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

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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

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Phosphorylation
Nitric Oxide Synthase Type III
Bradykinin
Endothelial cells
Endothelial Cells
Chemical activation
Phosphatidylinositol 3-Kinase
Nitric Oxide
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
AMP-Activated Protein Kinases
Calcineurin
Cyclosporine
Shear stress
Intercellular Signaling Peptides and Proteins
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Harris, M. B., Ju, H., Venema, V. J., Liang, H., Zou, R., Michell, B. J., ... 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

Reciprocal Phosphorylation and Regulation of Endothelial Nitric-oxide Synthase in Response to Bradykinin Stimulation. / Harris, M. Brennan; Ju, Hong; Venema, Virginia J.; Liang, Haiying; Zou, Rong; Michell, Belinda J.; Chen, Zhi Ping; Kemp, Bruce E.; Venema, Richard C.

In: Journal of Biological Chemistry, Vol. 276, No. 19, 11.05.2001, p. 16587-16591.

Research output: Contribution to journalArticle

Harris, MB, Ju, H, Venema, VJ, Liang, H, Zou, R, Michell, BJ, Chen, ZP, Kemp, BE & Venema, RC 2001, 'Reciprocal Phosphorylation and Regulation of Endothelial Nitric-oxide Synthase in Response to Bradykinin Stimulation', Journal of Biological Chemistry, vol. 276, no. 19, pp. 16587-16591. https://doi.org/10.1074/jbc.M100229200
Harris, M. Brennan ; Ju, Hong ; Venema, Virginia J. ; Liang, Haiying ; Zou, Rong ; Michell, Belinda J. ; Chen, Zhi Ping ; Kemp, Bruce E. ; Venema, Richard C. / Reciprocal Phosphorylation and Regulation of Endothelial Nitric-oxide Synthase in Response to Bradykinin Stimulation. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 19. pp. 16587-16591.
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