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.

Oxidant generation in anoxia-reoxygenation and ischemia-reperfusion was compared in isolated rat lungs. Anoxia-reoxygenation was produced by N2 ventilation followed by O2 ventilation. After anoxia, lung ATP content was decreased by 59%. Oxygenated ischemia was produced by discontinuing perfusion while ventilation with O2 was maintained. With anoxia-reoxygenation, oxidant generation, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 3.6-fold, lung thiobarbituric acid reactive substances (TBARS) increased 342%, conjugated dienes increased 285%, and protein carbonyl content increased 46%. Pretreatment of lungs with 100 μM allopurinol inhibited the reoxygenation-mediated increase in lung fluorescence by 75% and TBARS by 69%. Oxygenated ischemia resulted in an approximately eightfold increase in lung H2DCF oxidation and a fourfold increase in TBARS, but allopurinol had no effect. On the other hand, 100 μM diphenyliodonium (DPI) inhibited the ischemia-mediated increase in lung fluorescence by 69% and lung TBARS by 70%, but it had no effect on the increase with anoxia-reoxygenation. Therefore, both ischemia-reperfusion and anoxia-reoxygenation result in oxidant generation by the lung, but a comparison of results with a xanthine oxidase inhibitor (allopurinol) and a flavoprotein inhibitor (DPI) indicate that the pathways for oxidant generation are distinctly different.

We hypothesize that lung ischemic injury is related to cessation of flow leading to endothelial cell membrane depolarization and activation of oxidant-generating systems. Cell membrane potential was assessed in isolated, oxygen ventilated, Krebs-Ringer bicarbonate buffer-dextran-perfused rat lungs by lung surface fluorescence after infusion of bis-oxonol or 5,5',6,6'-tetrachloro-1, 1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), voltage-sensitive dyes. Surface fluorometry showed increased bis-oxonol fluorescence (34.7 +/- 3.3% above baseline) and decreased JC-1 fluorescence (24.5 +/- 4.5% below baseline) with ischemia, compatible with membrane depolarization. Fluorescence change was initiated within 1-2 min of the onset of ischemia and was rapidly reversible with reperfusion. Fluorescence changes varied with perfusion flow rate; maximal increase occurred with the transition from 1.8 ml/min to zero flow. Elevation of static intravascular pressure resulted in only a minor increase of bis-oxonol fluorescence. In situ subpleural fluorescence microscopy showed that endothelial cells are the major site of the increased bis-oxonol fluorescence signal with ischemia. These results indicate that endothelial cell membrane depolarization represents an early event with lung ischemia. Since the adenosine triphosphate content of lung was unchanged with ischemia in the O2-ventilated lungs, we postulate that membrane depolarization results from elimination of shear stress, possibly via inactivation of flow-sensitive K+-channels.