Alternative Renin-Angiotensin System.

The renin-angiotensin system is the most important peptide hormone system in the regulation of cardiovascular homeostasis. Its classical arm consists of the enzymes, renin, and angiotensin-converting enzyme, generating angiotensin II from angiotensinogen, which activates its AT1 receptor, thereby increasing blood pressure, retaining salt and water, and inducing cardiovascular hypertrophy and fibrosis. However, angiotensin II can also activate a second receptor, the AT2 receptor. Moreover, the removal of the C-terminal phenylalanine from angiotensin II by ACE2 (angiotensin-converting enzyme 2) yields angiotensin-(1-7), and this peptide interacts with its receptor Mas. When the aminoterminal Asp of angiotensin-(1-7) is decarboxylated, alamandine is generated, which activates the Mas-related G-protein-coupled receptor D, MrgD (Mas-related G-protein-coupled receptor type D). Since Mas, MrgD, and the AT2 receptor have opposing effects to the classical AT1 receptor, they and the enzymes and peptides activating them are called the alternative or protective arm of the renin-angiotensin system. This review will cover the historical aspects and the current standing of this recent addition to the biology of the renin-angiotensin system.

Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro.

Angiotensin AT2-receptor (AT2R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT2R-agonists is very limited due to the lack of high-throughput assays for AT2R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT2R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT2R-transfected CHO cells (AT2R-CHO) or in non-transfected CHO cells (Flp-CHO) using the fluorescent probe DAF-FM diacetate. It is run in 96-well plates and fluorescence signals are semi-automatically quantified. The assay was tested for sensitivity (recognition of true positive results), selectivity (recognition of true negative results), and reliability (by calculating the repeatability coefficient (RC)). The high-throughput, semi-automated method was proven suitable, as the NO-releasing agents C21, CGP42112A, angiotensin-(1-7) and acetylcholine significantly increased NO release from HAEC. The assay is sensitive and selective, since the established AT2R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT2R-CHO cells, while the non-AT2R-agonists angiotensin-(1-7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT2R-agonists, of which 39 met our requirements for reliability (RC ≤ 20% different from RC for C21). Our newly developed high-throughput method for detection of AT2R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT2R-agonists in future drug development programs.

Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT 2 -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 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.