Ascorbic Acid Enhances Endothelial Nitric-oxide Synthase Activity by Increasing Intracellular Tetrahydrobiopterin

Abstract

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...

Ascorbic Acid Prevents Blood–Brain Barrier Disruption and Sensory Deficit Caused by Sustained Compression of Primary Somatosensory Cortex

“…In addition, with regard to reactive nitrogen species, we found that the treatment of NO synthase inhibitor L-NAME reduced the extravasation of Evans Blue and IgG, despite no statistical significance ( Figures 4P and 5J). However, ascorbic acid, which protected the BBB in our model, has been shown to be capable of increasing the NO production and bioactivity without affecting the NO synthase expression (Heller et al, 2001;Huang et al, 2000). Actually, up to now, a number of reports on the role of NO in the BBB modulation are also contradictory (Boje and Lakhman, 2000;Kuhlmann et al, 2006;Wong et al, 2004).…”

Coenzyme Q10 improves endothelial dysfunction of the brachial artery in Type II diabetes mellitus

“…However, along with other studies [40,41], we found no evidence to support this finding. Other potential mechanisms whereby CoQ could have improved endothelial function in the brachial artery involve reduction in the cellular levels of asymmetric dimethyl-arginine and AGEs [31,42], as well as an increase in the bioavailability of tetrahydrobiopterin [43] and glutathione [44]. Normalization of mitochondrial superoxide production could be central to these mechanisms and to an anti-inflammatory effect of CoQ [45].…”

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…Inadequate levels of co-factors and L-arginine can result in NOS dysfunction and uncoupling, altering NOS output from NO to O 2 ·- (Vasquez-Vivar et al 1998;Huang et al 2000). Dysfunction of NOS also can occur with oxidation of the heme complex or BH 4 by ONOO -also resulting in uncoupled NOS (Figure 2.7, (Huang et al 2000;Harrison et al 2003b).…”