Cigarette smoke exposure dose‐dependently alters the activity and coupling of endothelial nitric oxide synthase in the endothelium.

A critical mechanism implicated in cigarette smoke (CS) induced cardiovascular diseases is the inhibition of endothelial nitric oxide synthase (eNOS). Here we show that CS extract (CSE) alters endothelial function by an eNOS‐dependent mechanism. We demonstrate that treatment with CSE impairs vessel relaxation; we show that NO generation from endothelial cells, as measured by EPR spin‐trapping, was decreased by 36%, 43%, or 48% with 10%, 20%, or 50% CSE treatment, respectively. Subsequent addition of tetrahydrobiopterin (BH4) restored eNOS activity in cells treated with >50% CSE, however the decrease in eNOS activity produced by higher doses of CSE produce was irreversible. The addition of BH4 to cells treated with >20% CSE actually led to an increase in eNOS activity. We found no change in eNOS protein levels in cells treated with low concentrations of CSE, while there was a significant decrease in eNOS protein following treatment with CSE concentrations >50%. Additionally, we found that Thr495, phosphorylation of which has been reported to negatively regulate eNOS, is time dependently dephosphorylated by treatment with CSE. Thus, low level CSE treatment leads to reversible inhibition of eNOS via oxidation of BH4, and exacerbates the potential eNOS‐dependent oxidative stress via dephosphorylation of Thr495. Conversely, high CSE concentrations irreversibly inhibit via a reduction in total eNOS protein.