Opposing effects of reactive oxygen species and cholesterol on endothelialnitric oxide synthase and endothelial cell caveolae

Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regul ated by agonists and blood flow and chronically by factors that regulate mR NA stability and gene transcription. Recently, localization of eNOS to spec ialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO p roduction, we first studied the effects of cholesterol loading on eNOS loca lization and NO production in cultured bovine aortic endothelial cells (BAE Cs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 mu g/mL cholesterol for 24 hour s stimulated significant increases in total eNOS protein expression (1.50-f oId), eNOS associated with caveolae-enriched membranes (2.23-fold), and cal cium ionophore-stimulated NO production (1.56-fold). Because reactive oxyge n species (ROS) contribute to endothelial dysfunction in hypercholesterolem ia, we next studied the effects of ROS on eNOS localization and caveolae nu mber. Treatment of BAECs for 24 hours with 1 mu mol/L LY83583, a superoxide -generating napthoquinolinedione, decreased caveolae number measured by ele ctron microscopy and prevented the cholesterol-mediated increases in eNOS e xpression. In vitro exposure of caveolae-enriched membranes to ROS (xanthin e plus xanthine oxidase) dissociated caveolin more readily than eNOS from t he membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the associa tion of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveola e formation, eNOS expression, and eNOS-caveolin interactions.