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Am. J. Respir. Cell Mol. Biol., Volume 19, Number 5, November, 1998 819-825

Effect of Cigarette Smoke Extract on Nitric Oxide Synthase in Pulmonary Artery Endothelial Cells

Yunchao Su, Weihong Han, Carlos Giraldo, Yong De Li, and Edward R. Block

Department of Medicine, University of Florida College of Medicine, and Medical Research Service of the Department of Veterans Affairs, Gainesville, Florida

Cigarette smoking is associated with impaired endothelium-dependent vasodilation and reduced nitric oxide (NO) in the exhaled air of smokers. To explore 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 PAEC with CSE resulted in a time- and dose-dependent decrease in eNOS activity. The inhibitory effect of CSE on eNOS activity was not reversible. Both gas-phase and particulate-phase extracts of CSE contributed to the inhibition 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, and dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of eNOS 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, time-dependent decrease in eNOS protein content detected by Western blot analysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione contents or on lactate dehydrogenase (LDH) release. These results indicate that exposure to CSE causes an irreversible inhibition of eNOS activity in PAEC, and suggest that the decreased activity is secondary to reduced eNOS protein mass and mRNA. The decrease in eNOS activity may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.




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