Superoxide-mediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers.

Tobacco smoke is known to cause nitric oxide ((*)NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between (.)NO and superoxide (O(2)(*-)) radicals and the consequent impact on (*)NO bioavailability and nitroxidative stress in bovine aortic endothelial cells exposed to cigarette smoke extract (CSE) and in smokers. Bovine aortic endothelial cells in the presence of CSE triggered O(2)(*-) production as indicated by spin-trapping electron paramagnetic resonance experiments. O(2)(*-) was produced both extracellulary (3.4 vs. 1.0 nmol.h(-1)*mg(-1); CSE vs. control; cytochrome c(3+) reduction assay) and intracellularly (40% inhibition of cytosolic aconitase). CSE also led to the production of peroxynitrite as evaluated by dihydrorhodamine oxidation and protein tyrosine nitration on cells. O(2)(*-) and peroxynitrite formation were decreased by ascorbate and alpha-tocopherol. Additionally, CSE led to the oxidation of endothelial nitric oxide synthase increasing the monomeric inactive form of endothelial nitric oxide synthase. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 IU all-rac-alpha-tocopherol every 12 h for 165 days. Smokers had endothelial dysfunction compared with control subjects (95% confidence interval: 2.5, 8.3 vs. 10.6, 14.2; P < 0.05) as assessed by flow-mediated dilation of the brachial artery, and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of flow-mediated dilation in smokers reverted after a long-term antioxidant supplementation (95% confidence interval: 13.9, 19.9; P < 0.05), reaching values comparable with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert (*)NO toward peroxynitrite by inducing O(2)(*-) production in vascular endothelial cells both in vitro and in vivo.