Abstract
BACKGROUND: The renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects. METHODS: The effect of Ang-(1-5) (1µM) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT (2) -receptor (AT (2) R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT (2) R-knockout mice. Binding of Ang-(1-5) to the AT (2) R was confirmed and the preferred conformations determined by in silico docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics. RESULTS: Key findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at (Ser1177) eNOS and (Tyr657) eNOS and thereby (2) increased NO release from HAEC and AT (2) R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT (2) R-KO or in PD123319-treated mice and which were more potent than effects of the established AT (2) R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT (2) R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT (2) R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease. CONCLUSIONS: Ang-(1-5) is a potent, endogenous AT (2) R-agonist.