a minor role in this pro­cess. However, gastric acid is of great importance in the pathophysiology of many of the disorders affecting the upper gastrointestinal tract.

The studies described in this thesis were performed to further elucidate some aspects of the regulation of the human gastric function.

In chapter I an update is given of the current literature on the regulation of gastric acid secretion in man. The first part of it describes the different stimula­tory and inhibitory mechanisms and pathways. The second part is devoted to individual stimulatory and inhibitory substances. In chapter 2 an update is given of the current literature on the regulation of gastric emptying in man. In addition, effects of medical and surgical therapy on gastric emptying arc described.

Chapter 3 describes a study on the effect of histamine on bombesin-stimu­lated gastrin release in dogs. Several studies have shown that the histamine Hr receptor antagonist cimetidine increases gastrin release, though other studies have not confirmed this finding. Further the presence of histamine containing mast cells in the antrum suggests a possible role of histamine in the regulation of gastrin secretion. The hypothesis tested in this study was that histamine reduces gastrin release independent of change in intragastric pH.

Chapter 4 reports on a study on the effect of histamine on bombesin- and peptone-stimulated gastrin release in man. Based on the findings in chapter 3 and on some studies suggesting a stimulatory effect of cimetidine on meal-in­duced gastrin release the same hypothesis as in chapter 3 was tested in man.

In chapter 5 a study on the effect of selective and nonselective cholinergic blockade on bombcsin- and peptone-stimulated gastrin release in man is de­scribed. Muscarinic cholinergic nerves have an important role in the regulation of gastrin release. Comparing the effects of the specific muscarinic M 1-receptor antagonist pirenzepine and the nonselective muscarinic receptor antagonist atropine on gastrin release it was investigated whether M 1-receptors might have a specific role in the regulation of gastrin secretion.

1

Chapter 6 describes a study on the role of endogenous secrctin in acid-in­duced inhibition of human gastric function. It is well known that acidification of stomach and/or duodenum potently inhibits gastric acid secretion and gastric emptying, while at the same time it stimulates the release of secretin. Admin­istration of high doses of secretin, producing supraphysiological plasma concentrations, inhibits acid secretion, gastrin release and gastric emptying. In this study the effect of secretin at plasma concentrations in the physiological range on gastric acid secretion, gastrin release and gastric emptying of liquids was investigated, testing the hypothesis that acid-induced inhibition of gastric function is due, at least in part, to the release of secretin.

In chapter 7 a study on the effect of secretin at near-physiologic plasma con­centrations on the gastric emptying rate of solid food in man is reported, testing the hypothesis that secretin is involved in the inhibition of gastric emptying by duodenal acidification.

Finally chapter 8 describes a study on the effect of cholecystokinin at physi­ologic plasma concentrations on the gastric emptying rate of a firm semisolid meal in man. Instillation of fat into the duodenum is a potent stimulant of the release of cholecystokinin, while at the same time it inhibits gastric emptying. In this study the hypothesis was tested that cholecystokinin is a hormonal medi­ator of the inhibition of gastric emptying induced by intraduodenal nutrients.

2

CHAPTER 1

REGULATION OF GASTRIC ACID SECRETION

IN MAN: AN UPDATE

J. H. Klcibcukcr

Department of Gastrocntcrology, U nivcrsity Hospital, Groningcn.

INTRODUCTION Gastric acid secretion is one of the most extensively studied topics in gastro­

intestinal physiology and has been the source of many scientific controversies ( I ). Due to modern investigational methods our understanding of physiology and pathophysiology of acid secretion has greatly increased during the past few decades. The main functions of gastric acid, secreted in rcponse to a meal, arc addition of tluid to the meal to further liquify gastric contents and to acidify them. thereby activating the pepsinogcns. In the interprandial period gastric acid has an important function in maintaining a barrier against bacterial inva­sion and colonization of the upper gastrointestinal tract. The secretion of gas­tric acid is of only limited significance for the digestive process. Digestion and absorption of food arc undisturbed in subjects with achlorhydria and the size of food particles leaving the stomach through the pylorus is similar whether the stomach contents arc acidified or not (2, 3). Nevertheless, the important role of acid in the pathophysiology of gastric acid-related disorders makes a good un­derstanding of its regulation relevant.

This article presents an update of the regulation of postprandial gastric acid secretion. Although animal studies have contributed a great deal to our know­ledge of the physiology of acid secretion, this review deals largely with results from studies in humans. Results of animal studies, in vitro and in vivo, arc con­sidered only to a limited extent. The first part of this article discusses the differ­ent stimulatory and inhibitory mechanisms and pathways. The second part is devoted to individual stimulatory and inhibitory substances and, if applicable, their antagonists.

The postprandial stimulation of gastric acid secretion is classically divided in­to three phases: the cephalic, the gastric and the intestinal phase. These three do not act successively and independently but overlap each other and interact with each other. In addition to stimulatory mechanisms, at the same time inhibitory pathways are activated, which exert an equally important role in the regulation of postprandial gastric secretory function (Table I).

3

Table!. Gastric acid ,ecret1on: stimulatory and inhibitory mechani�m,

Pathway Mediator

Cephalic phase

- stimulatory direct\ agal acetylcholine vagal- ant rum ga�trin

Gastric phase

Di�tention vagovagal reflex acetylcholinc - stimulatory intramural reflex (acetylchol,ne)

(antral. adrenergic ga�trin)

Chemicals - stimulatory G-cell �t,mulation:

- protein digests

l - amines - coffee

ga,trin - beer. wine - calcium calcium

I '! alcohol

- inhibitory antral pH!. gasl.!"in ! . neural? ? . acetylcholine?

prostaglandin�

Intestinal phase

- stimulatory protein digests duodenal gastrin, in duodenum. absorbed amino acids, jejunum largely?

- inhibitory protein digests ?

in ileum fat neurotensin?

peptidcYY? both vagally dependent ( cholecy,tokinin?) absorbed fat

carbohydrate, enteroglucagon? notGIP

duodenal pH ? • somatostatin? not secretin

CEPHALIC PHASE

Gastric acid secretion in response to a meal is initiated by the first contact with the meal by seeing and smelling the food. Just thinking about and discuss­

ing appetizing food, without the sight and smell of it, are already potent stimu-

4

Ian ts of acid secretion ( 4). The cephalic stimulation of acid secretion has been extensively studied using the method of modified sham feeding. During this procedure subjects chew and subsequently spit an appetizing meal without swallowing any food, while at the same time gastric juice is being aspirated through a nasogastric tube. The induction of acid secretion by cephalic stimula­tion is mediated via the vagus nerves. On the one hand this is through direct neural stimulation of the acid secreting parietal cells, which is mediated by the neurotransmitter acetylcholine (5). On the other hand cephalic stimulation can induce gastrin release from the antral G-cells, provided that the antrum is not acidified (6). Since during and after a meal gastric acid is initially largely neutralized, due to the buffering capacity of the food, the cephalic stimulation of gastrin release takes part in the cephalic phase of acid secretion. Acetylcho­line does not mediate this stimulation. In fact acetylcholine has an inhibitory ef­fect on gastrin release (5). The neurotransmitter, responsible for the vagally in­duced gastrin release, has not been identified with certainty. Bombesin, a pep­tide originally isolated from the frog skin, is an important candidate. A bombe­sin-like peptide, named gastrin releasing peptide (GRP), has been shown to be present in nerves of the mammalian gastrointestinal tract, including nerve end­ings in the antral mucosa (7, 8). Bombesin and GRP are very powerful stimu­lants of gastrin release (9, 10). From animal studies evidence has been obtained that a bombesin-like peptide is the neurotransmitter mediating vagally induced gastrin release (11, 12, 13). When all mechanisms of meal-induced acid secre­tion act simultaneously the cephalic phase accounts for approximately one third of the total amount of acid secreted, exerting its effect mainly during the first postprandial hour (14).

GASTRIC PHASE Stimulation of acid secretion during this phase is due to gastric distention and

to chemical reactions of the food with the gastric mucosa.

Gastric distention Distention of the entire stomach by inert liquids elicits acid secretion (15).

The degree of this stimulation is not correlated with the volume of the liquids (15) or the intragastric pressure (16). A volume of 100 ml elicits as much acid se­cretion as 700 ml and at intragastric pressures of 10 and 15 cm H2O no more acid is secreted than at 5 cm H2O. This induction of acid secretion is mainly due to distention of the fundus. In healthy subjects antral distention does not elicit acid secretion (17), while fundic distention by means of a balloon does so (18). The acid stimulatory effect of distention is largely due to a vagovagal reflex and

5

to a lesser extent to an intramura l gastric reflex ( 1 9) . Distention by l iquids can

also induce a small increase in plasma gastrin concentration ( 1 6 , 20) . This in­

crease is poorly correlated with the acid �ccrction rate and is of minor signifi­

cance for the stimulation of it. The distention-induced gastrin release is medi­

ated by a bcta-adrcncrgic mechanism ( 2 1 ). Similar to sham feeding gastrin re­

lease by distention is enhanced by atropine (20) , indicating a cholincrgic inhib­

itory mechanism . Distention docs not only induce acid stimulatory mecha­

nisms . Fundic and antral distention both reduce the acid secretion rate el icited

by a submaximal dose of pcntagastrin (22 , 23) , showing that under certain con­

ditions distention has an inh ibitory effect. I t is estimated that ga�tric di�tcntion

contributes about one fifth to one fourth of total postprandial acid secretion

( 1 4) .

lntragastric chemicals.

A mixed meal in the stomach exerts a potent stimulatory effect on gastric acid

secretio n . independent of concomitant distention . Thi� is mainly due to stimu­

lation of gastrin release by certain food components (24 ), most probably

through a direct effect on the antral G-ccl ls (25 ) . The most well known and very

potent stimulants of gastrin secretion arc proteins and their digestive products ,

but also coffee (26) , decaffeinated or not, and beer and wine (27, 28) are capa­

ble in this respect . Also other commonly consumed beverages l ike tea , seven­

up and coke have acid stimulatory qualities (29. 30) . The most potent stimulant

of acid secretion of beverages tested is mi lk (29) . However, after its ingestion

mi lk init ial ly increases intragastric pH. rather than lowering it, due to its buffer­

ing capacity. In vivo and in vitro studies in rats showed that. besides amino

acids, amines, derived from these amino acids and present in regular food. arc

potent stimulants of gastrin release as well (3 1 , 32) . It may be that amino acids

are to be decarboxylatcd to amines intracel lularly to exert their gastrin releas­

ing action (3 1 ) . However, although these amines can be removed largely from

food by freeze drying it under alkaline conditions (32) , this procedure did not

dimin ish the gastrin releasing capacity of decaffeinated coffee (Kleibeuker,

Mogard, Reeve , Walsh , unpublished observations) . So probably also

substances other than amino acids and amines have gastrin releasing qualities.

The gastrin releasing capacity of amino acids in vitro is directly correlated with

their l ipid solubility (3 1 ) . In accordance with this, the amino acids with strong­

est stimulatory capacity in humans arc the aromatic ones, tryptophan and phe­

nylalanine (33) . Diluted alcohol can also stimulate acid secretion though less so

than wine (28) . This stimulatory action is independent of gastrin release. I ntra­

gastric ionized calcium potently induces acid secretion too (34, 35) . A single 0.5

g dose of the antacid calcium carbonate has already a stimulatory effect (36) .

6

Calcium also elicits gastrin release but this is only partly responsible for

its effect on acid secretion. The mechanisms through which alcohol and calcium stimulate acid production have not been clarified. The effect of calcium is not

caused by an increase of serum calcium.

When intragastric pH is being kept constant small doses of atropine inhibit

amino acid-induced gastrin release (37), while higher doses do not affect (37) or increase it (38). So cholinergic nerves seem to have a dual effect on meal-in­

duced gastrin secretion. Integrity of the vagal innervation of the parietal cell

area is not only essential for the cephalic and distention-induced stimulation of

acid secretion, but is also important for the effect of gastrin. This is exemplified

by the fact that after highly selective vagotomy the responsiveness of the parietal cells to stimulation by gastrin is greatly decreased (39).

Ingestion of a mixed meal increases intragastric pH, due to the buffering ca­

pacity of its constituents ( 40). When these buffering substances are gradually emptied by the stomach, while acid secretion rate is still considerable , intragas­

tric pH decreases. This causes an inhibition of gastrin release and consequently

of gastric acid secretion (4 1 ). This feedback mechanism has an important role in the regulation of acid secretion. Gastric luminal acidification not only inhib­its the chemically induced gastrin release but also that stimulated by sham

feeding (6), as mentioned previously. The pathway through which intraluminal

acid inhibits gastrin release has not been elucidated yet. Atropine can prevent acid-induced inhibition of sham feeding-stimulated gastrin release (5), so a

cholinergic mechanism seems to be involved. This is in accordance with results from studies in the isolated rat stomach ( 42), which suggest that intramural cho­

linergic and noncholinergic nerves are involved. In addition, prostaglandins

may have a role in this inhibition (43).

INTESTINAL PHASE

Intestinal contents can affect acid secretion. The different food components

have stimulatory and/or inhibitory effects. When peptides and/or amino acids

arc instilled into the duodenum or jejunum gastric acid is stimulated (44, 45). Instillation into the duodenum is the most potent of these two ( 46). The mecha­nisms through which these substances stimulate acid secretion have not been

fully elucidated. The most proximal part of the duodenum contains G-cells.

These release gastrin in response to a meal analogous to the antral G-cells ( 47). However, the stimulation of acid by digestive products of protein in the second

part of the duodenum and in the jejunum is independent of gastrin release (44-

46). Part of this stimulation may be exerted by absorbed amino acids. Intra­venous administration of amino acids induces acid production (48). Particularly

7

tryptophan and phenylalanine are potent in this respect, having stimulatory ef­fects at plasma concentrations which are found after a meal ( 49). However, this mechanism comprises only part of the total effect of intraluminal amino acids ( 46). The presence of digestive products of protein in the distal small bowel has an inhibitory effect on gastric acid secretion ( 45).

Both fat and carbohydrates, when present in the duodenum and/or small bow­el (45, 50), reduce acid secretion. The inhibition by fat is dependent on an in­tact vagal innervation of the parietal cells (50). Fat is a potent stimulant of the release of the gut hormones neurotensin (51) and peptide YY (52). These two hormones originate from endocrine cells in the distal part of the gastrointes­tinal tract (52, 53). They both inhibit acid secretion at plasma concentrations which can be found after a fatty meal or instillation of fat into the intestine (54, 55). The effect of neurotensin on acid production is dependent on intact vagal innervation of the proximal stomach (56) and in dogs it was found that the inhi­bition by peptide YY was vagally mediated (57). These results support the con­cept that these peptides are at least in part responsible for the fat-induced inhi­bition of acid secretion. They thereby probably fulfill the criteria for being en­terogastrones, i.e. intestinal hormones with a physiogical gastric inhibitory function. In addition, absorbed fat may also contribute to the inhibitory effect of fat, through a pathway, which is independent of neurotensin (51 ). Cholecys­tokinin does not seem to have an important role in the regulation of gastric se­cretory function (58). Although acute hyperglycemia reduces acid secretion (59), the mechanism through which intraintestinal glucose exerts its inhibitory effect on acid production is independent of blood glucose concentrations (60). Recently it was suggested that the gut hormone enteroglucagon might have a role in this process (61). Glucose also elicits the release of the gut hormone gas­tric inhibitory peptide also named glucosedependent insulinotropic peptide (GIP) , which at high doses reduces acid secretion. However, at concentrations seen in the normal postprandial period, GIP does not affect gastric acid (62).

Duodenal acidification is a potent inhibitor of gastric acid secretion (63). This acidification elicits the release of secretin (63-65). When administered at high doses secretin strongly reduces gastrin release and acid secretion (66, 67). However, secretin at low physiologic plasma concentrations does not contrib­ute to any extent to acid-induced inhibition of acid secretion or gastrin release (68). Circulating somatostatin does not seem to have a role in this inhibitory mechanism either (69). Moreover, whether the duodenal acidification, occur­ring under physiological conditions does affect gastric secretory function in man, has not been shown so far. Finally, the perfusion of the colon with hyper­tonic solutions inhibits pentagastrin-stimulated acid secretion (70). So it may be that the colon also has a role in the regulation of gastric secretory function.

8

Tahle I I . Ga�tric acid �ecrction: Mimulatory and inhihitory mediators

�uh�tance effect•

hbtaminc ,tim acetylcholine �tim ga�trin Mim �omato,tatin inh

neuroten,in'! tnh peptide YY'! tnh enteroglucagon'' inh metenkephalin '' ,tim

prostaglandins'! inh homhe,in ,tim

�tim �timulatory. inh = inhthllory

source

gastric mast cells v,,gu� nerve antral. duodenal G-cell, gastric D-celb. duodenal D-cells?

intcstinal endocrine cells

gastric nerve�. endocrine cells'! ga�tric muco�Jl cell� antral nerve�

route

paracrinc neural endocrine paracrine. endocrine

endocrine

neural paracrine? paracrine neural G-cell �timulation

INDIVIDUAL REGULA TORY MEDIA TORS OF GASTRIC ACID SECRETION

In the previous part of this article several substances involved in different mechanisms of the regulation of gastric acid secretion have been mentioned. In this part these and other regulatory mediators will be considered individually. The development of specific antagonists and agonists for some of these sub­stances has contributed greatly to the present knowledge about their physiolo­gical role (Table 11).

Histamine Histamine is produced and secreted in human gastric mucosa by mast cells

(71 ). It is a very potent stimulant of acid secretion. However, until the identifi­cation of a subclass of histamine receptors on the parietal cells, the histamine H2-receptors, and the concurrent development of specific competitive hista­mine Hz-receptor antagonists (72), there was strong doubt about the physiological role of histamine in gastric acid secretion. The inhibition of basal and stimulat­ed acid secretion, independent of the type of the stimulus, by Hz-receptor an­tagonists like cimetidine and ranitidine definitely proved that histamine has a physiological stimulatory role in acid secretion. The exact place of histamine in this respect has not been fully clarified yet. On the one hand histamine may have a permissive role in the stimulation of the parietal cells by other stimula­tory substances like gastrin and acetylcholine. On the other hand it may be that gastrin acts indirectly on the parietal cells by stimulating mast cells to release

9

histamine . There is strong evidence that in dogs gastrin directly stimulates pa­

rietal cells and does not induce histamine release (73-75) . In contrast , studies in

man showed that pentagastrin caused degranulation of gastric mast cells, a re­duction in tissue histamine concentration and an increase in gastric histamine

output (76, 77) . Cimetidine, though inhibiting pentagastrin-stimulatcd acid se­

cretion , did not affect gastric histamine output (77) . These results suggest that

in man, contrary to the situation in dogs, pentagastrin stimulates acid secretion

at least in part through the release of histamine. A recently developed mast cell

stabilizer, FPL 52694, inhibited pentagastrin-induced acid secretion (76 , 78) ,

which is in accordance with the above mentioned results. However, it did not

affect meal-stimulated acid secretion (78) . So more studies have to be done to

elucidate the role of histamine in human acid secretion.

The human antral mucosa also contains mast cells, producing histamine (71 ) .

So the question was raised whether histamine might affect gastrin release . Studies with histamine Hrreceptor antagonists yielded conflicting results in

this respect. Two recent studies could not show any effect of histamine on stim­

ulated gastrin release , thereby almost certainly excluding a role of histamine in

the regulation of gastrin secretion (79, 80) .

Acetylcholine

The induction of acid secretion by acetylcholine is mediated through musca­

rinic receptors, as it can be blocked by relatively low doses of atropine. Stimula­

tion of muscarinic receptors stimulates acid secretion by isolated human gastric

glands (81 ) , suggesting a direct effect of acetylcholine on the parietal cells. This

is in accordance with the finding of muscarinic receptors on isolated canine pa­

rietal cells (82) . Pirenzepine is a recently developed antimuscarinic drug. Re­

sults from studies with this agent provided evidence for the existence of musca­

rinic Ml - and M2-receptors , pirenzepine being an antagonist for the Ml -recep­

tors (83) . Like the classic anticholinergic agents, pirenzepine inhibits basal and

stimulated acid secretion (84, 85) . So Ml -receptors seem to be involved in acid stimulation . In vitro animal studies suggested that the parietal cells do not have

M l -receptors, but M2-receptors (86) . It is speculated that the vagus nerves sti­

mulate acid secretion through M l-receptors on neurons in the ganglia in the

gastric wall and subsequently through M2-receptors on the parietal cells (87).

Gastrin

Gastrin occurs in the circulation in two main molecular forms, gastrin- 1 7 or little gastrin , with 1 7 amino acids , and gastrin-34 or big gastrin , with 34 amino

acids. Until recently it was thought that gastrin- 1 7 was six to eight times more

potent than gastrin-34 in stimulating acid secretion. However, it has been

10

shown that the two have similar capacity in this respect (88). The plasma half lives arc markedly different: 8 minutes for gastrin-17 and 44 minutes for gas­trin-34 (88). From a study in nonopcrated persons and subjects with an antrec­tomy and gastroduodcnostomy (89) it was concluded that the duodenum is the main source of gastrin-34, while the antrum mainly releases gastrin-17. The authors calculated that the duodenum could account for 40-50% of circulating gastrin in the basal state and that up to one-third of gastrin released by a meal, could originate in the duodenum. Gastrin- 17 is degraded in vivo in several frag­ments. One of these is the NH2-terminal tridecapeptide of gastrin (90). It was first suggested that this peptide could inhibit gastric acid secretion (91), but a later study could not show any influence of this substance on acid production (92).

Somatostatin

The regulatory peptide somatostatin is produced and secreted by the endo­crine O-cclls in the mucosa of the whole gastrointestinal tract (53). These cells have cytoplasmic processes which make close contact with adjacent cells (93), e.g. in the stomach with the parietal cells in body and fundus and with the G­cells in the antrum. It is assumed that somatostatin, released from these cells, has a paracrine function , i.e . the released peptide molecules reach their target cells through diffusion in the interstitial fluid. Somatostatin generally exerts an inhibitory effect on its receptor cells. It is thought that somatostatin has an im­portant role in the fine tuning of secretory function. Many agents which stimu­late exocrine and/or endocrine secretions, do also induce release of somatosta­tin.

Extensive studies on the relationship between gastrin and somatostatin re­lease have been done in the isolated perfused rat stomach ( I 1, 1 2, 94). These suggest that vagal stimulation releases gastrin through a bombesin-like neuro­peptide and somatostatin through a cholinergic pathway. Studies with human antral cell columns are in accordance with this (95). This would explain the in­crease of gastrin release after atropine, by inhibiting somatostatin release. Somatostatin probably also has a hormonal function in human gastric acid se­cretion. After a meal plasma somatostatin concentrations increase (96-98). In­fusion of somatostatin, producing plasma concentrations mimicking those found postprandially, markedly inhibits meal-stimulated acid secretion, without affecting gastrin release (97-99). A study in anaesthesized rats strongly suggested that the inhibition of acid secretion by somatostatin was mediated by prostaglandins (100). This could not be confirmed in man (101, 102). It is not yet clear through which pathway(s) somatostatin release is induced by a meal

1 1

and what the source of it is. Studies in dogs suggest that somatostatin is a hormonal mediator of duodenal acidification-induced inhibition of gastric secretory function (1 03). So far this could not be found in humans (69).

Other mediators possibly involved in regulation of gastric secretory function

As mentioned previously distention-induced acid secretion is likely to be me­diated through a beta-adrenergic pathway (2 1 ). Specific beta-adrenergic mech­anisms are involved in the hypoglycemia-induced acid secretion and gastrin re­lease ( 1 04). Patients with a phaeochromocytoma and high circulating plasma adrenaline concentrations have elevated basal and meal-induced plasma gas­trin levels ( 105). Except for distention-induced gastrin release the adrenergic nerves arc not thought to have an important role in the regulation of postpran­dial gastric secretory function under normal physiologic conditions.

Endogenous opiates have a wide distribution in the gastrointestinal tract. in­cluding the stomach. These substances are found in endocrine cells as well as in peripheral nerves. The opiate receptor antagonist naloxonc inhibits basal and stimulated acid secretion ( 106, 1 07). The opiate metcnkephalin ( 108) but not beta-endorfin ( I 09), stimulates acid production. This suggests that endogenous opiates, particularly metenkephalin, may be involved in the regulation of gas­tric acid secretion. Neither naloxonc nor mctcnkcphalin affect gastrin release (106-108).

Thyrotropin releasing hormone (TRH) is present in human gastric mucosa, predominantly in the antrum ( 1 1 0). TRH reduces pentagastrin- and histamine­stimulated acid secretion ( 1 1 1 , 1 1 2). Calcitonin gene-related peptide is a re­cently discovered regulatory peptide, which is encoded in the same gene as cal­citonin. It can inhibit basal and stimulated acid secretion ( 1 1 3). Other peptides, capable to inhibit gastric secretory function are calcitonin ( 67. 1 1 4), corticotro­pin-releasing factor ( 1 15) and epidermal growth factor or urogastrone ( 1 1 6). For all these peptides a physiologic role in the regulation of acid secretion has not been established so far.

Prostaglandins, produced in the gastric mucosa, have local regulatory ef­fects. They are probably involved in the so-called cytoprotection. Exogenously administered prostaglandins and their analogues are able to inhibit gastric acid secretion ( 1 17, 1 1 8). Recent studies on the effect of indomethacin, an inhibitor of prostaglandin biosynthesis, on basal and stimulated acid secretion, yielded conflicting and variable results ( 1 0 1 , 1 1 9, 1 20). It is to be doubted whether pros­taglandins have a physiologic role in the regulation of acid production. As pre­viously mentioned, prostaglandins may be involved in acid-induced inhibition of gastrin secretion (43).

12

REFERENCES I . Baron JH. The discovery of gastric acid. Gastroenterology 1979; 76: 1 056-64. 2. Ohashi H. Meyer JH. Effect of peptide digestion on emptying of cooked liver in dogs. Gas­

troenterology 1980; 79: 305- 10. 3. Meyer JH. Ohashi H. Jchn D. Thomson JB. Size of liver particles emptied from the human

stomach. Gastroenterology 198 1 ; 80: 1489-96. 4. Feldman M. Rich,irdson CT. Role of thought. sight. smell. and taste of food in the cephalic

phase of gastric acid ,ecretion in humans. Ga,troenterology 1986; 90: 428-33 . 5 . Feldman M . Rich,irdson CT. Taylor IL. Wahh JH. Effect of atropine on vagal release of gas­

trin and pancreatic polypeptide. J Clin Invest 1979; 63: 294-8. 6. Feldman M. Wabh JH. Acid inhihition of sham feeding-stimulated gaMrin release and gas­

tric acid secretion: effect of atropine. Ga,troenterology 1980; 78: 772-6. 7. Dockray GJ. Vaillant C. Wabh J H. The neuronal origin of bomhcsinlike immunoreactivity

m the rat ga,trointe,tin,11 tract. Neuroscience I 979; 4 : 156 1-8. 8. Buffa R. Solovieva I . Fiocca R. Localization of homhesin and GRP (gastrin releasing pepti­

de) sequence, m gut nerves or endocrine cells. Histochemistry 1982; 76: 457-67. 9. Varner AA. Modlin IM. W,1lsh JH. High potency of hombesin for stimulation of human gas­

trin release and gastric acid secretion. Regul Pepi 198 1 ; I : 289-96. IO . Knigge U. Holst JJ. Knuhtsen S. Petersen B. Krarup T. Hobt-Pedersen J. Christiansen PM.

Gastrin releasing peptide: pharmacokinetics and effects on gastro-entero-pancrcatic hormo­nes and gastric secretion in normal man. J Chn Endocrinol Metab 1984; 59: 3 ! 0-5 .

1 1 . Martindale R. Kauffman GL. Levin S. Walsh JH. Yamada T. Differential regulation of gas­trin and somatostatin secretion from isolated perfused rat stomachs. Gastroenterology 1982; 83: 240-4.

1 2. Schuhert ML. Saffouri B. Walsh JH. Makhlouf GM. lnhihition of neurally mediated gastrin secretion hy bomhesin antiserum. Am J Physiol 1985; 248: G456-62.

13 . Knuhtsen S. Holst JJ. Knigge U. Olesen M. Nielsen OV. Radioimmunoassay. pharmacoki­netics and neuronal release of ga,trin-releasing peptide in anesthetized pigs. Gastroenterolo­gy 1984; 87: 372-8.

14. Richardson CT. Walsh JH. Cooper KA. Feldman M. Fordtran JS. Studies on the role of ce­phalic-vagal ,timulation in the acid secretory response to eating in normal human subjects. J Clin Invest 1977; 60: 435-4 1 .

15 . Feldman M. Comparison o f acid secretion rates measured b y gastric aspiration and b y i n vivo intra-gastric titration m healthy human subjects. Gastroenterology 1979; 76: 954-7.

16 . Soares EC. Zaterka S. Walsh JH. Acid secretton and scrum gastrin at graded intragastric pressures in man. Gastroenterology 1977; 72: 676-9.

17. Bergegardh S . Olhe L. Gastric acid response to ant rum distension in man. Scand J Gastroent 1975; IO : 171 -6 .

18. Grotzinger U. Bergegardh S . Olhe L. Effect of fundic distension on gastric acid secretion in man. Gut 1977; 18 : 105-10.

19. Grotzinger U. Bcrgegardh S . Olbe L. Effect of atropine and proximal gastric vagotomy on the acid response to fundic distension in man. Gut 1977; 1 8: 303- IO.

20. Schiller LR. Walsh JH. Feldman M. Distension-induced gastrin release . Effect of luminal acidification and intravenous atropine. Gastroenterology 1980; 78: 9 12-7.

2 1 . Peters MN, Walsh JH, Ferrari J, Feldman M. Adrenergic regulation of distention-induced gastrin release in humans. Gastroenterolgy 1982; 82: 659-63.

22. Grotzinger U. Bergegardh S. Olbe L. Effects of fundic distention on pentagastrin-stimulated gastric acid secretion in man. Gastroenterology 1977; 73: 447-52.

23. Schoon 1-M, Bergegardh S. Grotzinger U. Olbe L. Evidence for a defective inhibition of pen­tagastrin-stimulated gastric acid secretion by antral distension in the duodenal ulcer patient . Gastroenterology 1978; 75: 363-7.

24 . Feldman M. Walsh JH, Wong HC. Richardson CT. Role of gastric heptadecapeptide in the acid secretory response to amino acids in man. J Clin Invest 1978; 62: 308- 1 3 .

25 . Lichtenberger LM. Forssman WG, Ito S . Functional responsiveness of an isolated and enri­ched fraction of rodent gastrin cells. Gastroenterology 1980; 79: 447-59.

13

26. Feldman EJ. benherg J I . Gro,,man Ml . Ga,trn: acid and ga,trin rc,pon,e to decaffeinated coffee and a peptone meal. JAMA 198 1 : 246, 248-50.

27. Singer MV. Eysselein VE. Gochell H. Beer and wine hut not whi�ky and pure ethanol do ,ti­mulate relea,e of gaMrin in humans. Digestion 1983 : 26: 73-9.

28. Lenz HJ . Ferrari-Taylor J. benherg J I . Wine and five percent ethanol are potent ,timulants of gaMric acid ,ecretion in human,. Ga,troenterolog) 1983: 85 : 1082-7.

29. McArthur K. Hogan D. benherg J I . Relative ,timulatory dfcct, of commonly ingested hc­verage, on ga,tric acid ,ecrction m human,. Ga,troenterolog) 1982: 83: 199-20:l.

30. Du hey P. Sundram KR. Nundy S Effect of t.:,1 on ga,tnc acid ,.:cr.:tion. Dig Di, Sci 1984: 29: 202-6 .

3 1 . Lichtenhergcr LM. Delan,orne R. Graziani LA. 1 mportance of amino acid uptal-.e and de­carboxylation in gastrin relea�e from isolated G cell,. Nature 1982 : 295 : 698-700

32. Lichtenherger LM. Graziani LA. Duhinsl-.y WP. lmportanc.: of dietary a min.:, in meal-indu­ced ga,trin relea,e. Am J Phy,iol 1982: 24): G341-7.

33. Taylor IL. Byrne WJ . Chri,tic DL. Ament ME. Walsh JH. Effect of individual L-amino acid, on ga,tric ,1cid ,ccrction and ,erum ga,trin and pancreatic polypeptide n:lea,e in human,. Ga,troenterology 1982: 83: 273-8.

34. Barclay G . Maxwell V. Grossman Ml. Solomon TE. Effect, of graded amounts of 1ntraga,­tric calcium on acid ,ecr.:tion . g.iMrin relea,e . ,ind ga,tric emptying in normal and duodenal ulcer suhjccts. Dig Dis Sci 1983: 28: 385-9 1 .

35. Brassinne A . Wandj.i S . Effect o f intragaMric calcium on ga,triL acid ,ecretion and ga,trin re­lease in normal man and in certain gastroduodenal diwrder,. Ga-irocnterol Clin Biol 1983: 7: 659-6)

36. Levant JA. Walsh J H. bcnhcrg J I Stimul.1tion of ga,tric ,ccretion and gastrin relea,e hy ,in­gle oral dose, of calcium carhon.itc in man N Engl J Med 1973: 289: 555-8.

37. Schiller LR. Wabh JH. Feldman M. Effect of atropin.: on gastrin release stimulated hy an amino acid meal in humans. Gastroenterology 1982: 8): 267-72.

38. Lazzaroni M. Sangaletti 0. Del Soldato P. Bianchi Porro G. Effect of pirenzepine and atropi­ne on pep tone meal-stimulated ga,tric '<!Crcuon and plJsm.i ga,trin in h.:althy volunteer,. pa­tients with duodenal ulcer and , agotomizcd patients. Dige,tion 1985: )2: 267-72.

39. Blair AJ. R1chard,on CT. Wabh JH. Che\\ P. Feldman M. Effect of panetal cell vagotomy on acid �ecretory respons1vene�, to circulating ga,trin in human, Ga,troenterology 1986: 90: 1 ()() 1 . 7.

40. Fimmel CJ . Etienne A . Cilluffo T. v Ritter C. Ga,,er T. Rey J-P. Caradonna-Moscatelli P. Sabbatini F. Pace F. Buhler HW. Bauerfeind P. Blum A L. Long-term ambulatory gastric pH monitoring: validation of a new method and effect of H1-antagomst,. Ga,troenterology 1985: 88: 1842-5 1 .

4 1 . Walsh JH. Richardson CT. Fordtran J S . pH dependence of acid ,ecretion and ga,trin relea,e in normal and ulcer subject,. J Clin lnveM 1975 : 55: 462-8 .

42 . Saffouri 8 , Du Val JW. Makhlouf GM. Stimulation of gastrin secretion in vitro hy intralumi­nal chemicab: regulation by intramural chohncrgic and noncholinergic neurons . Ga,troente­rology 1984; 87: 557-6 1 .

43 Befrits R. Samuebson K. Johan»on C. G.i,tnc acid inhibition hy antral acidification medi­ated by endogenou� prostaglandin,. Scand J Gastroent 1984: 19: 899-904.

44. Isenberg J I . Ippoliti AF. Maxwell V. Perfusion of the proximal �mall intestine with peptone stimulates gastric acid secretion in man. Gastroenterology 1977: 73: 746-52.

45. Owyang C . Miller LJ. Malagelada J-R. Go VLW. Nutrient and bowel �egment dependency of human intestinal control of gastric secretion. Am J Physiol 1982: 243: G372-6.

46. Lenz HJ . Hogan WL. Isenberg J I . Intestinal phase of gastric acid secretion in human, with and without portocaval shunt. Gastroenterology 1985: 89: 79 1 -6.

47. Fritsch WP, Hauseman TU. Rick W. Gastric and extragastnc gastrin release in normal ,ub­jects, in duodenal ulcer patients. and in patients with partial gastrectomy (Billroth I ) . Gas­troenterology 1976; 7 1 : 552-7.

48. Isenberg J I . Maxwell V. Intravenous infusion of amino acids stimulates gastric acid secretion in man. N Engl J Med 1978; 298: 27-9.

14

49. McArthur KE. benherg J I . Hogan DL. Dreier SJ. Intravenous infusion of L-isomers of phe­nylalanine ,ind tryptophan stimulate gastric acid secretion at physiologic plasma concentra­tion, in normal suhjects and after pariewl cell vagotomy. J Clin l nveM 1983; 7 1 : 1254-62.

511. Kihl B. Olhe L. Fat inhihition of gastric acid ,ecn:tion in duodenal ulcer patients hefore and after proximal gastric vagotomy. Gut 1980; 2 1 : 1056-61 .

5 1 . Petersen B. Christiansen J. Rok.teus A . Rosell S. Effect of intravenous and intr.ijejunal fat infusion on gastric acid secretion and plai.ma neurotensin-likc immunoreactivity in man. Scand .J Ga,troent 1984; 19: 48-5 1 .

52. Adrian TE. Ferri G-L. B.tcarcse-Hamilton AJ. Fuessl HS. Polak JM. Bloom SR. Human dis­trihution and release of a putative new gut hormone. peptide YY. GaMroenterulogy 1985; 89: 1070-7.

53. Sjolund K. Sanden G. Hakani.on R. Sundler F. Endocrine celb in human intestine: an immu­nocytochemical !.tudy . GaMroenterology 1983; 85: 1 1 211-30.

54. Fletcher DR. Shull,.es A. Hardy KJ . The effect of neuroten,in and i.ecretin on gastric acid i.c­cretion and mucosal hlood flow in man . Regul Pept 1985; 1 1 : 2 1 7-26.

55. Adrian TE. Savage A P. Sagor GR. Allen JM. Bacarese-Hamilton AJ. Tatemoto K. Polak JM. Bloom SR. Effect of peptide YY on gastric. pancreatic. and hihary function in humans. Gastroenterology 1985; 89: 494-9.

56. Olsen PS. Pedersen JH. Kirkegaard P. Been H . Stadil F. Fahrenkrug ). Christiansen J. Neu­rotensin induced i nhihition of gastric acid secretion in duodenal ulcer patients hcfore and af­ter parietal cell vagotomy. Gut 1984; 25: 4R 1 -4.

57. Pappa� TN. De has HT. Taylor IL . Enterogastrone-like effect of peptide YY is vagally medi­ated in the dog. J Clin I nvest 1986; 77: 49-53 .

58. Ahrahm D. Mogard M. Maxwell V. Sankaran H. Deveney C. Wabh J H . Cholecystokinin in­hihition of meal-stimulated gastric acid secretion in man . Gastroenterology 1985; RR: 13()().

59. MacGregor IL. Deveney C. Way LW. Meyer J H . The effect of acute hyperglycemia on meal­stimulated gastric. hiliary. and pancreatic �ecretion . and �erum ga!.trin . Gastrocnterology 1976: 711: 197-202.

60. Moore JG, Crei.pin F. I n fluence of glucose on cephalic-vagal-stimulated gai.tnc acid ,ecreti­on in man . Dig Dis Sci 1980; 25: I I 7-22.

6 1 . Petersen B. Christiansen J. Holst JJ. A glucose-dependent mechanbm in jejcnum inhihib gastric acid secretion : a response mediated through enteroglucagon'? Scand J Ga,troenterol 1985 ; 20: 193-7.

62 . Maxwell V. Shulkes A . Brown JC. Solomon TE. Walsh J H . Grossman Ml. Effect of gastric inhihitory polypeptide on pentagai.trin-stimulated acid secretion in man . Dig Dis Sci 1980; 25: 1 13-22.

63. Ward AS. Bloom SR. The role of secret in in the inhihition of gastric secretion by intraduode­nal acid. Gut 1974; 15 : 889-97.

64. Greenherg GR. McCloy RF. Baron J H . Bryant MG. Bloom SR. Gastric acid regulates the release of plasma secretin in man . Eur J Clin Invest 1982; 12: 36 1-72.

65 . Fahrenkrug J. Schaffalitzky de Muckadell OB. Rune SJ. pH threshold for release of seeretin in normal suhjects and in patients with duodenal ulcer and patients with chronic pancreatitis . Scand J Gastroenterol 1978; 13: 1 77-86.

66. Londong W. Londong V. Hansen LE. Schwanner A. Gastric effecl!, and side effects of syn­thetic secretin in man . Regul Pept 198 1 ; 2 : 23 1 -44.

67. Jansen JBMJ. Lamers CBHW. Calcitonin and secretin inhibit bombe,in-stimulated serum gastrin and gastric acid secretion in man . Regul Pept 198 I ; I : 4 1 5-2 1 .

68 . Kleiheuker J H . Ey,selein VE. Maxwell V . Walsh J H . Role of endogenous secret i n i n acid-in­duced inhibition of human gastric function . J Clin I nvest 1984; 73 : 526-32.

69. Lucey MR. Was, JAH. Fairclough PD. O"Hare M. Kwasowski P. Penman E, Webb J. Rees LH . Does gastric acid release plasma somatostatin in man? Gut 1984; 25 : 1 2 1 7-20.

70. Jian R. Besterman HS. Sarson DL, Aymes C, Hostein J, Bloom SR. Ram baud JC. Colonic inhibition of gastric secretion in man. Dig Dis Sci 198 1 ; 26: 195-20 1 .

7 1 . Mohri K . Reimann H-J . Lorenz W , Troidl H . Weber D. H istamine content and mast cells in human gastric and duodenal mucosa. Agent, Actions 1978; 8: 372-5.

15

72. Blad, JW. Duncan WAM. Durant CJ. Ganellin CR. Par,on, EM. Definition and antagon­ism of histamine HJ-receptors. Nature 1972; 236: 385-90.

73. Soll AH. Amirian DA. Thoma, LP. ReedyTJ. Elashoff JD. Ga,trin receptors on isolated ca­nine parietal cell,. J Clin Invest 198-1: 73: 1-13-1--17.

74. Soll AH. Aminan DA. Thoma, LP. Park J. Ela,hoff J D. Beaven MA. Yamada T. Ga,tnn receptor, on nonparietal cell, isolated from canine fundic muco,a . Am J Phy,iol 198-1; 2-17: G7 1 5-23 .

75. Redfern JS. Thirlhy R. Feldman M. Richardson CT. Effect of pentaga,tnn on gastric muco­sa! histamine in dog,. Am J Physiol 1985: 2-18: G369-75.

76. Reimann H-J. Schmidt U. Ultsch B. Sullivan TJ. Wendt P. Action of FPL 5269-1 on gastric acid ,ecretion in the healthy human stomach. Gut 198-1; 25 : 1 22 1 --1 .

77. Man WK. lngoldhy CJH. Spencer J . b pentagastrin-,t1mulated secretion mediated hy hista­mine'? Gut 198-1; 25: 965-70.

78. Boyd EJS. Wilson JA. Worm,ley KG. Richard, MH. Langman MJS. Effect, of a ma,t cell ,tahili,er (FPL 52694) on human gastric secretion. Agent, Action, 1985; 16 : -162-7.

79. Richardson CT. Feldman M. Effect of hi,tamine and cimetidine on amino acid meal-stimula­ted gastrin release at a controlled intragastric pH in healthy human heings Regul Pept 1985 : IO : 339--1-1

80. Kleiheuker J H . Kooi H. Lamer, CBHW. Effect of histamine H,-receptor stimulation on homhesin- and peptone-stimulated gastrin release in man. Dig Db Sci 1986: 3 1 : I095-9.

8 1 . Haglund U. Elander B. Felleniu, E. Leth R. Rehnherg 0. Olhe L. The effects of secretago­gues on isolated human gastric glands. Scand J Gastroent 1982; 1 7: -155-60.

82. Soll A H . Secretagogue stimulation of "C-aminopyrinc accumulation hy isolated canine pa­rietal cells. Am J Physiol 1980; 238: G366-75.

83 . Hammer R. Berrie CP. Birdsall NJM. Burgen ASV. Hulme EC. Pirenzepine distinguishes between different suhcla"e' of mu,carine receptor,. Nature 1980; 283: 90-2 .

8-1. Lon dong W. Londong V. Prechtl R. Sch\\ anner A. Studies on the effectivenes, of cimetidi­ne. pirenzepine and synthetic ,ecretin on stimulated ga,tric acid secretion. Z Ga,troenterolo­gie 1980; 18 : 306- 1 3 .

85 . Procacciante F. Citone G. Montesam C. Rihotta G. Antisecretory activity o f pirenzepine ver,u, cimetidine in man: a controlled study. Gut 198-1; 25: 1 78-82.

86. Rosenfeld GC. Pirenzepine (LS 5 19) : a " cal,. in hi hi tor of acid secretion by isolated rat parie­tal cell, . Eur J Pharmacol 1983; 86: 99- 10 I .

87. Feldman M. Inhihition of gastric acid ,ecretion h} ,elective dnd nonselective anticholiner­gics. Gastroenterology 198-1; 86: 36 1 -6.

88. Ey,,elein VE. Maxwell V. Reedy T. Wiin,ch E. Wabh J H . Similar acid stimulatory poten­cies of synthetic human hig and little gastrins in man. J Clin Invest 198-1; 73: 1 284-90.

89. Lamers CB. Walsh J H . Jansen JB. Harri,on AR. lppoliti AF. van Tongeren JH. Evidence that ga,trin 34 is preferentially relea,ed from the humdn duodenum. Gastroenterology 1982: 83: 233-9.

90. Pauwels S. Dockray GJ. Walker R. Marcus S. Mctaholism of heptadecapeptide ga,trin in hu­mans studied by region-specific antisera. J Clin Invest 1985; 75 : 2006-1 3 .

91 . Petersen B . Chri,tiansen J . Rehfeld J F . The N-terminal tridecapeptide fragment o f gastrin-1 7 inhibits gastric acid ,ecretion . Regul Pept 1983; 7: 323-3-1.

92. Pauwels S. Dockray GJ. Walker R. Marcu, S. Metabolism of the 1 - 1 3 sequence of gastrin 1 7 in humans and failure to influence acid secretion. Gastroenterology 1985. 89: 49-56.

93. Larsson LI . Golterman N. DeMagistris L . Somatostatin cell processes as pathways for para­crine secretion. Science 1979; 205: 1 393-5.

94. Wolfe MM. Jain DK. Reel GM. McGuigan JE. Effects of carbachol on ga,trin and somato­statin release in rat antral tissue culture. Gastroenterology 1984; 87: 86-93.

95. Richelsen B. Rehfeld JF. Lar»on L I . Antral cell column: a method for studying relea,e of gastric hormones . Am J Physiol 1983; 2-15: G-163-69.

96. Penman E. Wass JAH. Medbak S. Morgan L. Lewi, JM. Bes,er GM. Rees LH . Re,ponse of circulating immunoreactive somatostatin to nutritional stimuli in normal subjects. Gastroen­terology 198 J'; 8 1 : 692-9.

1 6

97. Colturi TJ. Unger R H . Feldman M. Role of circulating somatostatin in regulation of gastric acid secretion. ga,trin release. and islet cell function. J Clin Invest 1984: 74: 4 1 7-23 .

98. Konturek SJ. Kwiecien N. Obtulowicz W. Bielan,1-.i W. Oleksy J. Schally AV. Effect, of ,o­m,ttostatin- 1 4 and som,itostatm-28 on plasma hormonal and g.i,tric secretory re,pon,es to ce­phalic and gastrointestinal stimulation in man. Scand J Ga,troenterol 1985; 20: 3 1 -8.

99. Loud FB . Hobt JJ. Egen,e E. Petersen B. Christiansen J. h ,omatmtatin a lrnmoral regula­tor of the endocrine pancreas and gastric acid secretion in man'! Gut 1985: 26: 445-9.

l !Kl. Ligum,ky M. Goto Y. Dcba, H. Yam.ida T. Prostaglandins mediate inhibition of gastric acid secretion by ,omato,tatin in the rat . Science 1983: 2 1 9 : 30 1 -3.

IO I . Feldman M. Col tun TJ . Effect of indomethacin on gastric .icid and bicarbonate secretion in human,. Gastrocnterolog} 1 984: 87: 1 339-43.

I02. Mogartl MJ-1. Maxwell V. Kovacs T. van Devcntcr G. Elashnff JD. Yamada T. Kauffman GL. Walsh JH. Somatostatin inhibits gastric acid secretion after gastric muco,al pro,ta­glandin ,ynthe,i, inhibition by indomcthacin in man. Gut 1985: 26: 1 1 89-9 1 .

103. B6ttcher W. Yamada T. Klcibeuker J. Kauffman GL. About the function of somatostatin and s.:crctin a, enteroga,trone, in the dog. Z Gastrocnterologie 1984: 22: 479.

104. Chri,ten,en KC. Specific beta-adrenergic mechanisms in the hypoglycaemic activation of gastrin and gastric acid secretion . Scand J Gastroenterol 1984; 19: 339-42.

I05. Tatsuta M. Baba M. ltoh T. Increased gastrin secretion in patients with phaeochromocyto­ma. Gastrocntcrology 1 983: 84: 920-3.

!06. Feldman M. Walsh J H. Taylor IL. Effect of naloxone and morphine on gastric acid secretion and on scrum gastrin and pancreatic polypeptide concentrations in humans . Gastroentcrolo­gy 1980: 79: 294-8.

!07. Feldman M. Cowley YM Effect of an opiate antagonist (naloxone) on the gastric acid secre­tory response to ,ham feeding. pentaga,trin and histamine in man. Dig Dis Sci I 982: 27: 308-IO.

108. Skov Oben P. Kirkegaard P. Petersen B. Lendorf A. Christiansen J. The effect of a synthetic mct-cnkcphalin analogue (FK 33-824) on gastric acid secretion and scrum gastrin in man. Scand J Gastrocntcrol 198 1 : 1 6: 53 1 -3.

109. Feldman M. Li CH. Effect of beta-endorphin on gastric acid secretion and scrum gastrin con­centration in human,. Regul Pept 1982; 4: 3 1 1 -5 .

I IO. Dolva LO. Hanssen KF. Aadland E. Sand T. Thyrotropin-releasing hormone immunorcacti­vity in the gastrointestinal tract of man. J Clin Endocrmol Metab 1983 ; 56: 524-9.

1 1 1 . Dolva LO. Hanssen KF. Bcrstad A. Frey HMM. Thyrotropin-releasing hormone inhibits the pentagastrin stimulated gastric secretion in m,m. A dose response Mudy. Clin Endocrinol 1979; 10 : 28 1 -6.

1 12 . Dolva LO. Hansscn KF. Flaten 0. Sl-.are S. Schrumpf E . Effect of thyrotropin-releasing hor­mone on gastric .icid secretion in man . Scand J G,1stroentcrol 1982: 1 7 : 775-80.

1 1 3. Kraenzlin ME. Ch"ng JLC. Mulderry PK. Ghatci MA. Bloom SR. Infusion of a novel pepti­de. calcitonin gene-related peptide (CGRP) in man . Pharmacokmctic, and effects on gastric acid secretion and on gastrointestinal hormones . Rcgul Pept 1985 , IO: 1 89-97 .

1 1 4. Bicberdorf FA. Gray TK. Walsh JH. Fordtran JS. Effect of calcitonin on meal-stimulated gastric acid secretion and scrum gastrin concentration. Gastroenterology 1974; 66: 343-6.

1 1 5 . Tachc Y. Goto Y. Gun ion M. Rivier J. Dcbas H. Inhibition of gastric acid secretion in rats and in dog, by corticotropin-releasing factor. Gastroenterology 1984; 86: 28 1 -6.

1 1 6. Elder JB. Ganguli PC. Gillespie IE. Gerring EL. Gregory H. Effect of urogastronc on gastric secretion and plasma gastrin level, in normal subjects. Gut 1975; 16 : 887-93 .

1 1 7. Robert A. Kane G. Recle SB. Dose response inhibition in man of meal-stimulated gastric acid secretion by lS(R)- 1 5-mcthyl pro,taglandin E�. given orally. Gut 198 1 ; 22: 728-3 1 .

1 18. Reele SB. Bohan D. Oral antisccrctory activity of prostaglandin E1 in man. Dig Dis Sci 1984; 29: 390-3.

1 19 . Levine RA. Schwartze! EH. Effect of mdomethacin on basal and histamine stimulated hu­man gastric acid secretion. Gut 1984; 25: 7 1 8-22.

1 20. Minuz P. Cavallini G. Bracco G. Degan M. Jeunct F. Kunovits G. Riela A. Velo GP . Effect of carprofen and indomethacin on gastric function and the content of pro,taglandins E1 and F1 • in human gastric juice. Hcpatogastroenterol 1 986; 33: 20-2 .

1 7

CHAPTER 2

REGULATION OF GASTRIC EMPTYING IN

MAN: PHYSIOLOGY AND EFFECTS OF

MEDICAL AND SURGICAL THERAPY.

AN UPDATE

J. H. Kleibeuker, C. B. H. W. Lamers

Department of Gastroenterology, University Hospital, Groningen; Depart­ment of Gastroenterology, University Hospital, Leiden , The Netherlands.

The major role of the stomach in the digestive process is exerted by its mo­toric function. Motility and emptying of the stomach have been studied exten­sively in animals and men. Due to the development of modern investigational methods our understanding of the physiology and pathophysiology of the gas­tric motoric function has greatly increased during the past few decades. In this article an update will be given about the regulation of gastric emptying in man. Although animal studies have contributed a great deal to our knowledge and understanding of gastric motility , this article will deal largely with results from studies in humans, while results from animal studies will be considered to a lim­ited extent only. In addition, the effects of medical and surgical therapy on gas­tric emptying will be mentioned where appropriate. New gastrokinetic drugs and new surgical techniques have been based on the better understanding of gastric physiology on the one hand , while on the other hand the effects of medi­cal and surgical interventions on gastric motility have greatly contributed to our knowledge of gastric function.

After a meal food is stored in the stomach and is then permitted to pass into the duodenum in small portions. Before being emptied digestible solid material is grinded and triturated to very small particles. This sequence is aimed to opti­mize the intestinal digestive and absorptive process. The stomach can be divid­ed into three functional motor regions: the fundus plus proximal corpus, in which food is stored and which has a major role in maintaining the gastroduode­nal pressure gradient; the distal corpus and the antrum, which have the main role in the grinding of food; and the gastroduodenal junction with the pylorus, which has an important role in the final emptying. The mechanisms through which the different components of food, namely liquids , digestible solids and indigestible solids , are emptied, are markedly different. The rate at which a

19

meal is emptied from the stomach is dependent on its composition, weight and volume, and in case of a solid meal on the size of the solid particles.

The gastric emptying of liquids is mainly a function of the proximal stomach ( 1 ) and, in addition, of the pylorus, whereas the antrum has only a minor role. When food is swallowed and enters the stomach the proximal part of it relaxes, so that despite the great increase of the intragastric volume the intragastric pressure is only modestly elevated, the so-called receptive and adaptive relaxa­tion (2). The emptying of liquids is largely determined by the pressure gradient between the stomach and the proximal duodenum (3). When the stomach is filled with normal 0. 1 5 M saline the emptying follows a monoexponential pat­tern ( 4), so with decreasing intragastric volume and intragastric pressure the emptying rate also decreases. However, when the liquid meals are caloric, acid­ic and/or are non-isotonic the emptying rate is slowed and a more linear pat­tern is observed (4, 5, 6). This is mediated through a decrease in fundic pressure on the one hand (5) and an increase in pyloric pressure on the other hand (7, 8). The importance of the pylorus for the emptying of liquids is exemplified by the effect of operations which disturb pyloric function. This was especially shown by Clarke and Alexander-Williams (9) who found that the addition of a pyloro­plasty to a highly selective vagotomy (HSY) significantly increased the empty­ing rate of a liquid meal, compared to HSY alone.

The emptying of digestible solid food is mainly a function of the distal part of the corpus and the ant rum ( 1 ) . The entering of solid food into the stomach initi­ates the postprandial pattern of regular circular contractions in the second part of the stomach, with a frequency of three to four cycles per minute ( 1 0). Based on studies in animals and man the sequence as described below is assumed to take place (2, 1 1 - 13). The contractions propagate as a peristaltic wave from the corpus towards the pylorus. Proximally they do not occlude the lumen, propel­ling the contents partly into the distal antrum, which is then relaxed, and partly back into the corpus. Depending on the pyloric diameter and the pyloric and in­traduodenal pressures part of the liquids and small particles are emptied at this stage. When the peristaltic wave approaches the distal antrum the contractions become nearly occlusive, thereby grinding larger particles. At this time the py­lorus closes and the retained material is retropelled forcefully to the proximal antrum, so further triturating the particles and mixing the gastric contents. A new peristaltic wave has then already started again , and the whole sequence is repeated. This process of grinding and sieving solid food prevents large parti­cles to be emptied and only particles of 1 mm or less are allowed to enter the du­odenum ( 1 4). Although gastric exocrine secretions aid in the grinding process, they are not essential for it (14-16). As expected, meals mainly composed of large digestible particles are emptied more slowly than when particles are mainly small sized (17, 18).

20

The importance of an intact antral function in the grinding and sieving of sol­id food is illustrated by the results of studies in operated patients. Mayer et al (19, 20) found that in patients with an antrectomy plus vagotomy solid food was emptied rapidly, and that a substantial part of the emptied particles was larger than 1 mm. In patients with a vagotomy plus pyloroplasty they also found a rapid precipitous emptying of solid food, but there was no persistent defect in the process of grinding and sieving (20).

Not all authors have found an increased emptying rate for solids after antrec­tomy (21, 22). From the various studies it can be concluded that the effect ofrc­scctive surgery on gastric emptying is variable and unpredictable. Studies are in progress to better define the factors, which may play a role in postrcscctivc gas­tric motility. The same variable patterns of gastric emptying, as seen after rc­sectivc surgery, have been shown after gastric bypass operation for morbid obesity (23). The effects of rcsectivc and of other types of gastric surgery arc summarized in table I.

Table I . Effects of several surgical techniques on gaMric emptying.

Type of ,urgery

highly ,elective vagotomy (HSY) HSY + pyloropla,ty truncal/,clective

vagotomy + pyloropla,ty

antrectomy/distal ga,trectomy (± vagotomy)

gastric hypa�s

Gastric emptying rate liquids solid.�

i i i t t

i

variable. f to ! .

often early precipitous variable, f to ! . often early precipitous variable, j to ! . often early precipitous

� increase, - = unchanged, ! = decrease gastric emptying rate; - = not studied

Many investigators have observed that the emptying of solid food begins af­ter a certain lag period after ingestion (24-26). It is assumed that this period is necessary for the grinding of food to sufficiently small-sized particles. How­ever, other authors did not find such a pattern and they think that the observa­tion of a lag phase is due to technical errors in the registration of the emptying (27). Differences between the test meals used may also contribute to the dis­crepancies in respect to the lag phase. Most authors agree that the emptying of solid food follows a nearly linear pattern.

21

Indigestible solid particles with a diameter more than 1 mm are largely re­tained during the normal postprandial emptying process. These particles are emptied during the interdigestive periods (28, 29), during which the gastric mo­tility follow� the pattern of the so-called interdigestive motor cycle (IMC) (30). During phase I of this cycle there is little peristaltic activity in the distal stom­ach. During phase II this activity gradually increases, while in phase III regular bursts of peristaltic waves are observed. The contractions are almost occluding from the start in the corpus and sweep any remaining solid debris out of the stomach. When after a meal the stomach has emptied most of its digestible con­tents there is a gradual transition of the postprandial motility pattern to that of phase II of the I MC.

The volume and the composition of ingested food are major determinants of gastric emptying rate. With increasing volume of a meal, liquid as well as solid (31, 32), the absolute emptying rate in terms of mass emptied per unit of time, increases. On the other hand the gastric emptying is slowed by an increase in ca­loric density of the meal (4, 32, 33), in such a way that after meals with similar volumes but different caloric densities a constant amount of calories is deliv­ered into the duodenum per unit of time. Intraduodenal fat potently inhibits the gastric emptying rate (34). Nonhydrolyzable fat did .oot exert any inhibition on the rate of the emptying (35). So digestion of fat seems essential for its inhib­itory effect. Duodenal acidification (36) and an increased osmolar load of the duodenum (6) also inhibit gastric emptying. Instillation of alcohol into the stom­ach has been shown to slow gastric emptying too (37). The retardation of gas­tric emptying by all these factors is mediated through stimulation of receptors in the duodenal and jejuna( mucosa. Hunt and associates (38, 39) have hy­pothesized that the above mentioned variety of constituents and properties of the intraluminal contents of the proximal intestine might affect the intercellular spaces of the cells lining the enteric lumen through variable mechanisms. Shrinkage of these spaces might excite receptors on adj a cent endocrine cells or sensory nerve endings .

lntraluminal factors in more distal parts of the gastrointestinal tract can also affect gastric emptying. Infusion of fat into the ileum ( 40) and painless rectal distention by a balloon (41) both retard the emptying rate.

The pathways through which the above described regulatory mechanisms of gastric motility and emptying are mediated have not yet been fully elucidated. Hormonal and intrinsic and extrinsic neural mechanisms are involved. The ex­trinsic innervation of the stomach is provided by the vagal and sympathetic nerves. In addition the stomach wall has an extensive intrinsic nervous system. The neural regulatory mechanisms of gastric motility are the result of a fine in­terplay between these interdependent systems. Information from a variety of

22

sensory neurons influences gastric function via intragastric intrinsic reflexes and via extrinsic, e.g. vagovagal, reflexes. In addition, other substances like circulating hormones may also affect the gastric nerves.

The extrinsic innervation of the proximal stomach is important for the above mentioned receptive relaxation after a meal. Subjects with a proximal gastric vagotomy show a markedly increased intragastric pressure after distention of the fundus, whereas rhytmic contractions are reduced in the proximal stomach ( 42). This abnormality results in an increased emptying rate for liquids in these patients (9, 43).

Several neurotransmitters are involved in the neural regulation of proximal gastric motility. Acetylcholine is an important transmitter for induction of contractions in the fundus. Relaxation of the proximal stomach is mediated through a vagal noncholinergic pathway. Dopamine (3) and particularly vaso­active intestinal peptide (VIP) (44) are candidates to function as neurotrans­mitter in the neurally induced fundic relaxation. Bombesin is another neuro­peptide shown to retard the emptying of liquids (45). Thyrotropin releasing hormone (TRH) has been found to increase fundic pressure, while reducing its rhytmic contractions similar to the effect of proximal gastric vagotomy (46). Both these substances are present in the gastric wall and may have a regulatory role in the motility of the proximal stomach.

As is the case in the proximal stomach a number of neurotransmitters have a role in the neural regulation of the motility of the distal stomach. In addition to acetylcholine substances like noradrenaline, dopamine, YIP, bombesin and endogenous opiates (47) may also be involved as neurotransmitter or neuro­regulator. Locally produced paracrine substances like prostaglandins ( 48) and histamine (49, 5 0) may be of importance too.

Several drugs act upon gastric motility and gastric emptying by interacting with neural regulatory mechanisms. The cholinergic drug bethanechol in­creases the number of antral contractions and the overall antral activity (5 1). Beta-adrenergic agonists and antagonists respectively retard and accelerate gastric emptying (5 2). The most extensively studied drugs in this respect are the gastrokinetic drugs metoclopramide (5 3) , domperidone (5 4) and, more recent­ly, cisapride. They all stimulate gastric motility and gastric emptying and are being used clinically in patients with gastric stasis due to a variety of disorders. Metoclopramide, the eldest of the three, affects the stomach through several mechanisms. It has a cholinergic effect, which is critically dependent on the presence of acetylcholine stores (5 5 ), probably by causing an increase of acetyl­choline release and by sensitizing the muscarinic receptors. In addition to this , metoclopramide has antidopaminergic properties (5 6). The results are a de­crease in the receptive relaxation, an increase in the size of antral contractions

23

and an improvement in the motoric gastroduodenal coordination (5 1, 57), thus causing an acceleration of gastric emptying. By crossing the blood brain barrier metoclopramide has also central nervous system effects. These contribute to its antiemetic property.

Domperidone is a specific dopamine Di-receptor antagonist. In contrast to metoclopramide it does not readily cross the blood brain barrier. Like metoclo­pramide domperidone decreases the receptive relaxation, stimulates antral peristaltic activity and gastroduodenal coordination (58, 59) and increases the gastric emptying rate. Cisapride is a recently developed drug which affects gas­tric motility probably through facilitation of acetylcholine release from the my­enteric nerves. Like the other two drugs it stimulates antral motility, improves gastroduodenal coordination and accelerates gastric emptying (59, 60).

The stimulatory effects of these three gastrokinetic drugs on gastric emptying are especially pronounced in patients with gastric stasis. In subjects with a nor­mal gastric motoric function the increase of gastric emptying rate by these sub­stances is only small and often not demonstrable (61 -65) .

Several gastrointestinal hormones affect the gastric emptying rate of solid and/or liquid food. Pentagastrin has been observed to retard gastric emptying (66 , 67). However, infusion of physiologic doses of the naturally occurring gas­trin-17 does not affect the emptying rate ( 45). Secretin and cholecystokinin are peptide hormones, which are produced and secreted by specific endocrine cells in the upperintestinal mucosa , respectively the S-cells and the I-cells. Secretin is released by duodenal acidification. There is strong evidence that this hor­mone inhibits gastric emptying at physiologic plasma concentrations (68-70). Cholecystokinin is released by intraduodenal fat and amino acids. Two recent studies have shown, that physiologic doses of cholecystokinin markedly slow the emptying of a test meal (71 , 72) which makes it very likely that cholecystoki­nin is a physiologic hormonal mediator of fat-induced inhibition of gastric emp­tying. Both secretin and cholecystokinin seem to affect the emptying rate by a decrease in fundic pressure and an increase in pyloric pressure (73-76). Neuro­tensin and peptide YY are produced and secreted by endocrine cells in the more distal bowel. Both have been shown to be able to slow gastric emptying (77, 78) and they may have some physiological role in the regulation of emptying through pathways originating in the lower gastrointestinal tract. A possible role of somatostatin in gastric motility has not yet been defined. In a recent study it has been shown that somatostatin increases the emptying rate of liquid meals, suggesting that somatostatin may have a role in opposing the action of other peptides known to delay gastric emptying (79). An other gut hormone, motilin, has also been shown to accelerate gastric emptying under certain conditions (80). It is thought however that motilin has some role in the regulation of the in-

24

terdigestive motor cycle, rather than in postprandial gastric emptying (8 1 ). Ta­

ble II summarizes the effects of different hormones and of other previously mentioned substances on gastric emptying.

Table 1 1 . GaMric emptying: stimulatory and inhibitory determinants and mechanisms.

Determinant

Food ,tim: quantity j inh: caloricden,ity r

l o,molarity • fat pH ! alcohol particle ,iLe j

Neuronal ,tim: acetylchol ine inh: dopamine

noradrenaline VIP

homhe,in cndogenou, opiate,

Paracrine inh: pro,taglandin,

hi,taminc"'

Hormones ,t im: �omato,tat in?

mot i l in'' inh: ga,trin'!

,ecretin cholccy,tokinin neurotensin? peptide YY''

Drugs slim: cholinergics

8-adrenergic antagonish metoclopramide

domperidone ci,apride

inh: anticholinergic, 8-adrenergic agonist levo-dopa

Mcchanbm

intragaMric pressure j duodenal receptor,. hormonal (secrettn . cholccystokinin) neuronal? ')

antropyloric sieve

contractions i fundic relaxation j .

contractions ! ? ., fundic relaxation j ? .,

'! .,

')

'! contractions l ,, fundic relaxation j . pyloric pressure l '! .,

like acetylcholine - noradrenaline acctylcholine release r . - dopamine ++dopamine acetylcholine release j - acetylcholine like noradrenaline l ike dopamine

Mim - stimulating. inh = inhibiting gaMric emptying: ? = not known ; ++ = antagonizing

25

In summary, the major role of the stomach in the digestive process is exerted

by its motoric function. The regulation of gastric motil ity is the result of a fine

interplay of the intrinsic and extrinsic neural system and of circulating gastroin­

testinal hormones. These regulatory mechanisms are greatly affected by the

physical and chemical properties of intragastric and intraintestinal contents .

Several drugs have been developed which increase gastric motility and are

being used in patients with gastric stasis . Gastric surgery can disturb the normal

gastric emptying process to a large extent.

REFERENCES 1. Kelly KA. Gastric emptying of liquids and solids; role of proximal and distal stomach. Arn J

Physiol 1980: 239: G71-G76. 2. Kelly KA. Motility of the stomach and gm,troduodenal junction. In Phy�iology of the Gastro­

intestinal Tract. LR Johnson (ed). Raven Pre�s. New York 1981. pp 393--H 0. 3. Valenzuela JE. Liu DP. The effect of variation� in intraga�tric pre��urc and ga�tric emptying

of a saline meal in humans. Scand J Gastroenterol 1982: 17 : 293-296. -1 . Brener W, Hendrix TR. McHugh PR. Regulation of the ga�tnc emptying of gluco�e. Ga�­

troenterology 1 983 ; 85: 76-82. 5. Dooley CP, Reznick JB. Valenzuela JE. Variations in ga�tnc and duodenal motility during

gastric emptying of liquid rneab in human�. Ga�troenterology 198-1; 87: 1 1 1-1- 1 1 19 . 6 . Meeroff JC. Go VLW, Phillip� SF. Control of gastric emptying hy o�rnolality of duodenal con­

tents m man. Gastroenterology 1975; 68: 1 14-1- 1 1 5 1 . 7 . White CM. Poxon V. Alexander-William� J . Effect� of nutrient liquids on human gastrodu­

odenal motor activity Gut 1983 ; 2-1; 1 109- 1 1 1 6. 8. Dooley CP, Reznick JB. Valenzuela JE. A contmuou� rnanornetric �tudy of the human pylo­

rus. Gastroenterology 1985; 89: 82 1-826. 9. Clarke RJ , Alexander-William� J. The effect of pre�erving antral innervation and of a pylor­

oplasty on gastric emptying after vagotorny in man. Gut 1973; 14: 300-307. 10 . Rees WD, Go VLW, Malagelada J-R. Antroduodenal motor response to solid-liquid and ho­

mogenized meals. Gastroenterology 1979; 76: 1-138- 14-12. I I . Weisbrod! NW. Basic control mechanism�. In Gastric and gastroduodenal motility . Akker­

rnans LMA. Johnson AG, Read NW (eds) . Praeger Publishers . New York 1984, pp 3-20. 12. Ehrle in HJ , Akkerrnans LMA . Gastric emptying. In Ga�tnc and gastroduodenal motility. Ak­

kerrnans LMA, Johnson AG. Read NW (eds). Praeger Publishers . New York 1984, pp 74-84. 1 3 . Schulze-Delrieu K . Ehrlein HJ . Blum AL Mechanb of the pylorus. In Gastric and gastrodu­

odenal motility. Akkerrnans LMA. Johnson AG. Read NW (eds). Praeger Publishers. Ne\\ York 1 984, pp 87- 102.

1 4. Meyer JH. Ohashi H. Jehn D. Thomson JB. S1zeof livcr particles ernptied from the human sto­mach. Gastroenterology 1 98 1 : 80: 1-189-1496

1 5. Ohashi H, Meyer J H. Effect of peptic dige�tion on emptying of cooked liver in dogs. Gastroen­terology 1980; 79: 305-3 1 0.

16. Arnold JG, Dubois A. In vitro studies of mtragastric digestion. Dig Dis Sci 1983; 28: 737-74 1 . 1 7. Weiner K , Graham LS. Reedy T. Elashoff J , Meyer JH. Sirnultaneous gastric ernptying of two

solid foods. Gastroenterology 198 1 ; 8 1 : 257-266. 18 . Holt S. Reid J. Taylor TV. Tothill P, Heading RC. Gastric emptying of solids in man. Gut

1982; 23: 292-296. 19. Mayer EA, Thomson JB, Jehn D. Reedy T. Elashoff J. Meyer JH. Gastric emptying and sie­

ving of solid food and pancreatic and biliary secretion after solid meals in patients with truncal vagotorny and antrectorny. Gastroenterology 1982; 83: 184-1 92 .

20. Mayer EA, Thomson JB. Jehn D. Reedy T, Elashoff J , Deveny C. Meyer JH. Gastric empty­ing and sieving of solid food and pancreatic and biliary secretions after solid meals in patients with nonresective ulcer surgery. Gastroenterology 1 984; 87: 1 264- 1 27 1 .

26

2 1 . Kalbasi H. Hudson FR. Herring A, Moss S. Glass HI . Spencer J . Gastric emptying following vagotomy and antrectomy and proximal gastric vagotomy. Gut I 975; I 6: 509-5 13.

22. Smout AJPM. Akkcrmans LMA. Roelofs JMM. Pasma FG. Oci HY. Wittcbol P. Gastric emptying in Billroth I I (Bi l ) patients: relationships with postcibal symptoms and comparison with duodenal ulcer patients (abstract). Neth J Med 1986; 29: 25 1 .

23. Horowitz M . Cook DJ . Collins PJ. Harding PE. Hooper MJ . Walsh JF. Shearman DJC. Measurement of gastric emptying after gastric bypass surgery using radionuclidcs. Br J Surg 1982; 69: 655-657.

2-1. Jacobs F. Akkermans LMA. Oci HY. HockMra A. Wittebol P. A radioisotope method to quantify the function of fundus. antrum and their contractile activity in gastric emptying of a se­mi-solid and solid meal. In Motility of the Digestive Tract. M Wienbeck (ed). Raven Press, New York 1982, pp 233-240.

25 . Collins PJ . Horowitz M. Cook DJ . Harding PE. Shearman DJC. Gastric emptying in normal subjects - a reproducible technique u�ing a �ingle �cintillation camera and computer system. Gut 1983: 24: 1 1 17-1 1 25.

26. Camilleri M. Malagelada J-R. Brown ML, Becker G. Zin,mei,ter AR. Relation between an­tral motility and gastric emptying of solids and liquid, in humans. Am J Phy�iol 1985; 249: G580-G585.

27. Moore JG, Chri>tian PE. Taylor AT. Alazraki N. Ga,tric emptying measurements: delayed and complex emptying pattern, without appropriate correction. J Nucl Med 1985; 26: 1 206-1 2 10.

28. Jian R, Assael T, Grall Y, Romary D, Jobin G. Valleur P. Dhamlincourt A-M, Bernier J-J . A comparative ,tudy of ga,tnc emptymg of digestible solid, and inert particles. in healthy and du­odenal ulcer patient�. Gastroenterol Clin Biol 1983; 7: 272-276.

29. Feldman M. Smith HJ . Simon TR. Gastric emptying of solid radiopaque markers: studies in healthy ,ubjccb and diabetic patients. Ga,troenterology 1984; 87: 895-902.

30. Sarna SK. Cyclic motor activity: migrating motor complex: 1985. Gastroenterology 1985 ; 89: 894-9 13.

3 1 . Hunt JN. Smith JL, J iang CL. Effect of meal volume and energy density on the gastric empty­ing of carbohydrates. Gastroenterology 1985 ; 89: 1326- 1 330.

32. Moore JG. Christian PE, Brown JA , Brophy C. Datz F. Taylor A. Alazraki N. Influence of meal weight and caloric content on gastric emptying of meals m man. Dig Dis Sci 1984; 29: 5 1 3-5 19.

33. Hunt JN . Stubbs DF. The volume and energy content of meals as determinants of gastric emp­tying. J Physiol 1975 ; 245: 209-225.

34. Collins P J. Heddie R. Horowitz M, Read NW. Dent J. Chatterton BE. The effect of intradu­odenal lipid on gastric emptying and intragastric distribution of a solid meal (abstract) . Gas­troenterology 1986; 90: 1 377.

35. Cortot A, Phillips SF, Malagelada J-R. Parallel gastric emptying of nonhydrolyzable fat and water after a solid-liquid meal in humans. Gastrocnterology 1982; 82: 877-88 1 .

36. Hunt J N . Knox MT. The slowing of gastric emptying by four strong acids and three weak acids. J Physiol 1972; 222: 1 87-208.

37. Jian R. Cortot A. Ducrot F. Jobin G, Chayvialle JA. Modigliani R. Effect of ethanol ingestion on postprandial gastric emptying and secretion. biliopancreatic secretions and duodenal ab­sorption in man. Dig Dis Sci 1986; 3 1 : 604-6 14.

38. Barker GR. Cochrane GMcL. Corbett GA, Dufton JF, Hunt JN . Kemp Roberts S. Glucose, glycine and diglycine in test meals as stimuli to a duodenal osmoreceptor slowing gastric empty­ing. J Physiol 1978; 283: 34 1 -346.

39. Hunt JN. Docs calcium mediate slowing of gastric emptying by fat in humans? Am J Physiol 1983; 244: G89-G94.

40. Read NW, McFarlane A. Kinsman RI , Bates TE. Blackhall NW. Farrar GBJ , Hall JC, Moss G, Morris AP, O'Neill B, Welch I, Lee Y. Bloom SR. Effect of infusion of nutrient solutions into the ileum on gastrointestinal transit and plasma levels of neutrotensin and enteroglucag­on. Gastroenterology 1984; 86: 274-280.

4 1 . Youle MS. Read NW. Effect of painless rectal distention on gastrointestinal transit of solid meal. Dig Dis Sci 1984; 29: 902-906.

27

42. Stadaas JO Intragastric pre,,ure/volume relation,hip hefore and after proximal ga,tnc vago­tomy. Scand J Ga,troenterol 1975: 1 0: 1 29- 1 34.

43. Lavigne ME. Wiley ZD. Martin P. W.iy LW. Meyer JH. Slei,enger MH. MacGregor IL. Ga,­tric. pancn:atic and hiliary M:cretion and the ratc of ga,tric emptying after parietal cell vagoto­my. Am J Surg I 979: 1 38: 644-65 1 .

44. Grider JR . Ca hie MB. Said SI. Mah.hlouf GM. Vasoacti, e intc,tinal peptide a, a neural medi­ator of ga,tric relaxation Am J Phy,iol 1985 : 248: G73-G78.

45. Wal,h JH. Max\\ ell V. Ferrari J . Varner AA. Bornhe,in ,tirnulate, human ga,tric function hy ga,tnn-dependent and independent rnechani,m,. Peptide, 1 98 1 : 2. ,uppl 2: 1 93-198.

46. Doi vi LO . Stada,I', JO Action� of thyrotropin rclea,ing hormone on ga,trointc,tinal funct ion, in man. Scand J Ga,troenterol 1979: 14: 419-423.

47. Edin R. Lundherg J . Tcreniu, L. Dahbtrorn A. Hoh.felt T. Kewenter J . Ahlman H. Evidence for vagal enkephahnerg1c neur.il control of the feline pyloru, and ,tomach. Ga�troenterolog) 1 980: 78: 492-497.

48. Sander, KM. Role.: of pro,taglandins in regulating ga,tnc rnot1hty. Am J Phy,iol 1984: 247: GI 1 7-G i 26.

49. Ricci D. Lange R . Mag) ar L. McCall um RW. Effect of hi,tamine receptor ,tirnulation on gas­tric empt) ing in man Clin Re, 1 983 : J I : 682A.

50. Du hob A. CaMell DO. Histamine H,-receptor involvement in the regulation of gm,tric ernpty­ing. Arn J Phy,iol 1986: 250: G244-G247.

5 1 . Fox S. Behar J. Pathogenesi, of diabetic gastropare,i,: a phMmarnlogic ,tudy. Ga,trocntero­logy 1980: 78: 757-763.

52. Rees MR. Clark RA. Holdsworth CD. Barher DC. Howlett PJ . Thc effect of B-adrenoceptor agonists and antagoni.t, on g.i,tnc emptying in man. Br J Clin Pharmac 1980: IO : 55 1 -554.

53. Albibi R. McCallum RW. Metocloprarnide: pharmacology and clinical application. Ann Int Med 1 983: 98: 86-95.

54. Brogden RN. Carmine A A . Heel RC. Spc1ght TM. A, ery GS. Dornperidonc . Drugs 1982 : 24: 360-400.

55 . Hay AM. Man WK. Effect of rnetoclopramide on guinea pig ,tomach. Ga,troenterology 1979: 76: 492-496.

56. Berkowitz DM. McCall um R W Interaction of levodopa and metoclopramide on gastric emp­tying. Clin Pharmacol Ther 1980: 27: 4 1 4-420.

57. Johnson AG. The actmn of rnetoclopramide on human gastroduodenal motility. Gut 197 1 : 1 2: 42 1 -426.

58. Weihrauch TR. Ehl W. Effect of dornperidone on the motility of antrurn. pyloru, and duode­num in man. Scand J Gastroenterol 198 1 : 16. suppl 67: 195- 1 98.

59. Schuurkes JAJ. Van Nueten JM. Control of ga,troduodcnal coordination: doparninergic and cholinergic pathways. Scand J Gastroenterol 1 984: 19 . suppl 92: 8- 1 2.

60. Baeyens R . Reyntjes A. Verlinden M. Ci,apride accelerate, ga,tric emptying and mouth-to­caecum transit of a barium meal. Eur J Clin Pharm 1984; 27: 3 1 5-3 1 8 .

6 1 . Hancock BD. Bowen-Jones E. Dixon R. Dymock IW. Cowley DJ. The effect of metoclopra· mide on gastric emptying of solid meab. Gut 1974: 1 5 : 462-467.

62. Metzger WH. Cano R. Sturdevant RAL. Effect of metoclopramide in chronic gastric retention after gastric surgery. Gastroenterology 1 976: 7 1 : 30-32.

63 Behar J . Ramsby G. Gastric emptying and antral motility in reflux e,ophagitis. Gastroentero­logy 1 978; 74: 253-256.

64. Akkerrnans LMA. Jacobs F. Oei HY. Wittebol P. Gastric emptying. function of proximal and distal stomach and the effect of peripheral dopamine blockade (abstract). Gastroenterology 1 983 ; 84: 1 089.

65. Jian R, Ducrot F. Piedeloup C. Mary JY. Na jean Y. Bernier JJ. Measurement of gastric emp· tying in dyspeptic patients: effect of a new gastrokinetic agent (cisapride) . Gut 1985: 26: 352-358.

66. Hamilton SG, Sheiner HJ , Quinlan MF. Continuous monitoring of the effect of pentagastrin on gastric emptying of solid food in man. Gut I 976; 1 7: 273-279.

67 McGregor IL. Wiley ZD, Martin PM. Effect of pentagastrin infusion on gastric emptying rate of solid food in man. Dig Dis Sci 1 978: 23: 72-75.

28

68. Valenzuela JE , Defillipi C. Inhibition of gastric emptying in humans by secretin, the octapep­tide of cholecystokinin and intraduodenal fat. Gastroenterology 1 98 1 ; 8 1 : 898-902.

69. Kleibeuker JH , Eysselein VE, Maxwell V, Walsh JH . Role of endogenous secretin in acid-in­duced inhibition of human gastric function . J Clin Invest 1984; 73: 526-532.

70. Kleiheuker JH, Beekhuis H, Piers DA, Schaffalitzky de Muckadell 0. Retardation of gastric emptying of solids hy secretin in man (ahstract). Neth J Med 1986; 29: 148.

7 1. Liddle RA, Morita ET, Conrad CK, Williams JA . Regulation of gastric emptying in humans hy cholecystokinin. J Clin Invest 1986: 77: 992-996.

72 . Kleiheuker JH, Beekhuis H , Jansen JBMJ , Lamers CBHW, Piers DA. Infusion of physiologic doses of cholecystokinin inhihits gastric emptying of food in man (ahstract). Gastroenterology 1986; 90: 1495.

73. Yamagishi T, De has HT. Cholecystokinin inhibits gastric emptying by acting on both proximal stomach and pylorus. Am J Phy�iol 1978: 234: E375-E378.

74. Geller LI , Petrenko VF. Effect of ,ecretin and pancreozymin on intracavitary pre��ure in the ,tomach and duodenum, evacuation from the �tomach. and the tone of the pyloric sphincter. Fiziologiya Cheloveka 1980; 6: I 28- 1 32.

75 . Scarpignato C, Zimbaro G, Vitulo F, Bertaccini G. Caerulein delay� gastric emptying of solids in man. Arch Int Pharmacodyn 1 98 1 : 249: 98- 105 .

76. Phao�awa,di K. Fi,her RS. Hormonal effects on the pyloru�. Am J Phy�iol 1 982; 243: 0330-0335 .

77 . Blackburn AM, Bloom SR, Long RO, Fletcher DR, Christofides ND. Fitzpatrick ML, Baron JH. Effect of neuroten�in on gastric function m man. Lancet 1980; I : 987-989.

78. Allen JM, Fitzpatrick ML, Yeat� JC, Darcy K. Adrian TE, Bloom SR. Effects of peptide YY and neuropeptide Y on ga,tric emptying in man. Digestion 1984; 30: 255-262 .

79. Mogard M, Maxwell V, Van Deventer G, Elashoff J, Yamada T, WalshJH. Somatostatin en­hances gastric emptying of liquid meals in man (abstract) . Gastroenterology 1984; 86: 1 1 86.

80. Chrbtofides ND, Long RO, Fitzpatrick ML, McGregor GP. Bloom SR. Effect of motilin on the ga�tric emptying of glucose and fat in humans. Gastroenterology 198 1 ; 80: 456-460.

8 1 . Vantrappen G. Jans�ens J , Peeters TL, Bloom SR, Christofides ND, HellemansJ. Motilin and the interdigestive migrating motor complex in man. Dig Dis Sci 1979; 24: 497-500.

29

CHAPTER 3

INTRA VENOUS HIST AMINE REDUCES

BOMBESIN-STIMULA TED GASTRIN RELEASE

IN DOGS

J. H. Kleibeuker, G. L. Kauffman, Jr., J. H. Walsh

Center for Ulcer Research and Education, Veterans Administration Wads­worth Hospital Center, University of California, Los Angeles, California, U.S.A.

ABSTRACT The effect of histamine on gastrin release was studied in 7 conscious mongrel

dogs with chronic gastric and duodenal fistulas. Histamine-2 HCI was infused in doses ofO (control), 20, 40, 80, and 160 µ.g.kg· 1 .h· 1 for2 h on separate days. Dur­ing the second hour, bombesin 500 ng. kg· 1 .h· 1 was infused intravenously. In­tragastric pH was constantly kept at 2.5 by intragastric titration during each test. Leakage of gastric contents into the duodenum was prevented by a prepy­loric balloon passed retrograde through a duodenal fistula. Gastrin release, as expressed by the integrated response during the last 50 min of the bombesin in­fusion, was significantly (P <0.05) decreased by all doses of histamine, com­pared to control. The infusion doses of histamine studied, 20, 40, 80, and 160 µ.g.kg· 1.h· 1 reduced bombesin-stimulatcd gastrin release 16%, 19%, 1 9%, and 30% , respectively. This effect was blocked by a histamine H-2 but not an H-1 receptor antagonist. We conclude that by an H-2 mechanism, exogenous hista­mine reduces bombesin-stimulated gastrin release in dog.

INTRODUCTION Histamine is one of the most potent physiological stimulants of gastric acid

secretion by the parietal cell ( 1). Local mast cells appear to be the main source of histamine in the fund us and corpus in man (2) and dog (3). The antral mucosa also contains histamine (2), though at a slightly lower concentration than the other parts of the stomach. The antrum plays an important role in the regula­tion of gastric acid secretion through antral mucosa! G-cells, the main source of circulating gastrin. Bombesin is a potent gastrin releasing peptide. A bombe­sin-like substance is present in the antral mucosa (4) and is thought to have a physiologic role in the regulation of gastrin release, possibly as a neurotrans-

31

mitter of some vagal nerve endings (5 , 6) . Feldman et al . (7) found that in man,

the H-2 receptor antagonist cimetidine increased gastrin release after a steak

meal , an effect which was independent of change in intragastric pH. This result

suggested an inhibitory effect of endogenous histamine, acting on H-2 recep­

tors , on food-stimulated gastrin release.

We studied the influence of intravenous histamine on bombesin-stimulated

gastrin release in conscious dogs, testing the hypothesis that exogenous hista­

mine administration causes a dose-related reduction in bombesin-stimulated

gastrin release independent of change in intragastric pH.

MATERIALS AND METHODS

Seven mongrel dogs, each weighing 20 kg, previously prepared with chronic

gastric and duodenal fistulas were studied. After an overnight fast , the dogs

were prepared with three intravenous lines, for the infusion of histamine and

bombesin and for blood sampling, respectively. A Foley catheter was passed

into the stomach retrograde through the duodenal fistula and the balloon was

inflated with 5 ml of water to prevent leakage of gastric contents to the duode­

num . A 200 ml solution 0. 10 M HCl titrated to pH 2.5 by the addition ofNaOH

was instilled into the stomach through the gastric fistula, and continuous perfu­

sion of the stomach with this solution carried out at a rate of 300 ml/min , using a

peristaltic pump (Harvard Apparatus) . The intragastric pH was kept constant

at 2.5 by continuous autotitration of the perfusion fluid using 0.5 M Na OH ti­

trant and did not vary more than 0.4 pH units from that endpoint. The volume

of stimulated gastric acid secretion was accommodated by placing a reservoir in

the system. Acid output was equivalent to the amount ofNaOH added per unit

time required to keep the pH constant. Studies were performed in each dog

with a minimum interval of two days between each test. After a 30-min period

of equilibration , an infusion of histamine-2 HCl (Vega) in 0. 1 5 M saline was

started . The doses of histamine on different test days were in random order: 20,

40, 80 and 1 60 µ.g. kg·' . h· ' . In control experiments, only 0. 1 5 M NaCl was given

intravenously. The dose of 80 µ.g.kg· 1 . h· 1 was combined on different days with

( 1 ) cimetidine (Smith , Kline and French) 10 mg/kg bolus + 5 mg.kg· 1 . h· 1 intra­

venous infusion 1 5 min prior to and during the infusion of histamine; (2) the H­

t receptor antagonist mepyramine (ICN Pharmaceuticals Inc.) 1 0 mg/kg intra­

muscularly 1 5 min prior to the start of the histamine infusion ; or (3) a combina­

tion of cimetidine and mepyramine in the above-mentioned doses. 60 min after

the start of the infusion of histamine, bombesin (Peninsula Laboratories) 500

ng. kg· 1 .h· 1 (in 0. 1 5 M NaCl , containing 1 % (v/v) dog serum) was begun and

continued for 60 min. This dose of bombesin has only a modest stimulatory ef­

fect on basal acid secretion in dog (8).

32

Two venous blood samples were taken during the pre-bombesin period and

one at I O min intervals during the bombesin infusion. The samples were collect­

ed in 1 0-ml tubes containing 1 0.5 mg EDT A and were kept on ice until centrifu­gation. Plasma was stored at -20°C until the assay was performed. Plasma gas­

trin concentrations were measured by a specific radioimmunoassay as de­

scribed previously (9) , using antibody 16 1 1 . Analysis of data. Mean acid output before bombesin was calculated as the

mean of the three J O-min periods just before bombesin infusion and the mean

acid output of the first three I 0-min periods was calculated during bombesin in­

fusion. The integrated responses of plasma gastrin during each test in each dog

during the last 50 min of the bombesin infusion were calculated. A paired com­

parison was made of the gastrin responses between each dose of histamine and

control studies, by the paired t-test. Differences were considered to be statisti­cally significant when P::;0.05.

RESULTS During all studies, the intragastric pH remained at 2.5. There was no spill of

the gastric contents into the duodenum since no fluid came through the open

duodenal fistula. The dogs did not exhibit any distress during the infusion of histamine at doses � 1 60 µ.g. kg· 1 . h· 1

• Gastric acid output increased with in­

creasing doses of histamine and was unaffected by bombesin infusion as shown

in Table I. During the infusion of all doses of histamine. there was a significant

inhibition of bombesin-stimulated gastrin release compared to control studies.

Table I . HCI output (mean ± S .E . ) µ.mol. lO min· ' for 30 min before (8) and during (D) bombesin infusion in each study (n)

B D

Control (8) 1 306 ± 275 1 1 27 ± 1 90 Hist 20 (4) 2763 ± 6 10 3266 ± 3 1 6 H ist 40 (8) 3482 + 43 1 324 1 ± 465 Hist 80 (8) 3800 ± 427 3678 ± 324 Hist 1 60 (8) 4336 ± 200 4252 ± 440 Hist 80 + M (8) 4079 ± 323 3583 ± 322 Hi�t 80 + C (8) 1 6 1 ± 8 1 292 ± 1 28 H i�t 80 + M + C (8) 0 0

Histamine infusion doses of 20, 40, 80, 1 60 µ.g.kg· 1 . h· 1 significantly reduced

bombesin-stimulated gastrin release 1 6% , 1 9% , 1 9% , and 30% , respectively

(Figs. 1 and 2). Higher doses of histamine were not studied because they are not

33

"i 2000

C: ·e 0 * E 1 600

* * C.

OJ * VI C: 0 C.

1 200 VI OJ

C: ·;: +-VI

C BOO C,

"O OJ +-C L. C, OJ

400 +-C:

e 0 1= � 0 lil ";!. 8

� :,: :,: :,: :,:

0

Figure I . Effect of histamine (H) 20. �0. 80. and 160 µg.kg 1 . h . compared to control on homhe�in-�tim­ulated (500 ng.kg· 1. h· 1) gastrin release (integrated gastrin response in pmol.min. l 1) . "P<0.05 by paired t analysis (mean ± S .E .). (n= 7).

'T 50

0 E ..9-

40 C: ·;: +-VI

C C,

30

20

10 t -60 0 20 40 60

time (m in )

Figure 2 . Time course of the effect o f histamine (80 µg.kg' 1 .h' 1) (::>) on mean ± S.E . bombesin-stimulated gastrin release (pmol .J· ') compared to control (•).

34

well tolerated. Cimctidine and the combination of cimetidine and mepyraminc

blocked the inhibitory effect of histamine on the gastrin release whereas mepy­ramine alone did not produce any effect (Fig. 3).

� 2000

C

-� 0

1 600 * a,

1 200 a,

C *

·.::::

800 Cl 1 a,

Cl a, 400 C

u :,:

§ g + + +

0 0 g a, a:, :,: :,: :,: :,:

0

Figure 3. Effect of cimctidine (C). mepyraminc (M). and cimetidine plus mcpyraminc (C + M) on the inhibi­tion of homhc�in �timulated ga�trin release hy hi�taminc (H,.1). 80 µg.kg · ' . h· ' (n=7) . ·P<0.05 hy paired t analysis (mean ± S .E.).

DISCUSSION

These observations suggest that the inhibition of bombesin-stimulated gas­

trin release, by histamine, may be a direct effect. Since the intragastric pH was kept constant, this inhibitory effect of histamine is not a function of antral acidi­

fication . In addition , with no leakage of intragastric contents into the duode­

num a duodenogastric neural or humoral negative feedback mechanism cannot explain these observations. Although gastric distention can cause gastrin re­

lease ( 10), the circulating fluid system used in these studies contains a reservoir which is designed to contain the added secretory volume during histamine ad­

ministration and minimize any change in intragastric pressure. The mechanism through which histamine inhibits gastrin release may be related to the H-2 re­

ceptor. Several authors ( 1 1 - 1 3) have recently reported a stimulatory effect of

cimetidine on gastrin release in the rat. In one study ( 1 1 ) another histamine H-2 receptor antagonist, YM- 1 1 1 70, did not influence plasma gastrin concentra­

tion . Since, in our study, histamine-induced inhibition of gastrin release by

35

bombcsin was blocked by cimetidine, it seems likely that in dogs, H-2 receptors arc involved.

Feldman et al. (7) reported that with the gastric luminal fluid kept constant at pH 5.0, cimetidinc (300 mg) increased the integrated gastrin response to a steak meal in humans from 6.63 to 9.80 ng.min/ml. This represents a 48% increase over control response and suggests a histamine H-2 mechanism which inhibits gastrin release. Our studies suggest a similar effect, a 30% reduction in inte­grated gastrin response to bombesin by simultaneously administering hista­mine. Our results however contrast to those reported by Schusdziarra (14) in which histamine H-2 receptor stimulation did not affect liver extract-stimulat­ed gastrin release in dogs. In the above-mentioned study a histamine H-2 re­ceptor agonist was given rather than histamine, gastrin release was produced by gastric luminal perfusion with liver extract rather than intravenous bombesin infusion and the intragastric pH was kept at 6.5 to 7 .0 rather than 2.5. The effect of histamine on gastrin release may be stimulant specific and pH dependent.

Based on the fact that histamine is present in mast ccHs in the antral mucosa (2), it may be hypothesized that histamine exerts its effect through a local mechanism, either directly on the gastrin producing G-cclls, or indirectly, via vagal nerve endings or the somatostatin producing D-cells. Schusdziarra et al. (15) reported that histamine may play a role in postprandial regulation of gas­tric somatostatin release in dogs. On the other hand Nandiwada et al. (16) found that, in dogs, histamine inhibited peripheral vagal transmission via a pre­synaptic action, as shown by the heart rate, and that this action was mediated through H-2 receptors. Since gastrin is partially under vagal control, the mech­anism of the inhibition of gastrin release by histamine may also be through an effect of histamine on the vagus more proximaHy.

In earlier papers, it has been reported that histamine may also have a role in the postprandial regulation of pancreatic somatostatin release (15) and in the release of pancreatic polypeptide (17) in dogs. Our findings and those of others suggest that histamine, in addition to its role in the stimulation of acid secre­tion, may also have a regulatory role in the release of gastrointestinal hormones. Further studies will be needed to define the physiological nature of these interactions and better define the mechanisms through which histamine may exert its effect.

REFERENCES I . Grossman Ml . Regulation of gastric acid secretion. In Phy�iology of the gastrointestinal tract.

LR Johnson (ed). Raven Press. New York. 198 1 . pp 659-67 1 . 2 . Mohri K . Reiman HJ . Lorenz W. Troidl H. Weber D . Hbtamine contentand mast cells in hu­

man gastric and duodenal mucosa. Agent� Action� 197 1 : 8: 372-375. 3. Soll AH. Lewin K . Beaven MA. bolat1on of hi�tamine-containing cell� from canine fundic

mucosa. Gastroenterology 1 979; 77: 1 283- 1 290.

36

-l . Walsh JH. Reeve JR. Jr. Vigna SR. Dbtributiun and molecular form, of mammali,111 humbe­,in . In Gut Hormone,. SR Bloom. JM Polack (eds). Churchill Livingstone. Edinburgh. 198 1 . pp -l 13--t l8 .

5. Martindale R. Kauffman GL. Levin S . Wabh JH. Yamada T. Differential regulation of gas­trin and ,omatmtntin relt:a,e from isol<1ted perfu,ed rat stomach,. Ga,troenterology 1982: 83: 2-l0-2-l4.

6. Modlin IM. Lamer, C. Walsh JH. Mechani,m of gastrin release by bomhesin and food. J Surg Re, 1980: 28: 539-546.

7. Feldman M. Richardson CT. Petersen WL. Walsh JH. Fordtran JS. Effect of low-dme pro­panthcline on food-stimulated gastric acid secretion. N Engl J Med 1977; 297: 1427- I-l30.

8 . Taylor I L. Walsh J H . Carter D. Wood J. Grossman Ml . Effects of atropine and bethanechol on bombcsin-stimulated release of pancreatic polypeptide and gastrin in dog. Gastroenterolo­gy 1979: 77: 7 14-7 18.

9. Ro,enqubt G L. Wabh JH. Radioimmunoa,,ay of ga,trin. In Gastrointe,tinal Hormone,. GB Jerzey Glass (ed) . Raven Pre,,. New York . 1980. pp 769-795.

IO . Debas HF. Walsh JH. Gros,man Ml . Evidence for oxyntopyluric reflex for release of ,intra! ga,trin. Ga,troenterolugy 1975; 68: 687-690.

1 1 . Ohe K, Nakamura M. Fujiwara T. Matsumoto H. Kohchi M. Miyoshi A . Effect ofH1-receptor antagonbh. cimetidinc Jnd YM- 1 1 1 70. on serum gastrin levels in lumen-perfused rats. Dig Dis Sci 1983; 28: 98 1 -989.

12 . Schu,dziarra V. Bender H . Pfeffer A . Pfeiffer EF. Modulation of acetylcholine-induccd secre­tion of gastric bombcsin-Iike immunoreactivity by cholinergic and hbtamine H1-reccptors. so­matostatin. and intragastric pH. Regul Pepi 198-t: 8: 189- 198.

1 3 . Tabata K. Oka be S. Gastric secretory condition, and pla,ma ga,trin lcveb in rat, after prolon­ged treatment with cimctidine. Dig Di, Sci 198-l ; 29: -l0--l5.

14 . Schusdziarra V. Stapelfeldt W. Klier M. Maier V. Pfeiffer EF. Effect of H1-rcceptor stimula­tion on postprandial pancreatic and ga,tric endocrine function in dog,. Re, Exp Med (Berl) 1982: 18 1 : 253-257.

1 5 . Schusdziarra V. Rouiller D. Harri, V. Unger RH. Role of histamine H,-receptor, in ga,tric and pancreatic rele.isc of somato,tatin-liJ..c immunoreactivity during the g,"tric phase of a meal. Regul Pept 198 1 ; 2: 353-363.

16 . Nandiwada PA. Lokhandwala MF. Jandhyala BS Modul,1tion by hi,tamine of peripheral va­gal tran,mi"ion in ane,thetized mongrel dog,. Eur J Pharmacul 1980; 63: 28 1-286.

1 7. Linn es tad P, Guldvog I , Schrumpf E. The effect of histamine, HI and H2 agonists and HI and H2 antagonists on postprandial pancreatic polypeptide release in dogs. Scand J Gastroenterol 1983; 1 8: 1 65- 1 68.

37

CHAPTER 4

EFFECT OF HISTAMINE Hr RECEPTOR

STIMULATION ON BOMBESIN- AND

PEPTONE-STIMULATED GASTRIN RELEASE

IN MAN

J. H. Klcibeukcr, H. Kooi, C. 8. H. W. Lamers

Departments of Gastroenterology and Nuclear Medicine, U nivcrsity Hospital, Groningen and Department of Gastroenterology, University Hospital, Lei­den, the Netherlands.

ABSTRACT This study was undertaken to determine whether histamine Hr receptors are

involved in the regulation of gastrin secretion in man. Since previous studies on the effect of histamine H2-receptor blockade on gastrin release are conflicting, we have studied the effect of histamine infusion (130 nmol/kg.h) with simulta­neous H 1-receptor blockade on gastrin release in healthy male subjects. Intra­gastric pH was maintained at 4 .5 by continuous intragastric titration during all studies. Histamine did not affect gastrin release stimulated by infusion of bom­bcsin (90 pmol/kg.h) or by a peptone meal. Integrated gastrin secretion during bombesin plus histamine was 767 ± 151 pmol.min/1 (± SEM), compared to 757 ± 144 pmol.min/1 during bombesin plus saline, whereas integrated meal­stimulated gastrin release was 1666 ± 456 pmol.min/1 during histamine and 1856 ± 492 pmol.min/1 during saline. It is concluded that histamine Hrrecep­tors do not seem to be involved in the regulation of gastrin secretion in man.

INTRODUCTION It is now well established that histamine is of physiologic importance for the

stimulation of gastric acid secretion (1 ). Histamine is produced and secreted by mast cells present in the gastric mucosa. The non-acid producing mucosa of the antrum also contains mast cells and histamine, though slightly less than the acid producing mucosa of corpus and fundus (2). One of the main secretory func­tions of the antral mucosa is the production and secretion of gastrin. Whether antral histamine has any role in the regulation of gastrin release is not known. Many studies on the effect of histamine Hr receptor blockade on gastrin release have been reported, but no final conclusions could be drawn from the results.

39

Several studies (3 , 4, 5) showed that after the administration of cimetidine gas­

trio release in response to a meal was significantly increased. When intragastric

pH was kept constant cimetidine did not affect gastrin release in some studies

(5, 6, 7), while in one study an inhibitory effect of histamine on gastrin secretion was suggested by the demonstration of a significantly enhanced gastrin re­sponse to a meal after Hr receptor blockade (8).

In a recent study in dogs we showed that infusion of histamine dose-depen­dently reduced bombesin-stimulated gastrin release (9) , supporting an inhibi­tory effect of histamine on gastrin secretion. However, others have found that Hi-receptor stimulation by impromidine did not affect liver extract-stimulated gastrin release in dogs (10). The results of these two studies in dogs suggest that

the effect of Hrreceptor stimulation is dependent on the type of stimulation of

gastrin secretion. The effect of Hi-receptor stimulation on gastrin secretion in

man is not well established. Recently it was reported (7) that a submaximal dose of histamine did not affect gastrin secretion stimulated by an amino acid meal

in man. However, the lack of effect of histamine on gastrin secretion in that

study might be due to a too low dose of histamine infused or to the type of stimu­lation of gastrin release used.

In view of the incomplete and conflicting results of previous reports. we have studied the effect of infusion of a maximal dose of histamine with simultaneous H 1 -receptor blockade on gastrin release in response to infusion of bombesin and to a peptone meal in healthy subjects.

MATERIALS AND METHODS

Eleven healthy male subjects, mean age 23 years (range 2 1 -34) and mean

weight 72 kg (range 62-86), participated in the study. All subjects gave fully in­formed consent and the study was approved by the Medical Ethical Committee

of the Groningen University Hospital.

The effect of histamine on bombesin-stimulated gastrin release was studied in five subjects. The studies were done after an overnight fast and each study lasted 90 minutes. The subjects were sitting in a comfortable chair in a slightly recumbent position. They were prepared with indwel ling needles in both arms,

one for sampling blood and the other for infusion of bombesin and of histamine or saline. A double lumen 18-Charriere nasogastric tube (Lavacuator®, Mal­

linckrodt) was passed into the stomach, its position checked by a water recov­

ery test ( 1 1). 5 00 ml 0.05 M acetate buffer at pH 4.5, supplemented with glucose to an osmolality of 290 mOsm/kg, was instilled intragastrically through the

tube. Acetate buffer was used instead of 0. 1 5 M saline or 5.8 g/100 ml glucose solution ( 12) to minimize pH fluctuations. Continuous automated intragastric

40

titration was then started and was continued during the whole test (13): 30 ml portions of the gastric contents were removed and reinstilled 8 to 10 times per minute with an automatic syringe, using a modified HR flow-inducer (Watson­Marlow); the gastric contents so flowed continuously past a pH and a reference electrode (Radiometer); the pH-meter (Radiometer) was connected with an automatic titrator ( Radiometer), which instilled 0.5 M NaOH from an auto­matic burette (Radiometer) through the second lumen of the tube to maintain the intragastric pH at 4.5. To ensure reliable continuous titration care was ta­ken to replenish the intragastric liquids at appropriate intervals. During each test the intragastric pH remained between 4.2 and 4.6 approximately 95% of the time. After the subject had got used to the intragastric titration for a couple of minutes infusion of 0.15 M saline with or without histamine was started. His­tamine acid phosphate was infused at a rate of 130 nmol/kg.h ( = 40 µ.g/kg.h), which elicits maximal gastric acid secretion ( 14). The histamine was given dur­ing the whole test in a volume of 500 ml, using an Ivac infusion pump (Stopler). Fifteen minutes prior to the start of the histamine administration 2 mg clemasti­num (Tavegyl®, Wander AG), a potent H 1 -receptor antagonist, was given in­travenously. During control experiments , as carried out in all five subjects, 500 ml of saline was infused without histamine, but with the administration of cle­mastinum. During all tests synthetic bombesin (UCB Bioproducts) was infused from 60 till 90 minutes at a dose of 90 pmol/kg.h. The bombesin was given in 30 ml 0.15 M saline with 0.25% (W/V) human serum albumin, using a syringe in­fusion pump (B.Braun). Blood samples for determination of plasma gastrin concentration were taken at -15, 60 , 65, 70 , 80 and 90 minutes. Samples were collected in 10 ml tubes containing 15 mg EDT A and were kept on ice till centri­fugation. Plasma was stored at -20°C until assay.

The effect of histamine on peptone-stimulated gastrin release was studied in eight subjects. The subjects were prepared as described above. Histamine was given at the same dose during 90 minutes and control studies were carried out in all eight. At 50 minutes gastric contents were aspirated and 500 ml 8% peptone solution (Protease peptone®, Difeo Laboratories) at pH 4.5 with an osmolality of 355 mOsm/kg was instilled intragastrically and intragastric titration was con­tinued during the rest of the test. Blood samples were taken at -15, 50, 60, 70, 80 and 90 minutes. The tests in each subject were performed at intervals of at least one week; in 9 of 13 paired tests the interval was two or more weeks.

In three subjects samples were taken from the histamine-containing infusion fluid for determination of histamine concentration. The samples were obtained from a three-way cock, which was positioned between the infusion line and the indwelling needle. Histamine concentrations were measured by stable isotope dilution gas chromatography - mass spectrometry as previously described ( 15).

41

Plasma gastrin was determined by radioimmunoassay according to Rosen­quist and Walsh ( 16) with some modifications. For labeling 6 µ,g synthetic hu­man gastrin-17 I (UCB Bioproducts) was dissolved in 25 µ,I 0.25 M phosphate buffer at pH 7.4, to which 10 µ,I Na m1 (100 mCi/ml) (Amersham) and 5 µ,g chlo­ramine-T (Merck) in 10 µ,I 0.25 M phosphate buffer were added. After a reac­tion time of 25 s 100 µ,g sodiummetabisulphite (Brocades) in 20 µ,I of the same buffer was added. For purification of the label DEAE-Sephadex A 25 (Phar­macia Fine Chemicals) was used (16). The specific activity of the label was 0.8 µ,Ci/pmol. The binding of the label to excess antibody (1 :2000) was almost 80% and the serial dilution curves of labeled and standard gastrin were parallel to each other. To each assay tube 2000 cpm of label was added. Counting time was 10 minutes. The assay buffer was 0.025 M veronal buffer (pH 8.6) with 2% (V/ V) pasteurized plasma protein solution (Centraal Lab Bloedtransfusie Dienst). Synthetic human gastrin-17 I (UCB Bioproducts) was used as standard. Char­coal stripped human plasma. 100 µ,l , was added to the tubes for the standard curve. From the unknowns 100 µ,l was added to each tube. Antibody 1611 (17) was used at a final titer of 1 :50,000. The incubation volume was 1 ml. The incu­bation time was 72 h at 4°C. For separation of bound and unbound antigen a suspension of 50 g/1 charcoal (Merck) and 5 g/1 Dextran T70 (Pharmacia Fine Chemicals) in veronal buffer with 10% (VN) pasteurized plasma protein solu­tion was used. 400 µ,I of the suspension was added to each tube. After mixing the tubes were incubated at 4°C during 5 minutes and then centrifuged at 3000 rpm during 10 minutes. The detection limit of the assay was 0. 7 pmol/1 of the re­action mixture, the concentration of unlabeled peptide required to reduce the binding of labeled peptide by 50% (1D50) was 5 pmol/1 of the reaction mixture. The nonspecific binding was 6% and the binding to antibody in the absence of unlabeled antigen 50%. Intraassay and interassay coefficients of variation were 7 and 11 % respectively at a plasma gastrin concentration of 56 pmol/1.

Analysis of data. From all subjects the integrated gastrin responses during the infusion of bombesin (60-90 minutes) and during the peptone meal (50-90 minutes) were calculated. Results of control experiments and those with hista­mine infusion were compared by Student's t-test for paired results.

RESULTS

Both the infusion of bombesin and the intragastric instillation of the peptone meal induced marked increases in plasmagastrin. The infusion of histamine did not affect the gastrin release as elicited by the infusion of bombesin (table I, figure 1). The mean (± SEM) integrated responses of gastrin during the bom­besin infusion were 757 ± 144 pmol. min/I during the control studies and 767± 151 pmol.min/1 during the infusion of histamine. Likewise, the admini-

42

Ta hie I. Effect of hbtamine on homhesin-stimulated plasma gastrin concentrations

Control Histamine

536 32 1

2 1 1 48 1 227

Subjects

3 426 606

4 1053 907

5 622 775

Mean ± SEM 757 ± 1 44 767 ± 1 5 1

Integrated ga,trin re,pon,e, (pmol.min/1) during infusion of bombesin. 90 pmol/kg.h for 3 0 mi­nute,. in control experiment, and during infusion of histamine. 1 30 nmol/kg.h . in 5 subject,.

:::::: 0 E a.

C r.... ...... VI Cl en

50

40

30

20

10

control

histamine

o ---..-- �--,.-----....------"T""----------0 60

Figure I .

70 80 90

time (mi nutes)

Plasma gastrin concentrations (mean ± SEM. n=5) in response to bombesin (90 pmol/kg.h) during histamine infusion tests ( 1 30 nmol/kg.h) and control studies.

stration of histamine did not affect the peptone-stimulated gastrin release (ta­

ble II , figure 2) . The integrated responses of gastrin during the peptone meals

were 1856±492 pmol.min/1 during the control studies and 1666±456 pmol.min/1

during the infusion of histamine. The small difference between these two values

was largely due to the variation in one subject.

The concentration of histamine in the infusion fluid during the three tests

studied were 91 , 95 and 106% of the expected concentrations. There was no dif­

ference between the concentrations in samples taken at 15 , 45 and 85 minutes.

43

Table II Effect of histamine on peptone-,t,mulatcd pla,ma ga,tnn conccntrat1om

Subject,

2 5 6 7 8 9 IO I I Mean + SEM Control I <Kl8 367 1 1 76 3702 690 376 1 2980 I 1 63 1 856 + -192 Histamine 1 372 366 1 1 1 6 -1056 562 1 6 1 5 3 1 79 !059 1666 ± -156

Integrated gaMrin rc,pon,c, (pmol.m1n/l) during pcptonc meals in control experimenb and during infu,ion of histamine. I 10 nmol/kg.h. in 8 ,ubject,.

BO --

C: 70 ·;:

60

50

40

30

20

10

0

Figure 2.

r 0 50 60 70 BO

control

histamine

90 time (minutesl

Plasma gastrin concentrations (mean ± SEM. n= 8) in response to8% peptone solution during his­tamine infusion tests ( 1 30 nmol/kg .h) and control studies.

During the infusion of histamine all subjects experienced flushing and a small

increase of the heart rate was observed. However, all felt comfortable during

each test .

44

DISCUSSION The present study shows that stimulation of histamine Hz-receptors does not

affect the secretion of gastrin, indicating that antral Hz-receptors are not in­volved in the regulation of gastrin secretion in man. Stimulation of H2-recep­tors was achieved by infusion of a maximal dose of histamine after blockade of the H 1-receptors by an H 1 -receptor antagonist, while gastrin release was stimulated by intragastric instillation of a meal and by infusion of bombesin. Food is a physiologic luminal stimulus for gastrin release, whereas bombesin is thought to be involved in gastrin release as a neurotransmitter localized in nerve endings in the antrum (18, 19).

The results of this study are in agreement with findings of others that Hz-re­ceptor blockade by cimetidine does not affect gastrin release in man directly (5, 6, 7), and also with the recent finding by Richardson and Feldman (7), that infu­sion of histamine did not affect amino acid meal-stimulated gastrin release. However, as these authors already pointed out, the Jack of effect of histamine in their study might have been due to a too low dose of histamine infused and/or to the stimulus of gastrin secretion applied. They infused 20 µ,g/kg.h histamine acid phosphate (7), which is little more than half of the lowest dose used in our study in dogs, namely 20 µ,g/kg.h histamine-2 HCI (9), which is equivalent to 33 µ,g/kg.h histamine acid phosphate. In the present study we administered hista­mine at a dose twice that given by Richardson and Feldman. Furthermore we validated our infusion rate by determination of the histamine concentration in the infusion fluid. Since this dose is known to elicit maximal gastric acid secre­tion (14) high tissue concentrations were apparently reached during our stud­ies.

An inhibitory effect of histamine on gastrin secretion in the dog has been demonstrated during bombesin infusion (9) but not during food stimulation (10). Therefore, we have studied the effect of histamine on gastrin secretion stimulated by bombesin as well as by intragastric administration of a peptone meal. The results of the present study contrast with our finding in dogs that his­tamine dose-dependently reduces bombesin-stimulated gastrin release. The dose given in the present study was higher than the lowest dose used in the study in dogs, which reduced gastrin release by 16%. Since the study protocols were similar to a great extent, we suggest that the different effects of histamine on gastrin release in man and dog are due to species differences. The results in man are also in contrast with those reported in rats. Several investigators (20, 21, 22) have found that in these animals Hz-receptor blockade by cimetidine does in­crease gastrin release.

In conclusion, the present study shows that stimulation of Hz-receptors by a maximal dose of histamine has no effect on the bombesin- and meal-stimulated

45

gastrin release in man. Therefore, antral Hrreceptors do not seem to have a role in the regulation of gastrin secretion in man.

REFERENCES I . Gro�sman M l . Regulation of gaMric acid �ecrct1on In Phy�iology of the gastrointe�tinal tract.

LR Johnson (ed) . Raven Press. New York . 1 98 1 . pp 659-67 1 . 2 . Mohri K . Reiman HJ . Lorenz W . Troidl H . Weber D Histamine content and ma�t celb in hu­

man gastric and duodenal mucosa. Agents Actions 1 97 1 ; 8: 372-375 . 3 . Logan RFA . Forre�t JAH. Mcloughlin GP. Lidgard G. Hedding RC. Effect ofcimetidine on

scrum gastrm and ga�tric emptying in man. Dige�tion 1978; 18: 220-226. 4 Longstreth GF. Go VLW. Malagelada J-R. Po�tprandial ga�tric. pancreatic. and biliary re�­

ponse to histamine H,-receptor antagonist� in Jctive duodenal ulcer Ga,troenterology 1977 : 72: 9- 13 .

5 Richard�on CT. Walsh JH. Hieb M l . The effect of c1metidinc. a ne,, hi,taminc H,-receptor antagonist. on meal-�t1mulated acid �ecretion . �erum gaMrin . an<l gm,tric emptying in patient, with duodenal ulcer. Ga�troenterology I 976: 7 1 : 1 9-23.

6. Henn RM . Isenberg J I . Maxwell V. Sturdevant RAL. I nhibition of gastric ,1cid �ecrction by ci­metidine m patients with duodenal ulcer. N Engl J Med 1975 : 293: 37 1 -375.

7 . Richardson CT. Feldman M. Effect of histamine and cimetidine on ammo acid meal-�timula­ted gastrin release at a controlled intragastric pH m health) human being�. Regul Peptide� 1 985 : IO: 339-344.

8. Feldman M, Richardson CT. Petersen WL. Wabh J H . Fordtran JS. Effect of low-do�e pro­pantheline on food-stimulated ga�tric acid secretion. N Engl J Med 1977; 297: 1427- 1 430.

9. Kleibeuker J H . Kauffman GL. Walsh JH. l ntravenou� histamine reduce� bombe�in-st1mula­ted gastrin release in dogs. Regul Peptides 1985: 1 1 : 209-2 1 5 .

I O . Schusdziarra V. Stapelfeldt W. Klier M. Maier V. Pfeiffer EF. Effect ofhi�tamine H,-receptor stimulation on postprandial pancreatic and gastric endocrine function in dogs. Re� Exp Med (Berl) 1982; 1 8 1 : 253-257.

1 1 . Hassan MA. Hobsley M. Positioning of subject and of nasogastric tube during a ga�tric secre­tion study. Br Med J 1 970: I : 458-460.

1 2. Maxwell V, Eysselein VE. Kleibeuker JH . Reedy T. Walsh J H . Glucose perfusion mtraga�tric titration. Dig Dis Sci 1 984; 29: 32 1 -326.

1 3 . Lam SK. Isenberg J I . Grossman Ml. Lane WH. Walsh JH. Gastric acid secretion is abnormal­ly sensitive to endogenous gastrin released after peptone test meals m duodenal ulcer patients J Clin Invest 1 980; 65: 555-562.

14. Lawrie JH . Smith GMR. Forrest APM. The histamine-infusion test. Lancet 1964: 2 . 270-273. 1 5 . Keyzer JJ. Wolthers BG. Muskie! FAJ . Breukelman H. Kauffman H. De Vries K. Measure­

ment of plasma histamine by stable isotope dilution gas chromatography - mass �pectrometry: methodology and normal values. Anal Biochem 1 984; 1 39: 474-48 1 .

16 . Rosenquist GL, Walsh JH. Radioimmunoassayof gastrin. I n Gastrointestinal Hormones. GB Jerzey Glass (ed). Raven Press. New York . 1 980. pp 769-795.

1 7 . Eysselein VE. Maxwell V. Reedy T. Wunsch E. Walsh JH. Similar acid stimulatory potencies of synthetic human big and l itt le gastrins in man. J Clin Invest 1 984; 73: 1 284- 1 290.

1 8 . Martindale R, Kauffman GL. Levin S. Walsh J H . Yamada T. Differential regulation of gastrin and somatostatin secretion from isolated perfused rat stomachs. Gastroenterology 1982; 83: 240-244.

19 . Schubert ML, Saffouri B. Walsh JH .MakhloufGM. I nhibition of neurally mediated gastrin se­cretion by bombesin antiserum. Am J Physiol 1 985; 248: G456-G462.

20. Schusdziarra V, Bender H . Pfeffer A, Pfeiffer EF. Modulation of gastric bombesin-like immu­noreactivity by cholmerg1c and histamine H"·receptors. somatostatin. and intragastric pH. Re­gul Peptides 1 984: 8 : 1 89- 1 98.

46

2 1 . Tahata K. Okabe S. Gastric secretory conditions and plasma gastrin levels in rats after pro­longed treatment with cimetidine. Dig Dis Sci 1984; 29: 40-45.

22. Ohe K . Nakamura M. Fujiwara T. Matsumoto H . Kohchi M. Miyoshi A . Effect ofH,-receptor antagonists. cimctidine and YM- I I I 70. on ,crum ga,trin level, in lumen-perfused rat,. Dig Di, Sci 1983; 28: 98 1-989.

47

CHAPTER S

EFFECT OF SELECTIVE AND NONSELECTIVE

CHOLINERGIC BLOCKADE ON BOMBESIN­

AND PEPTONE-STIMULA TED GASTRIN

RELEASE

J. H. Kleibeuker, C. 8. H. W. Lamers

Department of Gastrocnterology, University Hospital, Groningen; Depart­ment of Gastroenterology, University Hospital, Leiden, the Netherlands.

ABSTRACT The differential effects of the selective muscarinic M 1 -receptor antagonist

pirenzepine and the nonselectivc muscarinic antagonist atropine on gastrin re­lease arc not well established. The present study was undertaken to compare in healthy subjects the effects of pircnzcpine and atropine on gastrin release stim­ulated by bombesin and pcptonc meal, two well established specific stimulants of gastrin secretion. Pirenzcpine (i. v. bolus 0.6 mg/kg) and atropine (i. v. bolus of 0.0 1 5 mg/kg, followed by infusion of 0 .005 mg/kg.h) were given in doses equipotent in terms of reduction of gastric acid secretion. Intragastric titration was performed during all tests at pH 5.5. Neither pirenzepine nor atropine did affect bombesin- or peptone-stimulated gastrin release. It is concluded that muscarinic M 1-receptors are not involved in the cholinergic regulation of gas­trin release and that in contrast to previous suggestions reduction of acid secre­tion by pirenzepinc is not mediated through an inhibition of gastrin release .

INTRODUCTION Gastrin has an important role in the stimulation of gastric acid secretion. It is

the major mediator of induction of acid secretion by intragastric food compo­nents, especially amino acids ( 1 ). In addition it contributes to the acid secretion induced by cephalic stimulation (2) and to a minor degree by gastric distention (3). The different pathways through which the release of gastrin by the antral G-cells is being regulated, have been elucidated only in part. Cholinergic nerves certainly have an important role in this respect. Recently evidence has been provided for the existence of different subclasses of cholinergic muscari­nic receptors. The newly developed drug pirenzepine has been shown to be a specific antagonist of muscarinic M 1 -receptors ( 4), whereas atropine is a nonse-

49

lectivc muscarinic receptor antagonist. A comparison of the effects of atropine and pirenzepine on gastrin release might yield information on which muscarinic receptors are involved in the regulation of gastrin secretion. Recently such a comparison was made by Lazzaroni et al (5). Pirenzepine, I O mg, and atropine, 1 mg, were administered as an intravenous bolus. It was found that pirenzepine did not affect peptone-stimulated gastrin release. However, in another study (6) it was suggested that peptone-stimulated gastrin secretion was inhibited by pirenzepine in a dose-related manner, the inhibition becoming apparent only at a dose of 0.5 mg/kg intramuscularly, which is about threefold higher than the dose used by Lazzaroni ct al (5). The peptide bombesin is another strong stimu­lant of gastrin release. Both bombesin and the amino acids in the pcptonc meal are specific stimulants of the gastrin producing G-cells. A bombesin-like pep­tide is assumed to be involved as neurotransmitter in the vagal stimulation of gastrin release. The effect of pirenzepinc on bombesin-stimulated gastrin se­cretion has not been evaluated so far.

In order to further elucidate the cholinergic regulatory mechanisms of gas­trin release we studied the effect of a high dose of pirenzepine on peptone- and bombesin-stimulated gastrin secretion and have compared these effects with those of atropine, in a dose which was comparable with that of pirenzepinc in respect to reduction of gastric acid secretion.

MATERIALS AND METHODS Seven healthy male subjects, median age 25 yrs (range 23-35), mean weight

72 kg (range 62-82) participated in the study. All subjects gave informed con­sent and the study was approved by the Medical Ethical Committee of the Uni­versity Hospital and the State University of Groningen.

The effect of selective and nonselective cholinergic blockade on bombesin­induced gastrin release was studied in six subjects. All of them were studied three times on different days. The intervals between the tests were at least three days. Tests were done in random order. The studies were performed after an overnight fast and each study lasted 80 minutes. The subjects were sitting in a comfortable chair in a slightly recumbent position. They were prepared with an indwelling needle in one arm and, in case of atropine administration, with a se­cond needle in the other arm. A double lumen 1 8-Charriere nasogastric tube (Lavacuator®, Mallinckrodt) was passed into the stomach, its position checked by a water recovery test (7). Five hundred ml glucose solution, 58 g/1 at pH 5 .5 (8) was instilled intragastrically through the tube at time 0. Continuous auto­mated intragastric titration was then started and was continued during the whole test, as recently described (9). The volume of the titrant (0.5 M NaOH) used was registered graphically by a Titrigraph (Radiometer) to quantify gastric acid

50

secretion. To ensure reliable continuous titration care was taken to replenish

the intragastric liquids at appropriate intervals. During all tests synthetic bom­bcsin (UCB Bioproducts) was infused from 50-80 min after the start of the test at a dose of 135 pmol.kg· 1 .h· 1

• At this dose gastrin release is stimulated to the same extent as by a peptone meal. Bombesin was given in 30 ml 0. 1 5 M NaCl

with 0.25% (W/V) human serum albumin. During one test 0.6 mg/kg

pircnzcpine (Boehringer Ingelheim) was administered intravenously at 5 min. This timing of administration ensured a stable plasma concentration of pirenze­

pine during the infusion of bombesin ( 10). During the second test 0.0 15 mg/kg atropine was administered intravenously at 35 min, followed by an infusion of

atropine at a dose of 0.005 mg.kg· 1 . h· 1 in 50 ml 0. 1 5 M NaCl during the rest of

the test. Gastric acid secretion was measured during the infusion of bombcsin. Blood samples for determination of plasma gastrin concentrations were taken at 0, 50, 55, 60, 70 and 80 min.

The effect of selective and nonsclective cholincrgic blockade on peptonc-in­

duced gastrin release was studied in six subjects. The subjects were prepared as described above. Again, the subjects were studied three times on different days

in random order, giving pircnzepine in one, atropine in another and no drug in

the third study. Each test lasted 90 min. At 50 min gastric contents were aspirat­ed and a 500 ml 8% peptone meal (Protease peptone®, Difeo Laboratories) at

pH 5.5 with an osmolality of 355 mosm/kg was instilled intragastrically and in­tragastric titration was continued during the rest of the test. Gastric acid secre­

tion was measured from 50 till 90 minutes . Blood samples were taken at 0, 50,

60, 70, 80 and 90 min. Plasma gastrin concentrations were determined by a spe­cific and sensitive radioimmunoassay as previously described ( 1 1 ).

Analysis of data. All results are expressed as mean ± standard error of the

mean (SEM). Gastric acid secretion during bombesin infusion and after instil­lation of the peptone meal arc expressed as mmol H+/h. Results of control ex­

periments and those with pirenzepine and atropine were compared by Stu­dent's t-test for paired results. The integrated gastrin concentrations during the

infusion of bombesin (50-80 min) and during the peptone meal (50-90 min)

were calculated for all subjects. Results of control experiments and those

with pirenzepine and atropine were compared by Student's t-test for paired results. Differences were considered to be significant if p < 0.05.

RESULTS Gastric acid secretion could be evaluated in four out of six subjects during

bombesin infusion and in five out of six subjects during the peptone meals. One

subject showed variable duodenogastric reflux during at least one of the tests,

another one had marked hypochlorhydria. Acid secretion was significantly in-

5 1

hibited by both pirenzepine and atropine. During the infusion of bombesin gas­tric acid secretion was 1 5.0 ± 3.2 mmol/h during the control studies and was re­duced by pirenzepine to 4.6 ± 1 .4 mmol/h (3 1 % of control) and by atropine to 8.3 ± 0.9 mmol/h (55% of control). The difference between the amounts of acid secreted during the pirenzepine and atropine tests was not statistically signifi­cant. During the peptone meals gastric acid secretion was 21 .0 ± 4.0 mmol/h during the control studies and was reduced by pircnzcpinc to 8. 9 ± 2.5 mmol/h (42% of control) and by atropine to 9.2 ± 2.3 mmol/h (44% of control).

Neither pirenzcpine nor atropine did affect bombesin-stimulatcd gastrin re­lease. During the infusion of bombesin the integrated gastrin concentration was 3737 ± 850 ng. min/I during the control studies, whereas after pirenzepine it was 3865 ± 835 ng.min/1 and during atropine 3866 ± 937 ng.min/1 (figure 1) .

--200

C

C 1 60 . C:

1 20

80

40

0

Figure l .

i y:=--

-

0 50 60 70 80

t ime (minutes)

atropine pirenzepine

control

Mean (± SEM) plasma gastrin concentrations during bombcsin infusion ( 1 35 pmol/kg. h , 50-80 min) during control studies. after pirenzepme (i .v. bolus of0.6 mg/kg at 5 min) and during atropine ( i .v . bolus of 0.0 1 5 mg/kg at 35 min. followed by infusion of0.005 mg/kg. h) in 6 subjects.

Peptone-stimulated gastrin release was not affected by pirenzepine and atro­pine either. The integrated gastrin concentration during the peptone meal was 4570 ± 756 ng .min/1 during the control studies. After pirenzepine it was 4431 ±

617 ng.min/1 and during atropine 4622 ± 649 ng.min/1 (figure 2).

52

- 200

C

C 1 60 ·c::

1 20

80

40

0

Figure 2 .

�

� 0 50 60 70 80 90

time (m inutes)

atropine control pirenzepme

Mean ( ± SEM) plasma gastrin conccntrations during8% peptonc meals (50-90 min) during control studies. after pircnzepinc ( i .v . bolus of0.6 mg/kg at 5 min) and during atropine ( i .v . bolus of0.0 15 mg/kg at 35 min. followed by infu�ion of0.005 mg/kg. h) in 6 subjects.

DISCUSSION

This study shows that pirenzepine and atropine in the doses used are without any effect on peptone- and bombesin-stimulated gastrin release. The doses of the two drugs were about equipotent in respect to reduction of gastric acid se­cretion.

The finding that atropine at this dose does not affect peptone-induced gas­trin secretion is in accordance with findings in comparable studies of some (12, 13) though not of all authors (5). The present study is also in agreement with the two former ones in respect to the reduction of acid secretion by atropine. So it can be assumed reliably that at the dose used atropine does not affect peptone­induced gastrin release. However, Schiller et al ( 13) showed in their study that lower doses of atropine suppressed amino acid meal-stimulated gastrin secre­tion. The authors concluded from their results that cholinergic mechanisms mainly facilitate meal-induced gastrin release but that there may also be some cholinergic inhibitory pathway.

The lack of effect of pirenzepine on peptone-stimulated gastrin secretion contrasts with the findings of Mignon et al (6) who reported an inhibition of gas­trin release by pirenzepine at a dose of 0.5 mg/kg, which is slightly lower than our dose of 0.6 mg/kg. The discrepancy between the results of Mignon et al and

53

those of the present study is probably not due to the difference between the modes of administration of pirenzepine. Whereas we gave an intravenous bolus injection, the other investigators gave an intramuscular injection. However, serum concentrations of pirenzepine after an intramuscular injection follow about the same course in the time as after an intravenous bolus from thirty minutes onwards ( 10). Apart from the mode of administration the designs of our study and that of Mignon et al were comparable. Therefore the origin of the discrepancy between the two studies is not readily apparent.

Based on studies in the isolated perfused rat stomach it was recently pro­posed again that part of the acid inhibitory effect of pirenzepine might be due to reduction of gastrin release (14). The results of our study and the previously mentioned findings of Lazzaroni et al (5) strongly suggest however that musca­rinic M ,-receptors do not have a role in the cholinergic modulation of peptone­induced gastrin release and that other types of muscarinic receptors are rele­vant in this respect.

In a previous study atropine did not affect bombesin-stimulated gastrin re­lease ( 15). We could not show any effect of atropine either. In addition we did not find any influence of pirenzepine on bombesin-induced gastrin release. So it seems that the effect of bombesin on gastrin is not modulated by the cholinergic system. Some caution however should be expressed, since in the same study Fletcher et al (15) found that gastrin release induced by the mammalian analog of bombesin, gastrin releasing peptide, was enhanced by atropine.

We conclude that pirenzepine and atropine in the doses used do not affect peptone- and bombesin-stimulated gastrin release. In contrast to previous sug­gestions, cholinergic M 1-receptors are not involved in gastrin release in man. The inhibitory effect of pirenzepine on acid secretion is not mediated through reduction of gastrin secretion.

REFERENCES I . Feldman M . Walsh JH . Wong HC. Richardson CT. Role of gastric heptadecapeptide in the

acid secretory response to amino acids in man. J Clin Invest 1978; 62: 308-3 13 . 2 . Feldman M . Richardson CT. Taylor IL. Wabh J H . Effect of atropine on vagal release of gm,­

trin and pancreatic polypeptide. J Clin Invest 1979: 63: 294-298. 3. Schiller LR. Walsh JH. Feldman M. Distension-induced gastrin release. Effect of luminal aci­

dification and intravenous atropine. Gastroenterology 1980; 78: 9 1 2-9 17 . 4 . Hammer R, Berrie CP, Birdsall NJM , Burgen ASV. Hulme EC. Pirenzepine distinguishes be­

tween different subclasses of muscarine receptors. Nature 1 980; 283: 90-92 5 . Lazzaroni M, Sangaletti 0, Del Soldato P. Bianchi Porro G. Effect of pirenzepine and atro­

pine on peptone meal-stimulated gastric secretion and plasma gastrin in healthy volunteers. patients with duodenal ulcer and vagotomized patient�. Digestion 1 985; 32: 267-272 .

6 . Mignon M , Vatier J , Bauer P , Bonfils S . Effect o f pirenzepine o n meal-stimulated acid secreti­on and gastrin release in normal man. Scand J Gastroenterol 1 982; (suppl 72) : 1 45- 1 5 1 .

7. Hassan MA. Hobsley M. Positioning of subject and of nasogastric tube during a gastric secre­tion study. Br Med J 1970; I : 458-460.

54

8. Maxwell V. Ey,,eiein VE. Klc1bcuker J . Reedy T. Wabh J H . Glucose perfusion 111traga,tnc t i t ration. Dig Di� Sci 1 984; 29: 32 1 -326.

9. Kleiheuker J H. Kooi H. Lamer, CBHW. Effect of hi,t.iminc H,-receptor ,t imulation on hom­hc,in- and pepl<rne-,t 1mulated ga,trin rele,l"e in man. Dig Di, Sci 1 986; 3 1 : 1 095- 1099.

10. Hammer R. Bozler G. Zimmer A. Ko.,., FW. Ph.trmacokinct ic, ,111d metaholi�m of LS5 I 9-Cll

(pirenzcpin ) in man . Therapiewochc 1 977; 27: 1 575- 1 593. 1 1 . Jan,en JBMJ. Ltmer, CBHW. Effect of change, in scrum calcium on secretin-st 1mulated ,c­

rum g.iMrin m p.itieni.. with Zollinger-Ellison syndrome. Gastroenterology 1 982; 1 73- 1 78. 1 2. Konturek SJ . Bicrn,ll J. Olek,y J. Rehfeld JF. Stud i i F. Effect of .itropme on ga,trin and ga,tric

acid rc,pon,c to pcptonc mc,11. J Clin Invest 1 974; 54: 593 597. 1 3 . Schiller LR. W.ilsh J H . Feldman M. Effect of at ropine on gaMrin rclea,c Mimulated hy an ,1mi­

no acid meal in humans. Gastroenterology 1 982; 83: 267-272 1 4. Sue R. Toomey ML. Todbco A. Soll AH. Yamada T. Pirenzepine-,en,itive mu,carmic recep­

tor, regul,1te ga,tnc ,om,1t0Mat in ,md g,istnn. Am J Phy,iol 1 985 , 248: G 1 84-G 1 87. 15 . Fletcher DR. Shulkcs A . Bladin PHD. Hardy KJ . The effect of atropine on hombesin and gas­

t rin releasing peptide st imulated ga�trin. pancreatic polypeptide and neurotcn,in release in man. Regul Pep! 1 983; 7: 3 1 -40.

55

CHAPTER 6

ROLE OF ENDOGENOUS SECRETIN IN

ACID-INDUCED INHIBITION OF HUMAN

GASTRIC FUNCTION

J. H. Klcibcukcr, V. E. Ey��clcin, V. Maxwcll, J. H. Walsh

Center for Ulcer Research and Education, Veterans Administration Wads­worth Hospital Center, University of California, Los Angeles, California, U . S.A.

ABSTRACT The role of secrctin in the inhibition of gastric acid secretion that occurs dur­

ing acidification of the gastric lumen was studied in nine healthy men. Gastric acid secretion was stimulated by 500-ml meals of 8% peptone solution, and the pH of the stomach was maintained at 5.5, 2.5, or 2.0 by intragastric titration. The increase in plasma sccretin was measured, after extraction, by a new secre­tin radioimmunoassay. After determining the intravenous dose of secretin re­quired to reproduce plasma secretin concentrations achieved during pH 2.5 and 2.0 meals, similar doses were given during administration of a pH 5.5 pep­tone meal. The doses of secretin led to plasma secretin concentrations that averaged 3.4 pM, not different from the 3.2 and 3.9 pM concentrations a­chieved during acidified meals. However, exogenous secretin infusion failed to inhibit acid secretion or gastrin response to peptone, although significant inhi­bitions occurred in both during peptone meals given at pH 2.5 or 2.0. When sc­cretin infusions were given at fivefold higher rates, plasma gastrin responses again failed to demonstrate significant inhibition. Gastric emptying was inhib­ited significantly by both acidified peptone meals but only slightly (P = 0.053) during exogenous infusion of physiologic secretin doses . The decrease in acid secretion could be explained by decreased gastrin release, but neither of these findings could be explained by circulating secretin concentrations. These re­sults cast strong doubt on a physiological role of secretin in inhibition of acid se­cretion in man.

57

INTRODUCTION Secretin is released by endocrine cells in the mucosa of the duodenum and

upper jejunum when the intraduodenal pH falls below 3 to -1- ( 1 -3). The amount of secretin released probably is dependent on the total amount of titratablc acid delivered to the duodenum. It is now generally accepted that sccrctin is a physi­ologic stimulant of pancreatic water and bicarbonate secretion ( 4, 5). Whether it has any other functions has not yet been established. Exogenous secrctin in supraphysiologic doses inhibits acid secretion and gastrin release, basally as well as in response to a variety of stimuli (6- 1 0) .

Recent studies in the dog ( 1 1 , 12) indicate that secrctin is a physiologic inhibi­tor of gastric acid secretion and gastrin release. So far . this has not been report­ed in man. A recent study ( 13) showed that secretin in low doses inhibits the gastric emptying of liquid meals in humans. We have found that inhibition of peptone-stimulated acid secretion at low intragastric pH was due entirely to in­hibition of gastrin release ( 1 4).

We used a newly developed radioimmunoassay for sccretin to study whether circulating secretin is a mediator of this acid-induced inhibition of gastrin re­lease and acid secretion. In addition, we studied the gastric emptying rate.

MATERIALS AND METHODS

Nine healthy male subjects (mean age 38 yr, range 20-69 yr) without previous gastric or duodenal disorders were studied. All gave written informed consent. The study was approved by the Human Studies Committee at Veterans Ad­ministration Wadsworth Center.

After an overnight fast a nasogastric tube (AN 10, Andersen Samplers Inc. , Atlanta GA) was passed into the stomach. The position of the tube was checked by a water-recovery test ( 1 5) and, if necessary, by fluoroscopy. After complete emptying of the stomach by aspiration, 500 ml of a liquid meal, con­taining 8% peptone (wt/vol) (Bacto Peptone, Difeo Laboratories , Inc., Detroit, MI), was instilled intragastrically and acid secretion was measured dur­ing 1 h by automated intragastric titration (16). During the test the volume of the intragastric contents was measured at 5 , 15, 30 , 45, and 60 min, using the dye-dilution technique ( 17). Phenol red was used as marker. Its concentration was measured spectrophotometrically at pH 11 , at a wavelength of 560 nm (Model DU, Beckman Instruments , Inc. , Fullerton, CA). The duodenal acid load during meals at low pH was calculated according to Gross et al. ( 1 8). Titra­table acid of the gastric contents was measured by titration to pH 7.

Before and during tests blood samples were taken through a needle in the an­tecubital vein for measurement of plasma gastrin and secretin concentrations. Samples were collected in standard EDT A-containing tubes (10.5 mg/tube) to

58

which 200 µ,I Trasylol (aprotinin), 1 0,000 KIU/ml (Bayer AG, Bayerwerk, FRG) per 5 ml blood was added. Samples were refrigerated immediately after collection, centrifuged within 30 min, and the plasma was stored at -20°C until radioimmunoassays were performed.

For secrctin infusion pure natural porcine secretin (manufactured by Kabi Vitrum AB, Stockholm, Sweden) dissolved in 0. 1 5 M NaCl, containing 0.25 % human scrum albumin (wt/vol), was used. The infusion was administered through a needle in the other arm. A small portion of each infusion solution was stored at 20°C for measurement of the sccrctin concentration. The biologic ac­tivity of the secrctin was tested by infusing increasing doses intravenously in a dog with a chronic pancreatic fistula after an overnight fast, and measuring pan­creatic secretion in respect to volume and bicarbonate contents. Gastrin plas­ma concentrations were measured with a specific radioimmunoassay as previ­ously described (1 9), using antibody 1 6 1 1 .

Sccrctin plasma levels were measured with a newly developed radioimmuno­assay.

Antibody. Secret in antibodies were raised in rabbits, using synthetic secretin (E. R. Squibb & Sons, Princeton, NJ) as antigen. The secretin was conjugated to bovine scrum albumin with carbodiimide as described earlier ( 1 9). The con­jugation product was emulsified with complete Freund's adjuvant for the initial immunization, and with incomplete Freund's adjuvant for subsequent booster immunizations. Booster immunizations were given at 4-8 wk intervals after the previous immunization. Blood was obtained 7 d after an immunization. The an­tiserum used in this assay, 7842, was obtained after the fourth immunization.

Cross-reactivity of the antiserum was tested for glucagon, gastric inhibitory peptide, and vasoactive intestinal peptide and further for bombesin, cholecys­tokinin octapeptide, gastrin, insulin, motilin, neurotensin, pancreatic polypep­tide, and somatostatin at concentrations up to at least 1 0 pmol/ml.

The sensitivity was tested by determining the 1D50 , defined as the concentra­tion of antigen required to reduce the binding of labeled antigen by 5 0%, and by determining the detection limit, defined as the smallest concentration of added antigen, which produces a significant inhibition of binding (P < 0.05 by t test), when samples are tested in four replications.

Labeled antigen. Secretin was labeled by the chloramine-T method. Synthet­ic secretin was purchased from Research Plus, Na 1 25I (100 mCi/ml) from Amersham Corp. I ; chloramine-T from Eastman Kodak Co., Rochester, NY; sodium metabisulphite from J. T. Baker Chemical Co., Phillipsburg, NJ; Se­phadex G-1 0 and SP-Sephadex C-25 from Pharmacia Fine Chemicals, Piscata­way, NJ. Secretin, chloramine-T, and sodium metabisulphite were all dis­solved in 0.25 M sodium phosphate buffer at pH 7.4.

5 9

Secretin, 1 0-20 µ.g, was dissolved in 50 µ.I buffer. to which 1 0 µ.I Na 1 �51 and 20 µ.g chloramine-T in I O µ.I buffer. were added. After a reaction time of 60 s I 00 µ.g sodium metabisulphite in 20 µ.I buffer was added

For a first purification the sample was applied to a Sephadex G- 10 column, 20 x I cm, eluted with 0.05 M sodium acetate buffer pH 5. 0. containing 0.425% so­dium chloride (wt/vol) and 2% Plasmanate (human plasma protein fraction, Cutter Laboratories. Inc . • Emeryville . CA). The flow rate was I ml/min. and 2-ml fractions were collected. From the first peak the fractions with the highest radioactivity were pooled, and, for further purification. applied to a SP-Sepha­dex C-25 column. 20 x I cm. eluted with the same buffer as the first column, but with a concentration gradient from 0.05 M/0.425% to 0. 1 M/0.85% buffer and NaCl respectively over 12 h . at a flow rate of 0.2 ml/min. Fractions of 2 ml were collected. From the second high peak the fractions with the highest radioactivi­ty, containing the purified label. were pooled and after addition of O.S�o Trasy­lol, 10,000 KIU/ml (vol/vol). divided in 250-µ.I portions. which were stored at -20°C until use. Each portion was used for only one assay. Fractions preceding and following the labeled antigen containing peak were assayed to identify un­labeled secretin.

The specific activity of the label was determined by comparing the displace­ment of a known amount of labeled antigen by doubling amounts of the same label and known amounts of standard secret in.

The immunoreactivity was determined by comparing serial dilution curves of the labeled secretin and standard secretin. and by determining the binding of label to an excess amount of antibody (titer 1 :5 .000). To each assay tube 2,000 cpm of label in 200 µ.I were added. Counting time was I min

Standard secretin. Squibb synthetic secretin was used as standard. The dry powder was dissolved in the assay buffer to a concentration of 1 nmol/ml. 250-µ.I portions of this solution were stored at-20°C. each portion to be used for on­ly one assay.

Assay condition. The incubation volume was 1 ml. The assay buffer was 0.05 M ammonium acetate (J. T. Baker Chemical Co.) at pH 5.5 . containing 2% Plasmanate and 0 .5% Trasylol. In preliminary experiments we found that at higher pH (6.5, 7.4) the binding in absence of antibody exceeded 10%, which we found unacceptable.

The standard and unknown samples were preincubated at 4°C with the anti­body for 48 h, after which label was added. After another 24 h at 4°C, bound and unbound fractions were separated, using charcoal. For the separation 400 µ.I of a suspension of activated charcoal (Mallinckrodt Inc., St. Louis, MO), 50 g/liter, and dextran (Pharmacia Fine Chemicals), 5 g/liter, in 0.05 M ammo­nium acetate at pH 5.5, with 10% newborn calf serum (vol/vol) (MA Biopro-

60

ducts, Walkersville, MD), was added to each tube. After centrifugation the bound and unbound fractions were separated. In preliminary experiments it was found that by this incubation method the 1D50 decreased 70% compared with simultaneous incubation of antibody and label for 24 h.

Data fitting. For the standard curve, antigen concentrations of 1-1,000 fmol/ ml were used. A computer fit the four-parameter logistic curve to the bound counts with nonlinear regression. The computer was also used to obtain un­known values from the standard curve (20).

Reproducibility of the assay. This was determined by calculating the coeffi­cients of variation within the assay and between assays.

Preparation of plasma samples. Since physiologic plasma levels of secretin are low and plasma interference is often relatively high, extraction and concen­tration of plasma is necessary before assaying. For this purpose we used C 1 8 car­tridges (SEP-PAK, Waters Associates, Millipore Corp., Milford, MA), small, densely packed C 1 8 columns. After preparation of the cartridge with 1 0 ml ace­tonitrile (J. T. Baker Chemical Co) and 10 ml 0.05 M ammonium acetate at pH 5.5, the plasma sample was applied. The column was then washed with the same buffer and it was then eluted with 50% acetonitrile/50% ammonium ace­tate. After discarding the first 0.5 ml, 1 . 1 ml was collected in a tube containing 0.0 1 ml Plasmanate and 0.005 ml Trasylol. After evaporation of the acetonitrile 0.6 ml ammonium acetate buffer was left; 450 µ,I of the 600 was added to the as­say tube. From each plasma sample 2 x 5 ml was extracted and assayed as dupli­cates. Recovery of secretin from this procedure was tested by adding doubling doses of standard secretin to 5 ml of charcoal-stripped hormone-free plasma, at concentrations of 3 . 125-25 fmol/ml plasma and extracting and assaying the samples as mentioned before. To determine remaining interference after ex­traction, from each subject studied, 10 ml plasma was treated with charcoal and 2 x 5 ml was extracted and assayed with the other samples. The same was done with the plasma used for recovery tests. Concentrations of unknowns were cal­culated from the assay results by correction for the recovery, the volume ex­tracted (5 ml), the final fraction of the sample used in the assay (450/600), and for remaining interference after extraction.

Studies performed. Four studies were performed on separate days, the first three in random order. In these three the liquid meals were given at pH 2.0, 2.5, and 5.5, respectively, and intragastric titration was performed at the same pH

as the meals. A basal blood sample was taken before the meal while aspirating gastric contents, and during the test samples were taken at 15-min intervals. On the same day as the test with the meal at pH 5.5, 2 h after finishing that test, se­cretin was infused in increasing doses, respectively, 2.5, 8.0, and 25.0 pmol.kg· 1.h·1

• Each dose was infused during 30 min. At the end of each period

6 1

a blood sample was taken. During the infusion gastric contents were aspirated continuously.

In the fourth study, a liquid meal at pH 5.5 was again given, now along with intravenous infusion of secretin at a dose designed to match plasma levels found during the meals at low pH. The required dose was estimated on an indi­vidual basis by comparison of the plasma secretin found during the meals at low pH and the levels during the infusion of increasing doses of secretin. The secrc­tin infusion was started at the same moment as the instillation of the meal. Blood was collected at 1 5-min intervals for gastrin and at 30 and 60 min for se­cretin.

In four subjects , two additional studies were performed. In both, again a meal at pH 5.5 was given. In the first, secretin was infused only during the meal at a dose five- to sixfold higher than the dose given in the former study. In the second, secretin was infused at the same dose as in the first . but the infusion was started 1 h before the meal and continued throughout the test. Gastric empty­ing was not studied in these two tests. Blood samples were taken at 1 5-min in­tervals for gastrin and at 30-min intervals for secretin.

Data analysis. For each test in each subject the integrated responses for gas­trin and secretin were calculated. For gastrin the integrated response was calcu­lated using the natural logs of the responses. The integrated mean was obtained by dividing this value by the time (60 min). Two-way analysis of variance (sub­ject by condition) was performed on these integrated means. Gastrin values given are the mean logs. transformed back to the raw scale. For acid, total secretion during the test hour was used in the analysis of variance without considering basal acid secretion. For the gastric emptying the T½ was calculat­ed separately for each test of each subject using a nonlinear fit to the single ex­ponential (21). Two-way analysis of variance was calculated using these esti­mates of T½.

RESULTS

SECRETIN RADIOIMMUNOASSA Y

Antibody. The final titer of the antibody used in the assay was 1 :400 ,000. Glucagon at a concentration of 1 nmol/ml produced a 10% inhibition of binding of the label to the antibody. No other cross-reactivity was detected (Fig. 1).

Labeled antigen. The specific activity of the label was 2.5 JLCi/nmol. The di­lution curve of the label was parallel to the standard curve. In the presence of excess antibody 85% of the label was bound. Unlabeled secretin eluted on the SP-Sephadex C-25 column in the fractions preceding those containing the la­beled secretin.

62

B/F

1.0

0.5

0

•

Figure I .

1 10 102

VIP GIP

GL�

103

104

105

106

CONCENTRATION , pmol/l

Representative ,tandard curve of the radioimmunoassay for secretin : the concentration of \tand­;ird secret in (pmol/liter) (x - axis) plotted against ratio of bound-to-free labeled secret in (y . .ixis) . Dilution curve\ for gluc.igon . ga\tnc inhibitory peptide (GIP). and vasoactive inte,tim1l peptide (VIP) in the \amc J».iy ,y,tcm.

Assay. The detection limit of antigen added to assay buffer was 2.6± 1 . 4 fmol/ml, the 1D50 was 12.9±2.0 fmol/ml (mean ± SD). Fig. I shows a represent­ative standard curve. The addition of Trasylol to the assay buffer decreased the 1D50 by 20%. Binding of the label in the absence of antibody was 6.7± 1 . 8%. The ratio of bound over free label in the absence of unlabeled antigen was 1 .06±0.2.

Reproducibility. The coefficient of variation within assays was 7% at 1 6 fmol/ml, 15% at I O, and 13% at 5 fmol/ml, and between assays 16% at 1 6 and 2 1 % at 5 fmol/ml.

Table I . Recovery of standard �ecretin . added to hormone-free plasma. after extraction and con­centration from C i. cartridges.

Concentration 3 . 125 6.25 12.5 25.0 Secrctin . pM n = 7 n = 1 3 n = IO n = 4

Recovery. % (mean±SD) 62± 13 64± 10 67± 10 72:= 10

63

Preparation of plasma samples. The recovery from the extraction procedure was 66±7%. The recovery was slightly higher at higher concentrations. How­ever . the ranges were the same, and the differences were not significant (Table I ) . Interference after extraction of charcoal treated plasma was I . I ±0. 9 fmol/5 ml plasma.

- 50 .....

E Q. -C •

a,

40 a, lit •

0 •

E lit 0

<I 30

•

•

•

20

• •

10 Y= -0.28 +1.229x • r = 0.93

•

0 10 20 30 40

infusionrate ,pmol .kg-1. h-1

Figure 2. Correlation in nine healthy male subiects between infusion rate (pmol .kg· 1 .h-1 ) of pure porcine se­cretin, as determined from the rad,oimmunoassay measured concentrations of the hormone in the infusion solutions. and the increase from basal of the plasma secretin concentrations (pM), as measured by radioimmunoassay. usmg C18 cartridges for the extraction and concentration of secretin from plasma. P <0.00 1 .

64

HUMAN STUDIES Plasma secretin. During the meals at low pH a rise of plasma seeretin concen­

trations was seen during the first 30 min (peak concentration pH 2 .0 , 5 .7 fmol/

ml ; pH 2 .5 . 5 .4 fmol/ml) after which the concentrations fell to almost basal at 60

min. During the meal at pH 5 .5 plasma secretin did not increase . When secretin

was infused in increasing doses there was a good correlation (r = 0 .93) between

the increases of plasma sccrctin and the infusion rate of seeretin , as calculated

from the measured secrctin concentrations (Fig. 2) . The clearance rate calcu­

lated from these data ( infusion rate/plasma concentration) was 1 5 .2±6.6

ml . kg- 1 . min· 1 (mean ± SD) . The mean dose of secretin needed to match the

plasma concentrations found during the meals at low pH was 5 . 6 pmol .kg- 1 .h· 1

(range 2.3-9 .5 ) . The integrated means of plasma secretin concentrations during

the meals of low pH and the meal of pH 5.5 with sccrctin infusion did not

differ. The results arc summarized in Table II and Fig. 3 A .

Table 1 1. McJn acid ,ecrct1on ,ind mean pla\ma ga�trin and �ccretin concentrations (integrated re­sponse/60 min) during I h intrngastnc titration after ga�tric inMillation of 500 ml 8° ., peptone �olu­tion.

pH meal

5 . 5 5.5 plus secret in 2.5 2.0

Acid secretion 11 6

mmol/h

1 5.7 1 4.9 I0.6*:;: 7.9*:;:

*Significantly different from 5 . 5 +Significantly different from 5 .5 plus �ccretin

Mean gaMrin Mean ,ecn:tin n - 9 n 9

pM pM

33 0.6:j: 33 3 .4 20 :j: 3.2 18 t 3 _9•

Gastrin . Compared with the meal of pH 5 .5 there was a significant inhibition

of gastrin release during the meals of low pH. The infusion of secrctin during

the meal of pH 5 . 5 did not influence gastrin release (Fig . 3 B and Table 1 1 ) .

Acid secretion. Acid secretion was significantly lower during the meals of

low pH, compared with the meal of pH 5 .5 . Acid secretion could be evaluated

in only six subjects, because duodenogastric reflux of bicarbonate occurred in

one subject during secretin infusion and intragastric pH of 5 .5 and in two sub­

jects during intragastric pH of2.0 and/or 2 .5 , and caused sudden increases in in­

tragastric pH. No difference was seen between acid secretion during the meals

at pH 5.5 with or without concomitant secretin infusion (Table II) .

65

---E Cl.

z t­u.I 0:: u u.J Vl

---0 e C. ' z 0:: I­V') < l!J

6

4

2

0

80

40

0

Figure 3.

N=9

0 1 5 30 45 60

T I ME (M I NUTES ) AFTER INSTI LLATION MEAL

N: 9

0 15 30 45

5.5 +S

2.5

5.5

60

TIME (M INUTES) AFTER INSTI LLATION MEAL

5.S +S

5.5

2.5

Mean ( ± SE) basal and protein-stimulated (500 ml 8°0 peptone solution) plasma secret in and plas­ma gastrin concentrations (pM) during mtragastric titration at pH 2.5 and pH 5.5 and at pH 5 .5 with concomitant secretin infusion in a dose designed to match the plasma concentration found during the test at pH 2 .5 (pH 5 . 5 plus secretm) . in nine healthy male subjects Plasma secretin and gastrin concentrations were determined by radioimmunoassay.

66

Duodenal acid load. The maximal duodenal acid load during the meals of low

pH occurred during the first 15 min, and subsequently decreased (Table III).

Table I l l . Duodenal acid load (millimolc�) (mc,in ± SEM) during I h intrJgaMric titration after ga�tric instillation of 500 ml 8°0 peptonc �olution .it pH 2.0 and 2 .5 (n = 6)

pH me.ii

2.0 2.5

0- I S min

19.6+-l.7 1 9. 1 ±2.8

1 5-30min

1 2.3±3 .7 7 . 1 ±2 .3

30--lS min

1 1 .3± 3.3 1 1 .0±2.0

45-60 min

5 .9± 1 . 2 7. 1 ±2 .0

Gastric emptying. Gastric emptying rate was significantly slower at low intra­

gastric pH than during intragastric pH of 5 .5. The T½ during the meal of pH 5.5

with concomitant secretin infusion was longer than during the meal without se­cretin, but this difference was not quite significant (P = 0.053) (Table IV).

High dose secretin infusion. Similar mean secretin plasma levels during the

plateau phase were found during the 1 -h and the 2-h tests (33 and 36 pM, re­

spectively, mean dose 32 pmol .kg· 1 . h- 1 ). Neither during the 1 -h infusion nor

during the 2-h infusion was the mean plasma gastrin different from that during

the meal without sccretin (36 and 34 vs. 45 pM) . Acid secretion could not be measured during these tests because of duodenogastric reflux, which was ap­

parent since the intragastric pH fluctuated above 5.5 during most of the tests. Biologic activity of secretin. During the infusion of 2.5 , 8.0, and 25

pmol.kg· 1 .h- 1 secretin in 0. 1 5 M NaCl with I % bovine serum albumin into the

test dog, the volume and bicarbonate concentration of the pancreatic secretion

increased progressively from basal values of 2.2 ml/ 1 5 min and 61 mM to maxi­

mal values of 8.3 ml/1 5 min and 1 1 3 mM.

Table IV. Rate of gastric emptying during I h intragastric titration after gastric instillation of 500 ml 8'1/o peptone solution (n = 8).

pH meal

5 . 5 5.5 plusS 2 .5 2 .0

T1h. mean ± SEM

min

29±3 47±9:j: 74 ± 1 2* 79± 10·

T 'h = calculated time. at which the stomach is half empty; S. concomitant secretin infusion. *Significantly different from 5.5. :j:Not significantly different from 5.5. P = 0.053.

67

DISCUSSION

SECRETIN RADIOIMMUNOASSA Y The radioimmunoassay for secretin has characteristics similar to those of

other assays described in recent years ( I , 22-25). The 10,11 of 1 2. 9 fmol/ml com­pares well with other assays ; however, the detection limit of 2.6 fmol/ml is slightly higher than that reported in several other assays. Because of the low physiologic plasma levels it was therefore necessary to develop a method for ex­traction and concentration of plasma by which larger volumes per plasma sam­ple could be processed than with the alcohol extraction method, in order to be able to measure those low levels reliably. The method described in this study of­fers the possibility to measure plasma concentrations as low as 1 . 3 fmol/ml. The recovery of the secretin from the SEP-PAK, 66% , is about the same as found with the alcohol extraction method (23, 25) and similar to that reported earlier for similar extraction (26). Remaining interference after extraction of plasma has also been reported by others, and is quantitatively not different from other reported data (25). Although most investigators add Trasylol to the assay buff­er, only one reported its influence on the assay, saying that it did not interfere with the results (23). We found that the addition of 0.5% Trasylol 10,000 KIU/ ml consistently improved the 10511 of the assay by 20%. An explanation may be that Plasmanate used as protein in our buffer may contain enzymes and the use of bovine serum albumin might make the addition of Trasylol unnecessary. The finding of a close correlation between the secretin infusion rates and the increases of plasma concentrations is a firm validation of our assay and the extraction procedure. The metabolic clearance rate found is similar to what Schaffalitzky de Muckadell et al. found (4).

HUMAN STUDIES Based on the studies (7, 8) that showed that exogenous secretin can inhibit

acid secretion and gastrin release in man, it has been postulated that secretin may play a physiologic role in the inhibition of acid secretion and gastrin re­lease. However, in these studies unphysiologically high doses of secretin were infused. Since the development of reliable radioimmunoassays for secretin, it is known that basal plasma levels of secretin are very low and that after meals only small absolute increases occur. Recent studies ( 1 1 , 12) in dogs indicate that at these low levels secretin can inhibit acid secretion and gastrin release, and that in dogs secretin may play a physiologic role in the inhibition of acid secretion. In this study we found a strong inhibition of acid secretion and gastrin release by a low intragastric pH. The data on gastric acid secretion at pH 2.0 have to be con­sidered with caution , since intragastric titration at that pH may underestimate

68

the amount of acid produced by up to 25% depending on the concentration of

the secreted HCI (27). However, also at pH 2.5 a significant inhibition oc­

curred. In contrast, exogenous secretin producing plasma levels similar to those found during meals of low pH did not inhibit gastrin release and acid se­

cretion during a meal of pH 5 .5 . Although the peak levels of secretin during the

meals of low pH were slightly higher than the plateau levels during the secretin

infusion (5.5 vs. 4 .6 fmol/ml) , it is unlikely that this explains the lack of inhibito­ry effect by secretin , because the mean levels were similar in these studies. Sev­

eral facts may contribute to the differences found between humans and dogs.

Postprandial plasma levels of secretin , as measured by the same radioimmuno­

assay, are higher in dogs than in men (5, 24) . Further, dogs seem to be more

sensitive to the acid-inhibiting effect of secretin than men (7) . A possible

explanation for these differences could be a structural difference between hu­

man and porcine and/or canine secretin , that could produce falsely low radio­

immunoassay values in men and/or a lower biologic activity.

The doses of secretin infused during the meals of pH 5 .5 produce a marked

increase of pancreatic secretion, and are therefore thought to produce physi­

ologic plasma concentrations. It might thus be expected that the secretin given

in this study could inhibit the gastrin release if it plays a physiologic role in this

inhibition. Our results are in agreement with earlier reports in which it was

shown that secretin was not responsible for the inhibition of pentagastrin­

stimulated acid secretion by intraduodenal acidification (28-30) , nor was it

apparently the cause of inhibition of histamine-stimulated secretion (31 ) .

Apparently factors other than secretin are the mediators o f the acid-induced

inhibition of gastrin release, which we found to be responsible for inhibition of

acid secretion by low intragastric pH . Whether the mechanism of this inhibition

is through intragastric pathways or whether mechanisms through receptors in

the duodenal or intestinal wall are involved is an unsolved question . The inhibi­

tion by intraduodenal acidification is independent of gastrin (28) , and thus does

not seem to play a great role in the inhibition by low intragastric pH, which is

primarily an inhibition of gastrin release . Further studies are necessary to de­

termine the mechanisms involved in the acid-induced inhibition of acid secre­

tion. Hormonal , paracrine, and neural mechanisms and substances such as so­

matostatin , vasoactive intestinal peptide, and prostaglandins may be involved.

The finding that exogenous secretin at doses five- to sixfold higher than those

found during the meals at low pH does not significantly inhibit the gastrin re­

lease is not in disagreement with other studies, although a real difference might

have become apparent if larger numbers of subjects had been studied. Dalton

et al. (8) found that secretin in doses up to 0.5 CU.kg.- 1 . h- 1 equivalent to 41

pmol .kg- 1 .h-1 did not inhibit gastrin release in response to a steak meal , but

69

these authors did not add protein to the infusion solution so it may well be that they gave lower doses than they had intended. Londong et al. (9) found that peptone-stimulated gastrin release was inhibited by 0.5 CU .kg 1 .h- 1 secretin, but this effect first became apparent dunng the second half hour of the first hour after the start of the study, and the integrated mean over the first hour was probably not different whether they gave secretin or not. Whether these high levels of secretin inhibit gastrin release and/or acid secretion is probably not im­portant for an understanding of physiologic mechanisms, since these levels are never seen under pure physiologic conditions.

A low intragastric pH clearly inhibits gastric emptying. Although we did not find a significant inhibition by secretin, secretin may still play a role in the inhi­bition of gastric emptying as the findings of Valenzuela and Defilippi ( 1 3) sug­gest. We studied only eight subjects and the borderline significance (P = 0.053) may have been due to this small number.

We conclude that secretin does not play a role in the acid-induced inhibition of peptone-stimulated acid secretion and is unlikely to have a physiologic role in the inhibition of acid secretion in man. The role that secretin may play in the acid-induced inhibition of gastric emptying seems to be a minor one.

REFERENCES I . Greenberg GR. McCloy RF. Baron JH . Bryant MG. Bloom SR. Gastnc acid regulate� the re­

lease of plasma secretm in man Eur J Chn I nvest 1 982: 1 2 : 36 1 -372. 2 . Pelletier MJ. Chayvialle JAP. Minaure Y. Uneven and transient secretm release Jfter a liquid

test meal. Gastroenterology 1 978; 75; I 1 24- 1 1 32 3. Fahrenkrug J . Schaffalitzky de Muckadell OB . Rune SJ . pH threshold for relea�e of secret in in

normal subjects and in patients with duodenal ulcer and patients with chronic pancreatitis. Scand J Gastroenterol 1 978; 13: 1 77- 1 86.

4 . Schaffalitzky de Muckadell OB, Fahrenkrug] . Boolsen SW. Worning H Pancreatic response and plasma secretin concentration during mfu�ion of low dose secretin in man. Scand J Gas­troenterol 1 978; 1 3 : 305-3 1 1 .

5 . Chey WY. Kim MS. Lee KY. Chang TM. Effect of rabbit antisecretin serum on postprandial pancreatic secretion in dogs. Gastroenterology 1 979; 77: 1 266- I 275.

6 . Wald um HL. Walde N. Bu rho I PG . The effect of secret in on gastric H� and pepsm secretion and on urinary electrolyte elevation m man . Scand J Gastroenterol 198 1 ; 16: 999- 1004.

7 . Brooks AM, Grossman MI . Effect of secretm and cholecystokinm on pentagastrin-stimulated gastnc secretion in man. Gastroentcrology 1970; 59. I 1 4- 1 19 .

8 Dalton MD, Eisenstein AM. Walsh JH. Fordtran JS Effect of sccretin on gastric functton in normal subjects and m patients with duodenal ulcer. Gastroenterology 1 976; 7 1 : 24-29.

9. Londong W, Londong V. Hanssen LE. Schwanner A. Gastric effects and side effects of syn­thetic secretin in man. Regul Pept 1 98 1 , 2: 23 1 -244.

10. Jansen JBMJ , Lamers CBHW Calcitonm and secretm inhibit bombesin-st1mulated serum gastrin and gastric acid secretion in man. Regul Pepi 198 1 : I : 4 1 5-421

1 1 . Chey WY. Kim MS. Lee KY, Chang TM. Secretin is an enterogastrone in the dog. Am J Phys1-ol 1 98 1 ; 240: G239-G244.

12 . Kim YM. Lee KY, Chey WY. Role of secretin on postprandial gastrin release in the dog: A further study. Surgery (St. Louis) 1 98 1 ; 90: 504-508.

70

13 . Valenzuela JE, Defilippi C. Inhibition of gastric emptying in humans by secretin, the octapep­tide of cholecystokinin and intraduodenal fat. Gastroenterology 1981 ; 8 1 : 898-902.

14 . Eysselein VE, Kleibeuker JH. Maxwell V, Reedy T, Walsh JH. Inhibition of gastric acid secre­tion at low intragastric pH in man : relation to plasma gastrin. Gastroenterology 1983; 84: 1 1 47a (Abstr .) .

15 . Hassan MA, Hobsley M. Positioning of subject and of nasogastric tube during a gastric secre­tion study. Br Med J 1 970; I : 458-460.

16 . Lam SK, Isenberg JI, Grossman Ml, Lane WH, Walsh JH. Gastric acid secretion is abnormal­ly sensitive to endogenous gastrin released after peptone test meals in duodenal ulcer patients. J Clin Invest 1980; 65: 555-562.

17 . George JD. New clinical method for measuring the rate of gastric emptying: the double-sam­pling test meal . Gut 1 968; 9: 237-242.

18 . Gross RA, Isenberg JI, Hogan D. Samloff IM. Effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load, and serum gastrin in duodenal ulcer and normal subjects. Gas­troenterology 1 978; 75 : 357-362.

19 . Rosenquist GL, Walsh JH . Radioimmunoassay of gastrin . In Gastrointestinal Hormones. G.B. Jerzy Glass, editor. Raven Press, New York, 1 980, pp 769-795.

20. Rodbard D, et al. Radioimmunoassay Data Processing: Listings and Documentation, Third ed. The Logistic Method and Quality Control (PB246 or 224). U.S. Department of Commerce,

National Technical Information Service, Springfield, VA. 1975; Vol. 2. 2 1 . Elashoff JD, Reedy TR, Meyer JH. Analysis of gastric emptying data. Gastroenterology I 982;

83: 1 306- 1 3 1 2. 22. Bu rho I PG. Waldum HL. Radioimmunoassay of secret in in acidified plasma. Acta Hepatogas­

troenterol 1 975 ; 25: 474-48 1 . 23. Hanssen LE, Torjesen P . Radioimmunoassay of secret in in human plasma. Scand J Gastroen­

terol 1977; 12 : 481 -488. 24. Rominger JM, Chey WY, Chang TM. Plasma secret in concentrations and gastric pH in healthy

subjects and patients with digestive diseases. Dig Dis Sci 198 1 ; 26: 591 -597. 25. Schaffalitzky de Muckadell OB, Fahrenkrug J. Radioimmunoassay of secretin in plasma.

Scand J Clin Lab Invest 1 977; 37: 155- 1 62. 26. Yang R-K, Li H-R, Eng J , Greenstein R, Straus E, Yalow RS. Secretin responses to feeding

and acid load. J Lab Clin Med 1 983; 102: 1 7-23. 27. Spenney JG. Physical chemical and technical limitations of intragastric titration. Gastroente­

rology 1979; 76: 1 025- 1 036. 28. Ward AS, Bloom SR. The role of secret in in the inhibition of gastric secretion by intraduode­

nal acid. Gut 1 974; 15 : 889-897. 29. Wormsley KG. Response to duodenal acidification in man. I I . Effects on the gastric secretory

response to pentagastrin. Scand Gastroenterol 1970; 5: 207-2 15 . 30. Berstad A, Petersen H. Dose-response relationship of the effect of secret in on acid and pepsin

secretion in man. Scand J Gastroenterol 1970; 5: 647-654. 3 1 . Johnston D, Duthie HL. Inhibition of histamine-stimulated gastric secretion by acid in the du­

odenum in man. Gut 1 966; 7: 58-68.

71

CHAPTER 7

RETARDATION OF GASTRIC EMPTYING OF

SOLID FOOD BY SECRETIN

J. H. Kleibcukcr, H. Beekhuis, D. A. Piers, 0. B. Schaffalitzky de Muckadell

Departments of Gastroenterology and Nuclear Medicine, University Hospital, Groningcn , The Netherlands; Department of Clinical Chemistry , Bispebjerg Hospital, Copenhagen, Denmark.

ABSTRACT The effect of secretin at nearly physiologic plasma concentrations on gastric

emptying rate of solid food was studied in ten healthy men. A 99"'Tc-colloid la­beled pancake was used as test meal. Gastric emptying rate was measured dur­ing one hour using a dual headed gamma camera, and was expressed as the half time of the emptying curve. To prevent endogenous secretin release 400 mg ci­metidine was given prior to the meal. Subjects were studied under three condi­tions: (1) during infusion of saline; (2) during continuous infusion of secretin, 6.6 pmol/kg.h; and (3) during three intermittent 10 minutes periods of secretin infusion, 7.6 pmol/kg.h during each period. Both continuous and intermittent infusion of secretin increased half emptying time, by 133% and 55% respective­ly. Plasma secretin concentration during (1) was 0.8 pM ;_plateau concentration during (2) 9.8 pM; and integrated mean concentration during (3) 4.8 pM. It is concluded that secretin at approximately physiologic plasma concentrations re­tards gastric emptying of solid food in man.

INTRODUCTION Secretin is a polypeptide hormone, which is released by endocrine cells in the

mucosa of the duodenum and upper jejunum when the intraduodenal pH falls below 3 to 4 ( I , 2, 3). Secretin is now well recognized as a physiological stimu­lant of pancreatic bicarbonate secretion ( 4, 5). Other physiologic functions of secretin have not yet been established. Acidic solutions are known to inhibit the gastric emptying rate (6). The mediator of this inhibitory effect is not known. Se cretin is a candidate for this in view of its release by duodenal acidifi­cation. Several investigators indeed found that secretin inhibits the gastric emptying rate of liquid meals, not only at high doses (7, 8), but probably also at doses which result in physiological plasma concentrations of secretin (9, 10).

73

However the effect of secretin on the gastric emptying of solid foods in man has not been studied. We, therefore, studied the influence of near-physiological plasma secretin concentrations on the gastric emptying rate of a solid meal. To exclude the effect of duodenal acidification on endogenous secretin release and on gastric emptying, cimetidine was given previous to all tests performed to in­hibit gastric acid secretion.

MATERIALS AND METHODS Subjects. Twelve healthy male volunteers (mean age 32 years, range 29-41)

were studied. All subjects gave informed consent. The study was performed ac­cording to the guide-lines as layed down in the declaration of Helsinki.

Test meal. A 99mTc-labeled pancake was used as test meal ( 11). The meal was made of 45 g white flour, 10 g Nutrilose® milkpowder (Nutricia), 10 g marga­rine, 5 g sugar and 80 ml water and contained 7.9 g protein, 39 g carbohydrate and 9 .4 g fat, corresponding to 269 Kcal. About 10 MBq 99mTc-colloid was add­ed to the batter, just before baking. The effective radiation dose equivalent was 0.3 mSv ( = 30 mrem) per test. In order to measure the binding of the tracer under the influence of chewing, saliva and gastric juice a labeled pancake was divided in four equal portions. Three of them were chewed and spat out again. One portion was cut into small pieces. All four portions were than mixed with 75 ml fresh pentagastrin-stimulated human gastric juice (pH 1.2) and incubated at 37°C during 4 hours. Subsequently the portions were sieved. The radioactivi­ty of the so obtained fluid and that of the remaining solid material were deter­mined. It was equally divided over the four portions. The fluid obtained from the four portions contained only a mean of2% (range 0-6) of the total activity of each of them. From these results it was concluded that the Tc-colloid labeled pancake is a suitable test meal for the study of the gastric emptying of solid food. The pancake was eaten immediately after preparing it. Having finished the pancake the subjects drank 100 ml water, rinsing their mouth with the wa­ter. It took about 4 minutes to consume the meal.

Gastric emptying rate was measured using a dual headed gamma camera (Siemens ROT A), which was interfaced to a computer (DEC, gamma 11 ). Low energy all purpose collimators were used. The subjects were seated between the heads of the camera leaning back slightly. As soon as the test meal was fin­ished digital frames were recorded for one hour in one minute periods. A region of interest over the stomach was chosen using the sum image of all sixty frames, certifying that the whole gastric region was included. Then the time-ac­tivity curves for these regions on the anterior and posterior views were generat­ed. The final emptying curve was obtained by calculating the geometric mean of the anterior and posterior curves. This method guarantees that the resulting

74

emptying curve is independent of the changing depth of the meal-bound tracer due to the anatomical position of the stomach ( 12).

The rate of gastric emptying was expressed as the half emptying time (T 1h), which is the time necessary to empty half of the activity originally present in the stomach, beginning at the start of registration, i.e. at the end of the meal. The T 1h was determined by fitting a power exponential curve to the emptying curve, as recommended by Elashoff ct al (13). The use of this curve provides the possi­bility to give a reasonable good description of a variety of emptying patterns of different meals. The relation between the fraction of the meal still present in the stomach (f) and the time (t) is expressed by the formula

0 f = 2-(lffl/2)

in which the variable power exponent beta (B) determines deviations from a monoexponential decaycurve. The emptying curve for solid food often shows a lag phase i.e. a period during which no food is emptied from the stomach (14, 15). This phase probably represents the time to grind the food into smaller par­ticles (16). With an increasing length of the lag phase the value of the power ex­ponent beta will be progressively larger than 1.0. The power exponential curve was fitted to the emptying curve using a nonlinear least squares method. The quality of the fit is represented by the parameter R2 , which should approximate 1.0 ( 13).

Secretin assay. Concentrations of secretin in plasma and infusion fluids were determined by radioimmunoassay as described previously (17 , 18).

Tests performed. All tests were done after an overnight fast. During each test the subjects had an intravenous needle in each arm, one for blood sampling and the other for infusion of saline with or without secretin. Twentyfive minutes prior to the meal 400 mg cimetidine (Smith, Kline & French) was given intrave­nously to inhibit gastric acid secretion and thereby to prevent release of endo­genous secretin, in order to have completely controlled plasma secretin con­centrations during all tests. To test the release of secretin under these circum­stances three subjects were given an unlabeled pancake after cimetidine. Blood samples were taken before and at 5-minute intervals after the meal for plasma secretin determination. In previous studies cimetidine has been shown not to affect the gastric emptying rate in humans (19, 20, 21).

Gastric emptying rate was measured during saline infusion ( control) and dur­ing continuous and intermittent infusion of secretin. In the control study, as performed in all ten subjects , 0.15 M NaCl at a rate of 30 ml/h was being in­fused. Six subjects were studied while secretin was infused continuously during the whole test. Highly purified natural porcine secretin (Kabi Vitrum) was dis-

75

solved in 0.15 M NaCl, containing 0.25% (W/Y) human scrum albumin. Sccrc­tin was given at a dose of 6.6 ± 0.5 pmol/kg.h as measured by radioimmunoas­say. The infusion, at a rate of 30 ml/h, was started just before the meal was eaten. Nine subjects were studied while secretin was infused intermittently. Secretin was given during three periods of 10 minutes, from 7 .5 till 17 .5 , from 25 till 35 and from 42.5 till 52.5 minutes after finishing the meal. In the remaining time 0.15 M NaCl was given. The dose of secrctin during each period was 7.6 ± 1.4 pmol/kg.h .. The total volume infused was 30-40 ml. The tests were performed in a random order in each subject with an interval between the tests of at least one week.

Blood samples were taken during the control studies and the studies with continuous infusion of sccretin 15 minutes before and 30 and 60 minutes after the meal. During the intermittent administration of sccrctin samples were tak­en before and 17.5, 25 , 35, 42.5 and 52.5 minutes after the start of the registra­tion. Samples were collected into ice-chilled tubes and left on ice until centrifu­gation immediately after the end of the test. Plasma was stored at -20°C until radioimmunoassay. The samples were coded and assayed in random order.

Statistical analysis. Results arc given as mean ± standard deviation. The half emptying times and the power exponents (beta) during the continuous and in­termittent infusion of secretin were compared with their respective paired con­trol values by the Wilcoxon·s rank sum test for paired results. Differences were considered to be significant. when p < 0 05.

RESULTS The plasma secretin concentrations remained at fasting levels during the

whole hour after the ingestion of the unlabeled pancake in the three subjects from whom samples were taken at 5-minute intervals: mean concentration be­fore the meal 0.8 ± 0.4 pmol/1, postprandially 0.8 ± 0.5 pmol/1.

Fasting plasma secretin concentration in the twelve test subjects was 1.2 ± 1.8 pmol/1. The concentration did not increase during control studies. During continuous infusion of secretm plasma concentrat10ns were 10.2 ± 3.5 pmol/1 at 30 minutes and 9.4 ± 3.4 pmol/1 at 60 minutes. Continuous infusion of secretin increased the half emptying time significantly from 67 ± 13 minutes to 156 ± 92 minutes (figure 1, 2). The power exponent was not significantly affected by se­cretin: 0.9 ± 0.3 during secretin infusion versus 1.2 ± 0.3 during control stud­ies. During intermittent infusion of secretin plasma concentrations reached peak levels around 8.5 pmol/1 and fell to 2. 5 pmol/1 in between (figure 3). As­suming that plasma secretin started to rise from fasting level when the secretin infusion was first begun and that at 60 minutes the plasma concentration was similar to those at 25 and 42. 5 minutes an integrated mean concentration could

76

Tv, (minutes)

350

300

250

200

1 50

100

50 I

0

control

Figure 1 .

continuous

secrehn

Gastric half emptying time\ (T1h) of a wmTc-colloid labeled pancake during control studies and during continuous infusion of �ecretin (6.6 + 0.5 pmol/kg .h) in six healthy subjects (mean ± SD).

� 2-.. 100

� 75

control u 50

25

0 0 15 30 45 60

time (minutes)

Figure 2. Mean gastric emptying curves of a WmTc-colloid labeled pancake during control studies (T½ 67 min, power exponent 1 .2) and during continuous infusion of secretin (6.6 ± 0.5 pmol/kg.h) (T½ 1 56 min, power exponent 0.9) in six healthy subjects.

77

-- 12

1 0

8

6

4

I 2

0 I 0

secretin infusion -

I

15

- -

I

I I

30

I

45 60

time (minutes) Figure 3 . Plasma secret in concentrat1ons (mean ± SD) during intermillent infusion of �ecretin ( • ) (7 .6 ± I .� pmol/kg .h) and during control studies ( )) in nine healthy subjects .

be calculated for each subject. This mean integrated concentration was 4.8 ± 0.9 pmol/1 . Intermittent infusion of secretin significantly increased half empty­ing time from 55 ± 10 minutes to 85 ± 29 minutes (figure 4, 5). The power expo­nent was not affected by intermittent secretin administration: 1.3 ± 0.5 during secretin infusion versus 1.2 ± 0.2 during control studies.

The power exponential curve fitted well to the emptying curves: median R2

was 0.96 (range 0.69-0.99; R2 � 0.90 in 3/27 tests).

DISCUSSION Secretin mediates stimulatio_n of pancreatic fluid and bicarbonate secretion

in response to acidification of the duodenum. It has been hypothesized that in addition to this effect secretin may also have a role in the prevention of duode­nal acidification by inhibition of gastric acid secretion and gastric emptying rate. Although secretin is indeed a potent inhibitor of gastric acid secretion and gastrin release (22, 23), there is recent evidence that secretin does not have a physiological role in the regulation of these in man (9).

In respect to gastric emptying, two studies (9, 10) suggest that secretin may be a physiologically important inhibitor of the emptying rate of liquids in man.

78

T112 !minutes)

120

80

40

Figure 4.

I

control intermittent secretin

Gastric half emptying times (T1h) of a '"'mTc-colloid labeled pancake during control studies and dur­ing intermittent infusion of secret in (7 .6 + 1 .4 pmol/kg .h) in nine healthy subjects (mean ± SD).

� 100

�

V)

.!: 75

d QI

E

so C0'1!rol

25

0

0 15 30 45 60

time (minutes)

Figure 5 . Mean gastric emptying curves o f a 99mTc-colloid labeled pancake during control studies (T½ 55 min, power exponent 1 .2) and during intermittent infusion of sccretin (7.6 :I- 1 .4 pmol/kg.h) (T½85 min, power exponent 1 .3) in nine healthy subjects.

79

In this study we now show that secretin is also a potent inhibitor of gastric emp­tying of solids. The question is whether this capacity has any impact for the reg­ulation of gastric motility. Physiologic postprandial plasma concentrations of secretin, as measured by the assay used in this study, are 7 pmol/1 or less (24). So during the continuous infusion of secretin as described above, the concentra­tions were slightly above the physiological range. During the intermittent infu­sion the peak plasma secretin concentrations of about 8.5 pmol/1 were still somewhat above the upper limit of postprandial concentrations (24), but the estimated integrated mean concentration of 4.8 pmol/1 approximated those seen in the normal postprandial situation. In addition. intermittent infusion re­sulted in fluctuating plasma secretin concentrations, which are also seen under physiological conditions (2, 3, 24). So the marked retardation of the gastric emptying during the intermittent infusion. namely an increase by 55% of the half emptying time, is a strong indication that also at slightly lower plasma con­centrations, which are in the physiologic range . secretin is able to inhibit the emptying rate of solid food.

There was a large interindividual variation in the response of the gastric emp­tying rate to the secretin administration. This was not explained by differences in plasma secretin concentrations, since there was no correlation between these concentrations and the increase of the half emptying times (r = 0.51 , p > 0.05). So the observed variation seems to be due to differences in gastric sensitivity to secretin.

The mechanism through which secretin influences gastric emptying is not elucidated by this study. In previous studies in man it has been shown that high doses of secret in induce contraction of the pylorus (25, 26) and possibly reduce the intracavitary pressure of the stomach (26), so slowing the gastric emptying. Secretin may elicit these effects through direct action on the gastric smooth muscle similar to the neuropeptide vasoactive intestinal peptide (27), which is structurally related to secretin; or it may elicit the release of local substances like prostaglandins or enkephalins, which are present in the wall of the stomach and whose actions result in retardation of gastric emptying (28, 29).

Secretin is not the only intestinal hormone with an inhibitory effect on gastric motility. Neurotensin (30) and peptide YY (31) and the neuropeptide bombe­sin (32, 33) have all been shown to be able to retard the gastric emptying in man. The physiologic impact of all these effects have not yet been elucidated. Two recent studies (34, 35) indicate that cholecystokinin is a physiologic hormonal mediator of fat-induced inhibition of gastric emptying in man. From this pres­ent and previous studies it can be concluded that secretin too is a serious candi­date to be of physiological importance in the regulation of the gastric emptying rate of liquids as well as solid food in man.

80

REFERENCES I . Fahrenkrug J. Schaffalitzky de Muckadell OB. Rune SJ . pH thre�hold for releahe of secret in in

normal subjects and in patients with duodenal ulcer and patient� with chronic pancreatitis. Scand J GaMroenterol 1 978; 1 3: 1 77-86.

2. Greenberg GR. McCloy RF. Baron JH. Bryant MG. Bloom SR. Ga�tric acid regulates the re­le,1�c uf plasma ,ecretin in m,m . Eur J Clin lnvc,t 1 982; 1 2 : 36 1 -72.

3. Pelletier MJ. Chayvialle J AP. Minaure Y. Uneven and tranhient ,ecrctin release after a li4uid test meal. G,1stroenterolugy 1 978; 75: 1 1 24-32.

4. Chey WY. Kim MS. Lee KY. Chang TM. Effect of rabbit anti,ecretin serum on pu�tprandial pJncre,ltic secretion in dugs. Ga,troenterology 1 979; 77: 1 266-75.

5. Schaffalitzky de Muckadcll OB. Fahrenkrug J. Bool,en SW. Worning H. Pancreatic re,pon,e and plasma ,ecretin concentration during infusion of low do,e ,ecretin in man. Scand J Ga,­troenterol 1 978; 1 3: 305- 1 1 .

6. Hunt JN. Knox MT. The �lowmgof ga�tric emptying by four strong acid, and three weal,. acid�. J Physiol (London) 1 972; 222: 1 87-208.

7. Chey WY. Hitanant S. Hendricks]. Lorber SH. Effect of secrctin and cholecystokinin on gah­tric secretion in man. Gastroenterology 1 970; 58: 820-27.

8. Vagne M. Andre C. The effect of secretm on gastric emptying in man. Gastroenterology 1 97 1 ; 60: 42 1 -24.

9. Kleibeukcr JH. Eysselein VE. Maxwell V. Walsh JH. Role of endogenous secret in in acid-in­duced inhibition of human gastric function . J Clin Invest 1 984: 73: 526-32.

1 0. Valenzuela JE. Defilippi C. Inhibition of gastric emptying in humans by secret in. the octapep­tide of cholecystokinin and intraduodenal fat. Gastroenterology 1981 ; 8 1 : 898-902.

1 1 . Jacobs F. Akkermans LMA. Oei Hong Yoe. Hoekstra A. Wittebol P. A radioisotope method to quantify the function of fund us. ant rum and their contractile activity in gastric emptying of a semi-solid and solid meal. In: Wien beck M. ed. Motility of the digestive tract. New Yori,.: Ra­ven Press. 1 982: 233-40.

1 2 . Christian PE. Datz FL. Sorenson JA. Taylor A. Technical factor, in gastric emptying ,tudie,. J Nucl Med 1 982; 24: 264-68.

1 3 . Elashoff JD. Reedy TJ. Meyer JH. Analysis of gastric emptying data. Ga�troenterology 1982; 83: 1 306- 12.

1 4. Lavigne ME. Wiley ZD. Meyer JH. Martin P. Mc Gregor I L. Ga,tric emptying rate, of solid food in relation to body size. Gastroenterology 1 978; 74: 1 258-60.

1 5. Moore JG. Christian PE. Coleman RE Ga�tric emptying of varying meal weight and composi­tion in man. Dig Dis Sci 1 98 1 ; 26: 1 6-22.

16 . Meyer JH. Ohashi H. Jehn D. Thom,onJB. Size of liver particles emptied from the human sto­mach. Gastrocnterology 1 98 1 ; 80: 1489-96.

1 7. Schaffalitzky de Muckadell OB. Fahrenkrug J. Radioimmunoa,say of ,ecretin in plasma. Scand J Clin Lab Invest 1977; 37: 1 55-62.

1 8. Schaffalitzky de Muckadell OB. Fahrenl,.rug J . Nieben J. We,tphall I . Worning H. Meal-�tim­ulated secretin release in man: effect of ac,d and bile. Scand J GaMroentcrol 1981 ; 1 6: 98 1 -88.

1 9. Richardson CT. Walsh JH. Hicks Ml. The effect of cimetidine. a new histamine H�-receptor antagonist. on meal-stimulated acid secretion. scrum gastrin, and gastric emptying in patients with duodenal ulcer. Gastroenterology 1 976; 7 1 : 1 9-23.

20. Logan RFA. Forrest J AH. Mcloughlin GP. Lidgard G. Heading RC. Effect of cimctidine on scrum gastrin and gastric emptying in man. Digestion 1 978; 1 8: 220-26.

2 1 . Scarpignato C. Bertaccini G. Different effects of cimetidinc and ranitidinc on gastric emptying in rats and man. Agents Actions 1982; 12 : 1 72-73.

22. Jansen JBMJ. Lamers CBHW. Calcitonin and sccretin inhibit bombesin-stimulated serum gastrin and gastric acid secretion in man. Regul Pepi 1 98 1 ; I : 4 1 5-2 1 .

23. Londong W. Londong V. Hanssen LE. Sch wanner A . Gastric effects and side effects of �yn­thetic secretin in man. Regul Pep! 1 98 1 ; 2: 23 1 -44.

24. Schaffalitzky de Muckadell OB, Fahrenkrug). Secretion pattern ofsecretin in man: regulation by gastric acid. Gut 1978; 1 9: 8 1 2- 1 8 .

25. Fisher RS, Lipshutz W. Cohen S . The hormonal regulation o f pyloric sphincter function. J Clin Invest 1 973 ; 52: 1 289-96.

8 1

26. Geller LI. Petrenko VF. Effect of secretin and pancreozymin on mtracavitary pressure in the stomach and duodenum. evacuation from the stomach and the tone of the pyloric sphincter. Fi­ziologiya Cheloveka 1980: 6: 128-32.

27. Grider J R. Cable MB. Said SJ. Makhlouf GM. Vasoactive intestinal peptide as a neural medi­ator of gastric relaxation. Am J Physiol 1985; 248: G73-78.

28. Sanders KM. Role of prostaglandins in regulating gastric motility. Am J Physiol 1 98-1 : 247: Gl 1 7-26.

29 . Edin R. Lundberg J. Terenius L et al. Evidence for vagal enkephalinergic neural control of the felme pylorus and stomach. Gastroenterology 1980; 78: -192-97 .

30. Blackburn AM, Bloom SR. Long R et al. Effect of neurotensin on ga,tnc function in man . Lancet 1980; I : 987-89.

3 1 . Allen JM. Fitzpatrick ML. Yeats JC. Darcy K. Adrian TE. Bloom SR. Effects of peptide YY and neuropeptide Y on gastric emptying in man. Digestion 198-1; 30: 255-62.

32. Scarpignato C. Micali B. Vitulo F. Zimbaro G. Bertaccini G. I nhibition of gastric emptying by bombesin in man. Digestion 1982; 23: 1 28-3 1 .

33. Walsh JH. Maxwell V. Ferrari J. Varner AA. Bombesin Mimulates human gastric function by gastrin-dependent and independent mechanisms. Peptides 198 1 : 2. suppl 2: 193-98.

34. Liddle RA. Morita ET. Conrad CK. Williams JA. Regulation of gastric emptying in humans by cholecystokinin. J Clin Invest 1986; 77: 992-96.

35. Kleibeuker JH. Beekhu1s H. Jansen JBMJ. Lamers CBHW. Piers DA I nfusion of physiologic doses of cholecystokinin inhibits gastric emptying of food in man (abstr.). Gastroenterology 1 986; 90: 1-195.

82

CHAPTER S

CHOLECYSTOKININ IS A PHYSIOLOGIC

HORMONAL MEDIATOR OF FAT-INDUCED

INHIBITION OF GASTRIC EMPTYING

J. H. Kleibeuker, H. Beekhuis , J. B. M. J. Jansen, D. A. Piers, C. B. H. W. Lamers

Departments of Gastroenterology and Nuclear Medicine, University Hospital, Groningen; Department of Gastroenterology, University Hospital, Leiden, The Netherlands.

ABSTRACT

The effect of cholecystokinin-33 at physiologic plasma concentrations on gastric emptying rate of a semisolid meal was studied in 8 healthy men. A firm custard pudding, labeled with \l<JmTc-Chelex- 100 particles was used as test meal. Gastric emptying rate was measured during one hour, using a dual headed gam­ma camera and was expressed as the half time of the emptying curve. Plasma cholecystokinin concentrations were determined by radioimmunoassay. Sub­jects were studied under three conditions: ( 1) during infusion of saline (con­trol); (2) during cholecystokinin infusion, 0.375 IDU/kg.h; and (3) during chol­ecystokinin infusion, 0.75 IDU/kg.h. In addition, plasma cholecystokinin was determined after a regular meal. During the control studies plasma cholecysto­kinin increased only minimally. After the regular meal cholecystokinin in­creased to 6.5 pmol/1 at 30 min, decreasing to 5.3 pmol/1 at 60 min. During the lower and the higher dose of cholecystokinin infusion it increased to 4.5 and 7 .3 pmol/1 respectively. Both doses of cholecystokinin significantly (p < 0.05) in­creased the half emptying time, from 45 ± 8 min to 86 ± 17 min during the lower dose and to 198 ± 50 min during the higher dose. It is concluded that cholecystokinin most likely is a physiologic hormonal mediator of fat-induced inhibition of gastric emptying.

INTRODUCTION

The composition of food is a major determinant of the emptying rate of the stomach. The higher the energy content of a meal the slower is the gastric emp-

83

tying (1, 2). Especially fat, with its high energy content per gram, is a potent in­hibitor of gastric emptying. Fatty meals arc emptied slowly by the stomach (3) and intraduodcnal instillation of fat retards the emptying rate markedly (4). This inhibition is mediated through receptors in the duodenal and jejuna! mu­cosa. Since this inhibitory mechanism is still present after truncal vagotomy, hormonal mediators are probably involved (3).

Intraduodenal fat is the main stimulus of the release of the peptide hormone cholccystokinin (CCK) from endocrine I-cells in the duodenal mucosa (5). Cholccystokinin is a physiologically important mediator of the fat-induced stimulation of pancreatic enzyme secretion and gall bladder contraction (6, 7) . It has been suggested that cholecystokinin might also have a role in the fat-in­duced inhibition of gastric emptying. Several studies have indeed shown that cholccystokinin is able to retard gastric emptying (8, 9), although this could not be confirmed by others (10). However, in none of these studies plasma con­centrations of cholccystokinin were determined, so it could not be judged , whether physiological plasma concentrations were obtained during any of these studies.

In a recent study Liddle ct al (11) showed that infusion of the octapeptide of cholecystokinin, CCK-8, resulting in physiological plasma concentrations, markedly and significantly delayed the gastric emptying of a liquid test meal in humans. Plasma concentrations were determined by a specific and sensitive bioassay. Cholecystokinin-8 is one of the molecular forms of cholecystokinin, the others being CCK 33/39, an intermediate form with at least 14 amino acids , and a larger molecular form, possibly CCK-58 ( 12). All these forms are present in blood after a fatty meal (13). Based on earlier studies (14, 15) it was thought until recently , that CCK-8 was a major constituent of circulating cholecystoki­nin. However, more recent studies ( 13, 16, 17) showed that CCK-8 is present in the circulation in only low concentrations. The most prominent molecular forms in the blood are the ones larger than CCK-8. Although the different mo­lecular forms of cholecystokinin are about equipotent in respect to stimulation of pancreatic enzyme secretion and gall bladder contraction (18 , 19) , this may not be the case for its effect on gastric smooth muscle. So it may be that the physiologic role of circulating CCK in the regulation of gastric motility is being misjudged, when the octapeptide is used for infusion.

To give a more definite answer to the question about the role of cholecystoki­nin as a hormonal mediator of fat-induced inhibition of gastric emptying, we studied the effect of infusion of physiological doses of CCK-33 on the gastric emptying rate in humans. Instead of a liquid meal, as used by Liddle et al, we used a firm semisolid test meal. Plasma concentrations were determined by ra­dioimmunoassay.

84

MATERIALS AND METHODS Subjects. Eight healthy volunteers (mean age 33 years, range 30-36) were

studied. All subjects gave informed consent. The study was approved by the Medical Ethical Committee of the University Hospital and the State University of Groningcn.

Test meal. A 99mTc-labclcd custard pudding was used as the test meal. The pudding was made of 40 g custard powder, I O g sugar and 25 0 ml water and con­tained 44 g carbohydrate , corresponding to 176 Kcal . This was cooked to a firm homogcnous pudding with a final weight of 220 g. This meal was chosen, since it docs not contain any protein or fat and was thus expected to elicit hardly any re­lease of endogenous cholecystokinin, which was confirmed in preliminary ex­periments. The meal was labeled with 10 MBq 99mTc, which was bound to cati­on exchange resin particles Chelex-100 (Sigma), as described by Wirth ct al (20). The particles were added to the custard just before the cooking. To test the reliability of the labeling technique three validation studies were per­formed. (I) Labeled particles were incubated during 10 minutes in boiling wa­ter and for 24 hours in 0.1 M hydrochloric acid respectively. After both proce­dures the labeling index remained more than 97%. (II) A labeled meal was chewed and spat out. The resulting slurry was incubated with excess pentagastrin­stimulated human gastric acid during 4 hours at 37°C. The mixture was then centrifuged during 15 min at 5 00 rpm. In the supernatant fluid, which did not contain any Che lex particles, less than 3 percent of the radioactivity was found, showing the stability of the label under the experimental conditions. (III) A meal was chewed and spat out again. During this about 4 g of saliva was added to the meal. The resulting slurry was then sieved through three sieves with holes of 7, 2.5 and 1 mm respectively. The portion retained by the first sieve represent­ed 19% of the total weight and contained 19% of the total radioactivity. For the portion retained on the second sieve these values were 35 % and 33% re­spectively, for the portion retained on the third sieve 8% and 7% , and for the portion coming through the third sieve 38% and 41 % . These results show that after chewing a considerable part of the meal was liquified, in the sense that particles of less than 1 mm are being emptied along with liquids. They also show that the label is divided proportionally over the liquified fraction and the remain­ing solid particles, which were retained by the different sieves. It is concluded from these data that the Chelex-particles are reliable for labeling the custard pudding as used in this study. The effective radiation dose equivalent was 0.3 mSv ( = 30 mrem) per test.

Gastric emptying rate was measured using a dual headed gamma camera (Siemens ROTA), which was interfaced to a computer (DEC, gamma 11 ). Low energy all purpose collimators were used. The subjects were seated between

85

the heads of the camera leaning back slightly. As soon as the test meal was fin­

ished digital frames were recorded for one hour in one minute periods. A region

of interest over the stomach was chosen , using the sum image of all sixty frames,

taking care that the whole gastric region was included. Then the time-activity

curves for these regions on the anterior and posterior views were generated.

The final emptying curve was obtained by calculating the geometric mean of

the anterior and posterior counts at each time interval . The rate of gastric emp­

tying was expressed as the half emptying time (T½) , which is the time necessary

to empty half of the activity originally present in the stomach , beginning at the

start of the registration , i . e . at the end of the meal . The T 1h was determined by

fitting a power exponential curve to the emptying curve (2 1 ) . The course of the power exponential curve is determined by the two variables T 1h and the power

exponent, the latter of which determines deviations from a monoexponential

decay curve . The power exponential curve was fitted to the emptying curve

using a nonlinear least squares method. The quality of the fit is represented by

the parameter R2 which should approximate 1 .0 (2 1 ) .

Cholecystokinin assay. Concentrations of cholecystokinin i n plasma were

determined by radioimmunoassay as described previously (22, 23) . The anti­

body used in this assay was T204. This binds to all biologically active CCK pep­

tides containing both the carboxyterminus and the sulphated tyrosine region .

The detection limit of the assay is 0.5 pmol/1 plasma. The intraassay variation

ranged from 4.6 tot 1 1 .5% and the interassay variation from 1 1 .3 to 26 . 1 % .

Studies performed. All tests were done after an overnight fast. The emptying

studies were done single blind in a random order, with intervals between the

tests of at least three days. During each test the subjects had an intravenous

needle in each arm , one for blood sampling and the other for infusion of saline

with or without cholecystokinin . In this study natural porcine cholecystokinin ,

purified by the Karolinska Institute was used, which was obtained through Ka­

bi Vitrum . This preparation contains cholecystokinin-33 and does not contain

any other peptide in appreciable amounts (Kabi Vitrum, personal communica­

tion; 24) . Gastric emptying was studied in all subjects three times: ( 1 ) during saline in­

fusion (control) ; (2) during infusion of cholecystokinin at a dose of0.375 IOU/

kg.h ; and (3) during infusion of cholecystokinin at a dose of 0. 75 IDU/kg .h .

Cholecystokinin was dissolved in saline and was given in a volume of 30 ml per

hour. The infusion was started at the end of the meal , when the registration of

radioactivity was started. The meal was consumed in about four minutes . At

the end of the meal the subjects drank 100 ml water, rinsing their mouth with

the water. Blood samples were taken in the basal state and at 1 5-minutes inter­

vals during the studies. Samples were collected into ice-chilled tubes and left on

86

ice until centrifugation, immediately after the end of the test. Plasma was stored at -20°C until radioimmunoassay.

In addition to the emptying studies, plasma CCK concentrations were deter­

mined after ingestion of a regular meal in all eight subjects. The meal was eaten after an overnight fast. It consisted of2 slices of bread, two eggs, 25 g bacon , 1 5

g margarine and 200 ml milk, containing 29 g protein , 40 g fat and 3 6 g carbohy­

drate (622 Kcal). Blood samples were taken before the meal and at I S-minutes intervals after the meal.

Statistical analysis. Results are given as mean ± standard error of the mean (SEM). The half emptying times and the power exponents during the infusion

of cholecystokinin were compared with the results from the control studies by

the Student's t-test for paired results. Differences were considered to be signifi­cant when p < 0.05.

RESULTS

After the regular meal plasma CCK concentrations increased from a basal of

1 .6 ± 0.5 pmol/1 to a maximum of6.5 ± 0.8 pmol/1 at 30 min , decreasing to 5 . 3 ±

0.9 pmol/1 at 60 min (figure I ). After the custard pudding, during the control

studies, plasma CCK concentrations increased from 0.8 ± 0. 1 pmol/1 to maxi­mally 1 .6 ± 0.3 pmol/1 , confirming the minimal capacity of this meal to release

� 1 0 0 E

C

·c 8

6 0

E 4

2

0

Figure I .

0

.>----------- 0.75

----;'.'""----o-----0 0.375

-----t-----.. 1----.. ! control

15 30 45 60

time I minutes)

Plasma cholecystokinin concentrations after a regular meal and after a custard pudding along with infusion of saline (control) . 0.375 IDU/kg .h cholecystokinin-33 and 0.75 IDU/kg .h cholecystoki­nin-33 (n = 8. M ± SEM) .

87

cholecystokinin ( figure 1). During the infusion of the lower dose of cholecysto­kinin plasma CCK increased from 1.0 ± 0.2 pmol/1 to a plateau level of about 4.5 pmol/1 at 30 minutes (figure 1 ). During infusion of the higher dose plasma CCK increased from 1.4 ± 0.3 pmol/1 to about 7.3 pmol/1 at 30 minutes (figure 1 ).

The gastric emptying rate was markedly and significantly inhibited by the in­fusion of both the lower and the higher dose of cholecystokinin. The mean half emptying time during the control studies was 45 ± 8 min. During the low dose cholecystokinin infusion it was increased to 86 ± 17 min and during the high dose infusion to 198 ± 50 min (figures 2 and 3).

Cholecystokinin did not affect the power exponent. During the control studies the mean exponent was 0. 76 ± 0.07 , and during the low and high dose cholecystokinin infusion 0. 76 ± 0.10 and 0. 73 ± 0.13 respectively.

T 112 (m i nutes)

350

300

2 50

200

1 50

1 00

50

0

control

Figure 2.

446

0.375 0.75

Individual half emptying times of a ""mTc-labeled custard pudding during control studies and during cholecystokinin infusion, 0.375 IDU/kg.h and 0.75 IDU/kg.h

88

The power exponential curve fitted well to the emptying curves: median R2

was 0.95 (range 0.68 - 0.99; R2 � 0.90 in 4/24 tests).

� 100

.!:

·c "ci

C 50

0

Figure 3 .

0 30

0.75

0.375

control

60 minutes

Mean gastric emptying curves after a 99mTc-labeled custard pudding during control studies (T½ 45 min, power exponent 0. 76) and during cholecystokinin infusion, 0.375 IDU/kg.h (T1h 86 min, power exponent 0. 76) and 0. 75 IDU/kg.h (T½ 198 min, power exponent 0.73) ( n = 8).

DISCUSSION

In this study we show that cholecystokinin-33 at physiological plasma con­centrations markedly and significantly inhibits the gastric emptying rate of a firm semisolid meal in humans. The plasma concentrations of CCK during the infusion of cholecystokinin closely mimicked those found after a modest regu­lar meal, also in respect to the course of the concentrations during the hour stud­ied. Although the concentrations during the highest dose of cholecystokinin were slightly higher than those after the meal, they were still well below those found after intraduodenal instillation of fat, which reach a maximum of 10.2 pmol/1, as determined by the same radioimmunoassay (25). These results strongly suggest that under physiological conditions cholecystokinin is in­volved in the regulation of gastric emptying.

The results of this study are in agreement with those of Liddle et al (11). How­ever, these investigators infused the octapeptide of CCK, which contributes only little to circulating cholecystokinin under physiological conditions (13, 16, 17). In contrast, we infused cholecystokinin-33 , which is one of the major circu-

89

lating molecular forms of CCK in humans. So in our study the physiological

situation in respect to circulating molecular forms of cholecystokinin was mim­

icked more closely than in Liddle's study. Apparently this has not affected the

agreement of the results of the two studies, which suggests that the different

molecular forms are about equipotent in respect to their effect on gastric

smooth muscle .

Whereas Liddle et al used a liquid test meal , namely water, we used a firm

custard pudding, which could best be described as firm semisohd. The empty­

ing curve of the meal did not show the typical pattern as seen after a solid meal .

This is probably due to the fact that part of the meal is liquified during chewing

and is being emptied soon after entering the stomach , whereas the rest of the meal enters the stomach as solid particles, which are first triturated by the stom­

ach before being emptied (26) . It is well known that the mechanisms of emp­

tying of liquids and of more solid food are markedly different . The emptying of

liquids is mainly a function of the proximal stomach , while that of more solid

food largely depends on the distal stomach (27) . In respect to the type of meal

used , Liddle 's study and ours are thus complementary, showing that CCK af­

fects the emptying rate of liquids as well as of more solid food .

The mechanism through which cholecystokinin affects the gastric emptying has not been elucidated by this study. A study in dogs suggested that CCK in­

hibits it by acting on both the proximal stomach and the pylorus (28). Studies in

humans are in accordance with this. Infusion of cholecystokinin increased the

pyloric pressure in healthy subjects dose-dependently (29) . Administration of

caerulein , an analogue of cholecystokinin , caused a contraction of the pylorus

during radiological examination , while at the same time the corpus muscle was

relaxed (9) . In addition , after intragastric instil lation of fat a sustained rise in

pyloric basal pressure has been recorded (30) .

Cholecystokinin has been suggested to be a mediator for the induction of sa­

tiety . Infusion of the octapeptide in men decreased the spontaneous food in­

take (31 ) . I t may be that the inhibition of gastric emptying by cholecystokinin is

responsible for (a part of) this satiety inducing capacity (32) .

I n conclusion , we found that physiologic doses of cholecystokinin-33 mark­

edly and significantly inhibited the gastric emptying rate of a meal . Cholecys­

tokinin is very likely to be a physiologic hormonal mediator of fat-induced inhi­bition of gastric emptying in man .

REFERENCES 1 . Hunt JN, Stubbs DF. The volume and energy content of meals as determinants of gastric emp­

tying. J Physiol 1975; 245: 209-25. 2. Moore JG, Christian PE, Brown JA et al Influence of meal weight and caloric content on gas­

tric emptying of meals in man. Dig Dis Sci 1 984; 29: 5 13- 19.

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3. Kroop HS. Long WB. Alavi A. Hansell JR. Effect of water and fat ongastric emptying of solid meals. Ga�troenterology 1 979; 77: 997- 1000.

4. Collin� PJ , Heddie R . Horowitz M. Read NW. Dent J , Chatterton BE. The effect of intradu­odenal lipid on gastric emptying and intragastric distribution of a solid meal (abstract). Gas­troenterology 1 986; 90: 1 377.

5 . Hopman WPM. Jansen JBMJ , Lamers CBHW. Comparative study of the effects of fat , pro­tein and starch on plasma cholecystokinin in man. Scand J Gastroenterol 1 985; 20: 843-47.

6. Beglinger C. Fried M, Whitehouse I, Jansen JB, Lamers CB. Gyr K. Pancreatic enzyme re­sponse to a liquid meal and to hormonal stimulation. J Clin Invest 1 985; 75: 147 1 -76.

7. Hopman WPM, Kersten� PJSM. Jansen JBMJ. Rosenbusch G, Lamers CBHW. Effect of gra­ded physiologic do�c� of cholecystokinin on gallbladder contraction measured by ultraso­nography. Ga�trocnterology 1 985; 89: 1242-47.

8. Chey WY, Hitanant S, Hendricks J, Lorber SH. Effect of secret in and cholecystokinin on gas­tric emptying and gastric �ecretion in man. Gastroenterology 1970; 58: 820-27.

9. Scarpignato C. Zimbaro G. Vitulo F. Bertaccini G. Caerulein delays gastric emptying of solids in man. Arch Int Pharmacodyn 1 98 1 ; 249: 98-105.

10. Valenzuela J E. Defilippi C. Inhibition of gastric emptying in humans by secretin. the octapep­tide of cholecystokinin, and intraduodenal fat. Gastroenterology 1 98 1 ; 8 1 : 898-902.

1 1 . Liddle RA, Morita ET. Conrad CK, Williams JA . Regulation of gastric emptying in humans by cholecystokinin. J Clin Invest 1986; 77: 992-96.

12 . Eysselein VE, Reeve JR , Shively J E , Hawke D, Walsh JH. Partial structure of a large canine cholecystokinin (CCK,8) : amino acid sequence. Peptides 1982; 3: 687-9 1 .

13 . Jansen J BMJ . Lamers CBHW. Molecular forms of cholecystokinin in human plasma after in­gestion of fat (abstract) . Gut 1 985; 26: A 1 132-33.

14. Walsh JH. Lamers CB. Valenzuela JE. Cholecystokinin-octapeptidelike immunoreactivity in human plasma. Gastroenterology 1 982 ; 82: 438-44.

15 . Calam J. Ellis A. Dockray GJ . Identification and measurement of molecular variants of chole­cystokinin in duodenal mucosa and plasma. J Clin Invest 1982; 69: 2 1 8-25.

16. Liddle RA, Goldfine ID . Rosen MS, Taplitz RA. Williams JA. Choleeystokinin bioactivity in human plasma. J Clin Invest 1985; 75: 1 1 44-52.

1 7. Bacarese-Hamilton AJ. Adrian TE, Bloom SR. Plasma cholecystokinin response to food in healthy subjects (abstract). Regul Pepi 1984; 9: 322.

1 8. Solomon TE , Yamada T, Elashoff J, Wood J, Beglinger C. Bioactivity of cholecystokinin ana­logs: CCK8 is not more potent than CCK11. Am J Physiol 1 984; 247: Gl05-GI I I .

19 . Lamers CBHW. Poitras P , Jansen JBMJ. Walsh JH . Relative potencies of cholecystokinin-33 and cholecystokinin-8 measured by radioimmunoassay and bioassay . Scand J Gastroenterol 1983; 18 , suppl 82: 19 1-92.

20. Wirth N, Swanson D. Shapiro B et al. A conveniently prepared Tc-99m resin for semisolid gas­tric emptying studies. J Nucl Med 1 983; 24: 5 1 1 - 14.

2 1 . Elashoff JD, Reedy TJ. Meyer JH. Analysis of gastric emptying data. Gastroenterology 1 982; 83: 1 306- 12 .

22. Jansen JBMJ , Lamers CBHW. Radioimmunoassay of cholecystokinin in human tissue and plasma. Clin Chim Acta 1 983; 1 3 1 : 305- 16 .

23. Jansen JBMJ . Lamers CBHW. Radioimmunoassay of cholecystokinin: production and evalu­ation of antibodies. J Clin Chem Clin Biochem 1 983; 2 1 : 387-94.

24. Feurle GE. Neurotensin-like immunoreactivity in some preparations of secret in and cholecys­tokinin . Agents Actions. 1 985; 16: 4 1 1 - 14 .

25. Hopman WPM, Jansen JBMJ , Rosenbusch G, Lamers CBHW. Role of cholecystokinin and the cholinergic system in the intestinal phase of gall bladder contraction in man (abstract) . Gut 1 986; 27: A603.

26. Meyer JH , Ohashi H , Jehn D, ThomsonJB. Size of liver particles emptied from the human sto­mach. Gastroenterology 1 98 1 ; 80: 1 489-96.

27. Kelly KA. Gastric emptying of liquids and solids: role of proximal and distal stomach. Am J Physiol 1 980; 239: G7 1 -G76.

28. Yamagishi T. De bas HT. Cholecystokinin inhibits gastric emptying by acting on both proximal stomach and pylorus. Am J Physiol 1978; 234: E375-E378.

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29. Phaosawasdi K. Fisher RS. Hormonal effects on the pylorus. Am J Phy,iol 1 982: 2-13: G330-G335.

30. White CM . Poxon V. Alexander-Will iams J . Effects of nutrient l iquids on human gastrodu­odenal motor activity. Gut 1 983: 24: l l 09- l6 .

3 1 . Kissi leff HR . Pi-Sunyer FX. Thorton J . Smith GP. C-terminal octapeptide of cholecystokinin decrease, food intake in man. Am J Clin Nutr 1 98 1 : 3-1: 15-1-60.

32. Moran TH. Mc Hugh PR. Cholecystokinin �uppre��e� food intake liy inhibiting gastric empty· ing. Am J Physiol 1 982: 2-12: R-19 I -R-197.

92

SUMMARY

The stomach is one of the most extensively studied organs in gastrointestinal physiology. Gastric motility and the secretion of gastric acid have been studied for many years and have been the source of many scientific controversies. Due to modern investigational methods our understanding of gastric physiology and pathophysiology has greatly increased during the past few decades.

The role of the stomach in the digestive process can be divided into motoric and secretory functions. Gastric motility plays an important role in the diges­tion of food. After a meal food is stored in the proximal part of the stomach. Solid food is then grindcd and triturated by the distal stomach to very small particles. These arc being emptied along with liquids into the small intestine little by little in order to optimize further digestion and absorption. Gastric acid has only a minor role in the digestive process. However, it is an important factor in the pathophysiology of many of the disorders of the upper gastrointestinal tract.

In this thesis a number of studies arc described, which were performed to further elucidate some aspects of the regulation of gastric functions. In chapters I and 2 updates on the regulation of gastric acid secretion and gastric emptying arc given.

Histamine is a major stimulant of gastric acid secretion, by directly stimulat­ing the parietal cells through histamine Hrrcccptors. Gastric histamine is pro­duced and secreted by mast cells in the gastric mucosa. Since these mast cells arc also present in the antral mucosa the question was raised whether histamine might affect gastrin release. Studies on the effect of the histamine H2-rcccptor antagonist cimctidinc on gastrin release in man and animals have yielded con­flicting results. In chapter 3 the effect of intravenous histamine on bombcsin­stimulated gastrin release was studied in dogs. Bombcsin is a ncuropcptide, thought to be involved in the neural stimulation of gastrin release by antral G­cells. Histamine was found to cause by its stimulatory action on H2-reccptors a dose-related reduction in gastrin release, independent of changes in intragas­tric pH. Infusion doses of histamine-2HCI of 20 up to 160 µ,g/kg.h reduced gas­trin release by 1 6-30%. The results of this study suggest that histamine has a function in the regulation of gastrin release.

Chapter 4 describes the effect of histamine Hr receptor stimulation on bom­besin- and peptone-stimulated gastrin release in man. Peptone consists of ami­no acids and peptides and its intragastric instillation strongly induces gastrin re­lease. In all studies intragastric pH was kept constant at 4.5 by continuous intra­gastric titration. A histamine H 1-receptor antagonist was administered prior to all tests. Histamine was infused intravenously at a dose of 130 nmol/kg.h, which elicits maximal gastric acid secretion. Plasma gastrin concentrations were de-

93

termined by a newly validated radioimmunoassay. Histamine affected neither bombesin- nor peptone-stimulated gastrin release. It is concluded that his­tamine H2-receptors do not seem to be involved in the regulation of gastrin se­cretion in man.

Cholinergic nerves have an important role in the regulation of gastrin re­lease. It has recently been shown that there are different subclasses of cholinergic muscarinic receptors. The recently developed drug pirenzepine is a specific an­tagonist of muscarinic M 1-receptors . whereas atropine is a nonspecific musca­rinic receptor antagonist. In chapter 5 it was investigated whether M 1-receptors might be involved in the regulation of gastrin release in man by comparing the effects of pirenzepine and atropine on bombesin- and peptone-stimulated gas­trin secretion. The two drugs were given in doses, about equipotent in respect to the reduction of gastric acid secretion. Pirenzepine was given as an i.v . bolus of 0.6 mg/kg, and atropine as an i. v. bolus of 0.015 mg/kg, followed by an infu­sion at a dose of 0.005 mg/kg.h. Intragastric pH was kept constant by means of continuous automated intragastric titration. Neither pirenzepine nor atropine in the doses used affected bombesin- and peptone-stimulated gastrin release. It is therefore concluded that M 1-receptors arc not involved in the regulation of gastrin release in man, and that the reduction of acid secretion by pirenzepine is not due to inhibition of gastrin secretion.

Acidification of gastric contents is known to potently inhibit meal-stimulated gastric acid secretion and gastrin release. Acidification of the duodenum also causes a reduction of acid secretion. When acidified gastric contents enter the duodenum, lowering the intraluminal pH below 3.5, the peptide hormone se­cretin is released by endocrine S-cells in the duodenal mucosa. Administration of high doses of secretin inhibits gastric acid secretion and gastrin release. In chapter 6 it was investigated whether secretin might have a role in the acid-in­duced inhibition of gastric secretion in humans. In this study a newly developed radioimmunoassay for secretin was used. Its development and validation are described in this chapter. Healthy subjects were given peptone meals at low pH (2.0, 2.5) and at near neutral pH (5.5). Gastric acid secretion was determined by intragastric titration, keeping the intragastric pH constant at the pH level of the meal. Plasma concentrations of gastrin and secretin during the meals were determined. The acidified meals significantly reduced acid secretion and gas­trin release compared to the meal of pH 5 .5. During the latter plasma secretin remained at fasting levels , whereas it increased significantly during the meals of low pH. During a fourth study a meal at pH 5.5 was given along with intrave­nous infusion of secretin, producing plasma secretin concentrations similar to those seen during the acidified meals. Neither gastric acid secretion nor gastrin release were affected by exogenous secretin. It is concluded that secretin is un­likely to have a physiologic role in the inhibition of acid secretion in man.

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Duodenal acidification is known also to inhibit gastric emptying rate. In chapter 6 gastric emptying was investigated using the double-sampling dye-di­lution technique. The acidified meals were emptied significantly slower than the meals of pH 5.5. Exogenous secretin also decreased gastric emptying rate, but this inhibition just failed to reach statistical significance (p - 0.053). The ef­fect of secret in on the gastric emptying rate of solid meals in man was studied in chapter 7. The test meal was a pancake, labeled with tcchnetium-99m. Radio­activity within the stomach was determined by using a dual-headed gamma ca­mera, with the subjects sitting between the heads of it. The gastric emptying curve was obtained by calculating the geometric means of anterior and posteri­or counts, obtained during I minute periods during I hour. The gastric empty­ing rate was expressed as the half time of this curve. Healthy subjects were stud­ied during infusion of saline (control) and during intermittent and continuous infusion of sccretin , producing near-physiologic plasma secretin concentra­tions. Secrctin was given intermittently to induce a pattern comparable to post­prandial plasma sccretin secretion. Before each study cimetidinc was adminis­tered to inhibit gastric acid secretion, thereby preventing release of endogenous secretin. Both intermittent and continuous administration of sccretin signifi­cantly inhibited gastric emptying rate. The mean half emptying time increased from 55 minutes during the control studies to 85 minutes during intermittent sc­cretin infusion, and from 67 to 1 56 minutes during the continuous infusion. From the results of chapters 6 and 7 it is concluded that sccrctin is a serious can­didate to be of physiological importance in the regulation of gastric emptying in man.

It is well known that fat retards gastric emptying. Fatty meals are emptied slowly by the stomach. Instillation of fat into the duodenum potently inhibits gastric emptying rate. Intraduodenal fat is the main stimulus of the release of the peptide hormone cholecystokinin by the endocrine I-cells in the duodenal mucosa. In chapter 8 it was studied whether cholecystokinin might have a role in the fat-induced retardation of gastric emptying in man . The test meal used in this study was a semisolid custard pudding, labeled with technetium-99. This meal was chosen since it does not contain any protein or fat , thereby eliciting only minimal release of endogenous cholecystokinin. Gastric emptying rate was determined in the same way as in chapter 7. Healthy subjects were studied on three different days. During the control test only saline was infused, during the other two tests cholecystokinin-33 in two different doses was administered. Plasma concentrations of cholecystokinin during both infusions were in the physiological range, i.e. similar to those seen after a modest regular meal. Both doses of cholecystokinin significantly inhibited gastric emptying. The mean half emptying time during the control studies was 45 minutes, during the lower dose of cholecystokinin 86 minutes and during the higher dose 1 98 minutes. It is

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concluded that cholccystokinin is most likely to be a physiologic hormonal me­diator of the inhibition of gastric emptying by intraduodenal nutrients in man.

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SAMENV A TIING

De maag is een zecr intcnsicf bestudccrd orgaan. De motilitcit van de maag en de secretie van maagzuur zijn al vclc jaren onderwerp van studic en hebben talrijke wetenschappclijkc controvcrsen vcroorzaakt. De kennis en het be grip van de fysiologie en pathofysiologie van de maag zijn gcdurendc de afgelopen jaren dankzij modernc mcthoden van onderzoek in hoge mate toegcnomen.

De plaats van de maag in het verteringsproces wordt bepaald door zij n moto­rische en sccretoire functies. De motiliteit van de maag is van groot bclang voor de vertering van het voedsel. Na ccn maaltijd wordt hct voedsel ecrst gereti­neerd in het proximalc gedecltc van de maag. Vast vocdsel wordt vervolgcns door het distale dee I van de maag gedispergecrd tot zecr kleinc deeltjes. Deze decltjcs worden tczamen met vloeibare maaginhoud in klcinc portics in het du­odenum geledigd . opdat de verdere vertering en de resorptie in de dunne darm optimaal kunnen plaatsvinden. De betckenis van hct maagzuur voor hct vcrte­ringsproces is beperkt. Het maagzuur is van groot belang voor de pathofysiolo­gie en de etiologic van tal van aandoeningen van het bovcnste decl van hct maagdarmkanaal.

In dit proefschrift wordcn ecn aantal studies beschrevcn die wcrdcn vcrricht om de kennis van de regulatie van de maagfuncties te vermeerdercn. De cerste twee hoofdstukken gevcn cen overzicht van de huidigc kennis van de regclme­chanismen van de maagzuursecrctie en de maagontlcdiging.

Bij de stimulatie van de zuursecretie speclt histamine een belangrijkc rol. Via histamine H2-receptoren heeft het een direkte stimulerende invloed op de parietale cellen, die het maagzuur produceren. De mcstccllen in hct maag­slijmvlies zijn de bron van het histamine in de maag. Daar ook het antrumslijm­vlies mestcellen bevat, recs de vraag of histamine de sccretie van gastrine door de antrale G-cellen zou kunnen bei'nvloeden. Studies bij mensen en dieren naar het effect van de Hrreceptorantagonist cimetidine op de gastrincsecretie leverden tegenstrijdige resultaten op. In hoofdstuk 3 worden de resultaten bcschreven van een onderzoek bij honden naar het effekt van intravcneuze toediening van histamine op de secretie van gastrine tijdens stimulatie hiervan door born besine. Bombesine is een neuropeptide, dat, naar men aanneemt, be­trokken is bij de neurogene stimulatie van de gastrinesecretie. Het bleek dat histamine een dosis-afhankelijke reductie veroorzaakte van de gestimuleerde gastrinesecretie. lnfusie van histamine-2 HCL in doses van 20 tot 160 µ.g/kg.h reduceerde de secretie van gastrine met 1 6 tot 30%. Dit effect kwam tot stand via Hrreceptoren en was onafhankelijk van veranderingen van de pH van de maaginhoud. De resultaten van deze studie suggereren dat Hrreceptoren be­trokken zijn bij de regulatie van de gastrinesecretie.

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Hoofdstuk 4 beschrijft een studie naar het effect bij de mens van stimulatie van histamine Hrreceptoren op de secretie van gastrine onder invloed van bombesine en van een peptone-maaltijd. Peptone is een mengsel van aminozu­ren en peptiden en is een krachtige stimulans van de gastrinesecretie. De pH van de maaginhoud werd tijdens alle testen constant op 4,5 gehouden door middel van continue intragastrische titratie. V66r iedere test werd een specifie­ke histamine H 1 -antagonist toegediend. Histamine werd intraveneus gegeven in een dosis van 130 nmol/kg. h. Deze dos is veroorzaakt maximale zuursecretie. De concentraties van gastrine in het plasma werden bepaald met een radioim­munoassay, die nag niet eerder werd beschreven. Histamine had geen invloed op de gastrinesecretie tijdens stimulatie hiervan door bombesine en peptone. De eonclusie is dat histamine Hrreceptoren vrijwel zeker niet betrokken zijn bij de regulatie van de secretie van gastrine bij de mens.

Cholinerge zenuwen hebben een belangrijke functie bij de regulatie van het gehalte van gastrine in het plasma. Recent is aangetoond dat er verschillende subklassen van cholinerge muscarinereceptoren bestaan. Pirenzepine, een re­cent ontwikkeld medicament, is een specifieke antagonist voor de muscarine M 1 -receptoren, terwijl atropine een niet-selectieve muscarinereceptoranta­gonist is. In hoofdstuk 5 wordt een onderzoek beschreven naar de betrokken­heid van M 1-receptoren bij de regulatie van de secretie van gastrine bij de mens. Hiertoe werden het effect van pirenzepine en dat van atropine op de gas­trinesecretie onder invloed van bombesine en peptone met elkaar vergeleken. De twee medicamenten werden gegeven in doses die ongeveer gelijkwaardig waren wat betreft de remming van de zuursecretie . Pirenzepine werd toege­diend als een intraveneuze bolus in een dosering van 0,6 mg/kg. De dosis van atropine was 0,01 5 mg/kg, eveneens als een intraveneuze bolus, gevolgd door een infuus met 0,005 mg/kg.h. De pH van de maaginhoud werd constant ge­houden door middel van continue intragastrische titratie . Pirenzepine noch atropine hadden in de gebruikte hoeveelheden enige invloed op de gastrinese­cretie tijdens stimulatie hiervan door bombesine en peptone. Geconcludeerd wordt dat M 1-receptoren niet betrokken zijn bij de regulatie van de secretie van gastrine bij de mens en dat de rem ming van de zuursecretie door pirenzepine niet berust op een vermindering van de gastrinesecretie.

Verlaging van de pH van de maaginhoud veroorzaakt een krachtige rem­ming van de produktie van maagzuur en de gastrinesecretie na een maaltijd. Ook een verlaging van de intraduodenale pH remt de zuursecretie. Wanneer zure maaginhoud in het duodenum komt en daardoor de pH er daalt tot onder 3,5, wordt het hormoon secretine gesecerneerd door de endocriene S-cellen, die zich in het slijmvlies van het duodenum bevinden. Indien secretine in hoge doses wordt toegediend, remt het de secretie van maagzuur en van gastrine. In

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hoofdstuk 6 wordt een onderzoek beschreven naar de mogelijke functie bij de mens van secretine bij de remming van de zuursecretie die door een verlaging van de intragastrische en intraduodenale pH wordt teweeggebracht. Bij dit on­derzoek werd een radioimmunoassay voor secretine gebruikt, die in het kader van deze studie werd opgezet. De ontwikkeling van deze assay en de beoorde­ling van de validiteit ervan worden in dit hoofdstuk beschreven. Aan gezonde proefpersonen werden peptone-maaltijden toegediend met een (age pH (2,0, 2,5) en met een bijna neutrale pH (5,5). De maagzuursecretie werd gemeten d.m.v. continue intragastrische titratie. Hierbij werd de pH van de maagin­houd voortdurend gelijk gehouden aan de pH van de oorspronkelijk toege­diende proefmaaltijd. De concentraties van gastrine en secretine in het plasma tijdens de maaltijden werden bepaald. De zuursecretie en de secretie van gas­trine werden door de maaltijden met een (age pH significant geremd in vergelij­king tot de secretie tijdens de maaltijden met ecn pH van 5,5. De secretinecon­centratie in het plasma steeg duidelijk tijdens de maaltijden met (age pH, ter­wijl er geen stijging optrad tijdens de maaltijden met een pH van 5,5. Tijdens een vierde test wcrd opnieuween maaltijd met een pH van 5,5 gegeven. Hierbij werd tevens secretine ge"infundeerd, in een dosis die zodanig was gekozen , dat de verkregen concentratics van sccretine in het plasma ovcrecnkwamen met die, gemeten tijdens de maaltijden met een (age pH. De zuurproduktie noch de gastrinesecretie werden be"invloed door de toediening van secretinc. De con­clusie is dat secretine zeer waarschijnlijk geen fysiologische functie heeft bij de rem ming van de zuursecretie bij de mens.

Het is bekend dat de maagontlediging wordt vertraagd door een daling van de intraduodenale pH. Bij het onderzoek, beschreven in hoofdstuk 6, werd ook de snelheid van de maagontlediging onderzocht. Daarbij werd gebruik ge­maakt van een kleurstofverdunningsmethode. De maagontlediging tijdens de maaltijden met een lage pH was significant trager dan tijdens de maaltijden met een pH van 5,5. De toediening van secretine veroorzaakte 66k een vertra­ging, maar dit effect was juist niet statistisch significant (p = 0,053). Een onder­zoek naar de invloed van secretine op de maagontlediging bij de mens na ge­bruik van vast voedsel wordt beschreven in hoofdstuk 7. Als proefmaaltijd werd een pannekoek gebruikt, die gemerkt was met technetium-99m. Qe hoe­veelheid radioaktiviteit in de maag na de maaltijd werd geregistreerd in peri­oden van een minuut gedurende een uur m.b.v. een dubbelkops gammacame­ra, waarbij de proefpersonen tussen de koppen in zaten. De radioaktiviteit werd bepaald door het berekenen van het geometrische gemiddelde van de hoeveelheid counts gemeten aan voor- en achterzijde. Door de aktiviteit, ge­meten per minuut, uit te zetten tegen de tijd werd de maagontledigingscurve verkregen. De snelheid van de maagontlediging werd uitgedrukt als de half-

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waardetijd van deze curve. Gezonde proefpersonen werden bestudeerd tijdens de infusie van fysiologisch zout ( controle) en tijdens intermitterende en conti­nue infusie van secretine. Hierbij werden concentraties van secretine in het plasma bewerkstelligd, die vrijwel in het fysiologisch gebied lagen. Secretine werd intermitterend toegediend om het secretiepatroon, zoals dat normaal na een maaltijd wordt gezien, zo goed mogelijk na te bootsen. V66r iedere test werd cimetidine gegeven om de zuurproduktie te remmen en daardoor de se­cretie van endogeen secretine te voorkomen. Zowel tijdens de intermitterende als tijdens de continue toediening van secretme werd een significante rem ming van de maagontlediging waargenomen. De tijd . nodig om de maag voor de helft te ontledigen steeg van een gemiddelde van 55 minuten tijdens de contro­lestudies naar 85 minuten tijdens de intermitterende toediening van secretine en van 67 naar 156 minuten tijdens de continue toediening. Op grond van de re­sultaten beschreven in de hoofdstukken 6 en 7 wordt geconcludeerd,dat het zeer aannemelijk is dat secretine een functie heeft bij de regulatie van de maag­ontlediging bij de mens.

Het is een bekend fcit dat vet de maagontlediging vertraagt. Vette maaltij­den verlaten de maag maar langzaam. Wanneer vet wordt toegediend in het du­odenum wordt het tempo van de maagontlediging daardoor sterk geremd. In­traduodenaal vet is de belangrijkste stimulans voor de secretie van het peptide­hormoon cholecystokinine door de endocriene 1-cellen , die zich in het slijm­vlies van het duodenum bevinden. In hoofdstuk 8 wordt een onderzoek be­schreven naar de functie van cholecystokinine bij de vertraging van de maag­ontlediging door vet bij de mens. De proefmaaltijd die in deze studie werd ge­bruikt was een halfvaste custardpudding , die gemerkt was met technetium-99m. Voor deze maaltijd werd gekozen op grond van het feit dat deze geen ei­witten of vetten bevat en daardoor vrijwel geen secretie van endogeen chole­cystokinine veroorzaakt. De snelheid van de maagontlediging werd bepaald op dezelfde wijze als beschreven in hoofdstuk 7. Gezonde proefpersonen werden bestudeerd tijdens infusie van fysiologisch zout ( controle) en tijdens de infusie van cholecystokinine in twee verschillende doses. Tijdens deze infusies kwa­men de concentraties van cholecystokinine in het plasma nagenoeg overeen met die na een gewone maaltijd. Cholecystokinine veroorzaakte een signifi­cante remming van de maagontlediging. Tijdens de controlestudies was de tijd, nodig om de maag voor de helft te ontledigen. gemiddeld 45 minuten, tijdens de lage dosis cholecystokinine was deze 86 minuten en tijdens de hogere dosis 198 minuten. De conclusie is dat cholecystokinine bij de mens vrijwel zeker als hormoon van fysiologische betekenis is voor de vertraging van de maagontledi­ging onder invloed van voedingsbestanddelen in het duodenum.

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