Background-Our group has previously shown that human umbilical vein endothelial cells exposed to smokers' serum decreased nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in the presence of increased eNOS expression. In the present study, we examined whether these observations extended to human coronary artery endothelial cells (HCAECs). In addition, the role of reactive oxygen species in the observed alterations was examined. Methods and Results-HCAECs were incubated with serum from 10 nonsmokers and 15 smokers for 12 hours with or without the addition of either polyethylene glycol-superoxide dismutase (PEG-SOD, 300 U/mL), PEG-SODϩPEG-catalase (1000 U/mL), chelerythrine (3 mol/L), or tetrahydrobiopterin (20 mol/L). At the end of incubation, NO, eNOS protein, and eNOS activity were measured from the same culture. HCAECs incubated with smokers' serum alone showed significantly lower NO production (PϽ0.05) and eNOS activity (PϽ0.005) but higher eNOS expression (PϽ0.005) compared with nonsmokers. In smokers, addition of PEG-SOD, PEG-SODϩPEG-catalase, or tetrahydrobiopterin significantly (PϽ0.05) improved NO levels and eNOS activity. Interestingly, in the same smokers, a significant decrease in eNOS expression was only seen with the addition of PEG-SODϩPEG-catalase (PϽ0.05) and treatment with PEG-SOD alone insignificantly increased eNOS expression. Conclusions-The present study indicates that in vitro, HCAECs show similar changes in NO biosynthesis as human umbilical vein endothelial cells when exposed to smokers' serum and also confirms that oxidative stress plays a central role in smoking-mediated dysfunction of NO biosynthesis in endothelial cells. Furthermore, these data support other studies suggesting a role for hydrogen peroxide in the upregulation of eNOS.
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