Hideki Ukawa

Impaired duodenal bicarbonate secretion and mucosal integrity in mice lacking prostaglandin E–receptor subtype EP3

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.

Effects of Cyclooxygenase-2 Selective and Nitric Oxide-Releasing Nonsteroidal Antiinflammatory Drugs on Mucosal Ulcerogenic and Healing Responses of the Stomach

Effects of selective cyclooxygenase-2 (COX-2)inhibitors (NS-398) and nitric oxide (NO)-releasingaspirin (NO-ASA) on gastric ulcerogenic and healingresponses were examined in comparison with nonselective COX inhibitors such as indomethacin and aspirin(ASA). Hypothermic stress (28-30°C, 4 hr) inducedgastric lesions in anesthetized rats with an increase ofacid secretion. The lesions induced by hypothermic stress were markedly worsened by subcutaneousadministration of both indomethacin and ASA but were notaffected by either NS-398 or NO-ASA, although theincreased acid secretion during hypothermia was not affected by any of the drugs. On the otherhand, the healing of gastric ulcers induced in mice bythermal cauterization (70°C, 15 sec) wassignificantly delayed by daily subcutaneousadministration of indomethacin and ASA as well as NS-398, but not by NO-ASA.COX-2 mRNA was not detected in the intact mucosa but waspositively expressed in the ulcerated mucosa, mostpotently on day 3 after ulceration. Prostaglandin contents in the intact mouse stomach werereduced by indomethacin, ASA, and NO-ASA, while theincreased prostaglandin generation in the ulceratedmucosa was inhibited by all drugs including NS-398.After subcutaneous administration of NO-ASA topylorus-ligated rats and mice, high amounts ofNOx were detected in both the gastriccontents and serum. In addition, both NS-398 and NO-ASAshowed an equipotent antiinflammatory effect againstcarrageenan-induced paw edema in rats as compared withindomethacin and ASA. These results suggest that bothindomethacin and ASA not only increased the mucosalulcerogenic response to stress but impaired the healingresponse of gastric ulcers as well. The former actionwas due to inhibition of COX-1, while the latter effectwas accounted for by inhibition of COX-2 and was mimicked by the COX-2-selective inhibitorNS-398. NO-ASA, although it inhibited both COX-1 andCOX-2 activity, had no deleterious effects on gastriculcerogenic and healing responses.

The roles of prostaglandin E receptor subtypes in the cytoprotective action of prostaglandin E2 in rat stomach

Aim: To investigate the EP receptor subtype involved in the gastroprotective action of prostaglandin (PG) E2 using various EP receptor agonists in rats, and using knockout mice lacking EP1 or EP3 receptors.

Cyclo-oxygenase isozymes in mucosal ulcerogenic and functional responses following barrier disruption in rat stomachs

1 We examined the effects of selective and nonselective cyclo‐oxygenase (COX) inhibitors on various functional changes in the rat stomach induced by topical application of taurocholate (TC) and investigated the preferential role of COX isozymes in these responses. 2 Rat stomachs mounted in ex vivo chambers were perfused with 50 mM HCl and transmucosal potential difference (p.d.), mucosal blood flow (GMBF), luminal acid loss and luminal levels of prostaglandin E2 (PGE2) were measured before, during and after exposure to 20 mM TC. 3 Mucosal application of TC in control rats caused a reduction in p.d., followed by an increase of luminal acid loss and GMBF, and produced only minimal damage in the mucosa 2 h later. Pretreatment with indomethacin (10 mg kg−1, s.c.), a nonselective COX‐1 and COX‐2 inhibitor, attenuated the gastric hyperaemic response caused by TC without affecting p.d. and acid loss, resulting in haemorrhagic lesions in the mucosa. In contrast, selective COX‐2 inhibitors, such as NS‐398 and nimesulide (10 mg kg−1, s.c.), had no effect on any of the responses induced by TC and did not cause gross damage in the mucosa. 4 Luminal PGE2 levels were markedly increased during and after exposure to TC and this response was significantly inhibited by indomethacin but not by either NS‐398 or nimesulide. The expression of COX‐1‐mRNA was consistently detected in the gastric mucosa before and after TC treatment, while a faint expression of COX‐2‐mRNA was detected only 2 h after TC treatment. 5 Both NS‐398 and nimesulide significantly suppressed carrageenan‐induced rat paw oedema, similar to indomethacin. 6 These results confirmed a mediator role for prostaglandins in the gastric hyperaemic response following TC‐induced barrier disruption, and suggest that COX‐1 but not COX‐2 is a key enzyme in maintaining ‘housekeeping’ functions in the gastric mucosa under both normal and adverse conditions.

Gastric ulcerogenic responses following barrier disruption in knockout mice lacking prostaglandin EP1 receptors

Endogenous prostaglandins (PGs) are considered to play a pivotal role in maintaining the mucosal integrity of the stomach after injury. In the present study, we evaluated the mucosal ulcerogenic and mucosal blood flow (GMBF) responses in the stomach after damage by taurocholate (TC) in knockout mice lacking EP1 or EP3 receptors.

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.

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

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