Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s).We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time-and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO 2 ؊ ؉ NO 3 Ϫ ) production and L-arginine to L-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V max by ϳ50% without altering the K m for L-arginine. The effect of ascorbate was tetrahydrobiopterin (BH 4 )-dependent, because ascorbate was ineffective with BH 4 concentrations >10 M or in PAECs treated with sepiapterin to increase intracellular BH 4 . The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH 4 . These data suggest that ascorbic acid enhances NO bioactivity in a BH 4 -dependent manner by increasing intracellular BH 4 content.Nitric oxide is produced from L-arginine in the vascular endothelium by the endothelial isoform of nitric-oxide synthase (NOS).1 Endothelial production of NO is crucial in the control of vascular tone (1), arterial pressure (2-4), smooth muscle cell proliferation (5, 6), and platelet adhesion to the endothelial surface (7). Impaired endothelium-derived NO bioactivity is a common feature of many vascular diseases (8 -10) that is thought to contribute to their clinical manifestations (11,12).The action of NO is particularly sensitive to the local availability of superoxide. Both endothelial elaboration of NO and arterial relaxation in response to nitrovasodilators are dependent upon intact copper-zinc superoxide dismutase (SOD) activity (13,14). Animal models of hypercholesterolemia (15, 16) and hypertension (17) demonstrate an excess vascular superoxide flux that is linked to reduced NO bioactivity. Conversely, increasing vascular SOD activity enhances NO-mediated arterial relaxation in experimental models of atherosclerosis (18,19) and hypertension (17). Thus, scavenging superoxide has important implications for NO bioactivity under both normal and pathologic conditions. Ascorbic acid also efficiently scavenges superoxide (20) and numerous studies in a host of pathologic conditions such as diabetes (21), hypercholesterolemia (22), smoking (23), and hypertension (24) indicate that NO bioactivity is improved by parenteral ascorbic acid at supraphysiologic concentrations (ϳ10 mM). We have observed enhanced NO bioactivity in atherosclerotic patients aft...