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Effects of pituitary adenylate cyclase activating polypeptide-27 on alkaline secretory and mucosal ulcerogenic responses in rat duodenum
PI1
ELSEVIER
SOO243205(98)oo28o-
Life Scimca, Vd. 63, No. 5, pp. 317~325,~~ cqyright~1998ELcvicrsdeltccIllc. PrintedintbeUSA. AUtigbtsracmd 0024-32a5/!% 319.00 t .oo
EFFECTS OF PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE-27 ON ALKALINE SECRETORY AND MUCOSAL ULCEROGENIC RESPONSES IN RAT DUODENUM Koji Yagi, Koji Takehara, Motohiro Kitamura and Koji Takeuchi Department
of Pharmacology & Experimental Therapeutics Kyoto Pharmaceutical University Misasagi, Yamashina, Kyoto 607
Japan (Received in final form May l3,W98)
Summary Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on duodenal mucosal HCO,- secretion and ulcerogenic responses induced by mepirizole in anesthetized rats were examined and compared with those of vasoactive intestinal polypep tide (VIP). Animals were given mepirizole (200 mg/kg, s.c.) for induction of duodenal ulcers, and gastric acid and duodenal HCO; secretions were measured with or without pretreatment of PACAPor VIP Mepirizole increased acid secretion and induced hemorrhagic lesions in the proximal duodenum within 6 h. Intravenous bolus injection or infusion of PACAP(4 and 8 mnollkg or 8 rmrol/kg/h) increased duodenal HCO; secretion even in the presence of mepirizole. without effect on acid secretion, and significantly reduced the severity of duodenal lesions caused by mepirizole. In contrast, VIP (8 nmollkg, i.v.) given by bolus injection significantly decreased acid secretion induced by mepirizole, in addition to stimulation of HCO, secretion, and prevented duodenal lesions in response to mepirizole. These results suggest that PACAPincreases duodenal HCO; secretion and this action may be important in maintaining the duodenal mucosal integrity against acid, and VIP affords duodenal protection by both increasing duodenal HCO; secretion and decreasing acid secretion. The reason for the different effects of PACAP and VIP on acid secretion is unknown. Key Words: PACAP-27, mepirizole, duodenal lesion, bicarbonate secretion, acid secretion Pituitary adenylate cyclase activating polypeptide (PACAP) is a hypothalamic peptide showing 68% sequence homology with vasoactive intestinal peptide (VIP), which stimulates adenylate cyclase activity (1,2). Binding sites for PACAP are not limited to brain, but are widely distributed in the gastrointestinal tract including the stomach and the duodenum (3-S). Two biological active forms of the peptide have been identified, one major with 38 amino acids (PACAP-38) and the other with 27 residues (PACAP-27), sharing the same N-terminal 27 amino acids, and two types of PACAP receptors have been identified and cloned (6). Recently, we found that both forms of PACAPs (PACAPCorresponding Author: Dr. Koji Takeuchi, Department of Phaimacol. & Exp. Ther. Kyoto Pharmaceutical University, Kyoto 6078414, Japan Tel: 81-075-595-4679, Fax: 81-075-595-4774, E-mail: [email protected]
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27 and PACAP-38), similar to VIP, stimulate HCO,- secretion in the rat duodenum but not in the stomach (7), although the physiological significance of HCO,- stimulatory action of this peptide remains unknown. It is known that HCO; secretion by duodenal mucosa plays a pivotal role in maintaining the mucosal integrity against acid (8). Indeed, the pathogenic importance of the impaired HCO; secretion in duodenal ulcers has been demonstrated in various experimental animal models and humans (9- 11). Mepirizole, a basic nonsteroidal antiinflammatory drug, causes duodenal ulcers in rats, partly by stimulating gastric acid secretion and by reducing duodenal HCO, secretion, especially the acidinduced HCO,- response (12,13). In the present study, we examined the effects of PACAPon duodenal HCO; secretory and ulcerogenie responses induced by mepirizole in rats and investigated whether the HCO; stimulatory action of PACAPconfers protection on the duodenal mucosa against ulceration. In addition, we also examined the effects of VIP on these responses to mepirizole, in comparison with those of PACAP27. Materials and Methods Animals. Male Sprague-Dawley rats, weighing 250-300 g (Charles River, Shizuoka, Japan), were kept in individual cages with raised mesh bottoms to prevent coprophagy and deprived of food but allowed free access to tap water for 18 h before the experiments. Studies were carried out using 4-6 rats per group. General Protocols. Animals were anesthetized with urethane (1.25 g/kg) given i.p. and placed at room temperature (22rtl”C). Under these conditions, we measured gastric acid secretion, duodenal HCO; secretion, and duodenal ulcerogenic responses to mepirizole and examined the effects of PACAPand VIP on these responses. Determination of Gastric Acid Secretion. Acid secretion was measured in the acute fistula rat according to a previously published method (14). In brief, the abdomen was incised, and both the stomach and duodenum were exposed. An acute fistula (inside diameter: 3 mm) made with a polyethylene tube was inserted into the stomach from a small incision made in the duodenum and held in place by a ligature around the pylorus. The stomach was filled with 2 ml of saline (154 mM NaCl) through the fistula, and the solution was changed every 15 min. The collected samples were centrifuged at 3000 r.p.m. for 15 min and titrated to pH 7.0 against 100 mM NaOH using an autoburette (Hiranuma Comtite-8, Tokyo, Japan). Mepirizole was given S.C. in a dose of 200 mg/kg, and acid secretion was measured for 4 h. PACAP(8 nmollkg) or VlP(8 nmollkg) was given i.v. as a single injection 5 min before the administration of mepirizole. In some cases, PACAPwas given by a continuous i.v. infusion (8 nmol/kg/h) during mepirizole treatment. The doses of these peptides were selected to stimulate duodenal HCO; secretion in rats (7). Determinution of Duodenal HCO;Secretion. HCO; secretion was measured in the proximal loop of the rat duodenum as described (10). Briefly, the duodenal loop (1.7 cm) was made between the pyloric ring and the area just above the outlet of the common bile duct in order to exclude the influences of bile and pancreatic juice. Then, the loop was perfused at a flow rate of 1 ml/min with saline that was gassed with 100% 0,, heated at 37-C, and kept in a reservoir, and the HCO; secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCI to the reservoir. Approximately 1 h after basal secretion had stabilized, mepirizole was given S.C. in a dose of 200 mglkg. PACAP(4 and 8 nmollkg) or VIP (8 nmollkg) was given i.v. as a single injection 1 h after the administration of mepirizole. In another experiment, the effects of PACAP(2,4 and 8 nmol/kg) and VIP (8 nmollkg) on duodenal HCO; secretion were also examined in the absence of mepirizole.
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PACAP on DuodenalHCOj Secretion and Ulcers
In some cases, PACAP(8 nmollkglh) was continuously absence or presence of mepirizole.
319
infused i.v. into the tail vein for 5 h in the
Evaluation of Duodmal Mucosa. Animals were given mepitizole S.C.in a dose of 200 mg/kg, without any surgical manipulation, and sacrificed 6 h later (15). Then, both the stomach and duodenum were removed, inflated by injecting 10 ml of 1% formalin for 10 min to fix the tissue walls, and opened along the greater curvature (stomach) or along the mesenteric attachment (duodenum). The area (mm*) of hemorrhagic lesions developed in the stomach and duodenum was measured under a dissecting microscope with a square grid (x10). PACAP(2,4 and 8 nmollkg) and VIP (8 nmoll kg) were given i.v. as a bo s injection two times, 5 min before and 3 h after the administration of mepitizole. In some cases, l! ACAP(8 nmollkglh) was continuously infused iv. into the tail vein for 6 h, starting from 30 min before mepirizole treatment. preparation of Drugs. Drugs used were urethane (Tokyo Kasei, Tokyo, Japan), PACAP-27, VIP (Peptide Institute, Osaka, Japan) and mepirizole (Daiichi Pharm., Tokyo, Japan). Mepirizole was suspended in 0.5% carboxymethylcellulose solution (CMC), while other drugs were dissolved in saline. Each agent was prepared immediately before use and given in a volume of 0.5 ml per 100 g body weight in cases of i.p. and S.C.administration or in a volume of 0.1 ml per 100 g body weight in case of i.v. administration. Control animals received saline or CMC as the vehicle. St&stis. Data are presented as the means&E. from 4-6 rats per group. Statistical analyses were performed using a two-tailed Dunnett’s multiple comparison test, and values of PcO.05 were regarded as significant. Results
Effects of PACAPand VIP on Acid Secretion Gastric acid secretion was increased in response to mepirizole (200 mg/kg) from 5.7d.8 peql15 min to 19.2&Z. 1 peq115 min 3 h later, the total acid output for 3 h being 149.8*23.4peq/3 h. Pretreatment of the animals with PACAP(8 nmollkg) had no effect on the increased acid secretion caused by mepirizole, and the acid secretion was increased from 4.91to.6 peq115 min to a maximal value of 15.7i2.0 peq/15 min even in the presence of PACAPat 8 nmollkg; the total acid output for 3 h was 137.6k6.2 peq13 h, which is not significantly different from that of control animals given mepirizole (Fig. 1A and 1B). Similarly, the acid secretory response to mepirizole was not affected by i,v, infusion of PACAPat 8 nmol/kg/h (not shown). In contrast, the prior administration of VIP (8 nmollkg) significantly decreased the acid secretory response induced by mepirizole; the total acid output for 3 h was 79.h14.6 peq/3 h, which is significantly lower than that observed in the rats given mepirizole plus saline. Effects of PACAPand VIP on Duodenal HCO; Secretion Spontaneous rates of HCO,- secretion in the duodenum were significantly increased by i.v. administration of PACAPas well as VIP (Fig. 2A and 2B). The HCO; stimulatory effect of PACAPwas dose-dependent, and the net HCO; output was l.lN.3 peq/h, 4.W.9 peqlh and 7.2kl.2 peqlh, respectively, at 2,4 and 8 nmol/kg. The potency of HCO,- stimulatory action of VIP at 8 nmollkg was almost equivalent to that of PACAPat the same dose; the net HCO; output was 5.6rto.5 peql h. In addition, PACAPwhen infused i.v. at 8 nmol/kg/h also caused a persistent increase of HCO; secretion without showing any tachyphylaxis during 5 hours (Fig. 3). Duodenal HCO,- stimulatory effects of PACAPand VIP were similarly observed in the animals pretreated with mepirizole (Fig. 4A and 4B). The spontaneous rates of duodenal HCO,- secretion were not significantly affected by S.C. administration of mepirizole (200 mglkg). Even in the presence of mepirizole, these peptides caused a significant and persistent increase of HCO; secretion;
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PACAP on Duodenal HCOj Secretion and Ulcers
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N=56 ‘#P
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Time (min) Fig. 1. Effects of PACAPaud VIPou acid secretory respouse to mepirizde iu aoesthetizad rats. Mepirizole was administered S.C. in a dose of 200 m&kg. PACAP- (8 umollkg) aud VIP (8 muollkg) were administered i.v. 5 min before mepirizde treatment. Data are presented as the meaus~S.E. of values determined every 1.5 min from 5-6 rats. Figure B shows total acid output for 3 h after administration of mepirizole. and data are preseuted as the meaus~S.E. from 5-6 rats. Statistically significant difference at P
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(2.4 aud Data are Figure B
shows total HCO; output (LGncrease) for 1 h after admiuistratioa of PACAPor VIP, and data are presented as the meaus+S.E. from 5-6 rats per group. * Statistically significant difference from saline, at PcO.05.
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PACAP on Duodenal HCOi Secretion and Ulcers
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the net HCO,- output induced by PACAPand VIP at 8 nmollkg was 7.4kl.O peq/h and 5.2i 0.4 peq/h, respectively, which are not significantly different when compared to those induced by these peptides in the absence of mepirizole. Similarly, duodenal HCO,- secretion was persistently increased in response to i.v. infusion of PACAP(8 nmobkglh); the net HCO; output was much same in the presence or absence of mepirizole (Fig. 3).
Effecfs of PACAP- 7 and VIP on Duodenal Ukerogenic Response The animals developed hemorrhagic lesions in the proximal duodenum with less damage in the stomach when examined macroscopically 6 h after the administration of mepirizole (Fig. 5). The duodenal damage consisted of one or two round lesions, the lesion score being 21.7k6.0 mm* with some damage (8.0&J. 1 mm2) in the stomach. Prior administration of PACAPreduced the duodenal lesions in a dose-dependent manner, with no effect on the gastric damage, and a significant effect was observed at 4 and 8 nmol/kg; the lesion score was 7.91.9 mm* and 1.8rtl.S mm’, respectively. Similarly, i.v. infusion of PACAP(8 nmol/kg/h) was also effective in reducing the severity of duodenal but not gastric lesions induced by mepirizole, the duodenal lesion score being 0.8ti.3 mmZ. On the other hand, pretreatment of the animtiith VIP (8 nmol/kg) significantly reduced the severity of lesions induced by mepirizole, not only in theduodenum but also in the stomach; the lesion score in the duodenum and the stomach was 5.8k1.2 mn+and 2.0&S mm2, respectively. Discussion
The present study confirmed our previous findings that a hypothalamic peptide PACAPis a potent stimulator of duodenal HCO,- secretion in rats (7) and further showed that this peptide protects the duodenal mucosa from damage by duodenal ulcerogen mepirizole. In addition, the comparative data between PACAPand VIP suggested that the mechanisms involved in the PACAP action in the gastroduodenal mucosa arc different from those of VIP, possibly mediated by different receptors, although these peptides have a high sequence homology. Mepirizole, a basic antiinflammatory drug, causes duodenal ulcers in rats (12), and the pathogenic mechanism involves the increase of gastric acid secretion (16) and the impairment of duodenal HCO; secretion (13). These changes are similarly observed in rats following the administration of cysteamine, another duodenal ulcerogen (9,17). In particular, the HCO; secretion is the most important factor in protecting the duodenal mucosa against acid injury, and the impairment of this process has been demonstrated in duodenal ulcer patients as well as the pathogenesis of experimental duodenal ulcers (g-11,18). Duodenal HCO,- secretion was markedly increased by PACAPas well as VIP, in agreement with previous studies (7,19,20). As evidenced in this study, both PACAPand VIP stimulated duodenal HCO,- secretion in the presence of mepirizole. These results suggest that these peptides restore the impairment of duodenal HCO; secretion caused by mepirizole and prevent duodenal damage. It is known that prostaglandin E2 stimulates duodenal HCO; secretion, mediated intracellularly by adenosine 3’,5’-cyclic monophosphate (CAMP) (8), and protects the mucosa against mepirizole (13). Since PACAP increases CAMP by activating adenylate cyclase in various tissues, similar to VIP (2,20), and since the HCO; stimulating action of PACAPwas potentiated in the presence of a phosphodiesterase inhibitor (7), it is likely that prostaglandin Ez and PACAPshare the same mechanism in the mucosal protection, suggesting the importance of HCO; secretion in the duodenal protection against mepirizole. The mechanisms of acid-induced HCO,- secretion involve both humoral and neural factors as well as endogenous prostaglandins, although some controversy exists concerning the interaction of various mediators for eliciting full response of HCO,- secretion (21,22). Exposure of the duodenum to acid stimulates release of prostaglandin Ez from the mucosa (21). and the acid-induced HCO; secretion is blocked by indomethacin (10). VIP is the most likely humoral factor mediating the HCO,- secretory response to acid, because it is a potent stimulant of duodenal
PACAP on Duodenal HCO;
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Secretionand Ulcers
PACAP-27,
Vol. 63, No. 5, 1998
8 nmoVkg/h
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Fffccts of i.v. infusion of PACAP-
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Effectsof PACAP- and VIP on duodenal lesions induced by mepirizole in anesthetized rats. The animals were given (2.4 and 8 amollkg) or VIP (8 mnollkg) was mepirizole S.C. in a dose of 200 mglkg and killed 6 h later. PACAP(8 nmol/kg/%) was administered iv. twice 5 min before and 3 h after mepiri&e trwtment. In some auimals, PACApinfused i.v. for 5 h. starting 30 min before mepirizole treatment. Data are presented as the meausiS.E. from 5-6 rats. $ Statistically significant difference from the correqonding
saline group, at PcO.05.
HCO,- secretion and is released from nerve endings during the exposure of duodenal mucosa to acid (23,24). Arimura et al (25) demonstrated that PACAP-immunoreactive fibers are present in the stomach and duodenum of various species including the rat. It is therefore possible that PACAP is also involved in the mechanism of acid-induced HCO,- secretion, since this peptide shares the same type of receptors as VIP (6). In addition, the present results suggest that mepirizole inhibits the acidinduced HCO,- secretion by suppressing the release of such mediators but not by interfering at their receptor sites. It is well accepted that gastric acid participates in the pathogenesis of duodenal ulcers (26). Indeed, the duodenal ulcers induced by mepirizole are prevented by antisecretory agents and antacids (12). We observed in this study that mepirizole increased acid secretion to about four times the basal values. However, it is unlikely that the protective action of PACAPon the duodenal ulcers is due to suppression of acid secretion, because this peptide did not significantly affect the enhanced acid secretory response to mepirizole. Mungan et al (27) reported that PACAPhad no effect on basal acid secretion but suppressed against pentagastrin-induced acid secretion in rats. They further showed that this effect of PACAPwas dependent on the experimental model used to induce acid secretion; especially, PACAPhad an inhibitory effect on acid secretion stimulated by histamine or pentagastrin but not carbachol in pyloric ligated rats (28). Ozawa et al (29) reported that i.v. infusion of PACAPdid not affect pentagastrin-induced acid secretion but rather increased this secretion when given intracistemally, showing the different effects depending upon the route of administration. Thus, the effects of PACAPs on gastric acid secretion are controversial among investigators. On the other hand, VIP is known to decrease acid secretion by a direct inhibition at parietal cells (30) or by a release of somatostatin (3 1). Mungan et al (27), however, showed that VIP did not alter either basal or pentagastrin-stimulated acid secretion in conscious rats. The present study confirmed the antisecretory action of VIP and showed that this peptide significantly inhibited the increase of acid
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PACAP on Duodenal HCOj Secretion and Ulcers
Vol. 63, No. 5, 1998
secretion in response to mepirizole. Thus, it is assumed that VIP protected the duodenal mucosa against mepirizole by both stimulation of duodenal HCO; secretion and suppression of gastric acid secretion. There are at least three types of high-affinity receptors for PACAP on the basis of their relative affinities for PACAPand VIP(632). Type I receptors are PACAP-preferring receptors whereas type II and III receptors recognize PACAP and VIP with a similar affinity and appear to be identical with VIP1 and VIP2 receptors, respectively (32). The present results support our previous finding that the HCO; stimulatory action of PACAP may be mediated by PACAP receptors of type I and III (7). However, these two peptides showed different activities against mepirizole-induced gastric acid secretion; a significant inhibition was obtained with VIP but not PACAP-27. The reason for the different effects of VIPand PACAPon acid secretion remains unexplained, and further studies are needed to clarify this point. Taken together, the present results showed that PACAPis a potent stimulator of HCO; secretion in the rat duodenum and confers mucosal protection against duodenal ulcerogen, possibly by increasing HCO; secretion. Since the binding sites for PACAPare not limited to brain but are widely distributed in the gastrointestinal tract (4,5), PACAP as well as VIP may be physiologically important substances in maintaining the mucosal integrity of the duodenum against acid. References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
A. MIYATA, A. ARIMURA, R.D. DAHL, N. MINAMINO, A. UEHARA, L. JIANG, M.D. CULLER and D.H. COY, Biochem. Biophys. Res. Commun. 164 567-574 (1989) A. MIYATA, L. JIANG, R.D. DAHL, C. KITADA, K. KUBO, M. FUJINO, N. MINAMINO and A. ARIMURA, B&hem. Biophys. Res. Commun. 170 643-648 (1990) A. ARIMURA, Regulat. peptides 37 287-303 (1992) K. KOVES, A. ARIMURA, S. VIGH, A. SOMOGYVARI-VIGH and J. MILLER, Peptides 14 449-455 ( 1993) A. ARIMURA and S. SHIODA, Front Neuroendocrinol 16 53-88 (1995) J. CRISTOPHE, Biochim. Biophys. Acta. 1154 183-199 (1993) K. TAKEUCHI, K. TAKEHARA, S. KATO and K. YAGI, Am. J. Physiol. 272 G646-G653 (1997) G. FLEMSTROM and A. GARNER, Am. J. Physiol. 242 G183-G193 (1982) A. BRIDEN, G. FLEMSTROM and E. KIVILAAKSO, Gastroenterology 88 295-302 (1985) K. TAKEUCHI, 0. FURUKAWA, H. TANAKA and S. OKABE, Gastroenterology 90 636-645 (1986) J.L. ISENBERG, J.A. SELLING, D.L. HOGAN and M.A. KOSS, New Eng. J. Med. 316 374379 (1987) S. OKABE, Y. ISHIHARA, H. IN00 and H. TANAKA, Dig. Dis. Sci. 27 242-249 (1982) H. TANAKA, S. UEKI, T. OHNO, K. TAKEUCHI and S. OKABE, Jpn. J. Pharmacol. 42 3833% (1986). H. NIIDA, K. TAKEUCHI and S. OKABE, Eur. J. Pharmacol. 198 137-142 (1991) K. TAKKEUCHI, T. OHUCHI and S. OKABE, Dig. Dis. Sci. 40 670-677 (1995) K. UESHIMA, K. TAKEUCHI. T. OHUCHI and S. OKABE, Dig. Dis. Sci. 39 1625-1632 (1994) H. SELY E and S. SZABO, Nature 244 458-459 ( 1973) K. TABATA, E.D. JACOBSON, M.H. CHEN, R.F. MURPHY and S.N. JOFFE, Gastroenterology 87 3%~401 (1984) J.I. ISENBERG, B. WALLIN, C. JOHANSSON, B. SMEDFORS, V. MUTT, K. TATEMOTO and S. EMAS, Regulat. Peptides 8 3 15-3 19 ( 1984) B. SAFSTEN and G. FLEMSTROM, Acta. Physiol. Scand. 149 67-72 (1993)
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21. J.R. HEYLINGS, A. GARNER and G. PLEMSTROM, Am. J. Physiol. 246 G235G246 (1984) 22. D.L. HOGAN, B. YAO, H.H. STEINBACH and J.I. ISENBERG, Gastroenterology 105 410417 (1993) 23. S.R. BLOOM, S.J. MITCHELL, G.R. GREENBERG, W. CHRISTOPIDBS. I? DOMSCHKE, I? MITZNEGG and L. DEMLING, Acta. Hepato-Gastroenterol. 25 365-368 (1978) 24. M.C. ALGAZI, H.S. CHEN, M.A. KOSS, D.L. HOGAN, J. STEINBACH, S.J. PANDOL and J.I. ISENBERG, Am. J. Physiol. 256 G833-G836 (1989) 25. A. ARIMURA, A. SOMOGYVARI-VIGH, A. MIYATA, K. MIZUNO, D.H. COY and C. KITADA, Endocrinology 129 2787-2789 (1991) 26. S. SZABO, E.S. REYNOLDS, M.L. LICI-ITENBERGER, L.R. HAITH and V.J. DZAU, ChemPathol. Pharmacol. 16311-323 (1977) 27. Z. MUNGAN, V. OZMEN, A. ERTAN and A. ARIMURA, Regul. Peptides 38 199-206 (1992) 28. Z. MUNGAN, R.A. HAMMER, U.S. AKARCA, G. KGMAKl and A.ARIMURA, Peptides 16 1051-1056
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ELSEVIER
SOO243205(98)oo28o-
Life Scimca, Vd. 63, No. 5, pp. 317~325,~~ cqyright~1998ELcvicrsdeltccIllc. PrintedintbeUSA. AUtigbtsracmd 0024-32a5/!% 319.00 t .oo
EFFECTS OF PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE-27 ON ALKALINE SECRETORY AND MUCOSAL ULCEROGENIC RESPONSES IN RAT DUODENUM Koji Yagi, Koji Takehara, Motohiro Kitamura and Koji Takeuchi Department
of Pharmacology & Experimental Therapeutics Kyoto Pharmaceutical University Misasagi, Yamashina, Kyoto 607
Japan (Received in final form May l3,W98)
Summary Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on duodenal mucosal HCO,- secretion and ulcerogenic responses induced by mepirizole in anesthetized rats were examined and compared with those of vasoactive intestinal polypep tide (VIP). Animals were given mepirizole (200 mg/kg, s.c.) for induction of duodenal ulcers, and gastric acid and duodenal HCO; secretions were measured with or without pretreatment of PACAPor VIP Mepirizole increased acid secretion and induced hemorrhagic lesions in the proximal duodenum within 6 h. Intravenous bolus injection or infusion of PACAP(4 and 8 mnollkg or 8 rmrol/kg/h) increased duodenal HCO; secretion even in the presence of mepirizole. without effect on acid secretion, and significantly reduced the severity of duodenal lesions caused by mepirizole. In contrast, VIP (8 nmollkg, i.v.) given by bolus injection significantly decreased acid secretion induced by mepirizole, in addition to stimulation of HCO, secretion, and prevented duodenal lesions in response to mepirizole. These results suggest that PACAPincreases duodenal HCO; secretion and this action may be important in maintaining the duodenal mucosal integrity against acid, and VIP affords duodenal protection by both increasing duodenal HCO; secretion and decreasing acid secretion. The reason for the different effects of PACAP and VIP on acid secretion is unknown. Key Words: PACAP-27, mepirizole, duodenal lesion, bicarbonate secretion, acid secretion Pituitary adenylate cyclase activating polypeptide (PACAP) is a hypothalamic peptide showing 68% sequence homology with vasoactive intestinal peptide (VIP), which stimulates adenylate cyclase activity (1,2). Binding sites for PACAP are not limited to brain, but are widely distributed in the gastrointestinal tract including the stomach and the duodenum (3-S). Two biological active forms of the peptide have been identified, one major with 38 amino acids (PACAP-38) and the other with 27 residues (PACAP-27), sharing the same N-terminal 27 amino acids, and two types of PACAP receptors have been identified and cloned (6). Recently, we found that both forms of PACAPs (PACAPCorresponding Author: Dr. Koji Takeuchi, Department of Phaimacol. & Exp. Ther. Kyoto Pharmaceutical University, Kyoto 6078414, Japan Tel: 81-075-595-4679, Fax: 81-075-595-4774, E-mail: [email protected]
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27 and PACAP-38), similar to VIP, stimulate HCO,- secretion in the rat duodenum but not in the stomach (7), although the physiological significance of HCO,- stimulatory action of this peptide remains unknown. It is known that HCO; secretion by duodenal mucosa plays a pivotal role in maintaining the mucosal integrity against acid (8). Indeed, the pathogenic importance of the impaired HCO; secretion in duodenal ulcers has been demonstrated in various experimental animal models and humans (9- 11). Mepirizole, a basic nonsteroidal antiinflammatory drug, causes duodenal ulcers in rats, partly by stimulating gastric acid secretion and by reducing duodenal HCO, secretion, especially the acidinduced HCO,- response (12,13). In the present study, we examined the effects of PACAPon duodenal HCO; secretory and ulcerogenie responses induced by mepirizole in rats and investigated whether the HCO; stimulatory action of PACAPconfers protection on the duodenal mucosa against ulceration. In addition, we also examined the effects of VIP on these responses to mepirizole, in comparison with those of PACAP27. Materials and Methods Animals. Male Sprague-Dawley rats, weighing 250-300 g (Charles River, Shizuoka, Japan), were kept in individual cages with raised mesh bottoms to prevent coprophagy and deprived of food but allowed free access to tap water for 18 h before the experiments. Studies were carried out using 4-6 rats per group. General Protocols. Animals were anesthetized with urethane (1.25 g/kg) given i.p. and placed at room temperature (22rtl”C). Under these conditions, we measured gastric acid secretion, duodenal HCO; secretion, and duodenal ulcerogenic responses to mepirizole and examined the effects of PACAPand VIP on these responses. Determination of Gastric Acid Secretion. Acid secretion was measured in the acute fistula rat according to a previously published method (14). In brief, the abdomen was incised, and both the stomach and duodenum were exposed. An acute fistula (inside diameter: 3 mm) made with a polyethylene tube was inserted into the stomach from a small incision made in the duodenum and held in place by a ligature around the pylorus. The stomach was filled with 2 ml of saline (154 mM NaCl) through the fistula, and the solution was changed every 15 min. The collected samples were centrifuged at 3000 r.p.m. for 15 min and titrated to pH 7.0 against 100 mM NaOH using an autoburette (Hiranuma Comtite-8, Tokyo, Japan). Mepirizole was given S.C. in a dose of 200 mg/kg, and acid secretion was measured for 4 h. PACAP(8 nmollkg) or VlP(8 nmollkg) was given i.v. as a single injection 5 min before the administration of mepirizole. In some cases, PACAPwas given by a continuous i.v. infusion (8 nmol/kg/h) during mepirizole treatment. The doses of these peptides were selected to stimulate duodenal HCO; secretion in rats (7). Determinution of Duodenal HCO;Secretion. HCO; secretion was measured in the proximal loop of the rat duodenum as described (10). Briefly, the duodenal loop (1.7 cm) was made between the pyloric ring and the area just above the outlet of the common bile duct in order to exclude the influences of bile and pancreatic juice. Then, the loop was perfused at a flow rate of 1 ml/min with saline that was gassed with 100% 0,, heated at 37-C, and kept in a reservoir, and the HCO; secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCI to the reservoir. Approximately 1 h after basal secretion had stabilized, mepirizole was given S.C. in a dose of 200 mglkg. PACAP(4 and 8 nmollkg) or VIP (8 nmollkg) was given i.v. as a single injection 1 h after the administration of mepirizole. In another experiment, the effects of PACAP(2,4 and 8 nmol/kg) and VIP (8 nmollkg) on duodenal HCO; secretion were also examined in the absence of mepirizole.
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In some cases, PACAP(8 nmollkglh) was continuously absence or presence of mepirizole.
319
infused i.v. into the tail vein for 5 h in the
Evaluation of Duodmal Mucosa. Animals were given mepitizole S.C.in a dose of 200 mg/kg, without any surgical manipulation, and sacrificed 6 h later (15). Then, both the stomach and duodenum were removed, inflated by injecting 10 ml of 1% formalin for 10 min to fix the tissue walls, and opened along the greater curvature (stomach) or along the mesenteric attachment (duodenum). The area (mm*) of hemorrhagic lesions developed in the stomach and duodenum was measured under a dissecting microscope with a square grid (x10). PACAP(2,4 and 8 nmollkg) and VIP (8 nmoll kg) were given i.v. as a bo s injection two times, 5 min before and 3 h after the administration of mepitizole. In some cases, l! ACAP(8 nmollkglh) was continuously infused iv. into the tail vein for 6 h, starting from 30 min before mepirizole treatment. preparation of Drugs. Drugs used were urethane (Tokyo Kasei, Tokyo, Japan), PACAP-27, VIP (Peptide Institute, Osaka, Japan) and mepirizole (Daiichi Pharm., Tokyo, Japan). Mepirizole was suspended in 0.5% carboxymethylcellulose solution (CMC), while other drugs were dissolved in saline. Each agent was prepared immediately before use and given in a volume of 0.5 ml per 100 g body weight in cases of i.p. and S.C.administration or in a volume of 0.1 ml per 100 g body weight in case of i.v. administration. Control animals received saline or CMC as the vehicle. St&stis. Data are presented as the means&E. from 4-6 rats per group. Statistical analyses were performed using a two-tailed Dunnett’s multiple comparison test, and values of PcO.05 were regarded as significant. Results
Effects of PACAPand VIP on Acid Secretion Gastric acid secretion was increased in response to mepirizole (200 mg/kg) from 5.7d.8 peql15 min to 19.2&Z. 1 peq115 min 3 h later, the total acid output for 3 h being 149.8*23.4peq/3 h. Pretreatment of the animals with PACAP(8 nmollkg) had no effect on the increased acid secretion caused by mepirizole, and the acid secretion was increased from 4.91to.6 peq115 min to a maximal value of 15.7i2.0 peq/15 min even in the presence of PACAPat 8 nmollkg; the total acid output for 3 h was 137.6k6.2 peq13 h, which is not significantly different from that of control animals given mepirizole (Fig. 1A and 1B). Similarly, the acid secretory response to mepirizole was not affected by i,v, infusion of PACAPat 8 nmol/kg/h (not shown). In contrast, the prior administration of VIP (8 nmollkg) significantly decreased the acid secretory response induced by mepirizole; the total acid output for 3 h was 79.h14.6 peq/3 h, which is significantly lower than that observed in the rats given mepirizole plus saline. Effects of PACAPand VIP on Duodenal HCO; Secretion Spontaneous rates of HCO,- secretion in the duodenum were significantly increased by i.v. administration of PACAPas well as VIP (Fig. 2A and 2B). The HCO; stimulatory effect of PACAPwas dose-dependent, and the net HCO; output was l.lN.3 peq/h, 4.W.9 peqlh and 7.2kl.2 peqlh, respectively, at 2,4 and 8 nmol/kg. The potency of HCO,- stimulatory action of VIP at 8 nmollkg was almost equivalent to that of PACAPat the same dose; the net HCO; output was 5.6rto.5 peql h. In addition, PACAPwhen infused i.v. at 8 nmol/kg/h also caused a persistent increase of HCO; secretion without showing any tachyphylaxis during 5 hours (Fig. 3). Duodenal HCO,- stimulatory effects of PACAPand VIP were similarly observed in the animals pretreated with mepirizole (Fig. 4A and 4B). The spontaneous rates of duodenal HCO,- secretion were not significantly affected by S.C. administration of mepirizole (200 mglkg). Even in the presence of mepirizole, these peptides caused a significant and persistent increase of HCO; secretion;
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N=56 ‘#P
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shows total HCO; output (LGncrease) for 1 h after admiuistratioa of PACAPor VIP, and data are presented as the meaus+S.E. from 5-6 rats per group. * Statistically significant difference from saline, at PcO.05.
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PACAP on Duodenal HCOi Secretion and Ulcers
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the net HCO,- output induced by PACAPand VIP at 8 nmollkg was 7.4kl.O peq/h and 5.2i 0.4 peq/h, respectively, which are not significantly different when compared to those induced by these peptides in the absence of mepirizole. Similarly, duodenal HCO,- secretion was persistently increased in response to i.v. infusion of PACAP(8 nmobkglh); the net HCO; output was much same in the presence or absence of mepirizole (Fig. 3).
Effecfs of PACAP- 7 and VIP on Duodenal Ukerogenic Response The animals developed hemorrhagic lesions in the proximal duodenum with less damage in the stomach when examined macroscopically 6 h after the administration of mepirizole (Fig. 5). The duodenal damage consisted of one or two round lesions, the lesion score being 21.7k6.0 mm* with some damage (8.0&J. 1 mm2) in the stomach. Prior administration of PACAPreduced the duodenal lesions in a dose-dependent manner, with no effect on the gastric damage, and a significant effect was observed at 4 and 8 nmol/kg; the lesion score was 7.91.9 mm* and 1.8rtl.S mm’, respectively. Similarly, i.v. infusion of PACAP(8 nmol/kg/h) was also effective in reducing the severity of duodenal but not gastric lesions induced by mepirizole, the duodenal lesion score being 0.8ti.3 mmZ. On the other hand, pretreatment of the animtiith VIP (8 nmol/kg) significantly reduced the severity of lesions induced by mepirizole, not only in theduodenum but also in the stomach; the lesion score in the duodenum and the stomach was 5.8k1.2 mn+and 2.0&S mm2, respectively. Discussion
The present study confirmed our previous findings that a hypothalamic peptide PACAPis a potent stimulator of duodenal HCO,- secretion in rats (7) and further showed that this peptide protects the duodenal mucosa from damage by duodenal ulcerogen mepirizole. In addition, the comparative data between PACAPand VIP suggested that the mechanisms involved in the PACAP action in the gastroduodenal mucosa arc different from those of VIP, possibly mediated by different receptors, although these peptides have a high sequence homology. Mepirizole, a basic antiinflammatory drug, causes duodenal ulcers in rats (12), and the pathogenic mechanism involves the increase of gastric acid secretion (16) and the impairment of duodenal HCO; secretion (13). These changes are similarly observed in rats following the administration of cysteamine, another duodenal ulcerogen (9,17). In particular, the HCO; secretion is the most important factor in protecting the duodenal mucosa against acid injury, and the impairment of this process has been demonstrated in duodenal ulcer patients as well as the pathogenesis of experimental duodenal ulcers (g-11,18). Duodenal HCO,- secretion was markedly increased by PACAPas well as VIP, in agreement with previous studies (7,19,20). As evidenced in this study, both PACAPand VIP stimulated duodenal HCO,- secretion in the presence of mepirizole. These results suggest that these peptides restore the impairment of duodenal HCO; secretion caused by mepirizole and prevent duodenal damage. It is known that prostaglandin E2 stimulates duodenal HCO; secretion, mediated intracellularly by adenosine 3’,5’-cyclic monophosphate (CAMP) (8), and protects the mucosa against mepirizole (13). Since PACAP increases CAMP by activating adenylate cyclase in various tissues, similar to VIP (2,20), and since the HCO; stimulating action of PACAPwas potentiated in the presence of a phosphodiesterase inhibitor (7), it is likely that prostaglandin Ez and PACAPshare the same mechanism in the mucosal protection, suggesting the importance of HCO; secretion in the duodenal protection against mepirizole. The mechanisms of acid-induced HCO,- secretion involve both humoral and neural factors as well as endogenous prostaglandins, although some controversy exists concerning the interaction of various mediators for eliciting full response of HCO,- secretion (21,22). Exposure of the duodenum to acid stimulates release of prostaglandin Ez from the mucosa (21). and the acid-induced HCO; secretion is blocked by indomethacin (10). VIP is the most likely humoral factor mediating the HCO,- secretory response to acid, because it is a potent stimulant of duodenal
PACAP on Duodenal HCO;
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Secretionand Ulcers
PACAP-27,
Vol. 63, No. 5, 1998
8 nmoVkg/h
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Fffccts of i.v. infusion of PACAP-
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or presence of mepirizole. PACAPwas infused i.v. in a dose of 8 mnolikgh for 5 h. In half the number of animals, mepirizole (200 mglkg) was given S.C. 1 h before the onset of PACAPinfusion. Data are presented as the means&.E. of values determined every 5 min from 5 rats per group. * Statistically significant difference from the corresponding saline group, at P-=0.05.
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VoL63, No. 5,19!J8
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Effectsof PACAP- and VIP on duodenal lesions induced by mepirizole in anesthetized rats. The animals were given (2.4 and 8 amollkg) or VIP (8 mnollkg) was mepirizole S.C. in a dose of 200 mglkg and killed 6 h later. PACAP(8 nmol/kg/%) was administered iv. twice 5 min before and 3 h after mepiri&e trwtment. In some auimals, PACApinfused i.v. for 5 h. starting 30 min before mepirizole treatment. Data are presented as the meausiS.E. from 5-6 rats. $ Statistically significant difference from the correqonding
saline group, at PcO.05.
HCO,- secretion and is released from nerve endings during the exposure of duodenal mucosa to acid (23,24). Arimura et al (25) demonstrated that PACAP-immunoreactive fibers are present in the stomach and duodenum of various species including the rat. It is therefore possible that PACAP is also involved in the mechanism of acid-induced HCO,- secretion, since this peptide shares the same type of receptors as VIP (6). In addition, the present results suggest that mepirizole inhibits the acidinduced HCO,- secretion by suppressing the release of such mediators but not by interfering at their receptor sites. It is well accepted that gastric acid participates in the pathogenesis of duodenal ulcers (26). Indeed, the duodenal ulcers induced by mepirizole are prevented by antisecretory agents and antacids (12). We observed in this study that mepirizole increased acid secretion to about four times the basal values. However, it is unlikely that the protective action of PACAPon the duodenal ulcers is due to suppression of acid secretion, because this peptide did not significantly affect the enhanced acid secretory response to mepirizole. Mungan et al (27) reported that PACAPhad no effect on basal acid secretion but suppressed against pentagastrin-induced acid secretion in rats. They further showed that this effect of PACAPwas dependent on the experimental model used to induce acid secretion; especially, PACAPhad an inhibitory effect on acid secretion stimulated by histamine or pentagastrin but not carbachol in pyloric ligated rats (28). Ozawa et al (29) reported that i.v. infusion of PACAPdid not affect pentagastrin-induced acid secretion but rather increased this secretion when given intracistemally, showing the different effects depending upon the route of administration. Thus, the effects of PACAPs on gastric acid secretion are controversial among investigators. On the other hand, VIP is known to decrease acid secretion by a direct inhibition at parietal cells (30) or by a release of somatostatin (3 1). Mungan et al (27), however, showed that VIP did not alter either basal or pentagastrin-stimulated acid secretion in conscious rats. The present study confirmed the antisecretory action of VIP and showed that this peptide significantly inhibited the increase of acid
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secretion in response to mepirizole. Thus, it is assumed that VIP protected the duodenal mucosa against mepirizole by both stimulation of duodenal HCO; secretion and suppression of gastric acid secretion. There are at least three types of high-affinity receptors for PACAP on the basis of their relative affinities for PACAPand VIP(632). Type I receptors are PACAP-preferring receptors whereas type II and III receptors recognize PACAP and VIP with a similar affinity and appear to be identical with VIP1 and VIP2 receptors, respectively (32). The present results support our previous finding that the HCO; stimulatory action of PACAP may be mediated by PACAP receptors of type I and III (7). However, these two peptides showed different activities against mepirizole-induced gastric acid secretion; a significant inhibition was obtained with VIP but not PACAP-27. The reason for the different effects of VIPand PACAPon acid secretion remains unexplained, and further studies are needed to clarify this point. Taken together, the present results showed that PACAPis a potent stimulator of HCO; secretion in the rat duodenum and confers mucosal protection against duodenal ulcerogen, possibly by increasing HCO; secretion. Since the binding sites for PACAPare not limited to brain but are widely distributed in the gastrointestinal tract (4,5), PACAP as well as VIP may be physiologically important substances in maintaining the mucosal integrity of the duodenum against acid. References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
A. MIYATA, A. ARIMURA, R.D. DAHL, N. MINAMINO, A. UEHARA, L. JIANG, M.D. CULLER and D.H. COY, Biochem. Biophys. Res. Commun. 164 567-574 (1989) A. MIYATA, L. JIANG, R.D. DAHL, C. KITADA, K. KUBO, M. FUJINO, N. MINAMINO and A. ARIMURA, B&hem. Biophys. Res. Commun. 170 643-648 (1990) A. ARIMURA, Regulat. peptides 37 287-303 (1992) K. KOVES, A. ARIMURA, S. VIGH, A. SOMOGYVARI-VIGH and J. MILLER, Peptides 14 449-455 ( 1993) A. ARIMURA and S. SHIODA, Front Neuroendocrinol 16 53-88 (1995) J. CRISTOPHE, Biochim. Biophys. Acta. 1154 183-199 (1993) K. TAKEUCHI, K. TAKEHARA, S. KATO and K. YAGI, Am. J. Physiol. 272 G646-G653 (1997) G. FLEMSTROM and A. GARNER, Am. J. Physiol. 242 G183-G193 (1982) A. BRIDEN, G. FLEMSTROM and E. KIVILAAKSO, Gastroenterology 88 295-302 (1985) K. TAKEUCHI, 0. FURUKAWA, H. TANAKA and S. OKABE, Gastroenterology 90 636-645 (1986) J.L. ISENBERG, J.A. SELLING, D.L. HOGAN and M.A. KOSS, New Eng. J. Med. 316 374379 (1987) S. OKABE, Y. ISHIHARA, H. IN00 and H. TANAKA, Dig. Dis. Sci. 27 242-249 (1982) H. TANAKA, S. UEKI, T. OHNO, K. TAKEUCHI and S. OKABE, Jpn. J. Pharmacol. 42 3833% (1986). H. NIIDA, K. TAKEUCHI and S. OKABE, Eur. J. Pharmacol. 198 137-142 (1991) K. TAKKEUCHI, T. OHUCHI and S. OKABE, Dig. Dis. Sci. 40 670-677 (1995) K. UESHIMA, K. TAKEUCHI. T. OHUCHI and S. OKABE, Dig. Dis. Sci. 39 1625-1632 (1994) H. SELY E and S. SZABO, Nature 244 458-459 ( 1973) K. TABATA, E.D. JACOBSON, M.H. CHEN, R.F. MURPHY and S.N. JOFFE, Gastroenterology 87 3%~401 (1984) J.I. ISENBERG, B. WALLIN, C. JOHANSSON, B. SMEDFORS, V. MUTT, K. TATEMOTO and S. EMAS, Regulat. Peptides 8 3 15-3 19 ( 1984) B. SAFSTEN and G. FLEMSTROM, Acta. Physiol. Scand. 149 67-72 (1993)
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21. J.R. HEYLINGS, A. GARNER and G. PLEMSTROM, Am. J. Physiol. 246 G235G246 (1984) 22. D.L. HOGAN, B. YAO, H.H. STEINBACH and J.I. ISENBERG, Gastroenterology 105 410417 (1993) 23. S.R. BLOOM, S.J. MITCHELL, G.R. GREENBERG, W. CHRISTOPIDBS. I? DOMSCHKE, I? MITZNEGG and L. DEMLING, Acta. Hepato-Gastroenterol. 25 365-368 (1978) 24. M.C. ALGAZI, H.S. CHEN, M.A. KOSS, D.L. HOGAN, J. STEINBACH, S.J. PANDOL and J.I. ISENBERG, Am. J. Physiol. 256 G833-G836 (1989) 25. A. ARIMURA, A. SOMOGYVARI-VIGH, A. MIYATA, K. MIZUNO, D.H. COY and C. KITADA, Endocrinology 129 2787-2789 (1991) 26. S. SZABO, E.S. REYNOLDS, M.L. LICI-ITENBERGER, L.R. HAITH and V.J. DZAU, ChemPathol. Pharmacol. 16311-323 (1977) 27. Z. MUNGAN, V. OZMEN, A. ERTAN and A. ARIMURA, Regul. Peptides 38 199-206 (1992) 28. Z. MUNGAN, R.A. HAMMER, U.S. AKARCA, G. KGMAKl and A.ARIMURA, Peptides 16 1051-1056
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29. M. OZAWA, M. AONO, K. MIZUTA, M. MORIGA and M. OKUMA, Dig Dis Sci 42 25522559 (1997) 30. S.J. KONTUREK, A. DEMBINSKI, l? THOR and R, KROL, Pfluger. Arch. 361 175181(1976) 3 1. M.L. SCHUBERT, Gastroenterology 100 1195- 1200 (1991) 32. S.R. RAWLINGS, Mol. Cell. Endocrinol. 101 C5C9 (1994)