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

Abstract

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.

Figures

Fig. 1.

Angiotensin receptor binding in WT and Mas knockout (KO) mouse kidneys. (A) Nonspecific, total AT1, and total AT2 binding of 125I-[Sar-1, Ile-8]Ang II binding in WT (Upper) and Mas-knockout (Lower) kidneys. (B) Nonspecific and total 125I-Ang (1–7) binding in WT (Upper) and Mas-knockout (Lower) kidneys. (C) Specific binding of 125I-[Sar-1, Ile-8] Ang II to AT1 (AT1 R) and AT2 (AT2 R) receptors in WT (+/+) and Mas-deficient (-/-) kidneys (Left) and specific binding of 125I-Ang (1–7) binding to WT (+/+) and Mas-knockout (-/-) kidneys (Right). Bars represent mean of six kidneys ± SEM. (D) Representative autoradiographic localizations of 125I-angiotensin IV binding in WT and Masknockout mouse kidneys. Color bars (Right)reflect pseudocolor imaging of different levels of exposure of the autoradiogram converted to units of fmol/g as described in Materials and Methods. (Bar = 2 mm.)

Fig. 2.

(A) Saturation isotherm and scatchard plot (Inset) of specific 125I-Ang-(1–7) binding to Mas-transfected COS cells. Cells were incubated with increasing concentrations of 125I-Ang-(1–7). No specific binding was determined in the presence of 1 μmol/liter Ang-(1–7). These data are represented as mean ± SEM of three different experiments. In the conditions used, the nonspecific binding averaged 40–60% of the total binding. (B) Competition for 125I-Ang-(1–7) binding toMas-transfected CHO cells by Ang-(1–7) and receptor antagonists. Competition curves were generated by adding increasing concentrations of CV-9174, PD 123319, A-779, and Ang-(1–7) to the incubation buffer containing 0.4 nmol/liter of 125I-Ang-(1–7). Data are presented as mean ± SEM of three to six independent experiments.

Fig. 3.

Effect of the Ang-(1–7) antagonist A-779 on the Ang-(1–7)-induced [3H]AA release from Mas-transfected CHO cells. Data are presented as mean ± SEM of three to six independent experiments performed in triplicate. **, P < 0.01 compared with untreated Mas-transfected CHO, ANOVA followed by Newman–Keuls test; #, P < 0.05 compared with Ang-(1–7) 10-8 M, nonpaired t test.

Fig. 4.

Effect of the Ang-(1–7) antagonist A-779, irbesartan (AT1 receptor antagonist) or PD123319 (AT2 receptor antagonist) on Ang-(1–7)-induced [3H]AA release fromMas-transfected COS cells. Data are presented as mean ± SEM of three to six independent experiments performed in triplicate.

Fig. 5.

Antidiuretic effect of Ang-(1–7) and AVP in water-loaded mice. Male control (n = 25) and Mas-deficient (n = 24) mice (25–35 g) were used. (A) Effect of Ang-(1–7) on water diuresis. (B) Effect of Ang-(1–7) on urine osmolality. (C) Effect of AVP on water diuresis (the same experimental protocol was used). Data are presented as mean ± SEM. *, P < 0.05 compared with the vehicle-treated mice (ANOVA followed by Newman–Keuls test).

Fig. 6.

Vasodilator effect of Ang-(1–7) in endothelium-containing aortic rings from WT (control) and Mas-knockout mice (knockout). (A) Tracing illustrating the effect of Ang-(1–7) on preconstricted aorta rings. Vessels were preconstricted by incubation with 0.3 mM phenylephrine. Numbers below the arrows indicate log of the peptide concentration (0.0001–0.3 μM). The arrows without numbers indicate concentrations 3-fold higher than the previous addition. (B) Diagram summarizing the vasodilator effect of Ang-(1–7) in the aortic rings of both animal models. Each point represents mean ± SEM generated from five separated experiments. P < 0.001 (two-way ANOVA).