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Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis

By Stuart A. Ritchie, Christine F. Kohlhaas, Alasdair R. Boyd, Krishna C. Yalla, Kenneth Walsh, John M.C. Connell and Ian P. Salt

Abstract

Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFα (tumour necrosis factor α), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis

Topics: QH345
Publisher: Portland Press Ltd.
Year: 2010
OAI identifier: oai:eprints.gla.ac.uk:24883
Provided by: Enlighten

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Citations

  1. (2004). Acute activation and phosphorylation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors. doi
  2. (2000). Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo. doi
  3. (2007). AMP-activated protein kinase media tes VEGF-stimulated endothelial NO production. doi
  4. (2003). Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase. doi
  5. (2002). Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A. doi
  6. (2006). Differences in eNOS activity because of subcellular localisation are dictated by phosphorylation state rather than the local calcium environment. doi
  7. (2003). Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells. doi
  8. (2009). Effects of combined phosphorylation at doi
  9. (2005). Endostatin induces acute endothelial nitric oxide and prostacyclin release. doi
  10. (2009). Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets. doi
  11. (2004). G972R IRS-1 variant impairs insulin regulation of endothelial nitric oxide synthase in cultured human endothelial cells. doi
  12. (2003). High glucose inhibits insulin-stimulated nitric oxide production without reducing eNOS Ser1177 phosphorylation in human aortic endothelial cells. doi
  13. (1999). Identification of flowdependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002. doi
  14. (2002). Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. doi
  15. (2005). Insulin and IGF-I phosphorylate eNOS in HUVECs by a caveolin-1 dependent mechanism. doi
  16. (1996). Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. doi
  17. (1994). Insulinmediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. doi
  18. (2007). Interleukin-6 impairs the insulin signalling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells. doi
  19. (2007). Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation. doi
  20. (2007). Role of eNOS phosphorylation at Ser-116 in regulation of eNOS activity in endothelial cells. doi
  21. (2008). Rosiglitazone stimulates nitric oxide synthesis in human aortic endothelial cells via AMP-activated protein kinase. doi
  22. (2003). Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex. doi
  23. (1998). The vasodilating effect of insulin is dependent on local glucose uptake: a double blind, placebo-controlled study. doi
  24. (2001). TNF-alpha inhibits flow and insulin signaling leading to NO production in aortic endothelial cells.
  25. (2002). Tumor necrosis factor-alpha inhibits insulin-induced increase in endothelial nitric oxide synthase and reduces insulin receptor content and phosphorylation in human aortic endothelial cells. doi
  26. (2009). Vascular actions of insulin with implications for endothelial dysfunction. doi

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