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。来自饮食硝酸盐的唾液中的亚硝酸盐在胃中的酸性条件下被化学还原为NO。因此，胃肠道高度暴露于来自内源性和外源性来源的NO。近年来，胃组织内源性合成的NO被证实对胃粘膜血流量、胃酸和粘液分泌、胃运动产生生理和病理生理效应，并调节粘膜防御和损伤。在本研究中，我们探讨了NO在乙醇诱导的大鼠胃黏膜损伤中的作用。在乌拉坦麻醉下，将大鼠的胃安装在体外小室上，然后灌流生理盐水和43%乙醇水溶液。用自旋捕捉技术结合电子顺磁共振波谱分析胃组织中NO的产生。胃粘膜血流量、管腔pH、胃粘膜电位差和宏观黏膜损伤面积在给予或不给予一氧化氮合酶抑制剂N^G-硝基-L-精氨酸甲酯(L-NAME)的情况下进行评估。应用L-NAME可加重粘膜损伤，抑制粘膜血流量增加。综上所述，内源性NO通过调节胃粘膜血流量对乙醇所致的胃损伤具有重要的保护作用。此外，我们还研究了被认为是内源性NO供体分子之一的二亚硝基二硫代铁络合物(DNIC)的生理活性和体内分布。体外和体内EPR谱的结合表明，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.