Tetsuya Yasuhiro

Role of Nitric Oxide in Pathogenesis of Aspirin-Induced Gastric Mucosal Damage in Rats

We examined the effect of NO synthase inhibitor on the functional and ulcerogenic responses to aspirin (ASA) in rat stomach. The animals were given ASA (20–80 mM) orally with or without HCl (10–50 mM) and killed 2 h later. NG-nitro-L-arginine methyl ester (L-NAME) was given i.v. 5 min before aspirin. In the functional study, a rat stomach was mounted on an ex vivo chamber under urethane anesthesia, perfused with saline, and transmucosal potential difference (PD), luminal pH, acid secretion and mucosal blood flow (GMBF) were measured simultaneously. ASA alone caused gastric damage in a dose-related manner; mostly nonhemorrhagic lesions. Pretreatment with L-NAME worsened such lesions and caused severe hemorrhagic lesions. Coadministration of HCl with ASA also potentiated gastric lesions in a concentration-dependent manner, changing nonhemorrhagic into hemorrhagic damage, and the worsening effect of L-NAME disappeared when 80 mM ASA was given together with HCl at >20 mM. In chambered stomachs, the mucosal application of ASA (80 mM, 30 min) caused a marked reduction in PD and a slight decrease in acid secretion, with minimal change in GMBF. L-NAME blocked the reduced acid response following ASA and caused stimulation of acid secretion with no effect on PD and GMBF. These effects of L-NAME were all antagonized by coadministration of L-arginine and significantly mitigated by sensory deafferentation or pretreatment with cimetidine or FPL-52694. These results suggest that (1) intragastric administration of ASA causes a release of NO, which reduces the development of hemorrhagic lesions by decreasing acid secretion, and (2) L-NAME worsens gastric damage by increasing acid secretion in ASA-treated stomachs, the process being dependent on endogenous histamine and sensory neurons.

Role of Nitric Oxide in Mucosal Blood Flow Response and the Healing of HCI-lnduced Lesions in the Rat Stomach

The role of nitric oxide (NO) in the gastric mucosal blood flow response and the healing of HCl-induced gastric lesions was investigated in rats. After 18 h fasting rats were given 0.6 N HCl p.o. for the induction of gastric lesions, and 1 h later they were fed normally. After induction of gastric lesions, they were repeatedly administered the NO synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME 5-20 mg/kg p.o. twice daily) or aminoguanidine (20 mg/kg s.c. once daily) for 7 days. Gastric lesions caused by HCl healed almost completely within 5 days with granulation and to an extent with re-epithelialization. Repeated administration of L-NAME but not aminoguanidine significantly delayed the healing of gastric lesions in a dose-dependent manner. The damaged mucosa secreted less acid, but showed a marked rise in H+ permeability, resulting in luminal acid loss accompanied by an increase of mucosal blood flow. Aminoguanidine did not significantly affect any of these functional changes observed in the stomach after damage by HCl, whereas L-NAME treatment slightly reversed the decreased acid response, increased the luminal H+ loss, and totally inhibited the mucosal hyperemic response associated with luminal acid loss in the damaged mucosa. In addition, the deleterious influences of L-NAME on the mucosal blood flow response and the healing of gastric lesions were significantly antagonized by co-administration of L-arginine but not of D-arginine (500 mg/kg x 2, i.p.). Luminal output of NO2-/NO3- was significantly increased in pylorus-ligated stomachs in control rats on days 3 and 5 after damage, and such increases in gastric NO output were completely attenuated by L-NAME treatment. These results suggest that endogenous NO may contribute to the healing of acute gastric injury by mediating the mucosal hyperemic responses associated with acid back-diffusion and by facilitating acid disposal in the damaged mucosa. NO mediating such responses and participating in the healing aspect of gastric lesions may be produced by the constitutive type of NO synthase.

Role of nitric oxide in pathogenesis of gastric mucosal damage induced by compound 48/80 in rats.

We examined the effects of various nitric oxide synthase (NOS) inhibitors on development of gastric lesions induced by compound 48/80 (48/80) in rats and investigated the roles of NO and inducible NOS (iNOS) in inflammatory gastric responses. Animals were given 48/80 (1 mg/kg, i.p.) once daily for 4 days, and the stomachs were examined for lesions 24 h after the final administration. NOS inhibitors such as L-NAME, L-NMMA, aminoguanidine or dexamethasone were administered for 4 days during 48/80 treatment. The repeated administration of 48/80 caused damage in the stomach with severe edema in the submucosa. These lesions induced by 48/80 were dose-dependently prevented by concurrent administration of L-NAME. The protective effect of L-NAME on 48/80-induced gastric lesions was mimicked by L-NMMA, aminoguanidine as well as dexamethasone, and significantly antagonized by co-administration of L-arginine but not by D-arginine. Acid secretion was slightly decreased after 48/80 treatment, but was significantly augmented by the combined administration of L-NAME with 48/80. The mucosal MPO activity, TBA reactants and vascular permeability in the stomach were all increased after 48/80 treatment, but these changes were also significantly mitigated by co-administration of L-NAME. The Ca(2+)-independent NOS activity in the mucosa was increased four times during 48/80 treatment, and this change was also inhibited by dexamethasone. These results suggest that: 1) the repeated administration of 48/80 induced inflammatory gastric lesions in the rat stomach; 2) the pathogenic mechanism of these lesions involves endogenous NO produced by iNOS, in addition to oxyradical formation; and 3) the deleterious role of NO during 48/80 treatment may be accounted for by a cytotoxic action of peroxynitrite, which is formed in the presence of NO and superoxide radicals.

Mechanism of Acid Secretory Changes in Rat Stomach After Damage by Taurocholate Role of Nitric Oxide, Histamine, and Sensory Neurons

The present study was performed to investigatethe mechanism underlying the acid stimulatory responsein the stomach after damage under the inhibition ofnitric oxide (NO) production byNG-nitro-L-arginine methyl ester (L-NAME). A rat stomach wasmounted in an ex vivo chamber, perfused with saline, andthe potential difference (PD) and acid secretion weremeasured before and after the application of 20 mM taurocholate (TC) for 30 min. Exposure of thestomach to TC caused a PD reduction and a decrease ofacid secretion. Pretreatment with L-NAME did not affectbasal acid secretion but significantly enhanced the acid secretion in the stomach after damagewith TC, without any effect on the PD response. Thiseffect of L-NAME was antagonized by simultaneousadministration of L-arginine but not D-arginine. The luminal appearance of NO was significantlyincreased in the stomach after exposure to TC, and thischange was completely blocked in the presence of L-NAMEor when EGTA was applied together with TC. The enhanced acid secretory response to TC in thepresence of L-NAME was inhibited by pretreatment withcimetidine, FPL-52694 (a mast cell stabilizer), orspantide (a substance P antagonist) or by chemical ablation of capsaicinsensitive sensory neurons.Mucosal exposure to TC increased histamine output in thelumen and decreased the number of metachromaticallystaining cells in the stomach, and these changes were also significantly prevented by FPL-52694,spantide, or sensory deafferentation. These resultssuggest that 1) damage in the stomach may activate theacid stimulatory pathway in addition to the NO-dependent inhibitory mechanism, but the latter effectovercomes the former, resulting in a decrease in acidsecretion, 2) the acid stimulation in the damagedstomach may be mediated by histamine released from the mucosal mast cell which may interact withcapsaicin-sensitive sensory nerves, and 3) L-NAMEunmasks the acid stimulatory response by suppressing theinhibitory mechanism.

Effects of pantoprazole, a novel H+/K+-ATPase inhibitor, on duodenal ulcerogenic and healing responses in rats: a comparative study with omeprazole and lansoprazole.

Background: Pantoprazole, 2‐[(2‐pyridylmethyl) sulphinyl] benzimidazole, is a new substituted benzimidazole that inhibits the parietal cell H+/K+‐ATPase.

Effects of Diabetes mellitus on the Contractile Activity of Carbachol and Galanin in Isolated Gastric Fundus Strips of Rats

The role of the cholinergic and peptidergic pathways in the impairment of gastric motility associated with diabetic gastroparesis was assessed at the postsynaptic level using isolated fundus smooth muscle strips. Maximal contractile responses to carbachol and galanin were significantly decreased in fundus strips isolated from rats rendered diabetic by a single intraperitoneal injection of streptozotocin (STZ, 70 mg/kg) 1, 4 and 8 weeks before experiments. We also observed notable decrements in the slopes and Hill’s coefficients without conspicuous changes in the EC50 of the respective galanin concentration-response curves measured in strips obtained from STZ animals after 4 and 8 weeks. L-NAME reversed the above-mentioned alterations in an L-arginine-sensitive manner in STZ rats after 4 weeks but not in STZ rats after 8 weeks. The blood plasma nitrite/nitrate levels in STZ animals after 4 and 8 weeks were increased by 44.6 and 61.9%, respectively. Ca2+-independent nitric oxide synthase activity in gastric fundus strips and stomach corpus mucosa from STZ rats after 4 weeks was markedly enhanced by 37.4 and 31.9%, respectively, suggesting an enhanced nitric oxide production. In vivo insulin treatment prevented diabetes-induced alterations in smooth muscle contractility. We conclude that the smooth muscle dysfunction evoked by experimental diabetes causing diminished contractions of fundus strips to carbachol and galanin is at least partly due to the increased nitric oxide synthesis.

Gastric mucosal damage induced by compound 48/80: Roles of serotonin and nitric oxide

The roles of nitric oxide (NO) and serotonin (5‐HT) in the development of gastric mucosal lesions induced by compound 48/80 were investigated in rats. Repeated i.p. administration of 48/80 (1 mg/kg) produced damage in the stomach with severe oedema in the submucosa. The lesions induced by 48/80 were prevented by FPL‐52694 (a mast cell stabilizer) and methysergide but not tri‐pelennamine. The lesions were also inhibited by simultaneous administration of NG‐monomethyl‐L‐arginine (L‐NMMA), and this effect was mimicked by inducible NO synthase (iNOS) inhibitors, such as aminoguanidine or dexamethasone and significantly antagonized by coadministration of L‐arginine. The mucosal myeloperoxidase activity, thiobarbituric acid reactants and vascular permeability in the stomach were all increased after 48/80 treatment and the changes were also attenuated by cotreatment with L‐NMMA. Repeated s.c. treatment with 5‐HT (20 mg/kg) provoked similar gastric lesions, which were also prevented by methysergide and iNOS inhibitors, as well as antioxidative drugs, such as allopurinol (a xan‐thine oxidase inhibitor) and hydroxyurea (a neutrophil‐reducing agent). The Ca2+‐independent NO synthase (NOS) activity was increased in the gastric mucosa after administration of 48/80 or 5‐HT and this change was inhibited by dexamethasone. These results suggest that: (i) the repeated administration of 48/80 induced inflammatory gastric lesions in the rat stomach, mediated by endogenous 5‐HT; (ii) NO/iNOS is involved in the pathogenic mechanism of 48/80‐induced gastric lesions, in addition to oxyradical formation; and (iii) the deleterious role of NO in this lesion model can be accounted for by a cytotoxic action of peroxynitrite that is formed in the presence of superoxide radicals.

Changes in gastric HCO-3 secretory response to NG-nitro-L-arginine methyl ester in rats following repeated administration.

Abstract  The effect of repeated administration of the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME) on gastric HCO‐3 secretion was examined using ex vivo chambered stomachs of anaesthetized rats. Intravenous administration of l‐NAME (5 mg/kg) increased gastric HCO‐3 secretion with a concomitant rise in arterial blood pressure (BP). The HCO‐3 stimulatory action of l‐NAME diminished when rats were pretreated with l‐NAME (20 mg/kg, p.o., twice daily) for 1 or 3 days and an inverse relationship was found between the degree of secretory stimulation and the period of pretreatment. The increased BP response to l‐NAME was also significantly lessened following repeated pretreatment; basal BP showed a stepwise increase during repeated pretreatment and did not change at all in response to i.v. l‐NAME after 3 days pretreatment. When ΔHCO‐3 output induced by i.v. l‐NAME was plotted against ΔBP (from basal values) during repeated pretreatment with l‐NAME, a significant relationship was found between these two factors. The reduction in the HCO‐3 secretory response to l‐NAME was restored when animals were pretreated with l‐arginine (500 mg/kg, i.p., twice daily) together with l‐NAME. However, prostaglandin E2 (300 μg/kg, i.v.) caused a gastric HCO‐3 secretory response similar to l‐NAME, regardless of whether rats had been pretreated with l‐NAME or not. In contrast, the attenuation by l‐NAME of the acid (0.2 mmol/L HCl)‐induced gastric hyperaemic response was not influenced by repeated pretreatment with l‐NAME. We conclude that repeated p.o. pretreatment with l‐NAME reduces the HCO‐3 stimulatory action of i.v. l‐NAME and that this phenomenon may be explained by the lack of further elevation of BP in response to i.v. l‐NAME following repeated pretreatment with this agent. Thus, the stimulation of HCO‐3 secretion by i.v. l‐NAME may be causally related with increased BP in response to this agent.

Reduction in Gastric Bicarbonate Secretory Response Induced by NG-Nitro-l -Arginine Methyl Ester Following Repeated Administration in Rats

Gastric HCO 3 - secretory response induced by the nitric oxide (NO) synthase inhibitor, NG-nitro-targinine methyl ester (l-NAME), was examined in rats before and after the repeated administration of this agent. HCO 3 - secretion was determined in ex-vivo chambered stomachs of anaesthetized rats. Intravenous administration of l-NAME (5 mg/kg) increased gastric HCO 3 - secretion with a concomitant rise in arterial blood pressure (BP). The HCO 3 - stimulatory action of iv l-NAME diminished when they were pretreated with l-NAME (20 mg/kg × 2, po) for 1 or 3 days, and an inverse relationship was found between the degree of stimulation and the period of treatment. The increased BP response to iv l-NAME was also significantly lessened following repeated administration; the basal BP showed a step-wise increase during treatment and did not change at all in response to iv l-NAME after 3 days treatment. When ΔHCO 3 - output induced by iv l-NAME was plotted against ABP change during repeated treatment with l-NAME po, a significant relationship was found between these two factors. The reduction of HCO 3 - response to iv l-NAME was significantly restored when the animals were given l-arginine (500 mg/kg × 2 ip) simultaneously with pot-NAME. However, prostaglandin E2 (300 µg/kg iv) caused a gastric HCO 3 - response similar in degree regardless of whether the animals were pretreated with l-NAME po or not. These results suggest that: (I) the repeated po treatment with l-NAME diminishes the HCO 3 - stimulatory action of iv l-NAME; (2) this phenomenon may be explained by the lack of further elevation of blood pressure to iv l-NAME following the repeated treatment; and (3) the stimulation of HCO 3 - secretion by iv l-NAME may be causally related to the increased blood pressure response to this agent.

Stimulation of acid secretion in rat stomach following exposure to taurocholate in the presence of the nitric oxide synthase inhibitor

1. The role of nitric oxide (NO) in the acid secretory response of the rat stomach following damage was investigated. A rat stomach was mounted in an ex-vivo chamber, perfused with saline, and the potential difference (PD), luminal pH, acid and HCO3- responses were measured before and after the mucosal exposure to 20 mM taurocholate (TC) for 30 min, with or without pretreatment with NG-nitro-L-arginine methyl ester (L-NAME). 2. Exposure of the stomach to TC caused a reduction of PD, a decrease of acid secretion and an increase in luminal HCO-. Pretreatment with L-NAME did not affect such PD and HCO3- responses, but completely attenuated the decreased acid secretory response and rather enhanced this secretion. 3. These effects of L-NAME were significantly antagonized by the co-administration of L-arginine but not D-arginine. The enhanced acid secretory response in the presence of L-NAME was significantly inhibited by prior administration of cimetidine or FPL-52694 (a mast-cell stabilizer). 4. The mucosal exposure to TC significantly decreased the number of mucosal mast cells and increased the luminal histamine output. 5. Damage in the stomach may activate the histamine-dependent acid stimulatory pathway in addition to the NO-dependent inhibitory mechanism, although the latter effect overcomes the former, resulting in a decrease of acid secretion. L-NAME unmasks the stimulation of acid secretion by suppressing the inhibitory pathway.