Development and Biological Application of Highly Sensitive Detection Method for Endothelial Relaxing Factor : NO

一氧化氮(NO)与H_2O_2反应产生强烈的鲁米诺依赖的化学发光。这个新开发的水合发光系统可以检测到PM水平的NO。化学发光是基于该反应中过氧亚硝酸根的形成。我将这一反应应用于连续检测大鼠离体灌流肾释放的NO，并同时改变压力。在Wistar大鼠，巴塞尔NO释放量为85×11 fmol/min/g肾重量，乙酰胆碱(ACh)随压力降低而增加NO释放量，呈剂量依赖关系。NO合成抑制剂N^G-单甲基-L-精氨酸可抑制该化学发光。这种抑制作用可被L-精氨酸逆转。NO释放的这些变化总是与灌流压力的镜像变化有关。化学性内皮剥脱减弱了对ACh的反应，然而，注入真正的NO溶液导致了化学发光的增加和伴随的压力降低。灌流超氧化物歧化酶对灌流压力和化学发光均无明显影响。罂粟碱降低了压力，但不影响化学发光。没有亚硝酸盐和硝酸盐等衍生物显示出无排放。硫代亚硝基化合物的释放量比NO少104倍，对内皮或内皮衍生化合物具有生物活性的化合物没有排放。这些结果表明，该方法对NO的检测具有很高的灵敏度和实用性，可能为评价NO的病理生理作用提供一种新的方法。

Nitric oxide (NO) reacts with H_2O_2 to produce a rise in potent luminol dependent chemiluminescence. NO at pM levels was detectable by this newly developed aquachemiluminescence system. The chemiluminescence is based on the formation of peroxynitrite in this reaction. I applied this reaction to the continuous detection of NO released from the rat isolated perfused kidney with simultaneous pressure changes. In Wister rats, basel NO release was 85* 11 fmol/min/g kidney weight and acetylcholine (ACh) increased NO release dose-dependently with the pressure reduction. An NO synthesis inhibitor, N^G-monomethyl-L-arginine, abolished the chemiluminescence. This inhibition was reversed by the addition of L-arginine. These changes in the NO release were always associated with mirror image changes in the perfusion pressure. Chemial endothelial denudation diminished the responses to ACh, however, infusion of authentic NO solution caused an increase in the chemiluminescence and a concomitant decrease in the pressure. the infusion of SOD caused no significant change in either perfusion pressure or chemiluminescence. Papaverine reduced the pressure but did not influence the chemiluminescence. NO derivatives like nitrite and nitrate showed no emission. thio-nitroso compounds exhibited emission 104 times* less than NO.Compounds which are biological active on the endothelium or endothelium derived compounds exhibited no emmision. These results suggest that this method is highly sensitive and useful for detection of NO and that this system may provide a new method of evaluating the pathophysiological roles of NO.