title: Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status.
abstract: Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine-citrulline conversion assay and HPLC analysis, respectively. Over a period of 4h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization.
authors.affiliation: Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, 1090 Vienna, Austria.
authors.forename: Angela
authors.initials: A
authors.lastname: Ladurner
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Schmitt
authors.forename: Daniel
authors.initials: D
authors.lastname: Schachner
authors.forename: Atanas G
authors.initials: AG
authors.lastname: Atanasov
authors.forename: Ernst R
authors.initials: ER
authors.lastname: Werner
authors.forename: Verena M
authors.initials: VM
authors.lastname: Dirsch
authors.forename: Elke H
authors.initials: EH
authors.lastname: Heiss
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D014664:Vasodilation / Q000187:drug effects
chemical_list: D034741:RNA, Small Interfering; D001708:Biopterins; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; D055372:AMP-Activated Protein Kinases; D054648:Protein Phosphatase 2; C003402:sapropterin; D001205:Ascorbic Acid