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

Abstract

Capettini LS, Cortes SF, Gomes MA, Silva GA, Pesquero JL, Lopes MJ, Teixeira MM, Lemos VS. Neuronal nitric oxide synthasederived hydrogen peroxide is a major endothelium-dependent relaxing factor. Am J Physiol Heart Circ Physiol 295: H2503-H2511, 2008. First published October 24, 2008 doi:10.1152/ajpheart.00731.2008.-Endothelium-dependent vasorelaxation in large vessels is mainly attributed to N -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. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 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 -nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOSNO2 -L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-Arg NO2 -L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 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 H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-Arg NO2 -L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 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 H2O2 in mediating vasodilatation. vasorelaxation; acetylcholine; antisense; knockdown NEURONAL NITRIC OXIDE (NO) synthase (nNOS) is a Ca 2ϩ / calmodulin-dependent isoform of NO synthase (NOS) constitutively expressed in neurons and in many other different tissues (17,32,36,50,57). In the cardiovascular system, nNOS is expressed in vascular smooth muscle cells (6, 7), vascular endothelium (1, 23), and cardiac myocytes (53). nNOS has a physiologically relevant role in modulating cardiac function (10), myogenic tone (16), systemic arterial pressure (28), and cerebral blood ...

Estradiol attenuates high glucose-induced endothelial nitrotyrosine: role for neuronal nitric oxide synthase

“…We found that all three NOS isoforms were expressed in HUVECs (Fig. 4A), similar to previously published data from several different groups (4,9,16). Interestingly, nNOS demonstrated a prominent nuclear localization, similar to the findings by several other groups (43,48), the significance of which in endothelial cells (such as on the regulation of protein expression) is currently being examined in our laboratory.…”

“…There is increasing evidence that nNOS exerts a beneficial effect under a number of inflammatory pathologies. Endothelial nNOS activity can have antiinflammatory and antiatherogenic effects, while nNOS-induced hydrogen peroxide can contribute to lowering of blood pressure (7,9,40). Given the emerging role for nNOS in maintaining cardiovascular health, any impairment of nNOS activity by E 2 under hyperglycemic conditions may actually worsen the cardiovascular outcome by making the vasculature more susceptible to inflammation and vasoconstriction despite offering some protection against glucose-induced peroxynitrite.…”

“…Three distinct isoforms of NOS are present in the body, namely, endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) (34). Classically, eNOS has been considered the main source of NO in the vasculature, although there is increasing evidence that nNOS in the endothelium plays an important function (4,9,10,17). Recently, we have demonstrated the presence of endothelial nNOS in rat mesenteric arteries where this enzyme contributes to a vasorelaxant role (33).…”

Peripheral neuropathy in mice with neuronal nitric oxide synthase gene deficiency

“…One can not exclude that it is also expressed in vasa nervorum considering recent report of the presence of nNOS in vascular endothelium (41). Note, that nNOS is constitutively expressed in some types of neurons, and can be induced in others (42).…”

Molecular Biology of Atherosclerosis

“…Furthermore, its effects are cell and context specific with arterial endothelial cells being generally more resistant to adverse effects than a number of other cell types. H 2 O 2 is also a major endothelial-derived hyperpolarizing factor (EDHF) mediating vasodilation by stimulation of a VSMC potassium channel (255,256). Nevertheless, ROS may promote activation of other pathways that interact with NF-B, such as the ASK1-JNK pathway as illustrated in FIGURE 5.…”