Fig. 3.

Functional analysis of AT₁R-MasR and AT₂R-MasR complexes in HEK-293T cells. HEK-293T cells were transfected with the cDNAs for MasR (1 μg) and for either AT₁R (1 μg) or AT₂R (1 μg) and, in assays of Ca²⁺ determination, with the cDNA for an engineered calcium sensor, GCaMP6 (1 μg). Transfected cells were pretreated with the selective antagonists (300 nM candesartan for AT₁R, 1 μM PD123319 for AT₂R, and 500 nM A779 for MasR) and subsequently treated with selective agonists (100 nM Ang II for AT₁R, 300 nM CGP-42112A for AT₂R, and 500 nM Ang [1–7] for MasR). Thereafter, cytosolic calcium increases (A, B), intracellular cAMP levels (C, D), ERK1/2 phosphorylation (E, F), and the time-dependent DMR signal (G, H) were determined. Values are the mean ± SEM of 6 independent experiments performed in triplicate. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment in cAMP determinations or versus vehicle in pERK and DMR assays