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Control of pepsin secretion by regulatory peptides in the rat stomach: Comparison with acid secretion
Peptides.Vol. 13, pp. 233-239, 1992
0196-9781/92$5.00 + .00 Copyright © 1992PergamonPressLtd.
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Control of Pepsin Secretion by Regulatory Peptides in the Rat Stomach: Comparison With Acid Secretion KHADDOUJ
T A Z I - S A A D , J A C Q U E S C H A R I O T A N D C L A U D E ROZI~ ~
I N S E R M U239, Facult~ de MOdecine X. Bichat, 16 R u e H Huchard, F75018 Paris, France Received 24 July 1991 TAZI-SAAD,K., J. CHARIOT AND C. ROZI~.Controlof pepsin secretion by regulatorypeptides in the rat stomach: Comparison with acid secretion. PEPTIDES 13(2) 233-239, 1992.--Previous studies of the control of pepsin secretion by neurohumoral agents showed some discrepancies between in vitro (isolated cells) and in vivo experiments. In the present work, the effects on pepsin secretion of CCK, pentagastrin, secretin, VIP, neurotensin, histamine, and methacholine were reinvestigatedin conscious gastric fistula rats, in comparison to acid secretion. EDs0's and doses inducing maximal responses were measured to directly compare the potency and efficacy of these substances. Methacholine was the most efficient (maximal response = 4.5 × basal level, EDs0 = 1.3 umol/kg, h) and CCK the most potent (EDs0 = 1.9 nmol/kg, h) stimulant, whereas secretin was a potent (EDs0 = 0.22 nmol/kg • h) and efficientinhibitor (maximal response = 100%inhibition). These three substancesare likelyto be important regulators of pepsin secretion in the rat. Pentagastrin and histamine did not stimulate pepsin output, as found by others with isolated chief cells in vitro. Neurotensin and large doses of VIP marginally inhibited pepsin secretion. Pepsin
Gastrin
CCK
Secretin
VIP
Neurotensin
WHEREAS the measurement of gastric acid secretion in conscious fistula rats is a widely used method in digestive physiology and pharmacology, fewer studies have been devoted to the factors controlling pepsinogen secretion. From classical studies, it is generally admitted that the main acid secretagogues gastrin and histamine are mild stimulants of pepsin output in vivo, whereas cholinergic agents possess a greater efficacy on chief cells. Discrepancies between the results obtained in isolated and intact preparations have occurred for gastrin and other peptides (CCK, VIP, secretin, etc.) (9). The purpose of this work was to reinvestigate the effect of some peptides and neurotransmitters on pepsinogen secretion in the intact rat stomach, in order 1) to directly determine their relative potencies and efficacies, and 2) to measure their relative potency compared to that for gastric acid secretion. This should help to gain understanding of the physiological role of each substance. Since all substances which may affect gastric secretion could not be tested, we selected the three main stimulants of parietal cells, gastrin, histamine, and methacholine, and four peptides for which effects on gastric secretion are not completely established, CCK, secretin, VIP, and neurotensin.
Methacholine
Histamine
Gastric secretion
maintained in the laboratory on a commercial autoclaved pelleted chow (UAR 113, Alimentation Rationnelle, Villemoisson, F-91360 Epinay sur Orge).
Gastric Chronic Fistula After premedication with 10 mg/kg of acepromazine IP (Vetranquil®, Clin Midy, F-45142, St Jean de la Ruelle), the rats were anesthetized with 50 mg/kg ketamine IP (Imalgene 500®, Rhrne M~rieux, F-69002 Lyon). A gastric fistula was set up following the method of Emas et al. (6): a seromuscular purse was prepared in the ruminal part of the stomach, and a Thomas cannula was inserted and secured in place with the help of a square pad of synthetic felt 1.5 × 1.5 cm (Dacron felt, USCI, USA).
Secretory Tests Two weeks of postoperative recovery were allowed before the secretory tests were begun. During this period, the rats were trained to stand quiet in the Bollman cages during three sham experiments, without any treatment or gastric juice collection. Secretory tests were then performed no more than once a week. After a 24-h fasting period with drinking water ad lib, the gastric cannula was opened and the stomach was washed with water prewarmed at 37°C. A plastic prolongator was screwed upon the gastric cannula, and a small diameter polyethylene catheter with lateral holes was introduced in the cannula. This
METHOD
Animals Male Wistar rats (Iffa Credo, Les Oncins, F-69210 L'Arbresle), weighing 300 _+ 20 g, were used in these studies. They were Requests for reprints should be addressed to Claude Roz6.
233
234 catheter helped to safely collect the gastric juice, by guiding it drop by drop into the collecting tube. The rats were placed in Bollman cages, and an infusion catheter was inserted in a tail vein and secured with adhesive tape. A venous infusion of NaC1 0.9% was then begun. Gastric secretion was collected as 40-min samples in tared tubes. The weight of gastric juice was measured, and accepted as a measure of gastric volume, assuming that 1 ml = 1 g. Two forty-rain samples were collected before injecting any substance and were taken to be representative of basal secretion. The substances tested were intravenously infused, using a pattern of increasing doses in the same rat, each dose being infused for 40 rain. No more than four successive doses were infused in each animal. The dose-response curves comprising more than four points were constructed by adding the results of a low dose range and a high dose range group of rats.
TAZI-SAAD, CHARIOT AND ROZ[~ analog of CCK8 whose activity is equal to that of CCK8 on all models tested to date, 0.6 to 60 nmol/kg, h (=0.69 to 69 ~g/ kg. h); pentagastrin (Peptavlon® ICI Pharma, France) 0.13 to 41.1 nmol/kg- h (=0.1 to 31.6 #g/kg. h); secretin (Cambridge Research Biochemicals, Cambridge, UK) 0.006 to 3 nmol/kg, h (=0.018 to 9.16 #g/kg. h); vasoactive intestinal peptide (Neosystem, Strasbourg, France) 0.125 to 12.5 nmol/kg, h (=0.416 to 41.6 #g/kg. h); neurotensin(1-13) (Sigma, St. Louis, MO, USA) 0.6 to 19 n m o l / k g . h (=1 to 31.6 #g/kg.h); histamine dihydrochloride (Sigma) 0.54 to 17.1 #mol/kg. h (=0.1 to 3.16 mg/kg-h); methacholine (acetyl-beta-methylcholine chloride) (Pharmacie Centrale des Hopitaux, Paris, France) 0.31 to 9.71 #mol/kg. h (=0.06 to 1.9 mg/kg, h). Peptide solutions were prepared in 0.9% NaC1 with 0.3% of bovine serum albumin added. Histamine and methacholine were dissolved in 0.9% NaCI.
Acid Secretion Acid was titrated to pH = 7.0 with NaOH 0.004 M, on 0.1 ml samples, using an automatic titrator (Metrohm AG, 9100 Herisau, Switzerland). When the pH of gastric samples was greater than or equal to 5.0, the samples were immediately acidified with 20 ul HC1 6 M per ml of gastric juice, in order to protect pepsin activity, which rapidly fades in solutions of pH >5.
Statistics The results are expressed as means + one standard error of the mean (SEM). EDso'S were calculated from the linear portion of the regression curve of the effect on log dose, as described in Tallarida and Murray (40). Statistical significance of the differences between means was tested by ANOVA followed by a post hoc test (Dunnett or Newman-Keuls).
Pepsin Secretion Pepsin activity was determined by a semiautomated method derived from that proposed by Anson and Mirsky (2), which comprises two successive steps: 1) Pepsin present in the samples was allowed to digest an aqueous solution of 2.5% hemoglobin (Sigma, USA), pH = 1.7, for 3 min at 37°C. The reaction was then stopped by adding 5% trichloroacetic acid (Prolabo, France), and soluble hydrolysis products were separated by centrifugation. 2) Soluble hydrolysis products were determined with the FolinCiocalteu reagent (Merck, USA) in a Technicon autoanalyzer, using tyrosine as a standard. Samples were 50 ttl for the dosage and 50 ul for the blank tube without hydrolysis. Peptic activity was determined by comparison to a standard scale of hog pepsin (Fluka, ref 77152, Buchs, Switzerland) and expressed as peptic units (PU). One unit corresponds to the amount of pepsin releasing 1 #mol tyrosine per rain in the conditions used.
Collection of Salivary Secretion In experiments with methacholine and VIP, salivary secretion was measured in separate experiments, to correct for a possible contamination of gastric secretion by salivary secretion. Under ether anesthesia, a polyethylene catheter (Biotrol® No. 13) was introduced in the lower esophagus through a ruminal incision, secured in place by two ligatures at the level of the cardia and tureen, and conducted through the abdominal wall. The muscle and skin were sutured, the rat was placed in a Bollman cage, and an infusion catheter was inserted in a tail vein. After two hours of postanesthesia recovery, saliva was collected as 40-rain fractions in 5-ml graduated tubes. Methacholine and VIP were infused in successive increasing doses in the same conditions as in gastric fistula rats. The volume of saliva samples was measured to the nearest 0.1 ml. Bicarbonate was determined with an autoanalyzer method (4).
Substances Tested The following substances were used: CCK ([Boc-Nle28Nle3J]CCK8) (a gift ofJ. Martinez, Centre CNRS-INSERM de Pharmacologie Endocrinologie, Montpellier, France), a synthetic
RESULTS The dose-responses curves obtained with the substances tested are shown in Figs. 1 to 3. The EDso'S corresponding to these data are grouped in Table 1.
CCK CCK induced a dose-related increase of pepsin output (Fig. l). Maximal stimulation was obtained with 19 nmol/kg, h of CCK, and peaked at about 3 times the basal pepsin output. The dose-response curve was biphasic, with supramaximal doses inducing a submaximal pepsin response. CCK also produced a dose-related stimulation of gastric juice volume and acid (Figs. 2 and 3). Maximal stimulation of volume and acid was obtained with 6 nmol/kg, h. A biphasic dose-response curve was also observed, with supramaximal doses inducing submaximal volume and acid responses.
Pentagastrin Pepsin output was poorly changed by pentagastrin. Only an insignificant tendency to increase from a basal level of 14 UP/ 40 rain to 20 UP/40 rain was found after 1.3 to 41 nmol/kg, h of pentagastrin (Fig. 1). Pentagastrin induced a dose-related stimulation of gastric juice volume and acid output (Figs. 2 and 3). The maximal stimulation of volume and acid was obtained with 4 nmol/kg, h of pentagastrin, and no supramaximal inhibition was observed.
Secretin Secretin induced a potent inhibition of pepsin output, which reached 100% after 3 nmol/kg, h of secretin (Fig. l). Pepsin concentration did not differ significantly from controls, except after the large dose of 3 nmol/kg, h (Table 2). The volume and acid output were also potently inhibited by secretin: the volume was reduced by 74% and the acid output by 100% with 3 nmol/kg, h of secretin.
CONTROL OF PEPSIN SECRETION
235
TABLE 1 EDso'sOF THE DIFFERENTSUBSTANCESUSED Volume Substance CCK (nmol/kg. h) Pentagastrin (nmol/kg • h)
Stimul.
Methacholine (~tmol/kg. h)
Stimul.
Pepsin lnhib.
Stimul.
0.36 (0.08/1.55)
0.22 (0.05/1.00)
0.08 (0.03/0.25) ND
0.32 (0.10/1.01)
2.08 (1.31/3.28) 0.23* ND
0.33 (0.11/0.99)
0.55 (0.27/1.13)
0.48 (0.14/1.59) 1.53 (1.09/2.08) 0.08* ND
lnhib.
1.86 (1.15/3.00) NS
0.98 (0.69/1.39) 0.47 (0.18/1.16) 0.29 (0.06/1.40)
Neurotensin (nmol/kg • h) Histamine (umol/kg • h)
Inhib.
0.72 (0.39/1.33) 0.40 (0.66/2.47)
Secretin (nmol/kg. h) VIP (nmol/kg- h)
Acid
NS 1.26 (0.87/1.83)
NS: nonsignificant stimulation. ND: nondetermined for technical reasons (dispersion, number of points, etc.). * Corrected for saliva contamination. In parentheses: 95% confidence interval limits. n = 6 to 12 rats per substance. VIP
The VIP effect on pepsin output was biphasic (Fig. 1). Small VIP doses induced a modest increase of pepsin output, peaking at 1.5 times the basal level with 1.25 nmol/kg, h of VIP. Larger VIP doses induced a progressive reduction of pepsin output, which decreased below the basal level. Pepsin concentration decreased in a uniformly dose-related way (Table 2). Salivary secretion was poorly stimulated by VIP: only the large dose of 12.5 nmol/kg-h of VIP had a significant effect (Table 3). No correction of gastric volume and acid for salivary secretion was thus necessary. The dose-response curves of gastric juice volume and acid output in VIP-infused animals were also biphasic. Small VIP doses (0.12 to 1.25 nmol/kg • h) increased the gastric juice volume and the acid output in a dose-related way (Figs. 2 and 3). Maximal stimulation was obtained with 1.25 nmol/kg, h of VIP, producing twice and 2.7 times the basal level, respectively, for volume and acid output. The volume of gastric juice tended to decrease with supramaximal VIP doses, but this was not significant. Supramaximal VIP doses induced submaximal acid responses. Neurotensin
Neurotensin induced a progressive dose-related inhibition of pepsin output. The maximal inhibition was about 60% and was obtained with 19 nmol/kg, h. Neurotensin also induced a progressive dose-related inhibition of volume and acid output. The maximal inhibition of gastric juice volume was obtained with 19 nmol/kg, h and was about 70% of the basal level (Fig. 2). The maximal inhibition of acid output was about 60% (Fig. 3) and was obtained with 6 nmol/ kg. h. Histamine No significant stimulation of pepsin output was obtained after histamine infusion, but the largest dose of histamine used (17.1 /~mol/kg. h) significantly decreased pepsin output (Fig. 1).
Histamine induced a dose-related increase of gastric juice volume (Fig. 2) and acid output (Fig. 3). Maximal stimulation occurred at the dose of 5.4 t~mol/kg, h and peaked at 1.7 and 2.7 times the basal level, respectively, for volume and acid output. Methacholine
Methacholine significantly stimulated pepsin secretion and was the most efficient of the stimulant agents used: the maximal pepsin output was 4.5 times the basal level and was greater than any maximal response obtained with the other substances (Fig. 1). Since methacholine significantly stimulates bicarbonate-rich salivary secretion, which was collected together with the gastric juice in our method, the values obtained for volume and acid need to be corrected from the salivary contamination. This has been done in Table 4, which includes the measured gastric juice values, the amount of saliva collected in similar conditions, and the corrected gastric juice values. After this correction, the maximal volume increase was 2.35 times the basal level (Fig. 2), and the maximal acid output was 2.3 times the basal level (Fig. 3). DISCUSSION In these experiments, methacholine was the most efficient stimulant of pepsin secretion, while it was moderately efficient on acid output. This result is in agreement with the literature data, since cholinergic agents are the most generally recognized potent stimulants of pepsin secretion, in conscious (37) and anesthetized rats (34) and on dispersed isolated rat gastric glands (29). Muscarinic receptors have been characterized on isolated guinea pig chief cells (38). Since salivary secretion was potently stimulated by methacholine, we had to correct gastric juice values from salivary output, so that acid output and volume may be somehow approximate. Saliva, however, does not hinder the accurate determination of pepsin output, as long as one takes care to keep the pH of gastric juice samples below 5 to avoid pepsin inactivation.
236
TAZI-SAAD, CHARIOT AND ROZI~
Pepsin output (UP/40 min
30
S
20
.
~
~
CCK
10
~
- ~ ~ , , ,
O,
,01
,1
m-
10 100 Dose(nmol/kg.h)
1
1 ETHAOH
Pepsin output (UP/40 min)
60-
40
.
20 ; O"
probably interacts on chief cells with CCK receptors of low affinity for gastrin, large concentrations ofgastrin are required to produce an effect. This effect can be demonstrated in appropriate conditions, but is probably of little physiological importance. In our experiments, 1.86 nmol/kg, h CCK produced a half-maximal stimulation of pepsin output (Table 1). Cherner et al. (5), using dispersed guinea pig chief cells, showed that it takes approximately 1000 times more gastrin than CCK to get half the maximal pepsin response to CCK. Assuming that rat chief cells would behave in the same manner, it would take 1.86 ~mol/ kg. h gastrin to stimulate half-maximally pepsin output, whereas the largest dose ofpentagastrin we used was only 40 nmol/kg, h. Acid and pepsin outputs were dose-dependently stimulated by CCK. The CCK8 analog [Boc-Nle2S-Nle3]]CCK8 was used in our experiments. More stable than CCK8, this analog displays in several in vitro and in vivo systems the same affinity and biological activity as CCK8 (27,28,31) and can thus be reliably used as a CCK receptor agonist. CCK stimulation of pepsin secretion has been reported in the anesthetized rat in vivo (34) and in several in vitro preparations [rabbit gastric glands (10,13), guinea pig dispersed chief cells (30), canine cultured chief cells (32)]. CCK receptors have been described on isolated guinea pig chief cells (5) and membranes (24). They seem more closely related to CCKA than to gastrin receptors, but their relative affinities for CCK analogs suggest that they might be different from both pancreatic and brain CCK receptors (24). The doseresponse curve for CCK-stimulated pepsin secretion was biphasic, indicating a supramaximal inhibition. This suggests that chief cell CCK receptors may take at least two different confor-
•
0
~7~'--~.~-4-~'4l PG l 'r ....
.~
,1
. . . . .
"1
1
. . . . . .
q
10
. . . . . .
I
......
"~! HIST "~
......
-q
. . . . .
=1
100 1000 10000100000 Dose(nmol/kg.h)
FIG. 1. Effect of several substances on the gastric pepsin output in conscious rats. Top panel: secretin (S), vasoactive intestinal peptide (VIP), [Boc-Nle2S-Nle3~]CCK8 (CCK), neurotensin (NT). Lower panel: pentagastrin (PG), methacholine (METHACH), histamine (HIST). Each substance was intravenously infused at increasing doses (each dose for 40 min). Mean _+ SEM; n = 6 to 12 rats per substance; the basal level (B) is the average of the two first 40-rain samples, collected during NaCl infusion; *p < 0.05, **p < 0.01, ***p < 0.001 compared to the basal level (Dunnett's test). Histamine did not increase pepsin output in the dose range which stimulated acid secretion, and even decreased pepsin output when administered in supramaximal doses for acid secretion. These data differ from some reports indicating stimulatory effects in anesthetized (34) or conscious rats (18,20), whereas in vitro, no effect [rabbit gastric glands (16)] or a moderate stimulation [rabbit gastric glands (13), canine cultured chief cells (32)] has been reported. Pentagastrin had no significant effect on pepsin output, even in doses up to 50 nmol/kg, h, which maximally stimulated acid output. These data are at variance with papers indicating some stimulation of pepsin secretion by gastrin or pentagastrin in rats. Most stimulant effects were obtained on denervated [Heidenhain pouch (12)] or isolated preparations [isolated perfused stomach (15,33)] or in anesthetized rats (34), whereas only Kowalewski (18) described some pepsin increase in conscious rats under longlasting (24 h) and large volume (120 ml/kg. 24 h) pentagastrin infusions, with gastric juice collected over 24 h. Discrepancies also exist in vitro, since some groups reported a stimulation of pepsin secretion by gastrin [isolated dog stomach (21), isolated rabbit glands (10,13), isolated perfused rat stomach (33), guinea pig dispersed chief cells (5)] whereas others found no effect [cultured canine chief cells (32), isolated perfused rat stomach (i 5), isolated perfused mouse stomach (7)]. Since gastrin
Volume (Id/40 min) 3000. B
2000.
***
1000-
O0
,01
,1
10
1
100
Dose (nmol/kg.h)
Volume (~I/40 rain)
3000 2000 i
~
,"~
PG ~ * *
METHACH HIST
1000, 00
,1
1
10
100 1000 10000100000 Dose (nrnol/kg.h)
FIG. 2. Effect of several substances on the volume of gastric juice in conscious rats. Groups and symbols as in Fig. 1.
CONTROL OF PEPSIN SECRETION
237
Acid output (Ixmol/40 rnin)
TABLE 2 EFFECTOF SECRET1NAND VIP ON PEPSINCONCENTRATION IN GASTRICJUICE
400
Secretin
300 200
VIP
Dose (nmol/kg.h)
Pepsin (PU/ml)
Dose (nmol/kg•h)
Pepsin (PU/ml)
0 0.006 0.06 0.6 3
10.1 _+0•85 10.0 _+ 1•20 10.6 _+0.52 9.2 _+ 1.20 0 +- 0t
0 0.125 0.396 1.25 3.96 12.5
10.8 _+0.69 12.4 +__2.77 9.1 _+ 1.14 7.7 _+0.49* 6.5 +_0.541" 4.0_ 1.08-~
100 0
0
,01
,1
1
10
100
Dose (nmol/kg.h)
PU = peptic units. Mean _+SEM; n = 6 to 12 rats per group. *~fCompared to the basal level, *p < 0.01, fp < 0.001. Acid output (l~mOl/40 min) 400m
300-
HIST
20O -
METHACH 100 0-
0
,1
1
10
100
1000
10000 100000
Dose (nmol/kg.h)
FIG. 3. Effect of several substanceson the gastric acid output in conscious rats. Groups and symbols as in Fig. I.
mations with different affinities, that might be related to stimulation and inhibition of secretion, as reported in the case of pancreatic acini (11,27). Secretin and VIP, a hormone and a neuropeptide that usually interact with closely related receptors, displayed clearly different effects in our conscious fistula rats: VIP produced a biphasic dose-response curve with a stimulation followed by an inhibition, whereas secretin was a pure, potent, and efficient inhibitor of acid and pepsin secretions. Since VIP is known to potentiate acetyicholine-induced salivary secretion (22), we sought a possible stimulation of salivary secretion in our conditions, which might explain the VIP-induced decrease of pepsin concentration. The very weak stimulatory effect of VIP on salivary secretion makes this hypothesis unlikely• The effects of VIP on acid and pepsin secretion in vivo are controversial and variable according to the species. In the rat, no effect of peripheral injections of VIP on acid secretion was reported in pylorus-ligated rats (8), with a large dose of 100 ug/ kg = 30 nmol/kg, but this was likely supramaximal. In vitro, VIP increased pepsinogen release by isolated gastric glands or dispersed chief cells from rats (29) or guinea pigs (39), and potentiated the stimulant effect of CCK in isolated rabbit (3) or rat gastric glands (29). Four classes of binding sites have been described for VIP and secretin on guinea pig isolated chief cells, and occupation of two of these classes correlated with the abilities of VIP and secretin to increase cellular cAMP and pepsinogen secretion (39). In the isolated luminally perfused mouse stomach, Schubert (35) recently evidenced that the effect of VIP on acid secretion depended on the balance between direct stimulation of acid se-
cretion and concurrent release of somatostatin. Somatostatin secretion was more sensitive to VIP than acid secretion. If such a dual mechanism also exists in the rat, it might account for the biphasic dose-response curve found here. In species other than the rat, many data indicate that secretin increases pepsin secretion although inhibits acid secretion (9), whereas no characterization of secretin receptors on isolated rat chief cells has yet been published. In anesthetized rats with gastric lumen perfusion, Schmidt-Wilcke and Nawrath (34) described a stimulation of pepsin output, but this was with extractive secretin, which might be impure• In conscious rats, Kowalewski (19) found no effect of secretin on pepsin output or a tendency to decrease under long-lasting (24 h) secretin infusions. The total inhibition of pepsin output that we observed under secretin infusion was not only the consequence of the potent inhibition of gastric juice volume, since 3 nmol/kg, h secretin suppressed totally pepsin output in our experiments, whereas gastric juice volume decreased only by 74%. Also, pepsin concentration in gastric juice never increased after any secretin dose (Table 2), although fluid secretion was markedly inhibited, but sharply decreased after 3 nmol/kg- h secretin. A direct or indirect inhibitory effect of secretin on chief cells should thus have occurred, a situation that seems different from what exists in guinea pig chief cells• Neurotensin inhibited basal acid and pepsin secretions maximally by about 60% at the largest dose of 19 nmol/kg, h. The effect of neurotensin on pepsin secretion in the rat has not been reported previously. The range of active doses suggests that neurotensin inhibition of gastric secretion may be pharmacological rather than physiological, since these doses are larger than those affecting duodenal bicarbonate (25) and pancreatic (26) secre-
TABLE 3 EFFECTSOF VIP ON SALIVARYVOLUMEAND BICARBONATEOUTPUT VIP (nmol/kg.h)
Volume (~1/40 rain)
BicarbonateOutput (~mol/40 rain)
0 1.25 4.0 12.5 40.0
256 +_93 240 ___34 276 + 82 475 + 58* 258 + 17
7.0 + 3.7 6.9 ___1.2 7.1 + 2.2 13.7 + 2.5* 6.2 + 0.8
* p < 0.05 compared to the basal level; n = 5 rats.
238
TAZI-SAAD, C H A R I O T A N D ROZI~
TABLE 4 METHACHOL1NESTIMULATIONOF GASTRICAND SALIVARYVOLUME,OF GASTRICACID OUTPUT, AND OF SALIVARYBICARBONATEOUTPUT Volume (~1/40 min)
Acid or Bicarbonate (umol/40 min)
Methacholine (gmol/kg. h)
Gastric (Total)
Salivary
Gastric (Corrected)*
Gastric Acid
Salivary Bicarbonate
Gastric Acid (Corrected)*
0 0.31 1.0 3.1 10
1100-+217 1231 -+211 1906 -+ 286 4419 -+ 467 6315 -+ 461
70_+ 26 60-+ 30 360 -+ 80 2260 -+ 300 3560 -+ 230
1030-+227 1171 -+213 1546 -+ 297 2159 -+ 555 2755 -+ 515
75.2_+25.1 88 -+29.7 91.9 -+ 6.8 91.7 -+ 17.4 9.0 -+ 3.9
2.1 _+ 1.2 1.6-+0.9 12.2 -+ 3.0 76.2 -+ 8.6 142.6 + 9.4
73.1+28.0 89.6_+29.7 104.1 _+ 10.7 167.9 _+ 19.4 151.6 + 10.2
* "Corrected" gastric volume and acid output = measures on total gastric juice contaminated by saliva, minus salivary secretion determined in separate experiments. n - 8 to 12 rats per group.
tions in the rat. However, some data obtained with a neurotensin antiserum suggest that neurotensin might mediate the gastric acid inhibitory effect of intestinal infusions of oleic acid in anesthetized rats (36). The mechanism of neurotensin inhibition of gastric secretion is unclear and probably indirect. Neurotensin receptors have not been characterized on isolated parietal cells (14), whereas no data have been published for chief cells. Neurotensin increases the postprandial release of pancreatic polypeptide (17) and may increase prostaglandin generation (23), whereas the necessity of intact vagal nerves for neurotensin inhibition of gastric secretion has been successively claimed (1) and challenged (23). In conclusion, this reinvestigation of some modulators of pepsin secretion in the rat stomach in vivo was aimed to gain understanding of the physiological role of each substance, based
on the assessment that a peptide should be both potent and efficient to be of real physiological importance. A m o n g the substances tested, muscarinic agonists and C C K are the main stimulants of pepsin secretion. The stimulants of parietal cells, histamine and pentagastrin, have few effects, if any, on pepsin output, whereas secretin is a potent inhibitor of gastric secretion, including pepsin. Most of these data are closer to the recent results obtained with in vitro isolated cell preparations than to older results obtained in vivo, sometimes with peptide samples of doubtful purity. ACKNOWLEDGEMENTS Partial financial support was provided by the Conseil Scientifique ot Facult6 Xavier Bichat and by Association Charles Debray.
REFERENCES 1. Andersson, S.; Chang, D.; Folkers, K.; Rosell, S. Inhibition of gastric acid secretion in dogs by neurotensin. Life Sci. 19:367-370; 1976. 2. Anson, M. L.; Mirsky, A. E. The estimation of pepsin with hemoglobin. J. Gen. Physiol. 16:59-63; 1933. 3. Ballantyne, G. H.; Zdon, M. J.; Zucker, K. A.; Tyskoff, M.; Fratesi, G. R.; Schafer. D. E.; Modlin, I. M. Vasoactive intestinal peptide augmentation of cholecystokinin-8-stimulated pepsinogen secretion: Evidence for modulation of chief cell function. J. Surg. Res. 40:617623; 1986. 4. Chariot, J.; Rozr, C. Drtermination automatiske de trrs faibles concentrations de bicarbonates. Application au suc pancrratique. Ann. Biol. Clin. 34:269-272; 1976. 5. Cherner, J. A.; Sutliff, V. E.; Grybowski, D. M.; Jensen, R. T.; Gardner, J. D. Functionally distinct receptors for cholecystokinin and gastfin on dispersed chief cells from guinea pig stomach. Am. J. Physiol. 254:GI51-G155; 1988. 6. Emas, S.; Nylander, G.; Wallin, B. Comparison of the dose-response curves for acid output to pentagastrin determined by two techniques in chronic gastric fistula rats. Digestion 22:94-100; 1981. 7. Fimmel, C. J.; Blum, A. L. Pepsin secretion: Neurohumoral regulation and drug effects. Scand. J. Gastroenterol. 19(Suppl. 101):3946; 1984. 8. Hernandez, D. E.; Jennes, C.; Emerick, S. G. Brain vasoactive intestinal peptide: A potent stimulant of gastric acid secretion. Brain Res. 420:129-134; 1987. 9. Hersey, S. J. Pepsinogen secretion. In: Johnson, L. R.; Christensen, J.; Jackson, M. J.; Jacobson, E. D.; Walsh, J. H., eds. Physiology of the gastro-intestinal tract, 2nd ed. New York: Raven Press; 1987: 947-957.
10. Hersey, S. J.; May, D.; Schyberg, D. Stimulation ofpepsinogen release from isolated gastric glands by cholecystokinin-like peptides. Am. J. Physiol. 244:GI92-GI97; 1983. 11. Jensen, R. T.; Wank, S. A.; Rowley, W. H.; Sato, S.; Gardner, J. D. Interaction of CCK with pancreatic acinar cells. Trends Pharmacol. Sci. 10:418-423; 1989. 12. Johansson, I.; Lundell, L.; Rosengren, E.; Svensson, S. E. Gastric secretion and its facilitation as related to gastric mucosal histamine. J. Physiol. (Lond.) 266:431-452; 1972. 13. Kasbekar, D. K.; Jensen, R. T.; Gardner, J. D. Pepsinogen secretion from dispersed glands from rabbit stomach. Am. J. Physiol. 244: G392-G396; 1983. 14. Kitabgi, P.; Kwan, C.; Fox, J.; Vincent, J. Characterization of neurotensin binding to rat gastric smooth muscle receptor sites. Peptides 5:913-917; 1984. 15. Kleveland, P. M.; Haugen, S. E.; Sandviks, S.; Waldum, H. L. The effect of pentagastrin on the gastric secretion by the totally isolated vascularly perfused rat stomach. Scand. J. Gastroenterol. 21:379384; 1986. 16. Koelz, H. R.; Hersey, S. J.; Sachs, G.; Chew, C. S. Pepsinogen release from isolated gastric glands. Am. J. Physiol. 243:G218--(3225; 1982. 17. Konturek, S. J.; Bilski, J.; Tasler, J.; Laskiewicz, J. Gut hormones in stimulation ofgastroduodenal alkaline secretion in conscious dogs. Am. J. Physiol. 248:G687-G691; 1985. 18. Kowalewski, K. Effect of nicotine hydrogen tartrate on gastric secretion of normal and vagotomized rats stimulated with histamine, pentagastrin and bethanechol chloride. Digestion 10:393-401; 1974. 19. Kowalewski, K. The effect of secretin on pentagastrin stimulated secretion of gastric pepsin and acid in rats. Arch. Int. Physiol. Biochim. 83:255-259; 1975.
C O N T R O L OF PEPSIN SECRETION 20. Kowalewski, K.; Chmum, G. Determination of histamine dose causing maximal gastric secretion. Study in rats with gastric fistulas. Am. J. Dig. Dis. 13:753-760; 1968. 21. Kowalewski, K.; Kolodej, A. Correlation between the outputs of pepsin and HCI by isolated, ex vivo perfused, canine stomach stimulated with humoral and cholinergic drugs. Pharmacology 9:325335; 1973. 22. Lundberg, J. M. Evidence for coexistence of vasoactive intestinal polypeptide (VIP) and acetylcholine in neurons of cat exocrine glands. Morphological, biochemical and functional studies. Acta Physiol. Scand. 496:1-57; 1981. 23. Mate, L.; Doyle, H. R.; Sakamoto, T.; Courtney, M.; Greeley, G. H.; Thompson, J. C. The mechanism of the inhibitory action of neurotensin on pentagastrin-stimulated gastric secretion in dogs. Surg. Gynecol. Obstet. 80:1064-1067; 1988. 24. Matozaki, T.; Sakamoto, C.; Nagao, M.; Nishisaki H.; Konda, Y.; Nakano, O.; Matsuda, K.; Wada, K.; Suzuki, T.; Kasuga, M. Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes. Biochem. Biophys. Res. Commun. 174:1055-1063; 1991. 25. Merlin, D.; Tazi-Saad, K.; Nagain, C.; Chariot, J.; Rozr, C. Action de la neurotensine sur la srcrrtion bicarbonatre duodrnale chez le rat. Comparaison avec les srcr&ions pancrratique et gastrique. Gastroenterol. Clin. Biol. 15:574-579; 1991. 26. Nagain, C.; Chariot, J.; Rozr, C. Mechanism of pancreatic stimulation by neurotensin in the rat. Gastroenterology 100:A291; 1991 (abstract). 27. Nagain, C.; Mendre, C.; Rodriguez, M.; Bernard, C.; Martinez, J.; Rozr, C. External pancreatic secretion in the rat: Supramaximal inhibition induced by the cholecystokinin octapeptide [CCK(2633)] and analogs altered on the 28-29 bond. Peptides 10:1041-1047; 1989. 28. Nagain, C.; Rodriguez, M.; Martinez, J.; Rozr, C. In vivo activities of peptide and pseudo-peptide analogs of the C-terminal octapeptide of cholecystokinin on pancreatic secretion in the rat. Peptides 8: 1023-1028; 1987. 29. Raufman, J. P.; Kasbekar, D. K.; Jensen, R. T.; Gardner, J. D. Potentiation of pepsinogen secretion from dispersed glands from rat stomach. Am. J. Physiol. 245:G525-G530; 1983.
239 30. Raufman, J. P.; Sutliff, V. E.; Kasbekar, D. K.; Jensen, R. T.; Gardner, J. D. Pepsinogen secretion from dispersed chief cells from guinea pig stomach. Am. J. Physiol. 247:G95-GI04; 1984. 31. Rodriguez, M.; Lignon, M. F.; Galas, M. C.; Mendre, C.; Martinez, J. Synthesis of pseudo-peptide analogues of the C-terminal heptapeptide of cholecystokinin and preliminary results on their in vitro activity on stimulation of amylase release from pancreatic acini. In: Theodoropoulos, T., ed. Peptide 1986. Proceedings ot the 19th European Peptide Symposium. Berlin: W. de Gruyter; 1986:455-458. 32. Sanders, M. J.; Amirian, D. A.; Ayalon, A.; Soil, A. H. Regulation of pepsinogen release from canine chief cells in primary monolayer culture. Am. J. Physiol. 245:G641-G646; 1983. 33. Sandvik, A. K.; Kleveland, P. M.; Waldum, H. L. Stimulated pepsin secretion after omeprazole-induced acid suppression in the totally isolated, vasculady perfused rat stomach. Scand. J. Gastroenterol. 22:362-366; 1987. 34. Schmidt-Wilcke, Von, H. A.; Nawrath, G. D. Die sfiure-pepsinsekretion der ratte unter dem einfluss verschiedener gastrointestinaler hormone. Arzneimittelforschung 25:1596-1600; 1975. 35. Schubert, M. L. The effect of vasoactive intestinal polypeptide on gastric acid secretion is predominantly mediated by somatostatin. Gastroenterology 100:1195-1200; 1991. 36. Seal, A.; Liu, E.; Buchan, A.; Brown, J. Immunoneutralization of somatostatin and neurotensin: Effect on gastric acid secretion. Am. J. Physiol. 255:G40-G45; 1988. 37. Seidel, E. R.; Johnson, L. R. Ontogeny of gastric mucosal muscarinic receptor and sensitivity to carbachol. Am. J. Physiol. 246:G550G555; 1984. 38. Sutliff, V. E.; Rattan, S.; Gardner, J. D.; Jensen, R. T. Characterization of cholinergic receptors mediating pepsinogen secretion from chief cells. Am. J. Physiol. 257:G226-G234; 1989. 39. Sutliff, V. E.; Raufman, J. P.; Jensen, R. T.; Gardner, J. D. Actions of vasoactive intestinal peptide and secretin on chief cells prepared from guinea pig stomach. Am. J. Physiol. 251:G96-G102; 1986. 40. Tallarida, R. J.; Murray, R. B. Manual of pharmacological calculations with computer programs. New York: Springer Vcrlag: 1984: 14-19.
0196-9781/92$5.00 + .00 Copyright © 1992PergamonPressLtd.
Printedin the USA.
Control of Pepsin Secretion by Regulatory Peptides in the Rat Stomach: Comparison With Acid Secretion KHADDOUJ
T A Z I - S A A D , J A C Q U E S C H A R I O T A N D C L A U D E ROZI~ ~
I N S E R M U239, Facult~ de MOdecine X. Bichat, 16 R u e H Huchard, F75018 Paris, France Received 24 July 1991 TAZI-SAAD,K., J. CHARIOT AND C. ROZI~.Controlof pepsin secretion by regulatorypeptides in the rat stomach: Comparison with acid secretion. PEPTIDES 13(2) 233-239, 1992.--Previous studies of the control of pepsin secretion by neurohumoral agents showed some discrepancies between in vitro (isolated cells) and in vivo experiments. In the present work, the effects on pepsin secretion of CCK, pentagastrin, secretin, VIP, neurotensin, histamine, and methacholine were reinvestigatedin conscious gastric fistula rats, in comparison to acid secretion. EDs0's and doses inducing maximal responses were measured to directly compare the potency and efficacy of these substances. Methacholine was the most efficient (maximal response = 4.5 × basal level, EDs0 = 1.3 umol/kg, h) and CCK the most potent (EDs0 = 1.9 nmol/kg, h) stimulant, whereas secretin was a potent (EDs0 = 0.22 nmol/kg • h) and efficientinhibitor (maximal response = 100%inhibition). These three substancesare likelyto be important regulators of pepsin secretion in the rat. Pentagastrin and histamine did not stimulate pepsin output, as found by others with isolated chief cells in vitro. Neurotensin and large doses of VIP marginally inhibited pepsin secretion. Pepsin
Gastrin
CCK
Secretin
VIP
Neurotensin
WHEREAS the measurement of gastric acid secretion in conscious fistula rats is a widely used method in digestive physiology and pharmacology, fewer studies have been devoted to the factors controlling pepsinogen secretion. From classical studies, it is generally admitted that the main acid secretagogues gastrin and histamine are mild stimulants of pepsin output in vivo, whereas cholinergic agents possess a greater efficacy on chief cells. Discrepancies between the results obtained in isolated and intact preparations have occurred for gastrin and other peptides (CCK, VIP, secretin, etc.) (9). The purpose of this work was to reinvestigate the effect of some peptides and neurotransmitters on pepsinogen secretion in the intact rat stomach, in order 1) to directly determine their relative potencies and efficacies, and 2) to measure their relative potency compared to that for gastric acid secretion. This should help to gain understanding of the physiological role of each substance. Since all substances which may affect gastric secretion could not be tested, we selected the three main stimulants of parietal cells, gastrin, histamine, and methacholine, and four peptides for which effects on gastric secretion are not completely established, CCK, secretin, VIP, and neurotensin.
Methacholine
Histamine
Gastric secretion
maintained in the laboratory on a commercial autoclaved pelleted chow (UAR 113, Alimentation Rationnelle, Villemoisson, F-91360 Epinay sur Orge).
Gastric Chronic Fistula After premedication with 10 mg/kg of acepromazine IP (Vetranquil®, Clin Midy, F-45142, St Jean de la Ruelle), the rats were anesthetized with 50 mg/kg ketamine IP (Imalgene 500®, Rhrne M~rieux, F-69002 Lyon). A gastric fistula was set up following the method of Emas et al. (6): a seromuscular purse was prepared in the ruminal part of the stomach, and a Thomas cannula was inserted and secured in place with the help of a square pad of synthetic felt 1.5 × 1.5 cm (Dacron felt, USCI, USA).
Secretory Tests Two weeks of postoperative recovery were allowed before the secretory tests were begun. During this period, the rats were trained to stand quiet in the Bollman cages during three sham experiments, without any treatment or gastric juice collection. Secretory tests were then performed no more than once a week. After a 24-h fasting period with drinking water ad lib, the gastric cannula was opened and the stomach was washed with water prewarmed at 37°C. A plastic prolongator was screwed upon the gastric cannula, and a small diameter polyethylene catheter with lateral holes was introduced in the cannula. This
METHOD
Animals Male Wistar rats (Iffa Credo, Les Oncins, F-69210 L'Arbresle), weighing 300 _+ 20 g, were used in these studies. They were Requests for reprints should be addressed to Claude Roz6.
233
234 catheter helped to safely collect the gastric juice, by guiding it drop by drop into the collecting tube. The rats were placed in Bollman cages, and an infusion catheter was inserted in a tail vein and secured with adhesive tape. A venous infusion of NaC1 0.9% was then begun. Gastric secretion was collected as 40-min samples in tared tubes. The weight of gastric juice was measured, and accepted as a measure of gastric volume, assuming that 1 ml = 1 g. Two forty-rain samples were collected before injecting any substance and were taken to be representative of basal secretion. The substances tested were intravenously infused, using a pattern of increasing doses in the same rat, each dose being infused for 40 rain. No more than four successive doses were infused in each animal. The dose-response curves comprising more than four points were constructed by adding the results of a low dose range and a high dose range group of rats.
TAZI-SAAD, CHARIOT AND ROZ[~ analog of CCK8 whose activity is equal to that of CCK8 on all models tested to date, 0.6 to 60 nmol/kg, h (=0.69 to 69 ~g/ kg. h); pentagastrin (Peptavlon® ICI Pharma, France) 0.13 to 41.1 nmol/kg- h (=0.1 to 31.6 #g/kg. h); secretin (Cambridge Research Biochemicals, Cambridge, UK) 0.006 to 3 nmol/kg, h (=0.018 to 9.16 #g/kg. h); vasoactive intestinal peptide (Neosystem, Strasbourg, France) 0.125 to 12.5 nmol/kg, h (=0.416 to 41.6 #g/kg. h); neurotensin(1-13) (Sigma, St. Louis, MO, USA) 0.6 to 19 n m o l / k g . h (=1 to 31.6 #g/kg.h); histamine dihydrochloride (Sigma) 0.54 to 17.1 #mol/kg. h (=0.1 to 3.16 mg/kg-h); methacholine (acetyl-beta-methylcholine chloride) (Pharmacie Centrale des Hopitaux, Paris, France) 0.31 to 9.71 #mol/kg. h (=0.06 to 1.9 mg/kg, h). Peptide solutions were prepared in 0.9% NaC1 with 0.3% of bovine serum albumin added. Histamine and methacholine were dissolved in 0.9% NaCI.
Acid Secretion Acid was titrated to pH = 7.0 with NaOH 0.004 M, on 0.1 ml samples, using an automatic titrator (Metrohm AG, 9100 Herisau, Switzerland). When the pH of gastric samples was greater than or equal to 5.0, the samples were immediately acidified with 20 ul HC1 6 M per ml of gastric juice, in order to protect pepsin activity, which rapidly fades in solutions of pH >5.
Statistics The results are expressed as means + one standard error of the mean (SEM). EDso'S were calculated from the linear portion of the regression curve of the effect on log dose, as described in Tallarida and Murray (40). Statistical significance of the differences between means was tested by ANOVA followed by a post hoc test (Dunnett or Newman-Keuls).
Pepsin Secretion Pepsin activity was determined by a semiautomated method derived from that proposed by Anson and Mirsky (2), which comprises two successive steps: 1) Pepsin present in the samples was allowed to digest an aqueous solution of 2.5% hemoglobin (Sigma, USA), pH = 1.7, for 3 min at 37°C. The reaction was then stopped by adding 5% trichloroacetic acid (Prolabo, France), and soluble hydrolysis products were separated by centrifugation. 2) Soluble hydrolysis products were determined with the FolinCiocalteu reagent (Merck, USA) in a Technicon autoanalyzer, using tyrosine as a standard. Samples were 50 ttl for the dosage and 50 ul for the blank tube without hydrolysis. Peptic activity was determined by comparison to a standard scale of hog pepsin (Fluka, ref 77152, Buchs, Switzerland) and expressed as peptic units (PU). One unit corresponds to the amount of pepsin releasing 1 #mol tyrosine per rain in the conditions used.
Collection of Salivary Secretion In experiments with methacholine and VIP, salivary secretion was measured in separate experiments, to correct for a possible contamination of gastric secretion by salivary secretion. Under ether anesthesia, a polyethylene catheter (Biotrol® No. 13) was introduced in the lower esophagus through a ruminal incision, secured in place by two ligatures at the level of the cardia and tureen, and conducted through the abdominal wall. The muscle and skin were sutured, the rat was placed in a Bollman cage, and an infusion catheter was inserted in a tail vein. After two hours of postanesthesia recovery, saliva was collected as 40-rain fractions in 5-ml graduated tubes. Methacholine and VIP were infused in successive increasing doses in the same conditions as in gastric fistula rats. The volume of saliva samples was measured to the nearest 0.1 ml. Bicarbonate was determined with an autoanalyzer method (4).
Substances Tested The following substances were used: CCK ([Boc-Nle28Nle3J]CCK8) (a gift ofJ. Martinez, Centre CNRS-INSERM de Pharmacologie Endocrinologie, Montpellier, France), a synthetic
RESULTS The dose-responses curves obtained with the substances tested are shown in Figs. 1 to 3. The EDso'S corresponding to these data are grouped in Table 1.
CCK CCK induced a dose-related increase of pepsin output (Fig. l). Maximal stimulation was obtained with 19 nmol/kg, h of CCK, and peaked at about 3 times the basal pepsin output. The dose-response curve was biphasic, with supramaximal doses inducing a submaximal pepsin response. CCK also produced a dose-related stimulation of gastric juice volume and acid (Figs. 2 and 3). Maximal stimulation of volume and acid was obtained with 6 nmol/kg, h. A biphasic dose-response curve was also observed, with supramaximal doses inducing submaximal volume and acid responses.
Pentagastrin Pepsin output was poorly changed by pentagastrin. Only an insignificant tendency to increase from a basal level of 14 UP/ 40 rain to 20 UP/40 rain was found after 1.3 to 41 nmol/kg, h of pentagastrin (Fig. 1). Pentagastrin induced a dose-related stimulation of gastric juice volume and acid output (Figs. 2 and 3). The maximal stimulation of volume and acid was obtained with 4 nmol/kg, h of pentagastrin, and no supramaximal inhibition was observed.
Secretin Secretin induced a potent inhibition of pepsin output, which reached 100% after 3 nmol/kg, h of secretin (Fig. l). Pepsin concentration did not differ significantly from controls, except after the large dose of 3 nmol/kg, h (Table 2). The volume and acid output were also potently inhibited by secretin: the volume was reduced by 74% and the acid output by 100% with 3 nmol/kg, h of secretin.
CONTROL OF PEPSIN SECRETION
235
TABLE 1 EDso'sOF THE DIFFERENTSUBSTANCESUSED Volume Substance CCK (nmol/kg. h) Pentagastrin (nmol/kg • h)
Stimul.
Methacholine (~tmol/kg. h)
Stimul.
Pepsin lnhib.
Stimul.
0.36 (0.08/1.55)
0.22 (0.05/1.00)
0.08 (0.03/0.25) ND
0.32 (0.10/1.01)
2.08 (1.31/3.28) 0.23* ND
0.33 (0.11/0.99)
0.55 (0.27/1.13)
0.48 (0.14/1.59) 1.53 (1.09/2.08) 0.08* ND
lnhib.
1.86 (1.15/3.00) NS
0.98 (0.69/1.39) 0.47 (0.18/1.16) 0.29 (0.06/1.40)
Neurotensin (nmol/kg • h) Histamine (umol/kg • h)
Inhib.
0.72 (0.39/1.33) 0.40 (0.66/2.47)
Secretin (nmol/kg. h) VIP (nmol/kg- h)
Acid
NS 1.26 (0.87/1.83)
NS: nonsignificant stimulation. ND: nondetermined for technical reasons (dispersion, number of points, etc.). * Corrected for saliva contamination. In parentheses: 95% confidence interval limits. n = 6 to 12 rats per substance. VIP
The VIP effect on pepsin output was biphasic (Fig. 1). Small VIP doses induced a modest increase of pepsin output, peaking at 1.5 times the basal level with 1.25 nmol/kg, h of VIP. Larger VIP doses induced a progressive reduction of pepsin output, which decreased below the basal level. Pepsin concentration decreased in a uniformly dose-related way (Table 2). Salivary secretion was poorly stimulated by VIP: only the large dose of 12.5 nmol/kg-h of VIP had a significant effect (Table 3). No correction of gastric volume and acid for salivary secretion was thus necessary. The dose-response curves of gastric juice volume and acid output in VIP-infused animals were also biphasic. Small VIP doses (0.12 to 1.25 nmol/kg • h) increased the gastric juice volume and the acid output in a dose-related way (Figs. 2 and 3). Maximal stimulation was obtained with 1.25 nmol/kg, h of VIP, producing twice and 2.7 times the basal level, respectively, for volume and acid output. The volume of gastric juice tended to decrease with supramaximal VIP doses, but this was not significant. Supramaximal VIP doses induced submaximal acid responses. Neurotensin
Neurotensin induced a progressive dose-related inhibition of pepsin output. The maximal inhibition was about 60% and was obtained with 19 nmol/kg, h. Neurotensin also induced a progressive dose-related inhibition of volume and acid output. The maximal inhibition of gastric juice volume was obtained with 19 nmol/kg, h and was about 70% of the basal level (Fig. 2). The maximal inhibition of acid output was about 60% (Fig. 3) and was obtained with 6 nmol/ kg. h. Histamine No significant stimulation of pepsin output was obtained after histamine infusion, but the largest dose of histamine used (17.1 /~mol/kg. h) significantly decreased pepsin output (Fig. 1).
Histamine induced a dose-related increase of gastric juice volume (Fig. 2) and acid output (Fig. 3). Maximal stimulation occurred at the dose of 5.4 t~mol/kg, h and peaked at 1.7 and 2.7 times the basal level, respectively, for volume and acid output. Methacholine
Methacholine significantly stimulated pepsin secretion and was the most efficient of the stimulant agents used: the maximal pepsin output was 4.5 times the basal level and was greater than any maximal response obtained with the other substances (Fig. 1). Since methacholine significantly stimulates bicarbonate-rich salivary secretion, which was collected together with the gastric juice in our method, the values obtained for volume and acid need to be corrected from the salivary contamination. This has been done in Table 4, which includes the measured gastric juice values, the amount of saliva collected in similar conditions, and the corrected gastric juice values. After this correction, the maximal volume increase was 2.35 times the basal level (Fig. 2), and the maximal acid output was 2.3 times the basal level (Fig. 3). DISCUSSION In these experiments, methacholine was the most efficient stimulant of pepsin secretion, while it was moderately efficient on acid output. This result is in agreement with the literature data, since cholinergic agents are the most generally recognized potent stimulants of pepsin secretion, in conscious (37) and anesthetized rats (34) and on dispersed isolated rat gastric glands (29). Muscarinic receptors have been characterized on isolated guinea pig chief cells (38). Since salivary secretion was potently stimulated by methacholine, we had to correct gastric juice values from salivary output, so that acid output and volume may be somehow approximate. Saliva, however, does not hinder the accurate determination of pepsin output, as long as one takes care to keep the pH of gastric juice samples below 5 to avoid pepsin inactivation.
236
TAZI-SAAD, CHARIOT AND ROZI~
Pepsin output (UP/40 min
30
S
20
.
~
~
CCK
10
~
- ~ ~ , , ,
O,
,01
,1
m-
10 100 Dose(nmol/kg.h)
1
1 ETHAOH
Pepsin output (UP/40 min)
60-
40
.
20 ; O"
probably interacts on chief cells with CCK receptors of low affinity for gastrin, large concentrations ofgastrin are required to produce an effect. This effect can be demonstrated in appropriate conditions, but is probably of little physiological importance. In our experiments, 1.86 nmol/kg, h CCK produced a half-maximal stimulation of pepsin output (Table 1). Cherner et al. (5), using dispersed guinea pig chief cells, showed that it takes approximately 1000 times more gastrin than CCK to get half the maximal pepsin response to CCK. Assuming that rat chief cells would behave in the same manner, it would take 1.86 ~mol/ kg. h gastrin to stimulate half-maximally pepsin output, whereas the largest dose ofpentagastrin we used was only 40 nmol/kg, h. Acid and pepsin outputs were dose-dependently stimulated by CCK. The CCK8 analog [Boc-Nle2S-Nle3]]CCK8 was used in our experiments. More stable than CCK8, this analog displays in several in vitro and in vivo systems the same affinity and biological activity as CCK8 (27,28,31) and can thus be reliably used as a CCK receptor agonist. CCK stimulation of pepsin secretion has been reported in the anesthetized rat in vivo (34) and in several in vitro preparations [rabbit gastric glands (10,13), guinea pig dispersed chief cells (30), canine cultured chief cells (32)]. CCK receptors have been described on isolated guinea pig chief cells (5) and membranes (24). They seem more closely related to CCKA than to gastrin receptors, but their relative affinities for CCK analogs suggest that they might be different from both pancreatic and brain CCK receptors (24). The doseresponse curve for CCK-stimulated pepsin secretion was biphasic, indicating a supramaximal inhibition. This suggests that chief cell CCK receptors may take at least two different confor-
•
0
~7~'--~.~-4-~'4l PG l 'r ....
.~
,1
. . . . .
"1
1
. . . . . .
q
10
. . . . . .
I
......
"~! HIST "~
......
-q
. . . . .
=1
100 1000 10000100000 Dose(nmol/kg.h)
FIG. 1. Effect of several substances on the gastric pepsin output in conscious rats. Top panel: secretin (S), vasoactive intestinal peptide (VIP), [Boc-Nle2S-Nle3~]CCK8 (CCK), neurotensin (NT). Lower panel: pentagastrin (PG), methacholine (METHACH), histamine (HIST). Each substance was intravenously infused at increasing doses (each dose for 40 min). Mean _+ SEM; n = 6 to 12 rats per substance; the basal level (B) is the average of the two first 40-rain samples, collected during NaCl infusion; *p < 0.05, **p < 0.01, ***p < 0.001 compared to the basal level (Dunnett's test). Histamine did not increase pepsin output in the dose range which stimulated acid secretion, and even decreased pepsin output when administered in supramaximal doses for acid secretion. These data differ from some reports indicating stimulatory effects in anesthetized (34) or conscious rats (18,20), whereas in vitro, no effect [rabbit gastric glands (16)] or a moderate stimulation [rabbit gastric glands (13), canine cultured chief cells (32)] has been reported. Pentagastrin had no significant effect on pepsin output, even in doses up to 50 nmol/kg, h, which maximally stimulated acid output. These data are at variance with papers indicating some stimulation of pepsin secretion by gastrin or pentagastrin in rats. Most stimulant effects were obtained on denervated [Heidenhain pouch (12)] or isolated preparations [isolated perfused stomach (15,33)] or in anesthetized rats (34), whereas only Kowalewski (18) described some pepsin increase in conscious rats under longlasting (24 h) and large volume (120 ml/kg. 24 h) pentagastrin infusions, with gastric juice collected over 24 h. Discrepancies also exist in vitro, since some groups reported a stimulation of pepsin secretion by gastrin [isolated dog stomach (21), isolated rabbit glands (10,13), isolated perfused rat stomach (33), guinea pig dispersed chief cells (5)] whereas others found no effect [cultured canine chief cells (32), isolated perfused rat stomach (i 5), isolated perfused mouse stomach (7)]. Since gastrin
Volume (Id/40 min) 3000. B
2000.
***
1000-
O0
,01
,1
10
1
100
Dose (nmol/kg.h)
Volume (~I/40 rain)
3000 2000 i
~
,"~
PG ~ * *
METHACH HIST
1000, 00
,1
1
10
100 1000 10000100000 Dose (nrnol/kg.h)
FIG. 2. Effect of several substances on the volume of gastric juice in conscious rats. Groups and symbols as in Fig. 1.
CONTROL OF PEPSIN SECRETION
237
Acid output (Ixmol/40 rnin)
TABLE 2 EFFECTOF SECRET1NAND VIP ON PEPSINCONCENTRATION IN GASTRICJUICE
400
Secretin
300 200
VIP
Dose (nmol/kg.h)
Pepsin (PU/ml)
Dose (nmol/kg•h)
Pepsin (PU/ml)
0 0.006 0.06 0.6 3
10.1 _+0•85 10.0 _+ 1•20 10.6 _+0.52 9.2 _+ 1.20 0 +- 0t
0 0.125 0.396 1.25 3.96 12.5
10.8 _+0.69 12.4 +__2.77 9.1 _+ 1.14 7.7 _+0.49* 6.5 +_0.541" 4.0_ 1.08-~
100 0
0
,01
,1
1
10
100
Dose (nmol/kg.h)
PU = peptic units. Mean _+SEM; n = 6 to 12 rats per group. *~fCompared to the basal level, *p < 0.01, fp < 0.001. Acid output (l~mOl/40 min) 400m
300-
HIST
20O -
METHACH 100 0-
0
,1
1
10
100
1000
10000 100000
Dose (nmol/kg.h)
FIG. 3. Effect of several substanceson the gastric acid output in conscious rats. Groups and symbols as in Fig. I.
mations with different affinities, that might be related to stimulation and inhibition of secretion, as reported in the case of pancreatic acini (11,27). Secretin and VIP, a hormone and a neuropeptide that usually interact with closely related receptors, displayed clearly different effects in our conscious fistula rats: VIP produced a biphasic dose-response curve with a stimulation followed by an inhibition, whereas secretin was a pure, potent, and efficient inhibitor of acid and pepsin secretions. Since VIP is known to potentiate acetyicholine-induced salivary secretion (22), we sought a possible stimulation of salivary secretion in our conditions, which might explain the VIP-induced decrease of pepsin concentration. The very weak stimulatory effect of VIP on salivary secretion makes this hypothesis unlikely• The effects of VIP on acid and pepsin secretion in vivo are controversial and variable according to the species. In the rat, no effect of peripheral injections of VIP on acid secretion was reported in pylorus-ligated rats (8), with a large dose of 100 ug/ kg = 30 nmol/kg, but this was likely supramaximal. In vitro, VIP increased pepsinogen release by isolated gastric glands or dispersed chief cells from rats (29) or guinea pigs (39), and potentiated the stimulant effect of CCK in isolated rabbit (3) or rat gastric glands (29). Four classes of binding sites have been described for VIP and secretin on guinea pig isolated chief cells, and occupation of two of these classes correlated with the abilities of VIP and secretin to increase cellular cAMP and pepsinogen secretion (39). In the isolated luminally perfused mouse stomach, Schubert (35) recently evidenced that the effect of VIP on acid secretion depended on the balance between direct stimulation of acid se-
cretion and concurrent release of somatostatin. Somatostatin secretion was more sensitive to VIP than acid secretion. If such a dual mechanism also exists in the rat, it might account for the biphasic dose-response curve found here. In species other than the rat, many data indicate that secretin increases pepsin secretion although inhibits acid secretion (9), whereas no characterization of secretin receptors on isolated rat chief cells has yet been published. In anesthetized rats with gastric lumen perfusion, Schmidt-Wilcke and Nawrath (34) described a stimulation of pepsin output, but this was with extractive secretin, which might be impure• In conscious rats, Kowalewski (19) found no effect of secretin on pepsin output or a tendency to decrease under long-lasting (24 h) secretin infusions. The total inhibition of pepsin output that we observed under secretin infusion was not only the consequence of the potent inhibition of gastric juice volume, since 3 nmol/kg, h secretin suppressed totally pepsin output in our experiments, whereas gastric juice volume decreased only by 74%. Also, pepsin concentration in gastric juice never increased after any secretin dose (Table 2), although fluid secretion was markedly inhibited, but sharply decreased after 3 nmol/kg- h secretin. A direct or indirect inhibitory effect of secretin on chief cells should thus have occurred, a situation that seems different from what exists in guinea pig chief cells• Neurotensin inhibited basal acid and pepsin secretions maximally by about 60% at the largest dose of 19 nmol/kg, h. The effect of neurotensin on pepsin secretion in the rat has not been reported previously. The range of active doses suggests that neurotensin inhibition of gastric secretion may be pharmacological rather than physiological, since these doses are larger than those affecting duodenal bicarbonate (25) and pancreatic (26) secre-
TABLE 3 EFFECTSOF VIP ON SALIVARYVOLUMEAND BICARBONATEOUTPUT VIP (nmol/kg.h)
Volume (~1/40 rain)
BicarbonateOutput (~mol/40 rain)
0 1.25 4.0 12.5 40.0
256 +_93 240 ___34 276 + 82 475 + 58* 258 + 17
7.0 + 3.7 6.9 ___1.2 7.1 + 2.2 13.7 + 2.5* 6.2 + 0.8
* p < 0.05 compared to the basal level; n = 5 rats.
238
TAZI-SAAD, C H A R I O T A N D ROZI~
TABLE 4 METHACHOL1NESTIMULATIONOF GASTRICAND SALIVARYVOLUME,OF GASTRICACID OUTPUT, AND OF SALIVARYBICARBONATEOUTPUT Volume (~1/40 min)
Acid or Bicarbonate (umol/40 min)
Methacholine (gmol/kg. h)
Gastric (Total)
Salivary
Gastric (Corrected)*
Gastric Acid
Salivary Bicarbonate
Gastric Acid (Corrected)*
0 0.31 1.0 3.1 10
1100-+217 1231 -+211 1906 -+ 286 4419 -+ 467 6315 -+ 461
70_+ 26 60-+ 30 360 -+ 80 2260 -+ 300 3560 -+ 230
1030-+227 1171 -+213 1546 -+ 297 2159 -+ 555 2755 -+ 515
75.2_+25.1 88 -+29.7 91.9 -+ 6.8 91.7 -+ 17.4 9.0 -+ 3.9
2.1 _+ 1.2 1.6-+0.9 12.2 -+ 3.0 76.2 -+ 8.6 142.6 + 9.4
73.1+28.0 89.6_+29.7 104.1 _+ 10.7 167.9 _+ 19.4 151.6 + 10.2
* "Corrected" gastric volume and acid output = measures on total gastric juice contaminated by saliva, minus salivary secretion determined in separate experiments. n - 8 to 12 rats per group.
tions in the rat. However, some data obtained with a neurotensin antiserum suggest that neurotensin might mediate the gastric acid inhibitory effect of intestinal infusions of oleic acid in anesthetized rats (36). The mechanism of neurotensin inhibition of gastric secretion is unclear and probably indirect. Neurotensin receptors have not been characterized on isolated parietal cells (14), whereas no data have been published for chief cells. Neurotensin increases the postprandial release of pancreatic polypeptide (17) and may increase prostaglandin generation (23), whereas the necessity of intact vagal nerves for neurotensin inhibition of gastric secretion has been successively claimed (1) and challenged (23). In conclusion, this reinvestigation of some modulators of pepsin secretion in the rat stomach in vivo was aimed to gain understanding of the physiological role of each substance, based
on the assessment that a peptide should be both potent and efficient to be of real physiological importance. A m o n g the substances tested, muscarinic agonists and C C K are the main stimulants of pepsin secretion. The stimulants of parietal cells, histamine and pentagastrin, have few effects, if any, on pepsin output, whereas secretin is a potent inhibitor of gastric secretion, including pepsin. Most of these data are closer to the recent results obtained with in vitro isolated cell preparations than to older results obtained in vivo, sometimes with peptide samples of doubtful purity. ACKNOWLEDGEMENTS Partial financial support was provided by the Conseil Scientifique ot Facult6 Xavier Bichat and by Association Charles Debray.
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