TNF-alpha potentiates protein-tyrosine nitration through activation of NADPH oxidase and eNOS localized in membrane rafts and caveolae of bovine aortic endothelial cells.
A major source of reactive oxygen species (ROS) in endothelial cells is the NADPH oxidase enzyme complex. The selective distributions of any enzyme within cells have important implications in regulating enzyme effectiveness through facilitation of access to local substrates and/or product targets. Because membrane rafts provide a spatially preferable environment for a variety of enzyme systems, we sought to determine whether NADPH oxidase is present and functional in this plasma membrane compartment in endothelial cells. We found that, in resting endothelial cells, NADPH oxidase subunits were preassembled and the enzyme functional in membrane rafts, specifically in caveolae. Stimulation with TNF-alpha induced additional recruitment of the p47(phox) regulatory subunit to raft-localized NADPH oxidase and enhanced ROS production within raft domains. TNF-alpha also induced nitric oxide production through activation of endothelial nitric oxide synthase (eNOS) present in the same membrane compartment. The dual activation of superoxide and nitric oxide-generating systems provided a spatially favorable environment for nitration of tyrosine-containing proteins localized to rafts. Perturbation of membrane raft structural integrity with cholesterol-sequestering compounds caused the delocalization of NADPH oxidase subunits and eNOS from the rafts and inhibited TNF-alpha-induced ROS production and protein tyrosine nitration. Together, these data provide evidence that membrane rafts and caveolae play a role in the spatial regulation of NADPH oxidase and subsequent ROS/reactive nitrogen species in endothelial cells.
Yang
Baohua
B
Cardiovascular Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine, MRB 826, 3420 North Broad St., Philadelphia, PA 19140, USA.
Rizzo
Victor
V
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D021962:Membrane Microdomains / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D002784:Cholesterol; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1