OBJECTIVES: Given the importance of endothelial nitric oxide synthase (eNOS) in regulating endothelium-dependent vasorelaxation, we investigated the effects of high-density lipoprotein in (HDL) on eNOS protein abundance in cultured human vascular endothelial cells. BACKGROUND: Endothelial dysfunction, characterized by decreased nitric oxide production, is one of the early features in the development of atherosclerosis. We have recently shown in vivo that niacin therapy increases plasma HDL concentration and improves endothelium-dependent vasorelaxation in patients with coronary artery disease. METHODS: Human vascular endothelial cells were cultured in the presence or absence of HDL or apolipoprotein (apo)A-I. The eNOS protein abundance was assessed by immunoblotting, and protein half-life was assessed by pulse-chase techniques. The eNOS messenger ribonucleic acid (mRNA) abundance was measured using real-time quantitative polymerase chain reaction. RESULTS: High density lipoprotein, or apoA-I alone, increased eNOS protein abundance by 3.5 +/- 0.7 and 2.7 +/- 0.5-fold, respectively (p < 0.05 for both). However, neither HDL nor apoA-I increased eNOS mRNA abundance. It was shown that HDL increased eNOS protein half-life up to 3.3 +/- 0.2-fold (p = 0.001). Both HDL and apoA-I activated mitogen-activated protein-kinase and phosphatidylinositol 3-kinase (PI3K) Akt-pathways in human arterial endothelial cells, and inhibition of either of these pathways by specific pharmacologic inhibitors abolished the effect of HDL on eNOS. CONCLUSIONS: We demonstrate that HDL activates both extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, resulting in enhanced eNOS protein stability and subsequent accumulation of eNOS protein. This posttranslational regulation represents a previously unrecognized mechanism for regulating eNOS.