Role of neuronal NO synthase in regulating vascular superoxide levels and mitogen-activated protein kinase phosphorylation

Aims The present study is designed to investigate the role of neuronal nitric oxide synthase (nNOS) in the regulation of vascular mitogen-activated protein kinase (MAPK) activity under basal and angiotensin II (Ang II)-stimulated conditions. Methods and results Incubation with a potent nNOS inhibitor (L-VNIO) significantly increased superoxide (O2−) levels, with increased MAPK phosphorylation, in isolated aorta and vascular smooth muscle cells (VSMCs) from wild-type mice. Both increases were inhibited by the superoxide dismutase mimetic, tempol, but not by the peroxynitrite scavenger, FeTPPS. The levels of O2− and MAPK phosphorylation were higher in aorta from nNOS−/− mice than from wild-type mice. These parameters were suppressed by tempol and oxypurinal (a xanthine oxidase inhibitor). In isolated VSMCs or aorta from wild-type mice, Ang II stimulation markedly increased the levels of O2− and MAPK phosphorylation. L-VNIO significantly reduced Ang II-induced increases of these parameters. Apocynin, an NAD(P)H oxidase inhibitor, further inhibited Ang II-induced increases of these parameters compared with the L-VNIO-treated group. FeTPPS did not suppress the Ang II-induced increase of O2− levels, but markedly inhibited Ang II-induced MAPK phosphorylation. In contrast to the wild-type, in isolated aorta or VSMCs from nNOS−/− mice, Ang II failed to increase O2− levels and MAPK phosphorylation. Conclusion Under basal conditions, nNOS-derived NO acting as antioxidant reduces O2− accumulation and suppresses vascular MAPK phosphorylation. Under Ang II-stimulated conditions, NAD(P)H oxidase-derived O2−, inducing nNOS uncoupling, potentiates the Ang II-induced increase of O2− generation. The generated O2− may react with NO to form peroxynitrite (ONOO−). Both O2− and ONOO− participate in Ang II-induced activation of vascular MAPK.