Fig. 2.

Coactivation of MOR and Gq-coupled muscarinic receptors reveals synergistic PLC activation. (A) HEK293 cells stably expressing flag-MOR were transduced with the DAG reporter and M1 receptor as in Fig. 1A and 1B. Cells were treated (compounds added at the dashed vertical line) with Gibco Dulbecco’s PBS, 100 nM carbachol, 500 nM DAMGO, 1 μM morphine, carbachol + DAMGO, or carbachol + morphine at the same concentrations. (B) To quantify these responses the area under the curve with vehicle subtracted for each curve was calculated; 1-way ANOVA F(4,10) = 11.5, P = 0.0009. (C and D) Stable MOR, HEK293 cells transduced, transfected, and treated as in (A) were treated for 16 hours without (C) and with (D) 100 ng/mL PTX for 16 hours. (E) Peak DAG production at each concentration of agonist relative to maximum PDBu-dependent DAG production was calculated from 3 independent experiments, each performed with 4 replicates. All data are mean ± S.E.M. P values were calculated with an ordinary 1-way ANOVA with Tukey’s post hoc test and 2-way ANOVA with Sidak’s post hoc test. *P < 0.05 and **P < 0.005.