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...

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Objective-To determine how hypochlorous acid (HOCl), the principal product of myeloperoxidase, modulates vascular function. Methods and Results-Rabbit arterial rings exposed to HOCl (0 to 500 mol/L) exhibited dose-and time-dependent impairment of endothelium-dependent arterial relaxation to acetylcholine and A23187, but not the NO donor, diethylamine NONOate, suggesting that HOCl targets the endothelium. This effect was not because of cytotoxicity, as HOCl treatment produced no significant change in endothelial cell morphology or lactate dehydrogenase release. We observed HOCl-mediated endothelial cell protein oxidation by immunoreactivity to HOP-1, a monoclonal antibody specific for HOCl-oxidized protein.In support of this notion, known HOCl scavengers, such as methionine and N-acetylcysteine, partially preserved endothelium-derived NO bioactivity in response to HOCl. In an unanticipated observation, HOCl-mediated impairment of NO bioactivity was prevented by manganese superoxide dismutase in a manner dependent on its enzymatic activity. Finally, we found that HOCl reduced endothelial nitric oxide synthase dimer stability, an effect that was also inhibited by superoxide dismutase. Conclusions-Taken together, these data indicate that HOCl imparts a defect in endothelial NO production due to a superoxide-dependent reduction in endothelial nitric oxide synthase dimer stability. These data provide another mechanism whereby myeloperoxidase-derived oxidants can contribute to the impairment of NO bioactivity that is characteristic of atherosclerosis. Key Words: oxidant stress Ⅲ nitric oxide Ⅲ endothelium Ⅲ superoxide A therosclerosis and its associated pathological conditions, such as hypercholesterolemia 1 and diabetes mellitus, 2 are characterized by abnormal endothelium-derived nitric oxide (EDNO) bioactivity. In atherosclerosis, vasodilatation to agonists for EDNO release, such as acetylcholine 3 or aggregating platelets, 4 is impaired or replaced by constriction. Endothelial dysfunction has been directly demonstrated in atherosclerotic human coronary arteries, 3 and there is strong evidence that it contributes to plaque activation and the pathogenesis of myocardial ischemia. 5 In fact, the presence of vascular dysfunction predicts the occurrence of clinical events in patients with atherosclerosis. 6 Available evidence suggests that vascular oxidative events play an important role in endothelial dysfunction. In animal models of atherosclerosis and hypercholesterolemia, the vascular production of superoxide anion radical is increased, 7 and this abnormality can decrease NO bioactivity through peroxynitrite formation. 8 Indeed, limiting superoxide production with oxypurinol, a xanthine oxidase inhibitor, partially restores EDNO bioactivity. 7 Similarly, in atherosclerotic rabbits, chronic treatment with polyethylene glycolatedsuperoxide dismutase (SOD) increases vascular SOD activity and partially restores NO-mediated relaxation to acetylcholine. 9 Later stages of atherosclerosis, however, are more complex. In Watan...

Hypertension is associated with low plasma ascorbic acid levels and impaired endothelial function. Recent evidence suggests that increased vascular oxidative stress contributes to the pathophysiology of endothelial dysfunction and hypertension. We recently showed that chronic oral ascorbic acid therapy lowers blood pressure in hypertensive patients. We hypothesized that it would also improve endothelial vasomotor function. In a randomized, double-blind, placebo-controlled study, we examined the effect of acute (2 g po) and chronic (500 mg/day for 1 mo) ascorbic acid treatment on brachial artery flow-mediated dilation in 39 patients with hypertension. Compared with 82 age- and gender-matched normotensive controls, these patients had impaired endothelium-dependent, flow-mediated dilation of the brachial artery [8.9 +/- 6.1 vs. 11.2 +/- 5.7% (SD), P < 0.04]. After therapy, plasma ascorbic acid concentrations increased acutely from 50 +/- 12 to 149 +/- 51 micromol/l and were maintained at 99 +/- 33 micromol/l with chronic treatment (both P < 0.001). As previously reported, chronic ascorbic acid therapy reduced systolic and mean blood pressure in these patients. However, acute or chronic ascorbic acid treatment had no effect on brachial artery endothelium-dependent, flow-mediated dilation or on endothelium-independent, nitroglycerin-mediated dilation. These results demonstrate that conduit vessel endothelial dysfunction secondary to hypertension is not reversed by acute or chronic treatment with oral ascorbic acid. The effects of this treatment on resistance vessel vasomotor function warrant further investigation.