title: NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells.
abstract: The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analysed the effect of laminar shear stress on .O(2)(-) formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced .O(2)(-) formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm(-2), 24 h) down-regulated .O(2)(-) formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox). In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of .O(2)(-) formation, gp91(phox) and p47(phox) expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates .O(2)(-) formation, gp91(phox) and p47(phox) expression in static cultures. In conclusion, our data suggest a transient activation of .O(2)(-) formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91(phox)/p47(phox)-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/.O(2)(-) balance and the vasoprotective potential of physiological levels of laminar shear stress.
authors.affiliation: University of Technology Dresden, Medical Faculty Carl Gustav Carus, Department of Vascular Endothelium and Microcirculation, Fetscherstr. 74, D-01307 Dresden, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Duerrschmidt
authors.forename: Claudia
authors.initials: C
authors.lastname: Stielow
authors.forename: Gregor
authors.initials: G
authors.lastname: Muller
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Pagano
authors.forename: Henning
authors.initials: H
authors.lastname: Morawietz
mesh_terms: D001485:Basement Membrane; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D013481:Superoxides / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D013481:Superoxides; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D010100:Oxygen; D019331:NG-Nitroarginine Methyl Ester