Angiotensin-(1-7) Through Receptor Mas Mediates Endothelial Nitric Oxide Synthase Activation via Akt-Dependent Pathways

Angiotensin-(1-7) [Ang-(1-7)] causes endothelial-dependent vasodilation mediated, in part, by NO release. However, the molecular mechanisms involved in endothelial NO synthase (eNOS) activation by Ang-(1-7) remain unknown. Using Chinese hamster ovary cells stably transfected with Mas cDNA (Chinese hamster ovary-Mas), we evaluated the underlying mechanisms related to receptor Mas–mediated posttranslational eNOS activation and NO release. We further examined the Ang-(1-7) profile of eNOS activation in human aortic endothelial cells, which constitutively express the Mas receptor. Chinese hamster ovary-Mas cells and human aortic endothelial cell were stimulated with Ang-(1-7; 10 mol/L; 1 to 30 minutes) in the absence or presence of A-779 (10 mol/L). Additional experiments were performed in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin (10 mol/L). Changes in eNOS (at Ser1177/Thr495 residues) and Akt phosphorylation were evaluated by Western blotting. NO release was measured using both the fluorochrome 2,3-diaminonaphthalene and an NO analyzer. Ang-(1-7) significantly stimulated eNOS activation (reciprocal phosphorylation/dephosphorylation at Ser1177/Thr495) and induced a sustained Akt phosphorylation (P