Rac and PI3 kinase mediate endothelial cell-reactive oxygen species generation during normoxic lung ischemia.
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.
Zhang
Qunwei
Q
Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6068, USA.
Chatterjee
Shampa
S
Wei
Zhihua
Z
Liu
Wei Dong
WD
Fisher
Aron B
AB
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
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