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 ϩ 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

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... . 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 mol/L PR-39. ROS production in lungs exposed to high K ϩ 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 ϩ -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 ϩ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium. (Circ Res. 1998;83:730-737.) Key Words: p47 phox Ⅲ gp91 phox Ⅲ bovine pulmonary artery endothelial cell Ⅲ diphenyliodonium Ⅲ PR-39