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

The angiotensin AT2 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 C21 (1µM) in the presence or absence of either PD123319 (10µM; AT2R-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. 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 w...
Source: Clinical Science - Category: Biomedical Science Authors: Tags: PublishAheadOfPrint Source Type: research