Robson A.S. Santos

Angiotensin-(1–7) is an endogenous ligand for the G protein-coupled receptor Mas

The renin–angiotensin system plays a critical role in blood pressure control and body fluid and electrolyte homeostasis. Besides angiotensin (Ang) II, other Ang peptides, such as Ang III [Ang-(2–8)], Ang IV [Ang-(3–8)], and Ang-(1–7) may also have important biological activities. Ang-(1–7) has become an angiotensin of interest in the past few years, because its cardiovascular and baroreflex actions counteract those of Ang II. Unique angiotensin-binding sites specific for this heptapeptide and studies with a selective Ang-(1–7) antagonist indicated the existence of a distinct Ang-(1–7) receptor. We demonstrate that genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene abolishes the binding of Ang-(1–7) to mouse kidneys. Accordingly, Mas-deficient mice completely lack the antidiuretic action of Ang-(1–7) after an acute water load. Ang-(1–7) binds to Mas-transfected cells and elicits arachidonic acid release. Furthermore, Mas-deficient aortas lose their Ang-(1–7)-induced relaxation response. Collectively, these findings identify Mas as a functional receptor for Ang-(1–7) and provide a clear molecular basis for the physiological actions of this biologically active peptide.

Angiotensin-(1-7): an update.

The renin-angiotensin system is a major physiological regulator of arterial pressure and hydro-electrolyte balance. Evidence has now been accumulated that in addition to angiotensin (Ang) II other Ang peptides [Ang III, Ang IV and Ang-(1-7)], formed in the limited proteolysis processing of angiotensinogen, are importantly involved in mediating several actions of the RAS. In this article we will review our knowledge of the biological actions of Ang-(1-7) with focus on the puzzling aspects of the mediation of its effects and the interaction Ang-(1-7)-kinins. In addition, we will attempt to summarize the evidence that Ang-(1-7) takes an important part of the mechanisms aimed to counteract the vasoconstrictor and proliferative effects of Ang II.

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; 10−7 mol/L; 1 to 30 minutes) in the absence or presence of A-779 (10−6 mol/L). Additional experiments were performed in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin (10−6 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<0.05). Concomitantly, a significant increase in NO release was observed (2-fold increase in relation to control). These effects were blocked by A-779. Wortmannin suppressed eNOS activation in both Chinese hamster ovary-Mas and human aortic endothelial cells. Our findings demonstrate that Ang-(1-7), through Mas, stimulates eNOS activation and NO production via Akt-dependent pathways. These novel data highlight the importance of the Ang-(1-7)/Mas axis as a putative regulator of endothelial function.

Recent advances in the angiotensin‐converting enzyme 2–angiotensin(1–7)–Mas axis

In the past few years, the classical concept of the renin–angiotensin system (RAS) has experienced substantial conceptual changes. The identification of: the renin/prorenin receptor; the angiotensin‐converting enzyme homologue, ACE2, as an angiotensin peptide‐processing enzyme and a virus receptor for severe acute respiratory syndrome, the Mas as a receptor for angiotensin (1–7) [Ang(1–7)], and the possibility of signaling through ACE have contributed to switch our understanding of the RAS from the classical limited‐proteolysis linear cascade to a cascade with multiple mediators, multiple receptors and multifunctional enzymes. With regard to Ang(1–7), the identification of ACE2 and of Mas as a receptor implicated in its actions contributed to decisively establish this heptapeptide as a biologically active member of the RAS cascade. In this review, we will focus on the recent findings related to the ACE2–Ang(1–7)–Mas axis and, in particular, on its putative role as an ACE–Ang II–AT1 receptor counter‐regulatory axis within the RAS.

Characterization of a new angiotensin antagonist selective for angiotensin-(1–7): Evidence that the actions of angiotensin-(1–7) are mediated by specific angiotensin receptors

In this study we describe a new angiotensin antagonist [Asp1-Arg2-Val3-Tyr4-Ile5-His6-D-Ala7, (A-779)] selective for the heptapeptide angiotensin-(1-7) [Ang-(1-7)]. A-779 blocked the antidiuretic effect of Ang-(1-7) in water-loaded rats and the changes in blood pressure produced by Ang-(1-7) microinjection into the dorsal-medial and ventrolateral medulla. In contrast, A-779 did not change the dipsogenic, pressor, or myotropic effects of angiotensin II (Ang II). Also, A-779 did not affect the antidiuretic effect of vasopressin or the contractile effects of angiotensin III, bradykinin, or substance P on the rat ileum. In the rostral ventrolateral medulla, the pressor effect produced by Ang-(1-7) microinjection was completely blocked by A-779 but not by AT1 or AT2 receptor antagonists (DUP 753 and CGP 42112A, respectively). Conversely, the pressor effect produced by Ang II was not changed by A-779 but was completely blocked by DUP 753. Binding studies substantiated these observations: A-779 did not compete significantly for 125I-Ang II binding to adrenocortical membranes at up to a 1 microM concentration. Low affinity binding was also observed in adrenomedullary membranes with an IC50 greater than 10 microM. Our results show that A-779 is a potent and selective antagonist for Ang-(1-7). More importantly, our data indicate that specific angiotensin receptors mediate the central and peripheral actions of Ang-(1-7).

Angiotensin-converting enzyme 2, angiotensin-(1–7) and Mas: new players of the renin–angiotensin system

Angiotensin (Ang)-(1-7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). Ang-(1-7) appears to play a central role in the RAS because it exerts a vast array of actions, many of them opposite to those attributed to the main effector peptide of the RAS, Ang II. The discovery of the Ang-converting enzyme (ACE) homolog ACE2 brought to light an important metabolic pathway responsible for Ang-(1-7) synthesis. This enzyme can form Ang-(1-7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1-9) with subsequent Ang-(1-7) formation by ACE. In addition, it is now well established that the G protein-coupled receptor Mas is a functional binding site for Ang-(1-7). Thus, the axis formed by ACE2/Ang-(1-7)/Mas appears to represent an endogenous counterregulatory pathway within the RAS, the actions of which are in opposition to the vasoconstrictor/proliferative arm of the RAS consisting of ACE, Ang II, and AT(1) receptor. In this brief review, we will discuss recent findings related to the biological role of the ACE2/Ang-(1-7)/Mas arm in the cardiovascular and renal systems, as well as in metabolism. In addition, we will highlight the potential interactions of Ang-(1-7) and Mas with AT(1) and AT(2) receptors.

Angiotensin-(1-7): Cardioprotective Effect in Myocardial Ischemia/Reperfusion

In this study we evaluate the effects of angiotensin-(1-7) on reperfusion arrhythmias in isolated rat hearts. Rat hearts were perfused according to Langendorff technique and maintained in heated (37±1°C) and continuously gassed (95% O2/5% CO2) Krebs-Ringer solution at constant pressure (65 mm Hg). The electrical activity was recorded with an ECG (bipolar). Local ischemia was induced by coronary ligation for 15 minutes. After ischemia, hearts were reperfused for 30 minutes. Cardiac arrhythmias were defined as the presence of ventricular tachycardia and/or ventricular fibrillation after the ligation of the coronary artery was released. Angiotensin II (0.20 nmol/L, n=10) produced a significant enhancement of reperfusion arrhythmias. On the other hand, Ang-(1-7) presented in the perfusion solution (0.22 nmol/L, n=11) reduced incidence and duration of arrhythmias. The antiarrhythmogenic effects of Ang-(1-7) was blocked by the selective Ang-(1-7) antagonist A-779 (2 nmol/L, n=9) and by indomethacin pretreatment (5 mg/kg IP, n=8) but not by the bradykinin B2 antagonist HOE 140 (100 nmol/L, n=10) or by L-NAME pretreatment (30 mg/kg IP, n=8). These results suggest that the antiarrhythmogenic effect of low concentrations of Ang-(1-7) is mediated by a specific receptor and that release of endogenous prostaglandins .by Ang-(1-7) contributes to the alleviation of reversible and/or irreversible ischemia-reperfusion injury.

Impairment of In Vitro and In Vivo Heart Function in Angiotensin-(1-7) Receptor Mas Knockout Mice

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas−/− mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4±0.09 versus 2.1±0.03 g in Mas+/+ mice), ±dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart.

Mas Deficiency in FVB/N Mice Produces Marked Changes in Lipid and Glycemic Metabolism

OBJECTIVE— Metabolic syndrome is characterized by the variable coexistence of obesity, hyperinsulinemia, insulin resistance, dyslipidemia, and hypertension. It is well known that angiotensin (Ang) II is importantly involved in the metabolic syndrome. However, the role of the vasodilator Ang-(1-7)/Mas axis is not known. The aim of this study was to evaluate the effect of genetic deletion of the G protein–coupled receptor, Mas, in the lipidic and glycemic metabolism in FVB/N mice. RESEARCH DESIGN AND METHODS— Plasma lipid, insulin, and cytokine concentrations were measured in FVB/N Mas-deficient and wild-type mice. A glucose tolerance test was performed by intraperitoneally injecting d-glucose into overnight-fasted mice. An insulin sensitivity test was performed by intraperitoneal injection of insulin. Uptake of 2-deoxy-[3H]glucose by adipocytes was used to determine the rate of glucose transport; adipose tissue GLUT4 was quantified by Western blot. Gene expression of transforming growth factor (TGF)-β, type 1 Ang II receptor, and angiotensinogen (AGT) were measured by real-time PCR. RESULTS— Despite normal body weight, Mas-knockout (Mas-KO) mice presented dyslipidemia, increased levels of insulin and leptin, and an ∼50% increase in abdominal fat mass. In addition, Mas gene–deleted mice presented glucose intolerance and reduced insulin sensitivity as well as a decrease in insulin-stimulated glucose uptake by adipocytes and decreased GLUT4 in adipose tissue. Mas−/− presented increased muscle triglycerides, while liver triglyceride levels were normal. Expression of TGF-β and AGT genes was higher in Mas-KO animals in comparison with controls. CONCLUSIONS— These results show that Mas deficiency in FVB/N mice leads to dramatic changes in glucose and lipid metabolisms, inducing a metabolic syndrome–like state.

Angiotensin(1-7) Blunts Hypertensive Cardiac Remodeling by a Direct Effect on the Heart

Angiotensin-converting enzyme 2 (ACE2) converts the vasopressor angiotensin II (Ang II) into angiotensin (1-7) [Ang(1-7)], a peptide reported to have vasodilatory and cardioprotective properties. Inactivation of the ACE2 gene in mice has been reported by one group to result in an accumulation of Ang II in the heart and an age-related defect in cardiac contractility. A second study confirmed the role of ACE2 as an Ang II clearance enzyme but failed to reproduce the contractility defects previously reported in ACE2-deficient mice. The reasons for these differences are unclear but could include differences in the accumulation of Ang II or the deficiencies in Ang(1-7) in the mouse models used. As a result, the roles of ACE2, Ang II, and Ang(1-7) in the heart remain controversial. Using a novel strategy, we targeted the chronic overproduction of either Ang II or Ang(1-7) in the heart of transgenic mice and tested their effect on age-related contractility and on cardiac remodeling in response to a hypertensive challenge. We demonstrate that a chronic accumulation of Ang II in the heart does not result in cardiac contractility defects, even in older (8-month-old) mice. Likewise, transgenic animals with an 8-fold increase in Ang(1-7) peptide in the heart exhibited no differences in resting blood pressure or cardiac contractility as compared to age-matched controls, but they had significantly less ventricular hypertrophy and fibrosis than their nontransgenic littermates in response to a hypertensive challenge. Analysis of downstream signaling cascades demonstrates that cardiac Ang(1-7) selectively modulates some of the downstream signaling effectors of cardiac remodeling. These results suggest that Ang(1-7) can reduce hypertension-induced cardiac remodeling through a direct effect on the heart and raise the possibility that pathologies associated with ACE2 inactivation are mediated in part by a decrease in production of Ang(1-7).