Motohiro Kitamura

Role of nitric oxide in regulation of gastric acid secretion in rats: effects of NO donors and NO synthase inhibitor

1 The role of nitric oxide (NO) in the regulation of acid secretion was examined in the anaesthetized rat. 2 A rat stomach was mounted in an ex vivo chamber, instilled with 2 ml of saline every 15 min, and the recovered sample was titrated at pH 7.0 against 0.1 N NaOH by use of an automatic titrator for acid secretion. Gastric mucosal blood flow (GMBF) was measured simultaneously by laser Doppler flowmeter. 3 Intragastric application of NO donors such as FK409 (3 and 6 mg ml−1) and sodium nitroprusside (SNP; 6 and 12 mg  ml−1) as well as i.p. administration of cimetidine (60 mg kg−1), a histamine H2‐receptor antagonist, significantly inhibited the increase in acid secretion in response to pentagastrin (60 μg kg−1 h−1, i.v.), in doses that increased gastric mucosal blood flow (GMBF). 4 Intragastric application of FK409 (6 mg ml−1) increased both basal and stimulated acid secretion induced by YM‐14673 (0.3 mg kg−1, i.v.), an analogue of thyrotropin‐releasing hormone (TRH), but had no effect on the acid secretory response induced by histamine (4 mg kg−1 h−1, i.v.). 5 Pretreatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 10 mg kg−1, i.v.) did not affect basal acid secretion, but significantly potentiated the increase in acid secretion induced by YM‐14673 and slightly augmented the acid secretory response to pentagastrin. 6 Both pentagastrin and YM‐14673 increased the release of nitrite plus nitrate (NOx), stable NO metabolites, into the gastric lumen, and these changes were completely inhibited by prior administration of L‐NAME (10 mg kg−1, i.v.). 7 Pentagastrin caused an increase in luminal release of histamine and this response was significantly suppressed by intragastric application of FK409 (6 mg ml−1). 8 These results suggest that either exogenous or endogenous NO has an inhibitory action on gastric acid secretion through suppression of histamine release from enterochromaffin‐like (ECL) cells.

Effects of selective cyclooxygenase-2 inhibitors on alkaline secretory and mucosal ulcerogenic responses in rat duodenum.

Effects of the selective cyclooxygenase-2 (COX-2) inhibitors such as NS-398 and nimesulide on duodenal HCO3- secretory and ulcerogenic responses to mucosal acidification were examined in rats, in comparison with indomethacin, a nonselective COX inhibitor. Duodenal HCO3- secretion in anesthetized rats was increased in response to mucosal acidification. The increased HCO3- response to acid was significantly suppressed by pretreatment with indomethacin (10 mg kg(-1), s.c.), while both NS-398 and nimesulide (10 mg kg(-1), s.c.) had no effect on this response. The luminal release of prostaglandin E2 (PGE2) was increased during and after mucosal acidification, and this response was significantly inhibited by indomethacin but not NS-398 or nimesulide. Indomethacin provoked hemorrhagic lesions in the duodenum when acid hypersecretion was concomitantly induced by histamine (8 mg kg(-1) hr(-1), i.v.), while either NS-398 or nimesulide did not cause damage in the duodenum. Either of these drugs had no effect on histamine-induced acid secretion. On the other hand, both NS-398 and nimesulide showed a significant suppression against carrageenan-induced rat paw edema, similar to indomethacin. The present study supports a mediator role for endogenous PGs in duodenal HCO3- secretion in response to mucosal acidification and suggests that COX-1 but not COX-2 is a key enzyme in regulating this process and maintaining the mucosal integrity against acid in the duodenum.

Stimulatory effect of nitric oxide on bicarbonate secretion in bullfrog duodenums in vitro.

The effect of nitric oxide (NO) on HCO^–₃ secretion was examined in vitro using an isolated preparation of bullfrog duodenum. The tissue was bathed in unbuffered Ringer’s solution gassed with 100% O₂ on the mucosal side and HCO^–₃ Ringer’s solution gassed with 95% O₂–5% CO₂ on the serosal side. The HCO^–₃ secretion was measured by the pH-stat method using 2 mmol/l HCl as the titrant to keep the mucosal pH at 7.4. (±)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamine (NOR3) was used as a NO donor and added to the serosal solution. To analyze the NOR3 action on HCO^–₃ secretion, the effects of dibutyryl adenosine-3′,5′-cyclic monophosphate (dbcAMP), dibutyryl guanosine-3′,5′-cyclic monophosphate (dbcGMP), methylene blue, and indomethacin on the HCO^–₃ response were also examined. NOR3 (1×10^–4 and 3×10^–4 mol/l) caused an increase in HCO^–₃ secretion in a dose-dependent manner, and this effect appeared with an about 30-min time lag, reaching the level of 1.5–2.5 times greater than basal values at 1–2 h later. Both dbcAMP (1×10^–3 mol/l) and dbcGMP (1×10^–3 mol/l) also caused a significant increase in HCO^–₃ secretion in bullfrog duodenums in vitro, although the onset of the HCO^–₃ response to dbcGMP was delayed as compared to the former. The stimulatory action of NOR3 on duodenal HCO^–₃ secretion was significantly attenuated by methylene blue (5×10^–5 mol/l) and indomethacin (1×10^–5 mol/l), the latter also inhibiting the HCO^–₃ response to dbcGMP. The release of prostaglandin E₂ in the serosal solution was significantly increased after addition of NOR3 (3×10^–4 mol/l) and dbcGMP (1×10^–3 mol/l) in an indomethacin-sensitive manner. These results suggest that the NO donor increases duodenal HCO^–₃ secretion in vitro, and this action of NO donor is cGMP-dependent and mediated by endogenous prostaglandins. Duodenal HCO^–₃ secretion may be regulated locally by NO/cGMP in addition to prostaglandin/cAMP.

Effects of pituitary adenylate cyclase activating polypeptide-27 on alkaline secretory and mucosal ulcerogenic responses in rat duodenum

Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on duodenal mucosal HCO3- secretion and ulcerogenic responses induced by mepirizole in anesthetized rats were examined and compared with those of vasoactive intestinal polypeptide (VIP). Animals were given mepirizole (200 mg/kg, s.c.) for induction of duodenal ulcers, and gastric acid and duodenal HCO3- secretions were measured with or without pretreatment of PACAP-27 or VIP. Mepirizole increased acid secretion and induced hemorrhagic lesions in the proximal duodenum within 6 h. Intravenous bolus injection or infusion of PACAP-27 (4 and 8 nmol/kg or 8 nmol/kg/h) increased duodenal HCO3- 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 nmol/kg, i.v.) given by bolus injection significantly decreased acid secretion induced by mepirizole, in addition to stimulation of HCO3- secretion, and prevented duodenal lesions in response to mepirizole. These results suggest that PACAP-27 increases duodenal HCO3- secretion and this action may be important in maintaining the duodenal mucosal integrity against acid, and VIP affords duodenal protection by both increasing duodenal HCO3- secretion and decreasing acid secretion. The reason for the different effects of PACAP and VIP on acid secretion is unknown.

Stimulatory effect of PACAP on gastroduodenal bicarbonate secretion in rats.

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO3- secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO3- secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO3- secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO3- secretion after intracisternal administration (1 nmol/rat). PGE2 (300 micrograms/kg, iv) significantly increased HCO3- secretion in both the stomach and the duodenum. The potency of duodenal HCO3- secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE2. The duodenal HCO3- secretory action of PACAP-27 as well as PGE2 was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 micrograms/kg, iv), the stimulator of adenylate cyclase, also increased HCO3- secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO3- secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO3- secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO3- secretion.

Prostaglandin E-type receptor subtypes and gastroduodenal bicarbonate secretion in rats

Abstract We investigated the relationship between prostaglandin E‐type receptor (EP receptor) subtypes and gastroduodenal HCO3‐ secretion in rats. Under urethane anaesthesia, a stomach mounted in an ex vivo chamber or a proximal duodenal loop was perfused with saline and the HCO3‐ secretion was measured at pH 7.0 using a pH‐stat method and by adding 10 mmol/L HCl. Prostaglandin E2 (PGE2, i.V.) increased HCO3‐ secretion in both the stomach and duodenum; this action was verapamil sensitive and only in the duodenum was potentiated by isobutylmethyl xanthine (IBMX). Duodenal HCO3‐ secretion was also stimulated by both sulprostone (EP1/EP3 agonist), enprostil (EP1/EP3 agonist), misoprostol (EP2/EP3 agonist), 11‐deoxy PGE1 (EP3/EP4 agonist) and ONO‐NT‐012 (EP3 agonist), but was not affected by either butaprost (EP2 agonist) or 17‐phenyl‐ω‐trinor‐PGE2 (EP1 agonist). In contrast, gastric HCO3‐ secretion was stimulated by sulprostone, enprostil and 17‐phenyl‐ω‐trinor‐PGE2, but not by misoprostol, butaprost, 11‐deoxy PGE1 or ONO‐NT‐012. The EP1 antagonist SC‐51089 inhibited the HCO3‐ stimulatory action of sulprostone in the stomach but not in the duodenum. Isobutylmethyl xanthine potentiated the HCO3‐ response to sulprostone in the duodenum, while verapamil reduced the response in both the stomach and duodenum. These results suggest that PGE2 stimulates HCO3‐ secretion via different EP receptor subtypes in the stomach and duodenum: in the former the EP1 receptors linked to Ca2+ and in the latter, the EP3 receptors coupled with both cAMP and Ca2+.