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

Abstract

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.

Cell-Specific Protective Signaling Induced by the Novel AT2R-Agonist NP-6A4 on Human Endothelial and Smooth Muscle Cells

“…AT2R activation is reported to increase eNOS expression and NO bioavailability in human endothelial cells ( Peluso et al, 2018 ). To determine whether NP-6A4 exerted similar effects in hCAECs, we investigated expression and phosphorylation levels of eNOS and NO generation in hCAECs treated with 5 μM NP-6A4.…”

“…Cardiac AT2R activation has been associated with NO-mediated downstream benefits ( Zhu et al, 2010 ), including vasodilation ( Yayama and Okamoto, 2008 ) and attenuation of fibrosis ( Kurisu et al, 2003 ). Compound 21 increased NO within the renal interstitial fluid in a model of type 1 diabetes ( Matavelli et al, 2015 ) and in human aortic endothelial cells ( Peluso et al, 2018 ).…”

Anti‐fibrotic mechanisms of angiotensin AT2‐receptor stimulation

“…It is also well known that AT 2 R couple to activation of endothelial nitric oxide synthase (eNOS), with subsequent generation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) . A recent study using endothelial cells indicates that the activation of eNOS is mediated through Akt‐mediated phosphorylation plus serine/threonine and tyrosine phosphatase‐mediated dephosphorylation events . This is important because eNOS activation and NO production can lead to inhibition of the synthesis of TGF‐β, which constitutes an AT 2 R‐mediated anti‐fibrotic mechanism .…”

NADPH Oxidases and Mitochondria in Vascular Senescence

“…Activation of AT 2 R stimulates vasodilation by increasing endothelial nitric oxide synthase (eNOS) activity, and nitric oxide (NO) production in endothelial cells [ 40 ], contributing to reduced blood pressure in spontaneously hypertensive rats [ 41 ]. The activation of eNOS by AT 2 R is mediated by increased phosphorylation of activating residues in eNOS induced by Akt, and dephosphorylation of inhibitory residues by phosphatases [ 42 ].…”