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.