Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells

Cigarette smoking is associated with impaired endothelium-dependent vasodil ation and reduced nitric oxide (NO) in the exhaled air of smokers. To explo re the mechanism for the impairment of NO-mediated vasodilation, we studied the effect of cigarette smoke extract (CSE) on NO synthase (eNOS) activity and content in pulmonary artery endothelial cells (PAEC). Incubation of PA EC with CSE resulted in a time- and dose-dependent decrease in eNOS activit y. The inhibitory effect of CSE on eNOS activity was not reversible. Both g as-phase and particulate-phase extracts of CSE contributed to the inhibitio n of eNOS activity. The protein kinase c (PKC) inhibitors staurosporine and chelerythrine did not affect the CSE-induced inhibition of eNOS activity. Catalase, superoxide dismutase (SOD), vitamin C, vitamin E, glutathione, an d dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of e NOS activity, and incubation of PAEC with 3 mM nicotine did not change the activity of eNOS. Treatment of PAEC with CSE also caused a nonreversible, t ime-dependent decrease in eNOS protein content detected by Western blot ana lysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione co ntents or on lactate dehydrogenase (LDH) release. These results indicate th at exposure to CSE causes an irreversible inhibition of eNOS activity in PA EC, and suggest that the decreased activity is secondary to reduced eNOS pr otein mass and mRNA. The decrease in eNOS activity may contribute to the hi gh risk of pulmonary and cardiovascular disease in cigarette smokers.