Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

<jats:p> Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO<jats:sup>−</jats:sup>) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the “hyperoxic state” of 21% O<jats:sub>2</jats:sub>, compared with more physiological O<jats:sub>2</jats:sub> tensions (Po<jats:sub>2</jats:sub>), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O<jats:sub>2</jats:sub><jats:sup>·−</jats:sup>) generation leading to ONOO<jats:sup>−</jats:sup> formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O<jats:sub>2</jats:sub> consumption rates of bovine aortic ECs sheared (10 dyn/cm<jats:sup>2</jats:sup>, 30 min) at 5%, 10%, or 21% O<jats:sub>2</jats:sub> or left static at 5% or 21% O<jats:sub>2</jats:sub>. Respiration was inhibited to a greater extent when ECs were sheared at 21% O<jats:sub>2</jats:sub> than at lower Po<jats:sub>2</jats:sub> or left static at different Po<jats:sub>2</jats:sub>. Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO<jats:sup>−</jats:sup> scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O<jats:sub>2</jats:sub><jats:sup>·−</jats:sup> production was higher in ECs sheared at 21% than at 5% O<jats:sub>2</jats:sub>, as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O<jats:sub>2</jats:sub> was modestly increased compared with ECs sheared at lower Po<jats:sub>2</jats:sub>, suggesting that eNOS activity may be higher at 21% O<jats:sub>2</jats:sub>. Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O<jats:sub>2</jats:sub><jats:sup>·−</jats:sup> production, leads to enhanced ONOO<jats:sup>−</jats:sup> formation intramitochondrially and suppression of respiration. </jats:p>