Al-Mehdi A B, Zhao G, Dodia C, Tozawa K, Costa K, Muzykantov V, Ross C, Blecha F, Dinauer M, Fisher A B
Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6068, USA.
Circ Res. 1998 Oct 5;83(7):730-7. doi: 10.1161/01.res.83.7.730.
We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.
我们之前已经证明，在培养的牛肺动脉内皮细胞（BPAECs）以及暴露于高钾和全肺缺血期间的离体灌注大鼠肺中会产生活性氧（ROS）。本研究评估了NADPH氧化酶途径作为这些模型中ROS来源的情况。通过荧光团二氯二氢荧光素的氧化检测到的ROS生成，在暴露于高（24 mmol/L）钾的BPAECs中增加了2.5倍，在大鼠或小鼠肺中增加了6倍。ROS生成受到黄素蛋白抑制剂二苯基碘鎓以及NADPH氧化酶组装抑制剂合成肽PR - 39的显著抑制，而别嘌呤醇则无作用。缺血（1小时）时，大鼠和小鼠肺中的ROS生成增加了7倍；PR - 39对ROS生成表现出浓度依赖性抑制，在3 μmol/L PR - 39时抑制率达50%。在gp91(phox)“敲除”的小鼠中，暴露于高钾或缺血的肺中的ROS生成基本被消除；这些肺在缺氧/复氧后的ROS生成增加与对照相似。PR - 39还抑制了缺血以及高钾介导的肺中硫代巴比妥酸反应性物质的增加。对BPAECs进行蛋白质免疫印迹以及对BPAECs和大鼠及小鼠肺进行免疫细胞化学分析显示，存在p47phox，它是NADPH氧化酶的一种胞质成分，也是PR - 39抑制的假定靶点。在完整肺中的原位荧光成像表明，在这些ROS生成模型中增加的二氯荧光素荧光主要定位于肺内皮。这些研究表明，暴露于缺血或高钾的肺中的ROS生成是由肺内皮的膜相关NAPDH氧化酶的组装和激活所致。
