Eitaro Aihara

Expression of vanilloid receptors in rat gastric epithelial cells: role in cellular protection.

Vanilloid receptors subtype 1 (VR1), a nonselective cation channel responsive to capsaicin, protons, and noxious heat, has been recently identified in not only neural but also non-neural cells. In the present study, we demonstrated the peripheral expression of VR1 in gastric mucosal epithelial cells and investigated the role of the receptor in cellular protection. The rat gastric mucosal epithelial cell line was used. The expression of VR1 was examined by Western blotting and RT-PCR. Cell damage was induced by immersion in 10% ethanol or acid (pH 4.0) for 30 min, and cell viability was determined by MTT assay. Capsaicin or resiniferatoxin was added 30 min before the challenge with ethanol or acid, while capsazepine or ruthenium red (a VR1 antagonist) was added simultaneously with capsaicin. The distinct expression of VR1 protein and mRNA was detected in rat gastric mucosal epithelial cell line as well as in the rat stomach and spinal cord by Western blotting and RT-PCR, respectively. The cDNA sequence of the PCR product was found to be almost identical to that of the authentic VR1 (99.8%) when the product was subcloned and sequenced. On the other hand, the cell damage induced by ethanol or acid was dose-dependently prevented by pretreatment with capsaicin. The protective effect of capsaicin was mimicked by resiniferatoxin and almost totally abolished by co-addition of capsazepine or ruthenium red. These findings suggest that VR1 is expressed peripherally in gastric mucosal epithelial cells and plays a cellular protective role.

Stimulation of duodenal HCO3− secretion by hydrogen sulphide in rats: relation to prostaglandins, nitric oxide and sensory neurones

Aim:u2002 We examined the effect of H2S on duodenal HCO3− secretion in rats and investigated the mechanism involved in this response.

Regulatory mechanism of duodenal bicarbonate secretion Roles of endogenous prostaglandins and nitric oxide

The secretion of HCO(3)(-) in the duodenum is increased by exogenous prostaglandin (PG) E(2) and mucosal acidification, the latter being accompanied by a rise in mucosal PGE(2) content and nitric oxide (NO) release. The stimulatory effect of PGE(2) is mediated intracellularly by both Ca(2+) and 3,5-adenosine cyclic adenosine monophosphate (cAMP), and this action is inhibited by EP3 and EP4 antagonists. The secretion is also increased by NOR3 (NO donor), and this response is mimicked by dibutyryl 3,5-cyclic guanosine monophosphate (dbcGMP) and attenuated by indomethacin. Mucosal acidification stimulates HCO(3)(-) secretion with concomitant increases in mucosal PGE(2) production and NO release. The effects on HCO(3)(-) secretion and PGE(2) production are inhibited by indomethacin [nonselective cyclooxygenase (COX) inhibitor] and SC-560 (selective COX-1 inhibitor) but not rofecoxib (selective COX-2 inhibitor). N(G)-nitro-l-arginine methyl ester [l-NAME: nonselective NO synthase (NOS) inhibitor], but not aminoguanidine [selective inducible NOS inhibitor], attenuates the acid-induced HCO(3)(-) secretion and NO release in an l-arginine-sensitive manner. In addition, the response to PGE(2) is potentiated by vinpocetine [phosphodiesterase (PDE) 1 inhibitor] and cilostamide (PDE3 inhibitor), while the response to NOR3 is increased by vinpocetine. We conclude that endogenous PGs and NO are both involved in the local regulation of acid-induced duodenal HCO(3)(-) secretion; COX-1 and constitutive NOS are key enzymes responsible for the production of PGs and NO, respectively; NO stimulates HCO(3)(-) secretion by increasing PG production; PGE(2) stimulates HCO(3)(-) secretion via activation of EP3/EP4 receptors; and both PDE1 and PDE3 are involved in the regulation of duodenal HCO(3)(-) secretion.

Mucosal irritative and healing impairment action of risedronate in rat stomachs: Comparison with alendronate

Background and Aim:u2002 We used alendronate and risedronate as bisphosphonates and examined whether or not these agents have a mucosal irritative action in the stomach and impair the healing of pre‐existing gastric ulcers in rats.

Impairment of Gastric Ulcer Healing by Alendronate, a Nitrogen-Containing Bisphosphonate, in Rats

Bisphosphonates such as alendronate have been developed as antiresorptive agents capable of treating diseases related to bone remodeling. In the present study, we examined the effect of alendronate on the healing of acetic acid-induced gastric ulcers in rats and investigated the mechanism involved in this action both in vivo and in vitro using the rat gastric epithelial cell line (RGM1). Acetic acid-induced gastric ulcers healed spontaneously, with up-regulation of COX-2/prostaglandin E2 production as well as expression of vascular endothelium-derived growth factor (VEGF) and basic fibroblast growth factor (bFGF) in ulcerated mucosa. The healing of ulcers was impaired by indomethacin (2xa0mg/kg, s.c.) or alendronate (60xa0mg/kg, p.o.) given once daily for 7 days, starting 3 days after acid application. Indomethacin, but not alendronate, inhibited mucosal prostaglandin E2 production. Alendronate as well as indomethacin decreased the protein expression of both VEGF and bFGF in ulcerated mucosa, resulting in a reduction of angiogenesis in the ulcer base. Supplementation of recombinant bFGF significantly reverted the delay in ulcer healing caused by alendronate. On the other hand, the size of cell-free areas in RGM1 cells in vitro decreased with time after wound induction, and this process was promoted by epidermal growth factor (EGF; 10xa0ng/ml). Co-incubation with alendronate (1xa0mM) did not affect the spontaneous healing but significantly suppressed the accelerated wound healing caused by EGF. These results suggest that alendronate impairs the healing of gastric ulcers in rats, and this effect may be related to down-regulation of VEGF and bFGF, the important growth factors for vascularization/granulation, as well as suppression of the stimulatory action of EGF on epithelial proliferation/migration.

Prophylactic effect of rebamipide against the irritative and healing impairment actions of alendronate in rat stomachs.

Abstract.We examined the effect of rebamipide, an antiulcer drug, on the mucosal irritative and the healing impairment action of alendronate in rat stomachs. Male SD rats were used to examine the effect of rebamipide in the following 3 experiments. 1) Alendronate applied topically to the chambered stomach under urethane anaesthesia produced a decrease in the transmucosal potential difference and an increase of gastric mucosal blood flow and luminal acid loss, resulting in slight damage to the mucosa. Pretreatment with rebamipide for 3 days had no effect on the changes induced by alendronate. 2) Oral administration of alendronate caused non-haemorrhagic lesions in the stomach within 4 hr. Rebamipide, given either as a single p. o. injection or repeatedly for 7 days, did not significantly affect the lesions induced by alendronate. 3) Alendronate, given p. o. once daily for 1 week, markedly delayed the healing of acetic acid-induced gastric ulcers with the suppression of basic fibroblast growth factor (bFGF) expression. Rebamipide, given twice daily for 7 days, significantly promoted the delayed ulcer healing caused by alendronate, with the concomitant recovery of bFGF expression. These results suggest that rebamipide does not affect the direct irritating action of alendronate in the gastric mucosa, but this agent antagonizes the healing impairment action of alendronate by counteracting the downregulation of bFGF expression in the ulcerated mucosa.

Gastric HCO3- secretion induced by mucosal acidification: different mechanisms depending on acid concentration.

We compared the HCO3− secretory responses induced by mucosal acidification at different HCl concentrations (100 and 200 mM HCl) in the rat stomach. Under urethane anesthesia, the stomach was mounted on an ex vivo chamber and perfused with saline under inhibition of acid secretion by omeprazole (60 mg/kg, i.p.). The HCO3− secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The acidification was performed by exposure of the mucosa to 100 mM or 200 mM HCl for 10 min. The secretion of HCO3− was increased by acidification of the mucosa at both 100 and 200 mM of HCl, and the maximal HCO3− response was 1.5-times greater at the latter concentration. The HCO3− responses induced by 100 and 200 mM HCl were both totally inhibited by prior administration of indomethacin, an inhibitor of prostaglandin (PG) production. The HCO3− stimulatory effect of 200 mM HCl was also significantly attenuated by pre-treatment with NG-nitro L-arginine methyl ester (L-NAME), the inhibitor of nitric oxide (NO) synthase, as well as chemical ablation of capsaicin-sensitive afferent neurons, whereas that of 100 mM HCl was affected by neither of these treatments. We conclude that the mucosal acidification stimulates gastric HCO3− secretion in different mechanisms, depending on the concentration of acid; the response caused by 100 mM HCl is mediated only by PGs, while that caused by 200 mM HCl is mediated by both capsaicin-sensitive afferent neurons and NO, in addition to PGs.

H2S-induced HCO3− secretion in the rat stomach – Involvement of nitric oxide, prostaglandins, and capsaicin-sensitive sensory neurons

Hydrogen sulfide (H2S) is known to be an important gaseous mediator that affects various functions under physiological and pathological conditions. We examined the effects of NaHS, a H2S donor, on HCO3(-) secretion in rat stomachs and investigated the mechanism involved in this response. Under urethane anesthesia, rat stomachs were mounted on an ex vivo chamber and perfused with saline. Acid secretion had been inhibited by omeprazole. The secretion of HCO3(-) was measured at pHu20097.0 using a pH-stat method and by the addition of 10 mM HCl. NaHS (0.5-10 mM) was perfused in the stomach for 5 min. Indomethacin or L-NAME was administered s.c. before NaHS treatment, while glibenclamide (a KATP channel blocker), ONO-8711 (an EP1 antagonist), or propargylglycine (a cystathionine γ-lyase inhibitor) was given i.p. before. The mucosal perfusion of NaHS dose-dependently increased the secretion of HCO3(-), and this effect was significantly attenuated by indomethacin, L-NAME, and sensory deafferentation, but not by glibenclamide or ONO-8711. The luminal output of nitric oxide, but not the mucosal production of prostaglandin E2, was increased by the perfusion of NaHS. Mucosal acidification stimulated HCO3(-) secretion, and this response was inhibited by sensory deafferentation, indomethacin, L-NAME, and ONO-8711, but not by propargylglycine. These results suggested that H2S increased HCO3(-) secretion in the stomach, and this effect was mediated by capsaicin-sensitive afferent neurons and dependent on nitric oxide and prostaglandins, but not ATP-sensitive K(+) channels. Further study is needed to define the role of endogenous H2S in the mechanism underlying acid-induced gastric HCO3(-) secretion.

Prophylactic Effect of Restraint Stress on Cerulein-Induced Pancreatitis in Rats: Role of Endogenous Glucocorticoids

Stress is reportedly known to affect the severity of acute pancreatitis, yet the effect has not been without controversy. We investigated the influence of restraint stress on cerulein-induced pancreatitis, especially in relation to endogenous glucocorticoids. In the present study, restraint stress significantly reduced the increase in serum amylase levels but not pancreas weight induced by cerulein, the effect being totally antagonized by pretreatment with mifepristone, a glucocorticoid receptor antagonist. The changes induced by cerulein were prevented by dexamethasone in a dose-dependent manner. Histologically, restraint stress suppressed the intralobular edema, similar to a low dose of dexamethasone, while the latter at a high dose prevented not only the intralobular but also the interlobular edema. These results suggest that restraint stress exerts a beneficial influence on the cerulein-induced pancreatitis, mainly mediated by endogenous glucocorticoids, and it is assumed that short-term steroid therapy has a potential of clinical application for treatment of pancreatitis.

No Role for Prostacyclin IP Receptors in Duodenal HCO3– Secretion Induced by Mucosal Acidification in Mice – Comparison with Capsaicin-Induced Response

Background/Aim: We investigated the role of prostacyclin (PGI₂) IP receptors in the acid-induced secretion of HCO₃^– using IP receptor knockout [IP (–/–)] mice, in comparison with capsaicin-induced secretion. Methods: Male C57/BL6 mice, both wild-type [WT] and [IP (–/–)], fasted for 18 h were used. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and the secretion of HCO₃^– was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The secretion was stimulated by exposure of the loop to 10 mM HCl, capsaicin, PGE₂ or cicaprost (a PGI₂ agonist) for 10 min. Results: PGE₂ stimulated HCO₃^– secretion in both WT and IP (–/–) mice, while cicaprost increased it in WT but not IP (–/–) mice. Luminal acidification increased the mucosal level of PGE₂ as well as 6-keto-PGF_1α, yet stimulated HCO₃^–secretion in both WT and IP (–/–) mice, in an indomethacin-inhibitable and sensory neuron-dependent manners. Perfusion of the duodenum with 20 mM HCl for 4 h caused severe damage in WT mice pretreated with indomethacin, but not in control WT or IP (–/–) mice. Capsaicin increased duodenal HCO₃^–secretion in WT mice, in an indomethacin-sensitive manner, yet no such response was observed in the animals lacking IP receptors. Conclusion: The presence of IP receptors is not essential for acid-induced HCO₃^– secretion and mucosal defense against acid injury in the duodenum, although activation of IP receptors results in stimulation of HCO₃^–secretion. Although duodenal HCO₃^– secretion induced by both acid and capsaicin depends on afferent neurons, it seems that the mode of interaction with the afferent neurons differs regarding dependency on the PGI₂/IP receptors.