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.
Departmento de Bioquímica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
Gonzalo
G
Peluffo
Pablo
P
Calcerrada
Lucia
L
Piacenza
Nelson
N
Pizzano
Rafael
R
Radi
D000328:Adult; D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage* / Q000378:metabolism / Q000493:pharmacokinetics; D001011:Aorta / Q000166:cytology; D001205:Ascorbic Acid / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics; D001916:Brachial Artery / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D030421:Peroxynitrous Acid / Q000378:metabolism*; D012907:Smoking / Q000009:adverse effects / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D055815:Young Adult; D024502:alpha-Tocopherol / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics
D000975:Antioxidants; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D024502:alpha-Tocopherol; D001205:Ascorbic Acid