Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase

The Angiotensin II type 2 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 Ser(1177), activation of phosphatases and dephosphorylation of eNOS at Tyr(657) and Thr(495) are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT(2)R-agonist Compound 21 (C21) (1 mu M) in the presence or absence of either PD123319 (10 mu M; AT(2)R antagonist), L-NG-Nitroarginine methyl ester (L-NAME) (10 mu M; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na3VO4) (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(1177)) or inhibitory eNOS residues (eNOS-Tyr(657), eNOS-Thr(495)) was determined by Western blotting. Phosphorylation of Akt at Ser(473) was measured to estimate Akt activity. AT2R 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-Ser(1177) and dephosphorylation of eNOS-Tyr(657) and eNOS-Thr(495). Phosphorylation at eNOS-Ser(1177) 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-Ser(1177)) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr(657), eNOS-Thr(495)) by serine/threonine and tyrosine phosphatases, thus increasing NO release.