FIGURE 5.

cAMP production and ERK-1/2 phosphorylation mediated by CB₂R-GPR55 heteromers in BT474 cancer cells. A and B, cAMP production in BT474-GPR55 cells was determined in cells not pretreated (A) or pretreated overnight (B) with 10 ng/ml PTX or with 100 ng/ml CTX prior to incubation with the CB₂R antagonists AM630 or SR144528 (SR2) or with the GPR55 antagonist HBA (A) and stimulated with HU-308, LPI, or both in the absence or in the presence of 0.5 μm FK. Values are mean ± S.E. of n = 3–9 and are expressed as a percentage of the FK-treated cells. C–E, ERK-1/2 phosphorylation was determined in BT474-GPR55 cells stimulated (7 min) with increasing concentrations of HU-308 or LPI (C), with 0.1 μm HU-308 or 1 μm LPI for different times (D), or pretreated or not treated with the CB₂R antagonists AM630 or SR144528 or with the GPR55 antagonist HBA prior to stimulation with HU-308, LPI, or both (E). Phosphorylation was expressed in arbitrary units (ALPHA counts, light emission at 520–620 nm). Values are mean ± S.E. of n = 3–9 and are expressed as a percentage of basal levels found in vehicle-treated cells. One-way ANOVA followed by Bonferroni post hoc test showed a significant effect over vehicle-treated cells (***, p < 0.001; **, p < 0.01), over FK-treated cells (A and B; ##, p < 0.01; ###, p < 0.001), or antagonist plus agonist over the agonist treatment (E; ###, p < 0.001).