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PNAS 100 (14): 8258-8263

Copyright © 2003 by the National Academy of Sciences.

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

Robson A. S. Santos*, Ana C. Simoes e Silva*, Christine Maric{dagger}, Denise M. R. Silva*, Raquel Pillar Machado*, Insa de Buhr{ddagger}, Silvia Heringer-Walther{ddagger}, Sergio Veloso B. Pinheiro*, Myriam Teresa Lopes*, Michael Bader§, Elizabeth P. Mendes*, Virgina Soares Lemos*, Maria Jose Campagnole-Santos*, Heinz-Peter Schultheiss{ddagger}, Robert Speth ,||, and Thomas Walther {ddagger},**

*Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, 31270, Minas Gerais, Brazil; {dagger}Department of Medicine, Georgetown University, Washington, DC 20057; {ddagger}Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Free University, 12200 Berlin, Germany; §Max Delbrück Center, 13125 Berlin, Germany; and Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164-6520


Figure 1
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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 Mas knockout 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.)

 

Figure 2
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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 to Mas-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.

 

Figure 3
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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.

 

Figure 4
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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 from Mas-transfected COS cells. Data are presented as mean ± SEM of three to six independent experiments performed in triplicate.

 

Figure 5
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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).

 

Figure 6
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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).

 


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