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.

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.

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.

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 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.

Initial arthritic lesions induced by immunization with type II collagen in Lewis rats.

Lewis rats were immunized with an intradermal injection of type II collagen to study the time course of arthritic lesions. Serum type II collagen antibody was detected 9 days after immunization. Increased paw volume in the hind limbs was noted on day 11. Histopathologically, proliferation of synovial lining cells was observed on day 11 and typical lesions similar to those of human rheumatoid arthritis were noted on day 18.

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 growth factors on acid-induced damage to rat gastric epithelial cells.

We previously established a model of cell damage induced by an acidified medium in the rat gastric epithelial cell line RGM1. Treatment of the cells with epidermal growth factor (EGF, 1-10 ng/ml) for 4 h prevented acid-induced cell damage in a concentration-dependent manner. In contrast, basic fibroblast growth factor (1-10 ng/ml) and platelet-derived growth factor BB (1-10 ng/ml) had no effect on cell damage. EGF did not affect DNA synthesis by the cells for 4 h. Pretreatment of the cells with cycloheximide (10 micrograms/ml) for 1.5 h before EGF treatment significantly attenuated the cytoprotective effect of EGF by > 50%. Replacement of Na+ with K+ in the acidified medium totally abolished the effect of EGF. Co-incubation with amiloride (1 mM) had no influence on the protective effect of EGF. These results indicate that the cytoprotective effect of EGF appears to involve both the activation of amiloride-resistant Na+/H+ exchangers and the synthesis of a new protein related to Na+/H+ exchangers.

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.

Characterization of a novel cell damage model induced by acid and pepsin using rat gastric epithelial cells: protective effect of sucralfate.

We have established a new model for rat gastric epithelial cell (RGM1) damage caused by both acid and pepsin. Exposure of RGM1 to an acidified medium (pH 3.5-5.0) for 10-50 min decreased cell viability in a time- and pH-dependent manner. Pepsin (0.5-1.0 mg/mL) at pH 4.5 potentiated cell damage in a concentration-dependent manner. Based on these results, two types of cell damage models caused by incubation of cells at pH 4.0 and with pepsin (0.75 mg/mL) at pH 4.5 for 30 min, respectively, were established. The intracellular pH (pHi) gradually decreased with a decrease in medium pH and an increase in exposure time. At pH < or = 4.0, pHi reached approximately pH 6.3. Pepsin at pH 4.5 caused a further reduction in pHi compared with the acidified medium alone. Pepsin pre-incubated with pepstatin did not induce any cell damage. Pretreatment with sucralfate (0.1-3 mg/ mL) for 2 h significantly prevented cell damage caused at both pH 4.0 and with pepsin at pH 4.5 in a concentration-dependent manner. Sucralfate (3 mg/mL) significantly prevented the reduction in pHi at pH 4.0 or with pepsin at pH 4.5. 16,16-Dimethyl prostaglandin E2 (30 micrograms/mL) had no effect on either cell damage or pHi. These cell damage models involving RGM1 are useful for studying the mechanism underlying cell damage and for the screening of cytoprotective drugs.

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.

Induction of duodenal ulcers in rats under water-immersion stress conditions. Influence of stress on gastric acid and duodenal alkaline secretion.

We investigated the influence of stress on gastric acid and duodenal HCO3- secretion in rats, and examined whether duodenal ulcers develop in rats under stress conditions in the absence or presence of acid hypersecretion caused by histamine. Either restraint alone or restraint plus water-immersion stress induced lesions in the stomach but not in the duodenum. However, subcutaneous administration of histamine dihydrochloride (40 mg/kg every 2.5 h for a total of three times) to stressed rats produced macroscopically visible damage in the proximal duodenum as well as in the stomach within 8 h of exposure to stress, and the incidence of duodenal lesions was 100% in the water-immersion group (24.8 +/- 3.8 mm2, n = 8). Histamine alone had no effect on either region. These lesions in the duodenum caused by water immersion plus histamine were prevented by subcutaneously administered cimetidine (30, 100 mg/kg) or 16,16-dimethyl prostaglandin E2 (10, 30 micrograms/kg) in a dose-related manner, but not by atropine (1, 3 mg/kg). Restraint decreased acid secretion by 40%, and additional water immersion restored the decreased acid secretion to normal levels. Basal duodenal HCO3- secretion was decreased to about 70% of normal values (5-6 microEq/15 min) in the restraint group, and after additional water immersion further declined to the values of 1.5-2 microEq/15 min. An increase of HCO3- secretion caused by acid was significantly inhibited by water immersion but not by restraint. Histamine significantly increased acid secretion but did not affect duodenal HCO3- secretion. In the rats treated with both water immersion and histamine, acid secretion was significantly reduced by either cimetidine (100 mg/kg) or 16,16-dimethyl prostaglandin E2 (30 micrograms/kg), whereas duodenal HCO3- secretion was significantly increased by 16,16-dimethyl prostaglandin E2. Atropine had little effect on either acid or HCO3- secretion. These results suggest that exposure of rats to stress decreases duodenal HCO3- secretion and increases the susceptibility of the mucosa to acid emptied from the stomach, thereby inducing duodenal ulcers if acid hypersecretion is concomitantly present.

Duodenal ulcers induced by indomethacin plus histamine in the dog. Involvement of the impaired duodenal alkaline secretion in their pathogenesis.

Mongrel dogs of either sex, weighing 14.0 +/- 0.7 kg, were given indomethacin orally in a dose of 70 mg/dog, and they were deprived of food thereafter. Twelve hours later, the animals were given histamine-2HCl intramuscularly 4 times every hour in a dose of 40 or 80 micrograms/kg. Indomethacin followed by histamine treatment produced well-defined ulcers in the proximal duodenum within 18 h with a few lesions in the stomach, although either of these agents alone did not induce any damage in the mucosa. Both the severity and incidence of the duodenal lesions were increased dose-dependently by histamine; the lesion index was 38.8 +/- 8.4 mm2 (n = 7) with an incidence of 100% at the dose of 80 micrograms/kg of histamine. The duodenal lesions mostly consisted of 2-4 round or elongated lesions which penetrated to the muscularis mucosae in some cases (42.8%). Histamine caused a marked increase in acid secretion in dogs with a vagally innervated total pouch, while indomethacin significantly inhibited the increased alkaline secretion caused by acid (50 mM HCl for 10 min) in the duodenal pouch (10 cm distal to the pylorus). Both cimetidine (20 mg/kg) and 16,16-dimethyl prostaglandin E2 (3 micrograms/kg), given subcutaneously, prevented these lesions in the duodenum as well as in the stomach by inhibiting acid secretion and/or increasing duodenal alkaline secretion. These results suggest that (a) indomethacin consistently produced ulcers in the duodenum of the dog when acid hypersecretion was induced by histamine, and (b) an impaired duodenal alkaline secretion may be an important pathogenetic element in this model.

Effects of prostaglandin biosynthesis inhibitors and ouabain on duodenal mucosa and HCO3- secretion in rats.

A single injection (s.c.) of prostaglandin biosynthesis inhibitors such as indomethacin (5 mg/kg), aspirin (200 mg/kg) and quinacrine (100 mg/kg) or a Na+.K+ ATPase inhibitor such as ouabain (10 mg/kg) significantly reduced the adaptive increase of HCO3- output caused by acid in the duodenum of anesthetized rats. These agents had no effect on basal duodenal HCO3- secretion and histamine-stimulated gastric acid secretion. Either of these agents, when given alone, had no effect on the duodenal mucosa of conscious rats, but produced damage in the proximal duodenum within 8 hr when given together with histamine (40 mg/kg X 3, s.c., every 2.5 hr). A significant relationship was found between the degrees of inhibition of acid-induced HCO3- output and the severity of duodenal lesions induced by these drugs (r = 0.8620, P less than 0.01). These results suggest that an impairment of the mechanisms related to acid-induced HCO3- secretion may be particularly relevant to the pathogenesis of duodenal lesions.

Cytoprotective action of histamine against 0.6 N HCl-induced gastric mucosal injury in rats; comparative study with adaptive cytoprotection induced by exogenous acid.

We examined the effects of histamine 2HCl (a stimulator of endogenous acid production) and exogenous acid on transmucosal potential difference (PD) and pH of anesthetized rat stomachs, in order to investigate the mechanism underlying the protective action of histamine against 0.6 N HCl-induced gastric mucosal injury in conscious rats. Subcutaneously administered histamine (3-20 mg/kg) dose-dependently produced a decrease in the PD and pH, and it reduced the severity of gastric mucosal injury caused by 0.6 N HCl. Both indomethacin (5 mg/kg, s.c.) and cimetidine (100 mg/kg, s.c.) completely reversed the protection afforded by histamine (20 mg/kg), although the decreased PD and pH responses were unaffected or inhibited, respectively, by indomethacin or cimetidine. Protective action of histamine was also partially mitigated by omeprazole (30 mg/kg, s.c.) which completely abolished histamine-induced acid secretion. On the other hand, exposure of the stomach for 10 min to exogenous acid (0.1-0.35 N HCl) caused a PD reduction and an increase of pH, in a concentration-related manner. The injury caused by 0.6 N HCl was prevented by prior exposure to these low concentrations of HCl, and the degrees of inhibition were associated with the concentration of HCl and the magnitude of PD reduction caused by HCl. The pretreatment with indomethacin, but not cimetidine or omeprazole, significantly antagonized the increased pH and mucosal protection induced by 0.35 N HCl. These results suggest that histamine protected the gastric mucosa against 0.6 N HCl-induced injury by two different ways, mediated with endogenous prostaglandins, (a) mainly through stimulation of H2-receptors and (b) partly through adaptive cytoprotection induced by acid.

Pathogenesis of digitoxin-induced duodenal ulcers in pregnant rats. Roles of gastric acid and duodenal alkaline secretion.

Late-stage pregnant rats (day 17) had higher rates of gastric acid secretion (45-55 mu eq/15 min) as compared to nonpregnant and middle-stage pregnant (day 10) rats (20-25 mu eq/15 min). In contrast, basal rates of duodenal alkaline secretion were significantly lower (2-3 mu eq/15 min) in pregnant rats (day 10 and 17) than those in nonpregnant rats (approximately 5 mu eq/15 min), although the duodenal mucosa responded to acid with a significant rise in HCO3- output in these three groups of rats. In pregnant rats (day 17), a single injection of digitoxin, a Na+ K+-ATPase inhibitor (10 mg/kg, subcutaneously), had no effect on basal acid and alkaline secretions, but significantly blocked the acid-induced HCO3- secretion for more than 18 hr from 6 hr after administration. This drug, when given once daily for four days (10 mg/kg, subcutaneously), produced well-defined ulcers in the proximal duodenum with few lesions in the stomach of female rats, and the severity and incidence were significantly higher in late-stage pregnant rats than in the other two groups of rats. Following repeated administration of digitoxin (10 mg/kg) to late-stage pregnant rats (days 17-20), acid secretion significantly declined after two days of treatment, while the acid-induced HCO3- secretion was significantly attenuated after one day of treatment and remained inhibited during the whole period. These results suggest that an impairment of the mechanisms related to acid-induced HCO3- secretion may be associated with the induction of duodenal ulcers caused by digitoxin in female rats, and the high incidence of these ulcers in late-stage pregnant rats may be due to acid hypersecretion.

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.

16,16-Dimethyl prostaglandin E2 aggravates gastric mucosal injury induced by histamine in rats. Possible role of the increased mucosal vascular permeability.

Histamine dihydrochloride (40 or 80 mg/kg, dissolved in 10% gelatin) subcutaneously administered to fasted rats induced few lesions in the gastric mucosa within 4 h. Pretreatment with subcutaneously administered 16,16-dimethyl prostaglandin E2 (dmPGE2; greater than or equal to 10 micrograms/kg) dose-dependently worsened mucosal injury induced by histamine, mostly with severe hemorrhage in the corpus mucosa along the greater curvature, although dmPGE2 alone did not induce any macroscopic damage. The mucosal vascular permeability as measured using Evans blue was slightly but significantly augmented by either dmPGE2 (30 micrograms/kg) or histamine (80 mg/kg) alone, but was markedly increased by histamine in the presence of dmPGE2. The increased vascular permeability occurred within 2 h, and preceded the appearance of hemorrhagic mucosal injury. Both the mucosal injury and the increased vascular permeability caused by histamine (80 mg/kg) in the presence of dmPGE2 (30 micrograms/kg) were significantly reduced by pretreatment with tripelennamine (30 mg/kg) and prednisolone (3 mg/kg), but not affected by atropine sulfate, cimetidine, methysergide, or indomethacin. The stimulation of acid secretion caused by histamine was significantly inhibited by dmPGE2 (30 micrograms/kg). Repeated administration of histamine (40 or 80 mg/kg) in the same area of the stomach in the presence of dmPGE2 (30 micrograms/kg), once or twice daily for 4 days to fed rats, induced more pronounced damage than single-dose treatment. These results suggest that dmPGE2 may aggravate gastric mucosal injury induced by histamine in rats probably due to potentiation of the increased vascular permeability caused by histamine through stimulation of H1-receptors.

Histopathological study of arthritic lesions induced by immunization with type II collagen in Lewis rats.

Lewis rats were immunized with an intradermal injection of type II collagen and autopsied 40 days later for histopathological examination of the limb joints. An increased in paw volume in the hind limbs was observed from 11 days after immunization until autopsy. The joints of the hind limbs were more frequently and more severely affected than those of the fore limbs. The tarsal joints were especially affected, with a 100% morbidity rate, and more frequently exhibited the most advanced stage of lesions.

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.