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

Abstract

The Angiotensin II type 2 receptor (AT₂R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT₂R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT₂R-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 AT₂R-agonist Compound 21 (C21) (1 µM) in the presence or absence of either PD123319 (10 µM; AT₂R 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 (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-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. 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. AT₂R 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: AT2-receptor; endothelial nitric oxide synthase; protein phosphatases; renin-angiotensin system.