The G protein-coupled receptor Mas was recently described as an angiotensin-(1-7) [ANG-(1-7)] receptor. In the present study we evaluated the anatomical localization of Mas using immunofluorescence in the central nervous system of adult male Wistar rats. An abundant labeling was found in the hippocampus, amigdala, anterodorsal thalamic nucleus, cortex, and hypoglossal nucleus. More importantly, a dense ANG-(1-7) receptor Mas immunoreactivity was observed in cardiovascular-related areas of the medulla and forebrain, shown in several previous studies as sites for the action of ANG-(1-7) in the brain. A strong staining was found in the nucleus of the solitary tract, caudal and rostral ventrolateral medulla, inferior olive, parvo and magnocellular portions of the paraventricular hypothalamic nucleus, supraoptic nucleus, and lateral preoptic area. Furthermore, Mas staining was predominantly present in neurons. At the medullary sites, a specific and high-intensity binding for rhodamine-ANG-(1-7) was also shown. The specific ANG-(1-7) binding was completely displaced by the anti-Mas antibody or by the ANG-(1-7) antagonist, A-779. The data presented provide the first anatomical basis for the physiological role of ANG-(1-7)/Mas axis in the modulation of different cardiovascular functions and give new insights for clarifying the role of ANG-(1-7) in the central nervous system.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous ligand of the Mas receptor and induces vasodilation, positive regulation of insulin, and antiproliferative and antitumorigenic activities. However, little is known about the molecular mechanisms behind these biological properties. Aiming to identify proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Our data suggest that the antiproliferative activity of Ang-(1-7) is due to the activation or inactivation of several target phosphoproteins, such as forkhead box protein O1 (FOXO1), mitogen-activated protein kinase 1 (MAPK), proline-rich AKT1 substrate 1 (AKT1S1), among others. In addition, the antitumorigenic activity of Ang-(1-7) is at least partially due to FOXO1 activation, since we show that this transcriptional factor is activated and accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7). Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, this study provides new concepts and new understanding of the Ang-(1-7) signal transduction, shedding light on the mechanisms underlying Mas activation.

Angiotensin-converting enzyme (ACE) inhibitors were developed based on proline-rich oligopeptides found in the venom of Bothrops jararaca (Bj) previously known as bradykinin-potentiating peptides (BPPs). However, the exact mechanism of action of BPPs remains unclear. The role of the ACE in the cardiovascular effects of two of naturally proline-rich oligopeptides (Bj-BPP-7a and Bj-BPP-10c) was evaluated in vitro and in vivo. Bj-BPP-7a does not potentiate the cardiovascular response to bradykinin and is a weak inhibitor of ACE C and N sites (K i ϭ 40,000 and 70,000 nM, respectively), whereas Bj-BPP-10c is a strong bradykinin potentiator and inhibitor of the ACE C site (K i ϭ 0.5 versus 200 nM for N site). Strikingly, both peptides, in doses ranging from 0.47 to 71 nmol/kg, produced long-lasting reduction (Ͼ6 h) in the mean arterial pressure of conscious spontaneously hypertensive rats (maximal change, 45 Ϯ 6 and 53 Ϯ 6 mm Hg for Bj-BPP-7a and Bj-BPP-10c, respectively). The fall in blood pressure was accompanied by variable degrees of bradycardia. In keeping with the absence of relationship between ACE-inhibitory and antihypertensive activities, no changes in the pressor effect of angiotensin I or in the hypotensive effect of bradykinin were observed at the peak of the cardiovascular effects of both peptides. Our results indicate that the antihypertensive effect of two Bj-BPPs containing the motif Ile-Pro-Pro is unrelated to their ability for inhibiting ACE or potentiating bradykinin (BK), indicating as a major component ACE and BK-independent mechanisms. These results are in line with previous observations suggesting ACE inhibition-independent mechanisms for angiotensin I-converting enzyme inhibitor.

The renin-angiotensin system is an important component of the central and humoral mechanisms of blood pressure and hydro-electrolytic balance control. Angiotensin II is a key player of this system. Twenty-five years ago the first manuscripts describing the formation and actions of another peptide of the RAS, angiotensin-(1-7), were published. Since then several publications have shown that angiotensin-(1-7) is as pleiotropic as angiotensin II, influencing the functions of many organs and systems. The identification of the ACE homologue ACE2 and, a few years later, Mas, as a receptor for angiotensin-(1-7) contributed a great deal to establish this peptide as a key player of the RAS. Most of the actions of angiotensin-(1-7) are opposite to those described for angiotensin II. This has led to the concept of two arms of the RAS: one comprising ACE/AngII/AT1R and the other ACE2/Ang-(1-7)/Mas. More recently, we have described the identification of a novel component of the RAS, alamandine, which binds to the Mas-related G protein coupled receptor D. This peptide is formed by decarboxylation of the Asp residue of angiotensin-(1-7), leading to the formation of Ala as the N-terminal amino acid. Alternatively, it can be formed by hydrolysis of Ang A, by ACE2. Its effects include vasorelaxation, central effects similar to those produced by angiotensin-(1-7), blunting of isoproterenol-induced heart fibrosis, and anti-hypertensive action in SHR. The putative enzyme responsible for alamandine formation from angiotensin-(1-7) is under investigation. The identification of this novel component of the RAS opens new venues for understanding its physiological role and opens new putative therapeutic possibilities for treating cardiovascular diseases.

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