Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells.
OBJECTIVE
Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis. Here we investigate the regulation of NADPH oxidase activity by nitric oxide (NO).


METHODS
Human cultured microvascular endothelial cells (HMEC-1) were treated with the NO donors, diethylenetriamine (DETA)-NONOate, S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 0.5-24 h. Superoxide production was measured by lucigenin chemiluminescence and dihydroethidium fluorescence, while NADPH oxidase subunit expression was measured via Western blotting. S-nitrosylation was assessed using the 2,3-diaminonapthalene (DAN) assay, and via immunoblotting with an anti-nitrosocysteine antibody.


RESULTS
Specific siRNA reduced Nox2 and Nox4 protein expression and markedly decreased superoxide production in HMEC-1. DETA-NONOate (10-300 micromol/L) suppressed superoxide production in HMEC-1 in a concentration- and time-dependent manner, which was not entirely attributable to stoichiometric reaction with NO, for the effect was observed more than 6 h after removing DETA-NONOate from solution. Similarly, sustained attenuation of superoxide production was achieved with SNP (10-100 micromol/L) and SNAP (10-100 micromol/L). The suppressive effect of NO was not dependent on (1) the sGC/cGMP/PKG pathway, (2) peroxynitrite-formation, (3) reduced protein expression of NADPH oxidase subunits or (4) dissociation of NADPH oxidase subunits. Treatment with NO caused S-nitrosylation of the crucial organizer subunit p47phox, and de-nitrosylation with UV light restored superoxide production.


CONCLUSIONS
NO causes sustained suppression of NADPH oxidase-dependent superoxide production in human endothelial cells by S-nitrosylation of p47phox. These findings highlight a novel approach by which vascular oxidative stress might be suppressed by NO donors.
Selemidis
Stavros
S
Bernard O'Brien Institute of Microsurgery, University of Melbourne, Fitzroy, Victoria 3065, Australia.
Dusting
Gregory J
GJ
Peshavariya
Hitesh
H
Kemp-Harper
Barbara K
BK
Drummond
Grant R
GR
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003864:Depression, Chemical; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D049449:Luminescence; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D015538:Nitrosation; D009603:Nitroso Compounds / Q000494:pharmacology; D018384:Oxidative Stress; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
D008562:Membrane Glycoproteins; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D034741:RNA, Small Interfering; C110881:S-nitro-N-acetylpenicillamine; D013481:Superoxides; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009599:Nitroprusside; C026373:2,3-diaminonaphthalene; D009569:Nitric Oxide; D015081:2-Naphthylamine; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C060505:neutrophil cytosolic factor 1; D010396:Penicillamine