Role of endogenous nitric oxide and prostaglandin in duodenal bicarbonate response induced by mucosal acidification in rats.

Duodenal HCO3- secretion increases in response to mucosal acidification by luminal acid. Although this process is known to be mediated by endogenous prostaglandins (PGs), the role of nitric oxide (NO) in this response has been little studied. We examined the effects of indomethacin and N(G)-nitro-L-arginine methyl ester (L-NAME) on the acid-induced HCO3- secretion in the rat duodenum, together with those on PGE2 generation as well as luminal release of NO metabolites (NOx). 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 mM HCl. Mucosal acidification was performed by exposing the loop to 10 or 100 mM HCl for 10 min. Acidification of the duodenal mucosa stimulated the HCO3- secretion, with concomitant increase of mucosal PGE2 contents and luminal release of NOx, the response being much greater in case of 100 mM HCl. Indomethacin significantly inhibited the acid-induced HCO3- secretion as well as the PGE2 biosynthetic response, without influence on the NOx release. Pretreatment of the animals with L-NAME attenuated both the increase of mucosal PGE2 contents and luminal release of NOx following the acidification, resulting in a marked inhibition of the acid-induced HCO3- response, and these effects were significantly antagonized by coadministration of L-arginine. Duodenal HCO3- secretion was also increased by mucosal exposure to NOR-3 (a NO donor), with concomitant increase of PGE2 generation, but these effects were mitigated in the presence of indomethacin. In addition, the duodenal damage caused by mucosal perfusion with 100 mM HCl for 4 hr was markedly aggravated by pretreatment with L-NAME as well as indomethacin. These results suggest that both endogenous NO and PGs are involved in the mechanism for the acid-induced duodenal HCO3- secretion, and that NO may increase the HCO3- secretion by stimulating PG generation.

Stimulation by nitric oxide of gastric acid secretion in bullfrog fundic mucosa in vitro.

We examined the effect of NO on acid secretion in vitro using isolated preparations of Bullfrog stomach. The bullfrog fundic mucosa was bathed in unbuffered Ringer solution gassed with 100% O2 on the mucosal side and HCO3- Ringer's solution gassed with 95% O2/5% CO2 on the serosal side, and the acid secretion was measured at pH 5.0 using the pH-stat method and by adding 5 mM NaOH. Serosal addition of a NO donor NOR-3 (10(-5) approximately 10(-3) M: (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexnamine) caused an increase of acid secretion in a dose-dependent manner, the effect lasting about 1 hr and reaching a maximal level of 2-fold the basal values. The acid stimulatory effect of NOR-3 was mimicked by another NO donor SNAP (10(-3) mol/L: S-nitroso-O-N-acetyl-penicillamine) and markedly and markedly inhibited by prior administration of cimetidine (10(-5) mol/L) as well as compound 48/80 (the mast cell degranulator). Likewise, the increased acid response to NOR-3 was significantly mitigatd by pretreatment with carboxy-PTIO (a NO scavenger) or superoxide dismutase (SOD), but not by indomethacin or methylene blue (a guanylyl cyclase inhibitor). Neoither L-NAME, L-arginine nor dibutyryl guanosine-3',5'-cyclic monophosphate (dbcGMP) has any effect on the basal acid secretion. Serosal addition of NOR-3 caused a significant increase in the luminal release of histamine, and this response was inhibited by pretreatment with either compound 48/80, carboxy-PTIO or SOD. These results suggest that the NO donor increases gastric acid secretion in the isolated frog stomach in vitro, and this action is mediated by endogenous histamine released from mast cells, the process being cGMP-independent but requiring the presence of superoxide radicals. In addition, it was speculated that the histamine releasing action of NO may be due to peroxynitrite produced by NO and superoxide radicals.

Effect of ecabet disodium, a novel locally-acting antiulcer drug, on epithelial restitution following injury by hypertonic NaCl in bullfrog stomach in vitro.

BACKGROUND: The antiulcer drug ecabet 2Na (12-sulfodehydroabietic acid disodium salt) exhibits a gastroprotective activity, mainly through a local action, involving endogenous prostaglandins (PGs) and nitric oxide (NO). In the present study, we examined the effect of ecabet on the epithelial restitution of the bullfrog gastric mucosa in vitro following injury by hypertonic NaCl. METHODS: Bullfrog fundic mucosa was mounted in an Ussing chamber. The tissue injury was induced by exposure of the mucosa to 1.25 M NaCl for 5 min, and transmucosal potential difference (PD) and electrical resistance (R) were measured during a 4-hour test period. Ecabet (3-30 mg/ml) was added to the luminal solution for 10 min before or after NaCl, while 16,16-dimethyl PGE(2) (dmPGE(2): 1x 10(-6) M) or NOR-3 (a NO donor: 1 x 10(-4) M) was added to the nutrient solution 10 min before NaCl. RESULTS: Mucosal application of 1.25 M NaCl caused an immediate reduction of PD and R, followed by a gradual normalization, reaching about 70% of the pre-exposure levels within 4 h. Ecabet, added before NaCl, significantly expedited the recovery of PD and R in a concentration-dependent manner; this effect was mimicked by posttreatment with ecabet and significantly mitigated by prior addition of indomethacin (1 x 10(-5) M) or N(G)-nitro-L-arginine methyl ester (L-NAME: 1 x 10(-3) M). The epithelial restitution was also significantly promoted by serosal application of either dmPGE(2) or NOR-3. The mucosal exposure to ecabet significantly increased the luminal release of PGE(2) and NO metabolites, the effects being attenuated by indomethacin and L-NAME, respectively. The mucous secretion was increased by ecabet as well as dmPGE(2) and NOR-3, and the effect of ecabet was significantly suppressed by both indomethacin and L-NAME. The inhibitory effects of L-NAME on the ecabet action were all significantly antagonized by concurrent addition of L-arginine. CONCLUSION: These results suggest that ecabet significantly expedited the restitution following gastric surface cell injury, and this action is mediated by endogenous NO as well as PGs and may be functionally associated with an increase of mucous secretion.

Interactive roles of endogenous prostaglandin and nitric oxide in regulation of acid secretion by damaged rat stomachs.

BACKGROUND: The acid inhibitory mechanism in the damaged stomach is known to involve endogenous nitric oxide (NO) as well as prostaglandin (PG). AIM: To investigate the interaction between PG and NO in regulation of acid secretion in the stomach following damage. METHODS: Under urethane anaesthesia, a rat stomach was mounted in an ex vivo chamber and perfused with saline. Acid secretion, luminal PGE2, NO metabolites (NOx) and histamine output were measured before and after application of 20 mM taurocholate Na (TC) for 30 min, with or without pre-treatment with indomethacin and/or N(G)-nitro-L-arginine methyl ester (L-NAME). RESULTS: Exposure of the stomach to TC caused a decrease in acid secretion, with concomitant increase of both luminal NOx and PGE2. Either L-NAME or indomethacin reduced the decrease in acid secretion in response to TC, but only L-NAME allowed acid secretion to increase over basal values. L-NAME prevented the increase of luminal NOx after TC treatment, while indomethacin inhibited PGE2 release during and after exposure to TC. The increase in acid secretion in the presence of L-NAME was prevented when indomethacin was given concomitantly. TC treatment increased histamine output in the lumen, a process that was enhanced by L-NAME but reduced by indomethacin. CONCLUSIONS: Damage to the stomach increases both NO and PG in the lumen, and decreases acid secretion. Inhibiting NO production increases acid secretion in the damaged stomach, but only when PG biosynthesis is intact. It is assumed that endogenous PG has a dual role in the regulation of acid secretion in the damaged stomach: an inhibitory effect at the parietal cell and an excitatory effect probably through enhancing the release of mucosal histamine.

Modulation by endogenous nitric oxide of acid secretion induced by gastric distention in rats: enhancement by nitric oxide synthase inhibitor.

The mechanism underlying acid hypersecretion induced by gastric distention was investigated in rats, especially in relation to endogenous nitric oxide (NO). Under urethane anesthesia, rat stomach was distended by instillation of saline (1-10 ml) through the acute fistula that was provided through a pylorus. Gastric samples were collected every 1 h, and the acid secretion was measured by titration with 100 mM NaOH. Gastric acid secretion was increased by distention, and the degree of stimulation was dependent on the volume of saline instillation; a maximal response occurred with 6-ml instillation, which maintained the intraluminal pressure of about 20 cm H(2)O. The increased acid secretory response induced by distention was completely blocked by omeprazole and significantly mitigated by vagotomy, sensory deafferentation, atropine, or famotidine but markedly enhanced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). On the other hand, the enhanced acid response in the presence of L-NAME occurred in an L-arginine-sensitive manner and was almost totally abolished by vagotomy and sensory deafferentation as well as by atropine. Gastric distention increased the release of NO metabolites and histamine into the gastric lumen. The NO metabolite release in the distended stomach was significantly decreased by vagotomy or L-NAME, whereas the histamine output was decreased by vagotomy but increased by L-NAME in an L-arginine-sensitive manner, respectively. These results suggest that 1) gastric distention increases acid secretion, initially through the perception by sensory neurons of the mechanical stimulation and mainly through the efferent vagocholinergic pathway, with the process being modified by endogenous NO, and 2) this molecule, released in a vagal-dependent manner, exerts a negative influence on acid secretion, at least in part by suppressing histamine release from the histamine-containing cells.

Ulcerogenic and healing impairing actions of monochloramine in rat stomachs: effects of zinc L-carnosine, polaprezinc.

Effects of a novel zinc compound (polaprezinc), N-(3-aminopropionyl)-L-histidinato zinc, on the mucosal ulcerogenic and healing impairing responses induced by monochloramine (NH2Cl) were examined in rat stomach. Oral administration of NH2Cl (> 60 mM) produced severe hemorrhagic lesions in unanesthetized rat stomachs with a marked increase of thiobarbituric acid reactants (TBAR). Pretreatment of the animals with polaprezinc (3 approximately 30 mg/kg, p.o.) showed a dose-dependent inhibition against gastric ulcerogenic and TBAR responses induced by NH2Cl (120 mM). Likewise, mucosal exposure to NH4OH (60 mM) in urethane anesthetized stomachs made ischemic by bleeding from the carotid artery (1 ml per 100 g body w.t.) resulted in severe gastric lesions. This ulcerogenic response caused NH4OH plus ischemia was also attenuated by prior application of polaprezinc as well as taurine (25 mg/ml, 1 ml). On the other hand, the healing of gastric mucosal lesions induced by NH2Cl occurred more slowly than of ethanol-induced lesions, and the latter was significantly delayed by the repeated administration of NH2Cl. Polaprezinc (> 10 mg/kg, p.o.) given twice daily for 7 days not only accelerated the healing of NH2Cl-induced gastric lesions but also antagonized the delayed healing of ethanol-induced lesions in the presence of NH2Cl as well. Polaprezinc showed a scavenging action against NH2Cl in vitro. These results suggest that NH2Cl caused deleterious action on the healing of pre-existing acute lesions as well as irritating action to the mucosa in the rat stomach. Polaprezinc not only protects the stomach against injury caused by NH2Cl but also promotes healing of NH2Cl-induced gastric lesions as well as the delayed healing of ethanol-induced lesions caused by NH2Cl. Although the detailed mechanisms underlying these actions of polaprezinc remain unknown, they may be partly attributable to a scavenging action of this agent against NH2Cl.

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 (1x10(-4) and 3x10(-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 (1x10(-3) mol/l) and dbcGMP (1x10(-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 (5x10(-5) mol/l) and indomethacin (1x10(-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 (3x10(-4) mol/l) and dbcGMP (1x10(-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.

Roles of endogenous prostaglandins and nitric oxide in gastroduodenal ulcerogenic responses induced in rats by hypothermic stress.

We examined the roles of endogenous prostaglandins (PGs) and nitric oxide (NO) in the gastroduodenal ulcerogenic responses to hypothermic stress (28 approximately 30 degrees C) in anesthetized rats. Lowering body temperature provoked damage in the gastroduodenal mucosa, with an increase of gastric acid secretion and motility. These responses were completely abolished by bilateral vagotomy or atropine, while 16,16-dimethyl PGE2 decreased the mucosal ulcerogenic response with no effect on acid secretion. The non-selective COX inhibitors, indomethacin or aspirin, worsened these lesions with enhancement of gastric motility and no effect on acid secretion, while the selective COX-2 inhibitor NS-398 did not affect any of these responses. On the other hand, the non-selective NOS inhibitor L-NAME but not aminoguanidine (a relatively selective inhibitor of iNOS), significantly potentiated the acid secretory and mucosal ulcerogenic responses in the stomach but reduced the duodenal damage in response to hypothermia, the effects being antagonized by co-administration of L-arginine. Hypothermia itself decreased duodenal HCO3- secretion under both basal and mucosal acidification-stimulated conditions. Both indomethacin and aspirin further decreased the HCO3- response to the mucosal acidification, while L-NAME significantly increased the HCO3- secretion even under hypothermic conditions, similar to 16,16-dimethyl PGE2. These results suggest that 1) hypothermic stress caused an increase of acid secretion and motility as well as a decrease of duodenal HCO3-secretion, resulting in damage in both the stomach and duodenum, 2) the COX-1 but not COX-2 inhibition worsened these lesions by enhancing gastric motility and further decreasing duodenal HCO3- response, 3) the cNOS but not iNOS inhibition worsened gastric lesions by increasing acid secretion but decreased duodenal damage by increasing HCO3- secretion. Thus, it is assumed that the gastroduodenal ulcerogenic and functional responses to hypothermic stress are modified by cNOS/NO as well as COX-1/PGs.

Effects of endothelin-1 on duodenal bicarbonate secretion and mucosal integrity in rats.

Effects of endothelin-1 on gastric acid secretion, duodenal HCO3- secretion, and duodenal mucosal integrity were investigated in anesthetized rats, in comparison with those of TY-10957, a stable analogue of prostacyclin. A rat stomach mounted on an ex-vivo chamber or a proximal duodenal loop was perfused with saline, and gastric acid or duodenal HCO3- secretion was measured using a pH-stat method and by adding 100 mM NaOH or 10 mM HCl, respectively. Duodenal lesions were induced by mepirizole (200 mg/kg) given subcutaneously. Intravenous administration of endothelin-1 (0.6 and 1 nmol/kg) caused an increase of duodenal HCO3- secretion with concomitant elevation of blood pressure; this effect was antagonized by co-administrahon of BQ-123 (ET(A) antagonist; 3 mg/kg, i.v.) and significantly mitigated by vagotomy. Likewise, endothelin-1 caused a significant decrease in histamine-stimulated acid secretion, and this effect was also significantly antagonized by BQ-123. Although TY-10957 (10 and 30 mg/kg, i.v.) produced a temporal decrease of blood pressure, this agent caused not only an increase of duodenal HCO3- secretion, independent of vagal nerves, but also a decrease of acid secretion as well. In addition, both endothelin-1 and TY-10957 significantly prevented mepirizole-induced duodenal lesions at the doses that caused an increase of duodenal HCO3- secretion and a decrease of gastric acid secretion. These results suggest that endothelin-1 affects the duodenal mucosal integrity by modifying both gastric acid and duodenal HCO3- secretions, the effects being mediated by ET(A) receptors.

Involvement of PACAP in acid-induced HCO3- response in rat duodenums.

Pituitary adenylate cyclase activating polypeptides (PACAP) stimulate duodenal HCO3- secretion in the rat. The present study was performed to determine whether endogenous PACAP is involved in the mechanism of acid-induced HCO3- response in the duodenum, using a PACAP antagonist, PACAP6-27. Under urethane anaesthetised conditions, a duodenal loop that was made between the pylorus and the area just above the outlet of the common bile duct 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. Duodenal HCO3- secretion was significantly stimulated by i.v. administration of PACAP-27 (8 nmol kg-1) as well as vasoactive intestinal polypeptide (VIP: 8 nmol kg-1). The effect of PACAP-27 (8 nmol kg-1) was equivalent to that induced by prostaglandin E2 (300 micrograms kg-1, i.v.) and significantly suppressed by either PACAP6-27 (40 nmol kg-1, i.v.) or VIP antagonist (Ac-Tyr1, D-Phe2-VIP: 40 nmol kg-1, i.v.). These peptide antagonists suppressed duodenal HCO3- secretory response to VIP but did not have any effect on either basal or PGE2-stimulated HCO3- secretion. On the other hand, the duodenal mucosa responded to acidification by increasing HCO3- secretion in a indomethacin-sensitive manner, and this process was also significantly suppressed by both PACAP6-27 and VIP-antagonist. Duodenal damage induced by acid perfusion (100 mM HCl for 4 h) was significantly worsened by PACAP6-27, VIP antagonist as well as indomethacin at the doses that suppressed acid-induced HCO3- secretion. These findings suggest that PACAP may play a role in local modulation of the duodenal mucosal integrity, by mediating the HCO3- secretory response induced by mucosal acidification.