Identification of protein phosphatase involvement in the AT(2)-receptor induced activation of endothelial nitric oxide synthase

Item Type:	Article
Title:	Identification of protein phosphatase involvement in the AT(2)-receptor induced activation of endothelial nitric oxide synthase
Creators Name:	Peluso, A.A. and Bertelsen, J.B. and Andersen, K. and Mortensen, T.P. and Hansen, P.B. and Sumners, C. and Bader, M. and Santos, R.A. and Steckelings, U.M.
Abstract:	The angiotensin AT2 receptor (AT(2)R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT(2)R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT(2)R-mediated phosphorylation of eNOS at Ser1177, activation of phosphatases and dephosphorylation of eNOS at Tyr657 and Thr495 are also involved. Human Aortic Endothelial Cells (HAEC) were stimulated with the AT(2)R-agonist C21 (1µM) in the presence or absence of either PD123319 (10µM; AT(2)R-antagonist), L-NAME (10µM; eNOS inhibitor), MK-2206 (100nM; Akt-inhibitor) sodium fluoride (1nM; serine/threonine-phosphatase inhibitor) or sodium orthovanadate (10nM; tyrosine-phosphatase inhibitor). NO release was estimated by quantifying DAF-FM fluorescence. The phosphorylation status of activating (eNOS-Ser1177) or inhibitory eNOS residues (eNOS-Tyr657, eNOS-Thr495) was determined by Western blotting. Phosphorylation of Akt at Ser473 was measured to estimate Akt activity. AT(2)R-stimulation significantly increased NO release from HAEC, which was blocked by PD123319, L-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT(2)R-stimulation resulted in phosphorylation of eNOS-Ser1177 and dephosphorylation of eNOS-Tyr657 and eNOS-Thr495. Phosphorylation at eNOS-Ser1177 was prevented by inhibition of Akt with MK-2206. From these data we conclude that AT2R-stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr657, eNOS-Thr495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.
Keywords:	Renin-Angiotensin System, AT2-Receptor, Endothelial Nitric Oxide Synthase, Protein Phosphatases
Source:	Clinical Science
ISSN:	0143-5221
Publisher:	Portland Press
Volume:	132
Number:	7
Page Range:	777-790
Date:	6 April 2018
Official Publication:	https://doi.org/10.1042/CS20171598
PubMed:	View item in PubMed

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