Angiotensin (Ang)-(1–7) has cardiovascular protective effects and is the opponent of the often detrimental Ang II within the renin–angiotensin system. Although it is well accepted that the G-protein–coupled receptor Mas is a receptor for the heptapeptide, the lack in knowing initial signaling molecules stimulated by Ang-(1–7) prevented definitive characterization of ligand/receptor pharmacology as well as identification of further hypothesized receptors for the heptapeptide. The study aimed to identify a second messenger stimulated by Ang-(1–7) allowing confirmation as well as discovery of the heptapeptide’s receptors. Ang-(1–7) elevates cAMP concentration in primary cells, such as endothelial or mesangial cells. Using cAMP as readout in receptor-transfected human embryonic kidney (HEK293) cells, we provided pharmacological proof that Mas is a functional receptor for Ang-(1–7). Moreover, we identified the G-protein–coupled receptor MrgD as a second receptor for Ang-(1–7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both Mas and MrgD . Mice deficient in MrgD showed an impaired hemodynamic response after Ang-(1–7) administration. Furthermore, we excluded the Ang II type 2 receptor as a receptor for the heptapeptide but discovered that the Ang II type 2 blocker PD123319 can also block Mas and MrgD receptors. Our results lead to an expansion and partial revision of the renin–angiotensin system, by identifying a second receptor for Ang-(1–7), by excluding Ang II type 2 as a receptor for the heptapeptide, and by enforcing the revisit of such publications which concluded Ang II type 2 function by only using PD123319.

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1h, 3h, 6h, and 9h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1h, 119 after 3h, 31 after 6h, and 86 after 9h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1h and 3h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7). The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.