Effect of N-methyl-d-aspartate receptor blockade on neuronal plasticity and gastrointestinal transit delay induced by ischemia/reperfusion in rats.

Intestinal ischemia impairs gastrointestinal motility. The aims of this study were to investigate the effect of intestinal ischemia on gastrointestinal transit and on the expression of enteric transmitters in the rat, and whether the glutamate N-methyl-d-aspartate receptors influence these effects. Ischemia (1 h), induced by occluding the superior mesenteric artery, was followed by 0 or 24 h of reperfusion. Normal and sham-operated rats served as controls. Serosal blood flow was measured with laser Doppler flow meter. Gastrointestinal transit was measured as time of appearance of a marker in fecal pellets. Immunohistochemistry was used to evaluate the number of neurons immunoreactive for neuronal nitric oxide synthase (NOS) or vasoactive intestinal polypeptide and the density of substance P immunoreactive fibers in the myenteric plexus. The N-methyl-d-aspartate receptors antagonist, (+)-5-methyl-10,11-dihydro-5HT-[a,b] cyclohepten-5,10-imine (MK-801) (1 mg/kg i.v.) or the NOS inhibitor, N-nitro-l-arginine (10 mg/kg i.v.) was administered prior to ischemia. Serosal blood flow was decreased by 70% during ischemia, but it was not altered in sham-operated rats. Gastrointestinal transit was significantly prolonged in ischemic/reperfused rats compared with controls. There was a significant increase in the number of vasoactive intestinal polypeptide and neuronal nitric oxide synthase immunoreactive neurons, and a marked decrease of substance P immunoreactive fibers in ischemia followed by 24 h of reperfusion animals compared with controls. These alterations were not observed in ischemia without reperfusion. A significant delay of gastrointestinal transit and increase of vasoactive intestinal polypeptide neurons were also observed in sham-operated rats. The changes in transmitter expression and gastrointestinal transit in ischemic/reperfused rats were prevented by pre-treatment with the NOS inhibitor, N-nitro-l-arginine or the N-methyl-d-aspartate receptors antagonist, MK-801. This study suggests an involvement of the glutamatergic system and its interaction with nitric oxide in intestinal ischemia/reperfusion. Ischemia/reperfusion might induce local release of glutamate that activates N-methyl-d-aspartate receptors leading to increased production of nitric oxide and adaptive changes in enteric transmitters that might contribute to gastrointestinal dysmotility.

Regional differences of H+, HCO3-, and CO2 diffusion through native porcine gastroduodenal mucus.

Gastroduodenal mucus may play a critical role in defending the epithelium from luminal acid and in the creation of a microenvironment suitable for H. pylori. We measured transmucus permeation of H+, HCO3-, and CO2 with an in vitro perfusion chamber through freshly harvested or partially purified porcine gastric mucin. pH and CO2 concentrations were measured with selective ion electrodes; HCO3- and CO2 concentrations were derived. Viscosity was measured by rotational microviscometry. Mucin viscosity was directly related to concentration. There was a large variation in viscosity among native mucus from antrum, corpus, and duodenum. The highest viscosity was found in the antral mucus; duodenal mucus had the lowest. Diffusion coefficients of duodenal mucus for H+ and HCO3- were significantly lower than those from corpus and antrum. CO2 diffusion coefficients were invariant. In conclusion, despite large variations in viscosity, antral and corpus gastric mucus were similar in terms of ion diffusion. Surprisingly, the low viscosity duodenal mucus was a more potent barrier to ion diffusion than was gastric mucus. Consequently, duodenal mucus may play a more important role in inhibiting ion diffusion than its gastric counterpart.

Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

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.

Acute adaptive cellular base uptake in rat duodenal epithelium.

We studied the role of duodenal cellular ion transport in epithelial defense mechanisms in response to rapid shifts of luminal pH. We used in vivo microscopy to measure duodenal epithelial cell intracellular pH (pH(i)), mucus gel thickness, blood flow, and HCO secretion in anesthetized rats with or without the Na(+)/H(+) exchange inhibitor 5-(N,N-dimethyl)-amiloride (DMA) or the anion transport inhibitor DIDS. During acid perfusion pH(i) decreased, whereas mucus gel thickness and blood flow increased, with pH(i) increasing to over baseline (overshoot) and blood flow and gel thickness returning to basal levels during subsequent neutral solution perfusion. During a second brief acid challenge, pH(i) decrease was lessened (adaptation). These are best explained by augmented cellular HCO uptake in response to perfused acid. DIDS, but not DMA, abolished the overshoot and pH(i) adaptation and decreased acid-enhanced HCO secretion. In perfused duodenum, effluent total CO(2) output was not increased by acid perfusion, despite a massive increase of titratable alkalinity, consistent with substantial acid back diffusion and modest CO(2) back diffusion during acid perfusions. Rapid shifts of luminal pH increased duodenal epithelial buffering power, which protected the cells from perfused acid, presumably by activation of Na(+)-HCO cotransport. This adaptation may be a novel, important, and early duodenal protective mechanism against rapid physiological shifts of luminal acidity.

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.

Prostaglandin E inhibits indomethacin-induced gastric lesions through EP-1 receptors.

BACKGROUNDS AND AIMS: We examined the effect of various prostaglandin E (PGE) analogs specific to EP receptor subtypes on indomethacin-induced gastric lesions in rats and investigated which EP receptor subtype is involved in the protective action of PGE(2) using EP-receptor knockout mice. METHODS: Gastric lesions were induced by subcutaneous administration of indomethacin (35 mg/kg). Gastric motility was measured using a balloon method, while neutrophil chemotaxis determined using a Boyden chamber. RESULTS: Indomethacin-induced gastric lesions were significantly prevented by PGE(2) as well as atropine, and the former effect was mimicked by sulprostone (EP(1)/EP(3)) and 17-phenyl PGE(2) (EP(1)) and antagonized by an EP(1) antagonist, ONO-AE-829. Neither butaprost (EP(2)), ONO-NT-012 (EP(3)) nor 11-deoxy PGE(1) (EP(3)/EP(4)) showed any protection on the lesions. Indomethacin caused a marked increase in gastric motility; the response preceded the onset of lesions and was inhibited by atropine as well as PGE derivatives acting as EP(1) receptors. Neutrophil chemotaxis was inhibited by PGE(2), butaprost and slightly by 11-deoxy PGE(1), but not by either 17-phenyl PGE(2), ONO-NT-012 or atropine. In addition, indomethacin caused damage similarly in both wild-type and knockout mice lacking EP(1) or EP(3) receptors, yet the protective action of PGE(2) was observed in wild-type and EP(3) receptor knockout mice but totally disappeared in mice lacking EP(1) receptors. CONCLUSION: PGE(2) inhibits indomethacin-induced gastric lesions, through EP(1) receptors, and this effect may be functionally associated with inhibition of gastric motility but not of neutrophil activation/migration.

Sensory pathways and cyclooxygenase regulate mucus gel thickness in rat duodenum.

We previously showed that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide. We then tested the hypothesis that similar factors regulate duodenal mucus gel thickness. Gel thickness was optically measured using in vivo microscopy in anesthetized rats. Duodenal mucosae were superfused with pH 7.0 buffer with vanilloid receptor agonist capsaicin, bradykinin, or PGE(2) injection or were challenged with pH 2.2 solution, with or without the vanilloid antagonist capsazepine, human CGRP-(8-37), N(G)-nitro-L-arginine methyl ester, and indomethacin. Other rats underwent sensory ablation with high-dose capsaicin pretreatment. Acid, bradykinin, capsaicin, and PGE(2) all quickly thickened the gel. Antagonism of vanilloid and CGRP receptors, inhibition of nitric oxide synthase, and sensory deafferentation delayed gel thickening, suggesting that the capsaicin pathway mediated the initial burst of mucus secretion that thickened the gel. Indomethacin abolished gel thickening due to acid, bradykinin, and capsaicin. Inhibition of gel thickening by indomethacin in response to multiple agonists suggests that cyclooxygenase activity is essential for duodenal gel thickness regulation. Duodenal afferent neural pathways play an important role in the modulation of cyclooxygenase-mediated physiological control of gel thickness.

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.

Stimulation by nitric oxide of HCO3- secretion in bullfrog duodenum in vitro--roles of cyclooxygenase-1 and prostaglandins.

The effect of nitric oxide (NO) on HCO3- secretion was examined in vitro using the isolated preparation of bullfrog duodenum, in relation to cyclooxygenase (COX) isozymes and endogenous prostaglandins (PGs). The tissue 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. The HCO3- secretion was measured by a pH-stat method using 10 mM HCl as the titrant to keep the mucosal pH at 7.4. NOR-3 [(+/-)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamine] was used as a NO donor and added to the serosal solution. To analyze the NOR-3 action, the effects of dibutyryl guanosine-3', 5'-cyclic monophosphate (dbcGMP), methylene blue, indomethacin (nonselective COX-inhibitor) and NS-398 (selective COX-2 inhibitor) on the HCO3- response were also examined. NOR-3 (1 x 10(-4) and 3 x 10(-4) M) caused an increase of HCO3- secretion in a dose-dependent manner, reaching the level of 2.5 times greater than basal values at 2 hr later. Likewise, dbcGMP (1 x 10(-3) M) also caused a significant increase of the duodenal HCO3- secretion. The HCO3- stimulatory action of NOR-3 was significantly attenuated by methylene blue (5 x 10(-5) M) and indomethacin (1 x 10(-5) M) but not by NS-398 (1 x 10(-5) M), and indomethacin also suppressed the HCO3- response to dbcGMP. The serosal release of PGE2 was significantly increased by both NOR-3 and dbcGMP, and these responses were inhibited by indomethacin but not NS-398. These results suggest that NO increases HCO3- secretion in Bullfrog duodenum in vitro, and this action is dependent on cGMP-related COX-1 activation and mediated by PGs.

Impaired duodenal bicarbonate secretion and mucosal integrity in mice lacking prostaglandin E-receptor subtype EP(3).

BACKGROUND & AIMS: To examine the involvement of EP(3) receptors in physiological regulation of duodenal HCO(3)(-) secretion, we disrupted the gene encoding EP receptors in mice by homologous recombination and evaluated acid-induced HCO(3)(-) secretion, which is physiologically important in the mucosal defense against acid injury, using EP(1)- and EP(3)-receptor knockout mice. METHODS: The experiments were performed in the following 3 groups of mice after 18 hours of fasting: wild-type [WT (+/+)] mice, EP(1)-receptor knockout [EP(1) (-/-)] mice, and EP(3)-receptor knockout [EP(3) (-/-)] mice. Under urethane anesthesia, the proximal duodenal loop was perfused with saline that was gassed with 100% O(2), heated at 37 degrees C, and kept in a reservoir, and HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 5 mmol/L HCl. RESULTS: The duodenum of WT (+/+) mice increased HCO(3)(-) secretion in response to luminal perfusion of prostaglandin E(2) and forskolin as well as mucosal acidification. The latter effect was significantly inhibited by prior administration of indomethacin. HCO(3)(-) response to acid was observed in EP(1) (-/-) mice but disappeared totally in EP(3) (-/-) animals, although the acidification increased mucosal PGE(2) generation by similar degrees in all groups. The HCO(3)(-) stimulatory action of PGE(2) was also absent in EP(3) (-/-) but not EP(1) (-/-) mice, but forskolin effect was observed in both groups of animals, similar to WT (+/+) mice. Perfusion of the duodenum with 20 mmol/L HCl for 4 hours caused severe damage in EP(3) (-/-) mice and WT (+/+) animals pretreated with indomethacin, but not in EP(1) (-/-) mice. CONCLUSIONS: The presence of EP(3)-receptors is essential for maintaining duodenal HCO(3)(-) secretion and mucosal integrity against luminal acid.

Effect of monochloramine on recovery of gastric mucosal integrity and blood flow response in rat stomachs--relations to capsaicin-sensitive sensory neurons.

Gastric mucosal blood flow (GMBF) response and the recovery of gastric mucosal integrity were investigated in anesthetized rat stomachs after damage by monochloramine (NH2Cl), in comparison with 20 mM taurocholate Na (TC). A rat stomach was mounted in an ex-vivo chamber, and the mucosa was exposed to 50 mM HCl during a test period. Mucosal application of 20 mM TC for 10 min caused a marked reduction of transmucosal potential difference (PD), but the PD recovered rapidly without development of gross lesions 90 min later. In contrast, the exposure of the mucosa to NH2Cl (5 to approximately 20 mM) produced a concentration-dependent decrease in gastric PD, and the values remained lowered even 90 min after removal of the agent, resulting in severe hemorrhagic damage in the stomach. TC caused a considerable H+ back-diffusion, followed by an increase in the GMBF. In the mucosa damaged by NH2Cl, such GMBF responses were not observed, except for the temporal increase during the exposure, although similar degrees of H+ back-diffusion were observed following NH2Cl treatment. In addition, the prior exposure of the mucosa to NH2Cl significantly attenuated gastric hyperemic response induced by capsaicin but not by misoprostol (a PGE1 derivative) or NOR-3 (a NO donor). Chemical ablation of capsaicin-sensitive sensory neurons had no effect on the PD reduction caused by TC but totally attenuated the GMBF response, resulting in hemorrhagic damage in the stomach. These results suggest that NH2Cl delayed the recovery of the mucosal integrity in the stomach after damage, and this effect may be attributable, at least partly, to the impairment of gastric hyperemic response associated with H+ back-diffusion, probably due to dysfunction of capsaicin-sensitive sensory neurons.

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.

Prostaglandin E receptor subtypes involved in stimulation of gastroduodenal bicarbonate secretion in rats and mice.

We investigated prostaglandin E (EP) receptor subtypes responsible for the HCO3- stimulatory action of prostaglandin E2 (PGE2) in the gastroduodental mucosa, by examining the effects of various prostanoids with subtype specific EP receptor agonists in rats and those of PGE2 in knockout mice lacking EP1 or EP3 receptors. In rats, gastric HCO3- secretion was stimulated by i.v. administration of PGE2, 17-phenyl PGE2 the selective EP1 agonist as well as sulprostone the EP1 and EP3 agonist, but was not affected by other EP agonists such as butaprost the selective EP2 agonist, ONO-NT-012 the selective EP3 agonist or 11-deoxy PGE1 the EP3 and EP4 agonist. In contrast, the HCO3- secretion in rat duodenums was stimulated by PGE2, sulprostone, ONO-NT-012 as well as 11-deoxy PGE1 but not affected by either 17-phenyl PGE2 or butaprost. The HCO stimulatory effect of sulprostone in the stomach was significantly inhibited by ONO-AE-829, the selective EP1 antagonist. On the other hand, PGE2 applied topically to the mucosa for 10 min caused a dose-dependent increase of HCO3- secretion in both the stomach and duodenum of wild-type mice. The HCO3- stimulatory action of PGE2 in the stomach was also observed dose-dependently in knockout mice lacking EP3-receptors but was absent in EP1-receptor knockout mice, while the stimulatory effect in the duodenum was observed in EP1-receptor knockout mice, similar to wild-type animals, but not in knockout mice lacking EP3-receptors. These results indicate that PGE2 stimulates HCO3- secretion via different EP receptor subtypes in the stomach and duodenum; the former is mediated by EP1-receptors, while the latter mediated by EP3-receptors.

Epidermal growth factor protects rat epithelial cells against acid-induced damage through the activation of Na+/H+ exchangers.

We examined the effect of epidermal growth factor (EGF) on acid-induced cell damage in rat gastric epithelial cells (RGM1) and investigated the mechanisms of this effect. Cells were incubated with EGF for 5, 15, 30, and 60 min, and then immersed in an acidified medium (pH 4.0) for 30 min to induce cell damage. EGF prevented cell damage in a concentration- and time-dependent manner. EGF reduced the effects of the acidified medium on the cells, preventing the reduction of intracellular pH. Replacement of Na+ with K+ in the acidified medium canceled the effect of EGF. Indomethacin and W-7 (a Ca-calmodulin inhibitor) did not alter the protective effect of EGF. In contrast, genistein (a tyrosine kinase inhibitor), amiloride (a Na+/H+ exchangers I and II inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) significantly decreased the effect of EGF. Expression of Na+/H+ exchangers type I and type II was confirmed by reverse transcription-polymerase chain reaction. Our results demonstrate that EGF prevents acid-induced cell damage, most likely through the activation of a Na+/H+ exchanger II via phosphatidylinositol 3-kinase.

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.

Effects of transforming growth factor alpha and hepatocyte growth factor on acid-induced damage in rat gastric epithelial cells.

We examined the effects of transforming growth factor alpha (TGF-alpha) and hepatocyte growth factor (HGF) on acid-induced damage in a rat gastric epithelial cell line. Pretreatment of cells for 30 min with TGF-alpha, but not HGF, significantly prevented the acid-induced cell damage in a concentration-dependent manner. Genistein significantly reduced the protective effect of TGF-alpha. DNA synthesis in RGM1 cells was increased when the cells were incubated with TGF-alpha and HGF for 24 hr. We conclude that the protective effect of TGF-alpha against acid-induced damage seems to be caused by the activation of Na+/H+ exchangers and not by enhanced DNA synthesis.

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.

Effects of sucralfate and its components on acid- and pepsin-induced damage to rat gastric epithelial cells.

We have established models of cell damage induced by acid and pepsin using rat gastric epithelial cells (RGM1). In the present study, the effects of aluminum hydroxide [Al(OH)3] and potassium sucrose octasulfate (KSOS), which are components of sucralfate, and sucralfate on cell damage and peptic activity of pepsin were examined. Pretreatment of cells with sucralfate (0.1-3 mg/ml) or Al(OH)3 (0.1-1 mg/ml) for 2 hr prevented both acid- (pH 4.0) and pepsin- (pH 4.5) induced cell damage. However, KSOS (0.1-1 mg/ml) did not show any effects on two different types of cell damage. The peptic activity of pepsin at pH 4.5 was about 10% of that at pH 2.0. Sucralfate and KSOS slightly inhibited peptic activity at pH 4.5. Al(OH)3 inhibited peptic activity by approximately 50%; however, no concentration-dependent pattern was observed. Pepstatin (0.003-0.1 mg/ml), a specific inhibitor of pepsin, inhibited the peptic activity in a concentration-dependent manner. Here, we confirmed that sucralfate and Al(OH)3 have cytoprotective effects against acid- and pepsin-induced cell damage. The mechanism behind the cytoprotective effects of sucralfate seems to relate to adhesion of the cell surface and neutralization of hydrogen ion by aluminum that prevents the penetration of hydrogen ions into the cells.

Cytoprotective effect of epidermal growth factor on acid- and pepsin-induced damage to rat gastric epithelial cells: roles of Na+/H+ exchangers.

We have previously established a cell damage model, with damage induced by either acid or pepsin treatment for 30 min, involving a rat gastric epithelial cell line (RGM1). In the present study, pretreatment of cells with epidermal growth factor (EGF; 0.1-10 ng/mL) or sucralfate (0.1-3 mg/mL) for 4 h prevented such cell damage in a concentration-dependent manner. Protection of cells by these drugs was not affected by pretreatment with indomethacin (10(-5) mol/L) for 4 h. Removal of Na+, but not Ca2+, from the acidified medium totally abolished the inhibitory effect of EGF, but not that of sucralfate. Genistein (a tyrosine kinase inhibitor) apparently reduced the inhibitory effect of EGF. DNA synthesis by RGM1 cells did not increase when cells were incubated with EGF for 4 h. We conclude that both EGF and sucralfate protect RGM1 cells from acid- and pepsin-induced damage and that the mechanism of protection by EGF against acid-induced damage seems to be via activation of Na+/H+ exchangers.