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J Physiol. 2006 Oct 15;576(Pt 2):557-67. Epub 2006 Jul 27.

NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells.

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University of Technology Dresden, Medical Faculty Carl Gustav Carus, Department of Vascular Endothelium and Microcirculation, Fetscherstr. 74, D-01307 Dresden, Germany.


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.

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