Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

<jats:p> Endothelium-dependent vasorelaxation in large vessels is mainly attributed to N<jats:sup>ω</jats:sup>-nitro-l-arginine methyl ester (l-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by l-NAME and N<jats:sup>ω</jats:sup>-nitro-l-arginine (l-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with l-Arg<jats:sup>NO2</jats:sup>-L-Dbu-NH<jats:sub>2</jats:sub> 2TFA (L-Arg<jats:sup>NO2</jats:sup>-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> production in endothelial cells that was prevented by the addition of l-NAME, l-NNA, L-Arg<jats:sup>NO2</jats:sup>-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>, reduced ACh-dependent relaxation and abolished ACh-induced H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-Arg<jats:sup>NO2</jats:sup>-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> in mediating vasodilatation. </jats:p>