Yumi Ohashi

Phosphodiesterase Isozymes Involved in Regulation of HCO3- Secretion in Isolated Mouse Stomach in Vitro

(±)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (NOR-3), a nitric-oxide (NO) donor, is known to increase \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion in rat stomachs, intracellularly mediated by cGMP; yet, there is no information about the phosphodiesterase (PDE) isozyme involved in this process. We examined the effects of various isozyme-selective PDE inhibitors on the secretion of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} in the mouse stomach in vitro and the type(s) of PDE isozymes involved in the response to NO. The gastric mucosa of DDY mice was stripped of the muscle layer and mounted on an Ussing chamber. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. NOR-3, 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), and various PDE inhibitors were added to the serosal side. Vinpocetine (PDE1 inhibitor) or zaprinast (PDE5 inhibitor) was also added serosally 30 min before NOR-3 or 8-Br-cGMP. Both NOR-3 and 8-Br-cGMP stimulated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion in a dose-dependent manner, and the response to NOR-3 was significantly inhibited by methylene blue. Likewise, the secretion induced by NOR-3 or 8-Br-cGMP was significantly attenuated by 6-((2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo(2.2.2)octan-2-yl)-5Z-hexenoic acid (ONO-8711), the PGE receptor (EP)1 antagonist, as well as indomethacin and potentiated by both vinpocetine and zaprinast at doses that had no effect by themselves on the basal secretion, whereas other subtype-selective PDE inhibitors had no effect. NOR-3 increased the mucosal PGE2 content in a methylene blue-inhibitable manner. These results suggest that NO stimulates gastric \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion mediated intracellularly by cGMP and modified by both PDE1 and PDE5, and this response is finally mediated by endogenous PGE2 via the activation of EP1 receptors.

W1724 Hydrogen Sulfide Stimulates Hco3- Secretion in Rat Stomach and Duodenum

Background/Aim: Hydrogen sulfide (H2S), generated endogenously from L-cysteine by cystationine-β-synthase (CBS) and cystathionine-g-lyase (CSE) in mammalian cells, has recently been recognized as an important mediator that affects various functions under pathophysiological conditions. However, the functional role H2S plays in the gastrointestinal tract remains largely unknown. In the present study, we examined the effects of NaHS, a H2S donor, on HCO3secretion in the rat stomach and duodenum and investigated the mechanism involved in these responses. Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, an ex-vivo chambered stomach or a duodenal loop was perfused with saline, and HCO3secretion was measured at pH 7.0 using a pH stat-method. NaHS (0.1~10 μmol/ ml) or HCl (mucosal acidification; 200 mM) was applied topically to the mucosa for 5 min or 10 min, respectively, while PGE2 (0.3 mg/kg) was given IV as a single injection. Indomethacin (5 mg/kg, SC), glibenclamide (KATP channel blocker; 10 mg/kg, IP) was given 30 min before NaHS, while L-NAME (nitric oxide (NO) synthase inhibitor; 20 mg/kg, SC) was given 3 h before. Propargylglycine (CSE antagonist; 10 mg/kg/h) was infused IV starting 1 h before mucosal acidification. Results: Intraluminal application of NaHS significantly and dosedependently increased the secretion of HCO3in both the stomach and duodenum. The HCO3stimulatory action of NaHS in these tissues were significantly attenuated by indomethacin and L-NAME as well as sensory deafferentation. Gastric but not duodenal HCO3 response was also attenuated by gibenclamide. PGE2 stimulated gastric and duodenal HCO3secretions, and these effects were both partially mitigated by glibenclamide but not indometahcin, L-NAME or sensory deafferentation. The acid-induced HCO3secretion was significantly mitigated by propargylglycine, the inhibitor of H2S production, in the duodenum but not the stomach. In addition, the duodenal lesions induced by acid perfusion (150 mM HCl for 4 h) was significantly aggravated by pretreatment with propargylglycine. Conclusions: These results suggest that H2S increases HCO3secretion in the rat gastroduodenal mucosa, and this action is partly mediated by PG, NO and sensory neurons in these tissue and partly associated with the activation of ATP-sensitive K+ channels in the stomach. In addition, it is assumed that endogenous H2S is involved in the regulatory mechanism of HCO3secretion and mucosal protection in the duodenum but not the stomach, because the acid-induced HCO3secretion was attenuated only in the duodenum by the inhibitor of H2S production.

W1542 Effect of Carbon Monoxide On HCO3- Secretion in Rat Stomachs: Involvement of Heme Oxygenase in Regulation of Gastric HCO3- Response

G A A b st ra ct s -/mice to wild type mice to determine if zinc works via the receptor or by direct interactions within the cytosol. Material and methods: Wild type and CaSR-deficient mice were fasted for 12-18 hours prior to sacrifice to ensure a consistent minimum of gastric acid secretion. Single gastric glands were hand-dissected from the corpus of the stomach. After incubation with the pH-sensitive dye, BCECF glands were transferred to a coverslip precoated with a biological cell adhesive, mounted in a chamber maintained at 37° and then imaged on a real time fluorescence digital system. For reproducible acidification, glands were exposed to a Na+-free NH4Cl prepulse and a 0 mM Na+ solution. Following acid loading and Na+removal, acid extrusion was monitored as rate of intracellular alkalinization. To examine proton extrusion by H+,K+-ATPase and to preclude contribution of Na+/H+ exchange, Na+free conditions were chosen. The rate of pH recovery, representing H+,K+-ATPase activity, was calculated as ΔpHi/Δt, using a high-K+/Nigericin calibration technique. In wild type mice Histamine or a potent calcimimetic activator of the CaSR, were used to stimulate acid secretion. In CaSR-deficient mice Histamine alone was used. In a subsequent series of experiments Zinc was added. Results: In the presence of functional CaSR the inhibitory effect of Zinc on gastric acid secretion was significantly higher than that seen in CaSRdeficient mice, suggesting the primary pathway for zinc action is at the level of the CaSR. Conclusion: This study shows the predominant role of the CaSR in Zinc-mediated inhibition of gastric acid secretion. Additionally this study suggests the importance of dietary zinc to modulate acid production. Failure to have sufficient zinc in the diet would result in reduced serum levels of zinc. This reduction in zinc could lead to an enhanced CaSR function and subsequent gastric acid hypersecretion even in the absence of hormonal or neuronal stimulation.

W1715 Effect of Carbon Monoxide/Heme Oxygenase On Duodenal HCO3- Secretion in Rats

Background/Aims: Carbon monoxide (CO) with free iron and biliverdin are generated from degradation of heme catalyzed by constitutive heme oxygenase (HO-2) and inducible HO1. Particularly, HO-1 is recognized as the stress protein, which is up-regulated by various stresses and exerts a protective action against tissue injury. By the way, HCO3secretion is one of protective mechanisms in the duodenum against acid stress. However, no information is available concerning the relation of HO-1/CO with HCO3secretion. In the present study, we examined the effect of a CO donor on HCO3secretion in the rat duodenum and investigated whether HO-1 is involved in the physiological regulation of this secretion. Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and HCO3secretion was measured at pH 7.0 using a pH-stat method. CORM-2 (a CO donor; 0.5-2 mM), biliverdin (10 mg/ml) or ruthenium (III) chloride (Ru; a negative control; 5 mM) was applied to the loop for 5 min. Mucosal acidification was performed by exposing the loop to 10 mM HCl for 10 min. Acetazolamide (an inhibitor of CA), indomethacin or L-NAME (a NOS inhibitor) was given SC 1 h or 3 h before the administration of agonists, while SnPP (an inhibitor of HO), CuPP (a negative inhibitor) or hemin was given IP 1 h before. Expression of HO protein was examined by western blot. Results: Topical application of CORM-2 significantly increased duodenal HCO3secretion in a concentration dependent manner, while neither Ru nor biliverdin have any effect. The stimulatory effect of CORM-2 was significantly attenuated by indoemthacin and acetazolamide but not L-NAME. The secretion of HCO3was increased by acidification of the mucosa, in an indomethacin-inhibitable manner. The acid-induced HCO3response was also markedly inhibited by SnPP but not CuPP. Furthermore, the inhibitory effect of SnPP was significantly reversed by the pretreatment with hemin. On the other hand, perfusion of the duodenal loop with 100 mM HCl for 2 h caused hemorrhagic lesions, and this response was significantly worsened by the prior administration of SnPP as well as indomethacin. In addition, the expression of HO-1 but not HO-2 protein was markedly up-regulated in the duodenum following acid perfusion for 4 h. Conclusions: These results suggest that CO stimulates HCO3secretion in the duodenum, and this effect is mediated by endogenous PGs and partly dependent on CO2 generated from the reaction of CO with O2. It is assumed that HO-1 plays an important role in maintaining the integrity of the duodenal mucosa when the tissue is exposed to acid stress.

W1716 Stimulation By Bradykinin of HCO3- Secretion in Rat Gastroduodenal Mucosa

BACKGROUND We examined the effect of bradykinin on gastroduodenal HCO(3)(-) secretion in rats and investigated the mechanisms involved in this action. MATERIAL/METHODS Under urethane anesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and the secretion of HCO(3)(-) was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. RESULTS Intravenous administration of bradykinin increased both gastric and duodenal HCO(3)(-) secretion in a dose-dependent manner. These effects were totally blocked by FR172357, the bradykinin B2 receptor antagonist, and significantly attenuated by indomethacin or L-NAME, although the degree of inhibition was much greater in the stomach than the duodenum. Likewise, the response to bradykinin in the stomach totally disappeared on the chemical ablation of capsaicin-sensitive afferent neurons, whereas this action in the duodenum was inhibited only partially by sensory deafferentation. Capsazepine, the antagonist of transient receptor potential vanilloid type 1 (TRPV1), did not significantly affect the HCO(3)(-) response to bradykinin in these tissues. CONCLUSIONS Bradykinin increases both gastric and duodenal HCO(3)(-) secretion through the activation of B2 receptors, and this action is mediated locally by endogenous prostaglandins (PGs) and nitric oxide (NO) as well as capsaicin-sensitive afferent neurons. It is assumed that bradykinin causes the release of PGs and NO as well as activation of afferent neurons via B2 receptors but not through the interaction with TRPV1, and these factors are all involved in the gastroduodenal responses, although the mode of interaction between these factors may be different in the stomach and duodenum.