DETERMINATION OF ENDOGENOUSLY PRODUCED NITRIC OXIDE (NO) IN RAT STOMACH : CYTOPROTECTIVE ROLE OF NO

一氧化氮（NO）是由三种形式的一氧化氮合酶（NOS）在胃中生物合成的。唾液中的亚硝酸盐来源于膳食硝酸盐，在胃的酸性条件下化学还原为NO。因此，胃肠道高度暴露于内源性和外源性一氧化氮。近年来，胃内源性合成NO已被证明对胃粘膜血流、胃酸和粘液分泌、胃运动、调节粘膜防御和损伤等方面发挥生理和病理生理作用。本研究探讨一氧化氮在乙醇致大鼠胃粘膜损伤中的作用。氨基甲酸乙酯麻醉下，将大鼠胃置于离体腔上；先灌注生理盐水，再灌注43%的乙醇水溶液。采用自旋捕获技术结合电子顺磁共振波谱分析了胃组织一氧化氮的产生。采用HPLC - griess反应法测定腔液中的亚硝酸盐和硝酸盐（NOx）。采用NOS抑制剂N^ g -硝基- l -精氨酸甲酯（N^ g - nitrol -arginine methyl ester, L-NAME）预给药/不给药，评价大鼠胃黏膜血流量、腔内pH、胃黏膜电位差及宏观粘膜损伤面积。L-NAME预用药加重了黏膜损伤，抑制了黏膜血流量的增加。综上所述，内源性NO通过调节胃黏膜血流量对乙醇性胃损伤起保护作用。此外，我们还研究了被认为是内源性NO供体分子之一的二硝基二硫代酸铁复合物（dinitrosyl dithiolato iron complex， DNIC）的生理活性和体内分布。体外和体内EPR光谱结合可以揭示DNIC不仅具有良好的膜通透性，而且对肝脏和肾脏具有明显的高亲和力。

Nitric oxide (NO) is biologically synthesized in stomach by three forms of nitric oxide synthase (NOS). Nitrite in saliva derived from dietary nitrate is chemically reduced to NO under the acidic conditions in the stomach. Thus, gastrointestinal tracts are highly exposed to NO derived from both endogenous and exogenous origins. Recently, endogenously synthesized NO in stomach has been demonstrated to exert physiological and pathophysiological effects on gastric mucosal blood flow, acid and mucus secretion, gastric motility, and regulates mucosal defense and injury. In this study we investigated the roles of NO on ethanol-induced gastric mucosal injury in rats. Under urethane anesthesia, a rat stomach was mounted on an ex vivo chamber ; saline and then aqueous ethanol (43%) solutions were perfused. NO production in stomach tissues was analyzed by spin trapping technique combined with electron paramagnetic resonance spectroscopy. Nitrite and nitrate (NOx) in the luminal fluid were determined by the Griess-reaction assay with HPLC.Gastric mucosal blood flow, luminal pH, gastric mucosal potential difference, and area of the macroscopic mucosal injury were evaluated with/without preadministration of NOS inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME). Preadministration of L-NAME aggravated mucosal damage and suppressed increase of mucosal blood flow. In conclusion, endogenously produced NO plays an important role in the protection against ethanol-induced gastric injury via regulation of gastric mucosal blood flow. Further, we investigated the physiological activities and in vivo distribution of dinitrosyl dithiolato iron complex (DNIC), which has been recognized as one of endogenous NO donor molecules. A combination of ex vivo and in vivo EPR spectrometry made it possible to reveal that DNIC not only possesses an excellent membrane-permeability but also has a markedly high affinity for the liver and kidney.