Fig. 5
AT1R-AT2R heteromer functionality in microglial primary cultures treated with LPS and IFN-γ. a–c Expression of AT1R/AT2R heteromers in primary microglial cultures were determined by PLA, which was performed using specific primary antibodies against AT1 and AT2 receptors (confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue)). Scale bar: 20 μm. d Bar graph showing the percentage of red dots/cell respect non-treated cells; mean ± S.E.M of counts in 5–7 different fields (n = 5; **p < 0.01; Student’s t test versus the control condition). e, f Microglial cultures were incubated for 48 h in the absence (left) or in the presence (right) of 1 μM LPS and 200 U/mL IFN-γ. Microglial cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R receptors) and subsequently with the specific agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R receptors). cAMP (e-f) and ERK1/2 phosphorylation (g-h) were subsequently measured. Values are the mean ± S.E.M. of 5 independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (+p < 0.05 versus Ang II treatment in pERK determinations; and *p < 0.05, **p < 0.01, ***p < 0.001; versus forskolin treatment in cAMP measurements or versus vehicle treatment (basal) in pERK measurements)