Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress

Superoxide (O-2(center dot-)) production by the NADPH oxidases is implicated in the pathogenesis of many cardiovascular diseases, including hypertension. We have previously shown that activation of NADPH oxidases increases mitochondrial O-2(center dot-) which is inhibited by the ATP-sensitive K+ channel (mitoK(ATP)) inhibitor 5-hydroxydecanoic acid and that scavenging of mitochondrial or cytoplasmic O-2(center dot-) inhibits hypertension. We hypothesized that mitoK(ATP)-mediated mitochondrial O-2(center dot) potentiates cytoplasmic O-2(center dot-) by stimulation of NADPH oxidases. In this work we studied Nox isoforms as a potential target of mitochondrial O-2(center dot-). We tested contribution of reverse electron transfer (RET) from complex II to complex I in mitochondrial O-2(center dot-) production and NADPH oxidase activation in human aortic endothelial cells. Activation of mitoK(ATP) with low dose of diazoxide (100 nM) decreased mitochondrial membrane potential (tetramethylrhodamine methyl ester probe) and increased production of mitochondrial and cytoplasmic O-2(center dot-) measured by site-specific probes and mitoSOX. Inhibition of RET with complex II inhibitor (malonate) or complex I inhibitor (rotenone) attenuated the production of mitochondrial and cytoplasmic O-2(center dot-). Supplementation with a mitochondria-targeted SOD mimetic (mitoTEMPO) or a mitochondria-targeted glutathione peroxidase mimetic (mitoEbselen) inhibited production of mitochondrial and cytoplasmic O-2(center dot-). Inhibition of Nox2 (gp91ds) or Nox2 depletion with small interfering RNA but not Nox1, Nox4, or Nox5 abolished diazoxide-induced O-2(center dot-) production in the cytoplasm. Treatment of angiotensin II-infused mice with RET inhibitor dihydroethidium (malate) significantly reduced blood pressure. Our study suggests that mitoKATP-mediated mitochondrial O-2(center dot-) stimulates cytoplasmic Nox2, contributing to the development of endothelial oxidative stress and hypertension.