title: Rac and PI3 kinase mediate endothelial cell-reactive oxygen species generation during normoxic lung ischemia.
abstract: Abrupt reduction of flow (ischemia) leads to endothelial cell membrane depolarization, NADPH oxidase activation, and reactive oxygen species (ROS) generation in isolated rat and mouse lungs and in flow-adapted endothelial cells in vitro. Here we evaluated the role of PI-3-kinase and rac in activation of endothelial NADPH oxidase. Endothelium of isolated perfused mouse lungs labeled with 2',7'-dichlorodihydrofluorescein (H(2)DCF) or hydroethidine (HE) showed increased ROS generation with ischemia; these results were supported by TBARS measurement in whole-lung homogenate and by in vitro studies using flow-adapted mouse pulmonary microvascular endothelial cells. Ischemia-induced ROS generation in intact lung or isolated cells was blocked by pretreatment with Clostridium difficile toxin B, a rac inhibitor, and by wortmannin or LY294002, PI3 kinase inhibitors. In cells, immunofluorescence and immunoblot after subcellular fractionation showed ischemia-induced translocation of rac, p47(phox), and p67(phox) to the plasma membrane. Increased extracellular K(+) also resulted in rac translocation, providing evidence that this pathway is sensitive to alterations of endothelial cell membrane potential. These results indicate that PI-3-kinase and the small G protein rac are involved in the activation of endothelial cell NADPH oxidase that is associated with the acute loss of shear stress.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6068, USA.
authors.forename: Qunwei
authors.initials: Q
authors.lastname: Zhang
authors.forename: Shampa
authors.initials: S
authors.lastname: Chatterjee
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Wei
authors.forename: Wei Dong
authors.initials: WD
authors.lastname: Liu
authors.forename: Aron B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry; D008168:Lung / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D008171:Lung Diseases / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D000730:Androstadienes; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002867:Chromones; D005452:Fluoresceins; D009025:Morpholines; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D013481:Superoxides; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; C079789:neutrophil cytosol factor 67K; C031815:toxB protein, Clostridium difficile; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C037631:2',7'-dichlorofluorescein; C060505:neutrophil cytosolic factor 1