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This Article Full Text Full Text (PDF) All Versions of this Article: 296/6/H1781    most recent 00930.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Peluffo, G. Articles by Radi, R. PubMed PubMed Citation Articles by Peluffo, G. Articles by Radi, R.

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

¹Departamento de Bioquímica and ²Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República; and ³Instituto Nacional de Cirugía Cardíaca, Montevideo, Uruguay

Submitted 25 August 2008 ; accepted in final form 30 March 2009

Tobacco smoke is known to cause nitric oxide (^·NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between ^·NO and superoxide (O₂^·–) 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 O₂^·– production as indicated by spin-trapping electron paramagnetic resonance experiments. O₂^·– was produced both extracellulary (3.4 vs. 1.0 nmol·h^–1·mg^–1; CSE vs. control; cytochrome c³⁺ 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. O₂^·– and peroxynitrite formation were decreased by ascorbate and -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--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 O₂^·– production in vascular endothelial cells both in vitro and in vivo.

superoxide; cigarette smoke extract; free radicals; antioxidants