Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT2-Receptor Agonist

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 AT2-receptor (AT2R) 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 AT2R-knockout mice. Binding of Ang-(1-5) to the AT2R 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 Ser1177eNOS and Tyr657eNOS and thereby (2) increased NO release from HAEC and AT2R-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 AT2R-KO or in PD123319-treated mice and which were more potent than effects of the established AT2R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT2R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT2R. (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 AT2R-agonist. ### Competing Interest Statement The authors have declared no competing interest. * ACE : Angiotensin Converting Enzyme Akt : Protein Kinase Akt Ang II : Angiotensin II Ang-(1-5) : Angiotensin-(1-5) Ang-(1-7) : Angiotensin-(1-7) AT2R : Angiotensin AT2-receptor AT2R : Angiotensin AT1-receptor ATP : Adenosine Triphosphate BP : Blood Pressure C21 : Compound 21 CHO : Chinese Hamster Ovary Cells CRC : Concentration-Response Curve EC50 : Half-maximum effective concentration Emax : Maximum effect HAEC : Human Aortic Endothelial Cells HR : Heart Rate iv : Intravenous L-NAME : NG-Nitro-L-Arginine-Methyl Ester MAP : Mean Arterial Pressure NO : Nitric Oxide NOS : Nitric Oxide Synthase RAS : Renin-Angiotensin System rhACE2 : Recombinant human Angiotensin Converting Enzyme type 2 Ser : Serine Tyr : Tyrosine