Wine stimulates gastric acid secretion in isolated rabbit gastric glands via two different pathways

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1 Aliment Pharmacol Ther 2; 16 (Suppl. 2): Wine stimulates gastric acid secretion in isolated rabbit gastric glands via two different pathways K. MATSUNO, K. TOMITA & S. OKABE Department of Applied Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan SUMMARY Background & aims: Alcoholic beverages such as beer and wine are well known to potently stimulate gastric acid secretion, most probably through an increase in circulating gastrin level. The present study examined whether or not wine stimulates gastric acid secretion by a direct effect on parietal cells, enterochromaffin-like (ECL) cells or both. Methods: Gastric mucosa was isolated from female Japanese white rabbits and gland specimens were prepared by the collagenase digestion method. Acid secretion was assessed by gland accumulation of [ 14 C] aminopyrine. The effects of red wine, ethanol, non-alcoholic wine and drugs were determined by incubating gastric glands with aminopyrine. Radioactivity in solubilized glands was determined by a liquid scintillation counting. Results: Neither wine nor ethanol (diluted 1 : 1 2 to 1:1 4 ) had any effect on gastric acid secretion, whereas non-alcoholic wine stimulated acid secretion in a dose-dependent manner. All substances, however, significantly stimulated gastric acid secretion in IBMX (phosphodiesterase inhibitor)-pretreated glands. S-9 (a CCK-2 receptor antagonist) and atropine had no effect on acid secretion stimulated by wine, ethanol or non-alcoholic wine in IBMX-pretreated glands. Famotidine and omeprazole significantly inhibited the acid secretion resulting from all of the above stimulants. BAPTA (an intracellular Ca 2+ chelator) inhibited acid secretion stimulated with wine or ethanol in a dosedependent manner, but did not inhibit secretion stimulated by non-alcoholic wine. Conclusions: Wine was found to stimulate gastric acid secretion in gastric glands via two pathways, by an ethanol-induced increase in the concentration of intracellular Ca 2+ in parietal cells, and by histamine release from ECL cells potentially induced by constituents present in wine. INTRODUCTION It is generally known that alcoholic beverages such as beer and wine, but not whisky and brandy, potently stimulate gastric acid secretion in humans. 1 6 The underlying mechanisms and active components have gradually been elucidated over the past two decades. 7 1 It has been found that circulating gastrin levels are regularly increased after beer or wine administration and that a selective CCK-2 receptor antagonist inhibits beer-stimulated acid Correspondence to: Dr S. Okabe, Department of Applied Pharmacology, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto , Japan. okabe@mb.kyoto-phu.ac.jp 11, 12 secretion in a dose-dependent manner in dogs. Accordingly, an increase in gastrin level has been postulated to be the main driving force behind increased acid secretion. Against this, however, several compounds, including maleic acid and succinic acid, in fermented alcoholic beverages, have been found to stimulate gastric acid secretion without increasing gastrin levels. 13 In addition, N-methyltyramine, an ingredient in beer, has been isolated and reported to stimulate gastric acid secretion via an increase in serum gastrin levels. 14 Accordingly, it has been postulated that in addition to a gastrin-induced pathway, a second pathway involved in alcoholic beverage-induced acid secretion exists. The present study was conducted to determine whether wine, its constituents, or both directly stimulate Ó 2 Blackwell Science Ltd 17

2 18 K. MATSUNO et al. parietal cells using isolated gastric glands. In addition, pharmacological analysis of the underlying mechanism of gastric stimulation by wine was attempted. METHODS Animals Female Japanese white rabbits weighing kg were used. Food was withheld for 48 h before gastric gland isolation, but water was freely provided. Both the maintenance of the animals and the experimental procedures were carried out in accordance with the guidelines of the Ethics Committee of Kyoto Pharmaceutical University. Materials S-9 was provided by Shionogi & Co. (Osaka, Japan). Omeprazole was provided by Nippon Chemiphar Co., Ltd. (Tokyo, Japan). Famotidine was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin (BSA), 3-isobutyl-1-methylxanthine (IBMX), and N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) were purchased from Nacalai Tesque (Kyoto, Japan). Atropine sulphate (atropine), collagenase and BAPTA/AM (an intracellular Ca 2+ chelator) were purchased from Wako Pure Chemicals (Osaka, Japan). Commercially available red wine (Mercian Co., Kanagawa, Japan) was used; the alcohol concentration was approximately 14%. Non-alcoholic wine, produced from Mercian red wine by evaporating the ethanol, was provided by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan). [ 14 C] aminopyrine was purchased from Muromachi Chem (Tokyo, Japan). Isolation of gastric glands Rabbit gastric glands were isolated according to the method of Berglindh et al. 15 with some additional modifications. Under pentobarbital ( mg/kg, i.v.) anaesthesia, the abdomen of each rabbit was opened and the aorta was catheterized. Each stomach was perfused in vivo under pressure with ml of phosphate-buffered saline (PBS). The corpus mucosa was stripped off with scissors and collected in cold buffer containing (in mmol/l) NaCl 132.4, KCl 5.4, Na 2 HPO 4 5., NaH 2 PO 4 1., MgSO 4 1.2, CaCl2 1., and HEPES., as well as 2. g/l glucose and 2. g/l BSA adjusted to ph 7.4. The tissue was rapidly minced into small pieces. The mucosal pieces were suspended and decanted in cold buffer three times until the supernatant remained clear. Collagenase was then added to a final concentration of.5 mg/ml and the mucosal suspension was incubated for 3 35 min under a 95% O 2 /5% CO 2 atmosphere. After incubation, the glands were filtered through nylon mesh (3 lm in diameter) and rinsed by sequential suspension and sedimentation in cold buffer. Aminopyrine accumulation Gastric acid secretion was monitored by [ 14 C] aminopyrine (AP) uptake in isolated glands (2 dilution of settled glands) in buffer. Aliquots of.5 ml were placed in a polypropylene Eppendorf tube (1.5 ml) and preincubated with lmol/l of 3-isobutyl-1-methylxanthine (IBMX), various inhibitors, or both for 15 min at room temperature. Thereafter, 1 ll of [ 14 C] AP (2 lci/ml) and 5 ll of wine, ethanol or nonalcoholic wine were added to each tube. The samples were subsequently incubated at 37 C in a shaking water bath for 45 min. After incubation, each suspension was centrifuged at 1 g for 15 s. The supernatant was then removed and each pellet was rinsed once with.5 ml of chilled PBS. The pellets were subsequently solubilized with.5 N of NaOH overnight. Gland radioactivity was determined with a liquid scintillation counter (Beckman Instruments Inc., Fullerton, CA, USA). Data analysis All data are presented as means ± S.E.M. Statistical analysis was performed using the two-tailed Dunnett s multiple comparison test and Student s t-test, with a P-value of <.5 regarded as significant. RESULTS Effects of wine and various drugs on gastric acid secretion Wine did not affect AP accumulation in the isolated glands at any concentration used in the study. Nonetheless, wine significantly stimulated AP accumulation in IBMX-pretreated glands. The maximal effect was obtained at a 1 : 1 2 dilution, at which accumulation rate was 22% greater than the control (Figure 1). Both

3 WINE-STIMULATED GASTRIC ACID SECRETION 19 S-9 and atropine at a concentration of 1 )4 mol/l 1 )5 mol/l had little or no inhibitory effect against the (1 : 1 2 dilution) wine-stimulated AP accumulation in IBMX-pretreated glands. In contrast, famotidine at a concentration of 1 )4 mol/l 1 )7 mol/l significantly inhibited AP accumulation. Inhibitory rate at 1 )4 mol/l was approximately 8% of the basal level (Figure 2). Effects of ethanol and various drugs on gastric acid secretion To investigate whether the acid stimulatory effect of wine was due to ethanol, ethanol was examined in isolation. Ethanol failed to stimulate AP accumulation in normal gastric glands, even at a 1 : 1 2 dilution, i.e. approximately.14% ethanol. Nonetheless, ethanol significantly stimulated AP accumulation in the IBMXpretreated glands, at a rate of accumulation 17% greater than the basal level even at a 1 : 1 2 dilution (Figure 3A). This increased accumulation was not affected by atropine even at a concentration of 1 )4 mol/l, yet was significantly inhibited by S-9 at a concentration of 1 )4 mol/l (Figure 3B). In contrast, famotidine significantly inhibited AP accumulation at a concentration of 1 )7 mol/l, but the degree of inhibition was not increased even with a dosage increase up to 1 )4 mol/l. Effects of non-alcoholic wine and various drugs on gastric acid secretion Non-alcoholic wine (1 : 1 2 dilution) significantly stimulated AP accumulation in both the normal (43% greater than basal level) and IBMX-pretreated glands (28% greater than basal level) (Figure 4A). Both S-9 and atropine had little or no effect on non-alcoholic wine-stimulated AP accumulation in the IBMX-pretreated glands. In contrast, famotidine significantly inhibited (1 : 1 2 dilution) non-alcoholic wine-stimulated AP accumulation in the IBMX-pretreated glands in a concentration-dependent manner; the rate of inhibition at a concentration of 1 )4 mol/l was nearly total (Figure 4B). 2 Wine Wine (+ IBMX) S-9 Famotidine Atropine Base Dilution rate (log) Figure 1. Concentration-response curves for the effect of winestimulated AP accumulation in normal and IBMX-pretreated rabbit gastric glands. The glands were incubated with wine for 45 min after preincubation with vehicle or IBMX. Note that wine significantly stimulated AP accumulation even at a 1 : 1 4 dilution in IBMX-pretreated glands. Data are expressed as percentage of basal accumulation and are presented as means ± S.E.M. for 4 6 samples. P <.5, significantly different from the basal level. Base Drug concentration (-log mol/l) Figure 2. Effects of S-9, famotidine and atropine on winestimulated (1 : 1 2 dilution) AP accumulation in IBMXpretreated gastric glands. Only famotidine markedly inhibited AP accumulation. Data are expressed as a percentage of accumulation and are presented as means ± S.E.M. for 4 8 samples. P <.5, significantly different from the basal level.

4 11 K. MATSUNO et al. (A) (B) 4 Ethanol + IBMX S-9 Famotidine Atropine 3 Base Base Dilution rate (log) Drug concentration (-log mol/l) Figure 3. Concentration-response curves for ethanol-stimulated AP accumulation in normal and IBMX-pretreated gastric glands (A); and the effects of drugs on ethanol (1 : 1 2 dilution)-stimulated AP accumulation in IBMX-pretreated glands (B). Only famotidine significantly inhibited AP accumulation. Data are expressed as a percentage of basal accumulation and are presented as means ± S.E.M. for 4 8 samples. P <.5, significantly different from the basal level. Effects of omeprazole on gastric acid secretion stimulated by wine, ethanol, and non-alcoholic wine Omeprazole at concentrations of 1 )6 mol/l and greater significantly inhibited AP accumulation increased by wine and non-alcoholic wine (1 : 1 2 dilution for both) in IBMX-pretreated glands, with a rate of inhibition of over 85% at a concentration of 1 )5 mol/l (Figure 5). In the case of ethanol stimulation, omeprazole significantly inhibited AP accumulation even at a concentration of 1 )7 mol/l. Effect of BAPTA on acid secretion stimulated by wine, ethanol and non-alcoholic wine The AP accumulation increased by wine or ethanol in the IBMX-pretreated glands was strongly inhibited by BAP- TA, with inhibition significant at concentrations greater than or equal to1 )5 mol/l (Figure 6). It should also be noted that in the case of ethanol, BAPTA inhibition at a concentration of 1 )4 mol/l was below the basal value. In contrast, BAPTA had little or no effect on nonalcoholic wine-stimulated AP accumulation. DISCUSSION This study clearly indicates that wine acts directly on isolated gastric glands, leading to stimulation of gastric acid secretion. This finding provides strong evidence that the acid stimulatory effects of wine result from at least two pathways, i.e. a gastrin-related and a gastrin-unrelated (direct) pathway. Current thought holds that parietal cells secrete 16, 17 gastric acid through two intracellular pathways. One pathway is mediated by an increase in intracellular camp concentration caused by activation of a Gs protein and adenylate cyclase via stimulation of histamine H 2 receptors. The other pathway is mediated by an increase in the intracellular Ca 2+ concentration subsequent to enhanced production of inositol-1,4,5-triphosphate and diacylglycerol via stimulation of CCK-2 or M 3 receptors. Many investigators consider that the above two pathways interact with each other. The theory that a maximal secretory response is uniquely obtained when increases in camp and the Ca 2+ concentration occur at the same time is widely accepted.

5 WINE-STIMULATED GASTRIC ACID SECRETION 111 (A) (B) Non-alcoholic wine + IBMX 4 3 Base S-9 Famotidine Atropine Base Dilution rate (log) Drug concentration (-log mol/l) Figure 4. Concentration-response curves for non-alcoholic wine-stimulated AP accumulation in normal and IBMX-pretreated gastric glands (A) and the effects of drugs on AP accumulation in IBMX-pretreated glands (B). Famotidine inhibited accumulation in IBMXpretreated glands to near completion. Data are expressed as a percentage of basal accumulation and are presented as means ± S.E.M. for 4 8 samples. P <.5, significantly different from control. As a preliminary investigation, we confirmed both that histamine stimulated AP accumulation in isolated gastric glands from rabbits, and that carbachol and pentagastrin had little or no effect on such accumulation. Nonetheless, it was found that both carbachol and pentagastrin stimulated AP accumulation when the gastric glands were preincubated with IBMX. These findings suggest that, at least in this gland model, an increased intracellular camp level is a prerequisite for acid stimulation by carbachol and pentagastrin. Similar to the findings with carbachol and pentagastrin, wine alone did not stimulate gastric acid secretion, but did significantly stimulate secretion in IBMX-pretreated glands. Until now, it has been thought that alcoholic beverage-induced stimulation of gastric acid secretion is principally mediated by circulating gastrin. Indeed, we have already reported that S-9 significantly inhibits gastric acid secretion induced by beer in Heidenhain pouch dogs. 11, 12 Nonetheless, in the present study, S-9 as well as atropine had little or no effect on wine-stimulated acid secretion in IBMX-pretreated glands. It is of interest that only famotidine significantly inhibited the wine-stimulated gastric acid secretion. These findings indicate that the stimulatory effect of wine on isolated gland preparations is not mediated by CCK-2 receptors, but rather by histamine H 2 -receptors. Singer et al. 3 reported that ethanol stimulates gastric acid secretion in humans without concomitant elevation of serum gastrin levels. In the present study, we demonstrated that although ethanol had no stimulatory effect on normal glands, it clearly stimulated acid secretion in IBMX-pretreated glands. Accordingly, as was the case for wine, an increase in intracellular camp level above a certain level appeared to be necessary for ethanol to stimulate acid secretion. Both S-9 and atropine had little or no effect on ethanol-stimulated acid secretion, suggesting that CCK-2 and M 3 receptors were not involved in the underlying mechanism. It is of interest that ethanol-stimulated acid stimulation was also significantly inhibited by famotidine, although the degree of inhibition was slightly less than that observed for wine. In any event, it is conceivable that the acid stimulatory effect of ethanol is partly mediated through H 2 receptors in parietal cells. The possibility remains that

6 112 K. MATSUNO et al. Wine Ethanol Non-alcoholic wine Base Cont Base Cont Base Cont (mol/l) Omeprazole Omeprazole Omeprazole Wine Ethanol Non-alcoholic wine IBMX mol/l IBMX mol/l IBMX mol/l Figure 5. Effect of omeprazole on AP accumulation in IBMX-pretreated gastric glands stimulated by wine, ethanol and non-alcoholic wine (1 : 1 2 dilution). Omeprazole potently inhibited AP accumulation. Data are expressed as a percentage of a accumulation and are presented as means ± S.E.M. for four samples. P <.5, significantly different from basal and # control (cont) level. ethanol acted on ECL cells, which would be followed by a release of histamine. We are currently investigating histamine levels in ethanol-exposed gastric mucosa. In other experiments, BAPTA was found to inhibit AP accumulation in a concentration-dependent manner in IMBX-pretreated glands treated with wine and ethanol. It is likely that ethanol stimulated AP accumulation not only through increased camp levels, but also by an elevation in the intracellular Ca 2+ concentration in parietal cells. Our results are supported by those of Mutsukawa et al. 18 who indicated that ethanol elevates the intracellular Ca 2+ concentration in rabbit parietal cells. It should be noted that BAPTA at a concentration of 1 )4 mol/l, or even 1 )5 mol/l, resulted in a decrease in AP accumulation below the basal level in both wine- and ethanol-treated glands. Such data strongly suggest that intracellular Ca 2+ plays an important role in basal acid secretion. Recently, it was reported that non-alcoholic components in alcoholic beverages also have stimulatory effects on gastric acid secretion. Accordingly, the present study also investigated the effects of non-alcoholic wine on gastric acid secretion using the isolated gland model. Unexpectedly, non-alcoholic wine strongly stimulated gastric acid secretion in gastric glands, regardless of whether or not pretreatment with IBMX was done. Although S-9 and atropine had no effect on non-alcoholic wine-stimulated acid secretion in IBMXpretreated glands, both famotidine and omeprazole significantly inhibited the acid secretion. It was also found that BAPTA, a potent inhibitor of wine- and ethanol-stimulated acid secretion, did not inhibit acid secretion stimulated in response to non-alcoholic wine. It remains possible that the mechanism by which nonalcoholic wine stimulates acid secretion does not involve the intervention of intracellular Ca 2+. Rather, nonalcoholic wine might stimulate histamine release from ECL cells, which in turn could lead to stimulation of histamine H 2 receptors on parietal cells. In fact, Intorre et al. 19 reported that red wine ( ml/dog) significantly increased plasma histamine concentration after intragastric ingestion, probably through participation of ECL cells. Although non-alcoholic wine and ethanol probably stimulate AP accumulation via different pathways, wine did not exhibit any synergistic or potentiated

7 WINE-STIMULATED GASTRIC ACID SECRETION 113 Wine Ethanol Non-alcoholic wine Base Cont Base Cont Base Cont (mol/l) BAPTA BAPTA BAPTA Wine Ethanol Non-alcoholic wine IBMX mol/l IBMX mol/l IBMX mol/l Figure 6. Effect of BAPTA on AP accumulation in IBMX-pretreated gastric glands stimulated by wine, ethanol, and non-alcoholic wine. BAPTA, even at a concentration of 1 )4 mol/l, had little or no effect on AP accumulation stimulated by non-alcoholic wine. Data are expressed as a percentage of accumulation and are presented as means ± S.E.M. for 3 4 samples. P <.5, significantly different from basal and # control (cont) level. properties. Indeed, non-alcoholic wine was a more potent stimulant than wine or ethanol. Niki et al. 2 have reported that ethanol inhibits histamine-stimulated AP accumulation in isolated parietal cells from guinea pigs. Accordingly, the stronger stimulatory effects of non-alcoholic wine were probably due to lack of inibition by the ethanol usually contained in wine. Certainly, the possibility remains that active ingredients present in wine are inactivated during the manufacturing process. In consideration of the above, we conclude that wine appears to stimulate gastric acid secretion in isolated gastric glands by two different pathways. One pathway involves an elevation of intracellular Ca 2+ concentration in parietal cells induced by the ethanol present in wine. The second follows from an increase in histamine release from ECL cells, induced by unidentified substances contained in wine. Concerning the unidentified substances, we found that nonalcoholic wine treated at C for 15 min could stimulate AP uptake, suggesting that these substances are heat-resistant. ACKNOWLEDGEMENTS We wish to thank C. J. Hurt (John Hopkins University School of Medicine, USA) for a critical reading of the manuscript and K. Oota and N. Matsuo for their technical assistance. REFERENCES 1 McArthur K, Hogan D, Isenberg JI. Relative stimulatory effect of commonly ingested beverages on gastric acid secretion in humans. Gastroenterology 1982; 83: Lenz HJ, Ferrari-Taylor J, Isenberg JI. Wine and five percent ethanol are potent stimulants of gastric acid secretion in humans. Gastroenterology 1983; 85: Singer MV, Leffmann C, Eysselein VE, Calden H, Goebell H. Action of ethanol and some alcoholic beverages on gastric acid secretion and release of gastrin in humans. Gastroenterology 1987; 93: Manfred V, Singer ST, Teyssen S, Eysselein VE. Action of beer and its ingredients on gastric acid secretion and release of gastrin in humans. Gastroenterology 1991; 11: Chari ST, Teyssen S, Singer MV. Alcohol and gastric acid secretion in humans. Gut 1993; 34:

8 114 K. MATSUNO et al. 6 Teyssen S, Lenzing T, Gonzalez-Calero G, Korn A, Riepl RL, Singer MV. Alcoholic beverages produced by alcoholic fermentation but not by distillation are powerful stimulants of gastric acid secretion in humans. Gut, 1997; 4: Peterson WL, Barnett C, Walsh JH. Effect of intragastric infusions of ethanol and wine on serum gastrin concentration and gastric acid secretion. Gastroenterology 1986; 91: Reichstein BJ, Okamoto C, Forte JG. Effect of ethanol on acid secretion by isolated gastric glands from rabbit. Gastroenterology 1986; 91: Izquierdo-Pulido ML, Vidal-Carou MC, Marine-Font A. Determination of biogenic amines in beers and their raw materials by ion-pair liquid chromatography with postcolumn derivatization. J AOAC Int 1993; 76: Teyssen S, Gonzalez-Calero G, Korn A, Singer MV. Action of ethanol and some alcoholic beverages on gastric acid secretion in anaesthetized rats. Alcohol Alcoholism 1997; 32: Sasaki N, Matsuno KS, Okabe S. Selective action of a CCK-B/ gastrin receptor antagonist, S-9, on pentagastrin-, peptone meal- and beer-stimulated gastric acid secretion in dogs. Aliment Pharmacol Ther ; 14: Matsuno K, Sasaki N, Okabe S. Gastric acid secretion in dogs in response to combinations of beer, ethanol and peptone meal the role of endogenous gastrin. Aliment Pharmacol Ther ; 14 (Suppl. 1): Teyssen S, Schimiczek M, Singer MV. Maleic acid and succinic acid in fermented alcoholic beverages are the stimulants of gastric acid secretion. J Clin Invest 1999; 13: Yokoo Y, Kohda H, Kusumoto A, et al. Isolation from beer and structural determination of a potent stimulant of gastrin release. Alcohol Alcoholism 1999; 34: Berglindh T, Obrink KJ. A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physiol Scand 1976; 96: Hirschowitz BI, Keeling D, Lewin M, et al. Phamacological aspects of acid secretion. Dig Dis Sci 1995; 4 (Suppl. 2): 3S 23S. 17 Sachs G, Modlin I. The regulation of gastric acid secretion. In: Acid-Related Diseases. Biology and Treatment. Schnetztor- Verlag GmbH D, Konstanz, 1998: Mutsukawa H, Zhao WL, Saitoh T, Natomi H, Sugano K. Ethanol elevates intracellular calcium in rabbit parietal cells. Gastroenterol Jpn 1991; 26(544): Intorre L, Bertine S, Luchetti E, Mengozzi G, Crema F, Soldani G. The effect of ethanol, beer and wine on histamine release from the dog stomach. Alcohol 1996; 13: Niki S, Kawai K. Effects of ethanol on acid secretion by isolated parietal cells from guinea pig. Nippon Shokakibyo Gakkai Zasshi 1991; 88: 294 (Abstract in English).