Reciprocal Phosphorylation and Regulation of Endothelial Nitric-oxide Synthase in Response to Bradykinin Stimulation

M. Brennan Harris, Hong Ju, Virginia J. Venema, Haiying Liang, Rong Zou, Belinda J. Michell, Zhi Ping Chen, Bruce E. Kemp, Richard C Venema

Research output: Contribution to journalArticle

309 Scopus citations

Abstract

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.

Original languageEnglish (US)
Pages (from-to)16587-16591
Number of pages5
JournalJournal of Biological Chemistry
Volume276
Issue number19
DOIs
StatePublished - May 11 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Harris, M. B., Ju, H., Venema, V. J., Liang, H., Zou, R., Michell, B. J., Chen, Z. P., Kemp, B. E., & Venema, R. C. (2001). Reciprocal Phosphorylation and Regulation of Endothelial Nitric-oxide Synthase in Response to Bradykinin Stimulation. Journal of Biological Chemistry, 276(19), 16587-16591. https://doi.org/10.1074/jbc.M100229200