Submitted on August 25, 2008
Revised on March 12, 2009
Accepted on March 30, 2009
Superoxide-mediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers
Gonzalo Peluffo, Pablo Calcerrada1, Lucìa Piacenza2, Nelson Pizzano3, and Rafael Radi1*
1 University Republica
2 Univeristy Republica
3 3Instituto Nacional de Cirugía Cardíaca
* To whom correspondence should be addressed. E-mail: rradi{at}fmed.edu.uy.
Tobacco smoke is known to cause nitric oxide (•NO) inactivation and endothelial dysfunction (ED). 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 (BAECs) exposed to cigarette smoke extract (CSE) and in smokers. BAECs 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 nmoles h-1 mg-1; CSE vs. control; cytochrome c3+ reduction assay) and intracellularly (40 % inhibition of cytosolic aconitase). CSE also lead to the production of peroxynitrite as evaluated by dihydrorhodamine oxidation and protein tyrosine nitration on cells. Superoxide and peroxynitrite formation were decreased by ascorbate and 
-tocopherol. Additionally, CSE lead to oxidation of endothelial nitric oxide synthase (eNOS) increasing the monomeric inactive form of eNOS. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 I.U all-rac--tocopherol every 12 hours for 165 days. Smokers had ED compared to controls (95% C.I: 2.5, 8.3 vs. 10.6, 14.2 P < 0.05) as assessed by flow mediated dilation (FMD) of the brachial artery and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of FMD in smokers reverted after long term antioxidant supplementation (95% C.I: 13.9, 19.9 P < 0.05) reaching values comparables with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert •NO towards peroxynitrite by inducing O2•- production in vascular endothelial cells both in vitro and in vivo.
