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Pubmed ID :29540539
Publication Date : //

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


The Angiotensin II type 2 receptor (ATR) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the ATR-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known ATR-mediated phosphorylation of eNOS at Ser, activation of phosphatases and dephosphorylation of eNOS at Tyr and Thr are also involved. Human aortic endothelial cells (HAEC) were stimulated with the ATR-agonist Compound 21 (C21) (1 µM) in the presence or absence of either PD123319 (10 µM; ATR antagonist), l-NG-Nitroarginine methyl ester (l-NAME) (10 µM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (NaVO) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser) or inhibitory eNOS residues (eNOS-Tyr, eNOS-Thr) was determined by Western blotting. Phosphorylation of Akt at Ser was measured to estimate Akt activity. ATR 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. ATR stimulation resulted in phosphorylation of eNOS-Ser and dephosphorylation of eNOS-Tyr and eNOS-Thr Phosphorylation at eNOS-Ser was prevented by inhibition of Akt with MK-2206. From these data, we conclude that ATR stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr, eNOS-Thr) by serine/threonine and tyrosine phosphatases, thus increasing NO release.

Authors : Peluso A Augusto , Bertelsen Jesper Bork , Andersen Kenneth , Mortsensen Tenna Pavia , Hansen Pernille B , Sumners Colin , Bader Michael , Santos Robson A , Steckelings Ulrike Muscha ,

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