Endothelial NADPH Oxidase as the Source of Oxidants in Lungs Exposed to Ischemia or High K ⁺

Abstract

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.

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…These NOX's appear to control several vascular processes, such as vascular tone, vascular cell growth and angiogenesis, and inflammation. Accordingly, pulmonary endothelial cells have been demonstrated to generate NADPH-derived oxidants in response to pulmonary ischemia (178,179) as well as hyperoxia (180), which is associated with membrane depolarization due to the activation of K(ATP) channels, and resulting in stimulated endothelial cell proliferation and NO production (178,181,182). Although these responses were in some cases inhibited by catalase, suggesting the involvement of H 2 O 2 (182), recent studies also suggest a role for O 2 •− in endothelial NOX signaling (77).…”

DCFH2 interactions with hydroxyl radicals and other oxidants – Influence of organic solvents

“…This method has been widely used in flow cytometry, fluorescence microplate assays, or fluorescence microscopy (Silveira et al, 2003;Tada et al, 2004;Watanabe 1998). Originally, it has been assumed that DCF-fluorescence appears as a consequence of the formation of superoxide anion radicals or hydrogen peroxide ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T 4 and, therefore, has been regarded as a measure for the generation of this reactive oxygen species (Al-Mehdi et al, 1998;Hempel et al, 1999;Kurose et al, 1997). (Warrington, PA, USA).…”

Intracellular Localization and Preassembly of the NADPH Oxidase Complex in Cultured Endothelial Cells

“…and H 2 O 2 , which may inactivate nitric oxide, may modulate redox-sensitive signaling pathways and gene expression, and are implicated in the pathophysiology of disorders such as hypercholesterolemia and atherosclerosis (1,2). A major source of ROS production in endothelial cells has recently been found to be a phagocyte-type NADPH oxidase (2)(3)(4)(5)(6)(7)(8). Several studies have suggested that, at a molecular level, the endothelial NADPH oxidase is analogous to the phagocyte NADPH oxidase complex in that all the main components of the phagocytic enzyme (i.e.…”

“…Several studies have suggested that, at a molecular level, the endothelial NADPH oxidase is analogous to the phagocyte NADPH oxidase complex in that all the main components of the phagocytic enzyme (i.e. gp91 phox , p22 phox , p47 phox , p67 phox , and Rac1) are detectable at both mRNA and protein level in endothelial cells (5,(7)(8)(9)(10)(11)(12). Furthermore, we and others have shown that the endothelial cell p22 phox and gp91 phox cDNA sequences are highly (Ͼ90%) homologous to published neutrophil sequences (8,12).…”