Fig. 3. Intracellular calcium determination assays in HEK-293T cells expressing AT₁ and/or CB₁ receptors.

HEK-293T cells transfected with the cDNAs for an engineered calcium sensor, 6GCaMP (1 μg), and AT₁R (1 μg) and/or CB₁R (1 μg) (A–C) were pretreated with ligand solvent (vehicle) or selective receptor antagonists (1 µM candesartan for AT₁R or 1 μM rimonabant for CB₁R) and subsequently treated with selective agonists (100 nM Ang II for AT₁R and/or 100 nM ACEA for CB₁R). Fluorescence measurements were taken every 5 s for a total time of 150 s. The fluorescence readings of a representative assay are shown in (A–C). The maximum fluorescence values for each condition have been normalized to the mean fluorescence values obtained in the Ang II condition in AT₁R-expressing cells (100%) (D–F). Values are the mean ± S.E.M. of five independent experiments performed in duplicates. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-hoc test (D, versus Ang II treatment ns. p > 0.05; ***p < 0.001; and F, versus Ang II treatment **p < 0.01; ***p < 0.001).