Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+
Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+
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DOI：
10.1161/01.res.83.7.730
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发表时间：
1998-10-05
影响因子：
20.1
通讯作者：
Fisher, AB
中科院分区：
文献类型：
--
作者：
Al-Mehdi, AB;Zhao, GC;Fisher, AB
我们以前已经证明了在培养的牛肺动脉内皮细胞（BPAECs），并在隔离灌注大鼠肺暴露于高K+和在全球肺缺血过程中的活性氧（ROS）的产生。本研究评估了NADPH氧化酶途径作为这些模型中ROS的来源。ROS的产生，检测氧化的荧光团，二氯二氢荧光素，增加2.5倍，在BPAEC和6倍，大鼠或小鼠肺暴露于高（24 mmol/L）K+。ROS的产生显着抑制二苯基碘，黄素蛋白抑制剂，和合成肽PR-39，NADPH氧化酶组装的抑制剂，而别嘌呤醇没有影响。随着缺血（1小时），大鼠和小鼠肺的ROS生成增加7倍; PR-39显示出ROS生成的浓度依赖性抑制，在3 μ mol/L PR-39时抑制率为50%。暴露于高K+或缺血的肺中的ROS产生基本上在gp 91（phox）的“敲除”小鼠中被消除，gp 91是NADPH氧化酶的膜定位细胞色素组分;缺氧/复氧后这些肺的ROS产生增加与对照相似。PR-39还抑制缺血和高K+介导的肺硫代巴比妥酸反应物质的增加。BPAECs和大鼠和小鼠肺的BPAECs和免疫细胞化学的蛋白质印迹法显示存在p47（phox），NADPH氧化酶的细胞质组分和PR-39抑制的假定靶点。在完整的肺原位荧光成像表明，增加的二氯荧光素荧光在这些模型中的活性氧生成主要局限于肺内皮。这些研究表明，暴露于缺血或高K+的肺中的活性氧产生是由肺内皮细胞膜相关NAPDH氧化酶的组装和激活引起的。
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 mu mol/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 p47(phox), 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.