Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

Glucagon-producing cells are increased in Mas-deficient mice.

It has been shown that angiotensin(1-7) (Ang(1-7)) produces several effects related to glucose homeostasis. In this study, we aimed to investigate the effects of genetic deletion of Ang(1-7), the GPCR Mas, on the glucagon-producing cells. C57BL6/N Mas-/- mice presented a significant and marked increase in pancreatic α-cells (number of cells: 146 ± 21 vs 67 ± 8 in WT; P < 0.001) and the percentage per islet (17.9 ± 0.91 vs 12.3 ± 0.9% in WT; P < 0.0001) with subsequent reduction of ß-cells percentage (82.1 ± 0.91 vs 87.7 ± 0.9% in WT; P < 0.0001). Accordingly, glucagon plasma levels were increased (516.7 ± 36.35 vs 390.8 ± 56.45 pg/mL in WT; P < 0.05) and insulin plasma levels were decreased in C57BL6/N Mas-/- mice (0.25 ± 0.01 vs 0.31 ± 56.45 pg/mL in WT; P = 0.02). In order to eliminate the possibility of a background-related phenotype, we determined the number of glucagon-producing cells in FVB/N Mas-/- mice. In keeping with the observations in C57BL6/N Mas-/- mice, the number and percentage of pancreatic α-cells were also significantly increased in these mice (number of α-cells: 260 ± 22 vs 156 ± 12 in WT, P < 0.001; percentage per islet: 16 ± 0.8 vs 10 ± 0.5% in WT, P < 0.0001). These results suggest that Mas has a previously unexpected role on the pancreatic glucagon production.