Journal
FREE RADICAL BIOLOGY AND MEDICINE
Volume 52, Issue 10, Pages 2082-2090Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2012.03.022
Keywords
Ascorbate; Endothelial NO synthase activity; Endothelial NO synthase phosphorylation; AMP-activated kinase; Protein phosphatase 2A; Free radicals
Funding
- Austrian Science Fund [J2957-B11, P22289, P23317]
- Hochschuljubilaumsstiftung der Stadt Wien [H-01509/2007]
- Austrian Science Fund (FWF) [P22289] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [P 23317, P 22289] Funding Source: researchfish
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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 4 h, 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-Ser(1177) and concomitantly decreased phosphorylation at eNOS-Thr(495), 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. (C) 2012 Elsevier Inc. All rights reserved.
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