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 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-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.

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“…Further, PP2A overexpression prevents the effects of ascorbate on eNOS phosphorylation and activity. These observations indicate that ascorbate stimulates eNOS activity by decreasing PP2A activity and thereby inhibiting the dephosphorylation of eNOS by PP2A (32).…”

“…Ascorbate stabilizes the eNOS cofactor BH4 (e.g., ascorbate reduces the products of BH4 oxidation) and thus prevents eNOS uncoupling in endothelial cells (25). But ascorbate also enhances eNOS activity in these cells through a rapid phosphorylation at eNOS-Ser1177 (32). eNOS activity is controlled by multiple kinases and phosphatases, including protein kinase C (PKC), Akt, AMPactivated kinase (AMPK), protein phosphatase 1, and PP2A (32).…”

“…But ascorbate also enhances eNOS activity in these cells through a rapid phosphorylation at eNOS-Ser1177 (32). eNOS activity is controlled by multiple kinases and phosphatases, including protein kinase C (PKC), Akt, AMPactivated kinase (AMPK), protein phosphatase 1, and PP2A (32). For the effect of ascorbate on eNOS phosphorylation, AMPK is the responsible upstream kinase (32).…”

“…eNOS activity is controlled by multiple kinases and phosphatases, including protein kinase C (PKC), Akt, AMPactivated kinase (AMPK), protein phosphatase 1, and PP2A (32). For the effect of ascorbate on eNOS phosphorylation, AMPK is the responsible upstream kinase (32). AMPK and eNOS are dephosphorylation targets of PP2A, and inhibition of PP2A by OA induces the same pattern of eNOS phosphorylation as does ascorbate (32).…”

“…For the effect of ascorbate on eNOS phosphorylation, AMPK is the responsible upstream kinase (32). AMPK and eNOS are dephosphorylation targets of PP2A, and inhibition of PP2A by OA induces the same pattern of eNOS phosphorylation as does ascorbate (32). Further, PP2A overexpression prevents the effects of ascorbate on eNOS phosphorylation and activity.…”