Suppression of NADPH oxidase activity by nitric oxide in human endothelial cells and arteries

Greg J Dusting, Stavros Selemidis, Hitesh Peshavariya, Tomasz Guzik2, Keith Channon2 & Grant R Drummond1, Bernard O’Brien Institute of Microsurgery, The University of Melbourne, 42 Fitzroy Street, Fitzroy 3065, Victoria, Australia 1Department of Pharmacology, Monash University, Clayton, Victoria2 Cardiovascular Medicine, University of Oxford, Oxford, UK.

The NADPH oxidase enzyme complex is expressed constitutively in the endothelium and is a major source of superoxide generated in blood vessels. Atherosclerosis, reperfusion injury and diabetes-associated vascular disease are accompanied by upregulation of NADPH oxidase activity, and this is the major cause of the oxidative stress associated with endothelial dysfunction and cell damage (Jiang et al., 2004). Since endothelium-derived nitric oxide (NO) reacts extremely rapidly with superoxide to form cytotoxic peroxynitrite, we considered whether release of NO might act in some way to limit superoxide production, so as to optimise its availability for immediate physiological functions. We therefore set out to investigate whether NO suppresses endothelial superoxide production by inhibiting NADPH oxidase activity.

NADPH oxidase-dependent superoxide production was detected by lucigenin chemiluminescence and dihydroethidium fluorescence in human cultured microvascular endothelial cells. Superoxide production was reduced or eliminated by superoxide dismutase and by the reputed NADPH oxidase inhibitors diphenylene iodonium (DPI) and apocynin, but was not affected by inhibitors of NO synthase (L-NAME), cyclo-oxygenase, lipoxygenase, cytochrome P450, xanthine oxidase or the mitochondrial respiratory chain. NADPH oxidase-dependent superoxide production was also suppressed by pre-treatment with the slow releasing NO donor, diethylaminotriamine (DETA)-NONOate (3-300 µM) in a concentration- and time- dependent manner. The suppression of superoxide production was not attributable to stoichiometric removal of superoxide for this effect was also observed for more than 6h after washing DETA-NONOate from the media. Similar, sustained inhibition of superoxide production was achieved with two other NO donors, sodium nitroprusside (1-100 µM) and S-nitroso-N-acetylpenicillamine (10-100 µM). The inhibitory effect of NO appeared not to depend upon the cyclic-GMP/protein kinase G pathway, peroxynitrite formation, or reduced expression of NADPH oxidase subunits. However, NO treatment clearly caused S-nitrosylation of the p47phox cytosolic subunit, and reversal of nitrosylation by brief exposure to intense cold light restored superoxide production. Finally, transfection of human internal mammary artery segments with an adenoviral vector containing neuronal nitric oxide synthase (nNOS) increased nNOS activity in the endothelial and adventitial layers, as well as reducing superoxide production, and this effect was still evident after short-term treatment with L-NAME (100 µM) in transfected arteries.

In conclusion, NO donors and nNOS transfection cause a sustained suppression of NADPH oxidase-dependent superoxide production in human endothelial cells and internal mammary artery. These findings highlight a novel approach to suppressing oxidative stress in arteries by the administration of NO donor drugs, and this may contribute to long term protection from vascular disease.

Jiang F, Drummond GR & Dusting GJ (2004) Endothelium 11:79-88.