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

• Published in: The Journal of biological chemistry Vol. 275; no. 23; pp. 17399 - 17406
• Main Authors: Huang, Annong, Vita, Joseph A., Venema, Richard C., Keaney, John F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.06.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Aorta; Arginine - metabolism; Ascorbic Acid - pharmacology; Atrial Natriuretic Factor - pharmacology; Biopterins - analogs & derivatives; Biopterins - metabolism; Biopterins - pharmacology; Calcimycin - pharmacology; Cattle; Cell Line; Cells, Cultured; Cyclic GMP - metabolism; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Kinetics; Nitric Oxide - metabolism; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type III; Nitroprusside - pharmacology; Pteridines - pharmacology; Pterins; Recombinant Proteins - metabolism; Spodoptera; Swine; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002248200

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 (NO2− + NO3−) production andl-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 Vmaxby ∼50% without altering the Km forl-arginine. The effect of ascorbate was tetrahydrobiopterin (BH4)-dependent, because ascorbate was ineffective with BH4 concentrations >10 μmor in PAECs treated with sepiapterin to increase intracellular BH4. 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 BH4. These data suggest that ascorbic acid enhances NO bioactivity in a BH4-dependent manner by increasing intracellular BH4 content.