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. 2018 Sep 26;9:1084. doi: 10.3389/fphar.2018.01084

FIGURE 2.

FIGURE 2

Quantification of intrinsic activity of SCBs at hCB1Rs by examining modulation of G-protein and adenylyl cyclase activity in CHO-hCB1 and non-transfected wild-type (CHO-WT) cells. G-protein activation in CHO-hCB1 (A) or CHO-WT (B) cell membranes by SCBs to compare CB1R potency and efficacy. Membranes (50 μg) prepared from CHO-hCB1 cells were incubated in the presence of 0.1 nM [35S]GTPγS with increasing concentrations (10-10 to 10-5 M) of CP-55,950 (filled squares), Δ9-THC (filled circles), AB-PINACA (open squares), 4OH-AB-PINACA (open circles) or 5OH-AB-PINACA (open triangles) (see section “Materials and Methods”). Data points presented are the mean ± SEM of basal G-protein activity in the presence of test compounds. Curve fitting of concentration-effect curves via non-linear regression was employed to determine the ED50 (measure of potency) and Emax (measure of efficacy) for G-protein activation by each agonist. The mean ± SEM and statistical comparison of ED50 and Emax values calculated for each SCB are presented in Table 2. Adenylyl cyclase activity in whole intact CHO-hCB1 (C) or CHO-WT (D) cells by SCBs to compare CB1R potency and efficacy. Following incubation of CHO-hCB1 cells with [3H]adenine for 4 h and washout, increasing concentrations (10-10 to 10-5 M) of CP-55,950 (filled squares), Δ9-THC (filled circles), AB-PINACA (open squares) or 4OH-AB-PINACA (open circles) were added to cells for 15 min (see section “Materials and Methods”). Data points presented are the mean ± SEM of basal intracellular cAMP in the presence of test compounds. Curve fitting of concentration-effect curves via non-linear regression was employed to determine the IC50 (measure of potency) and Imax (measure of efficacy) for adenylyl cyclase modulation by each agonist. The mean ± SEM and statistical comparison of IC50 and Imax values calculated for each SCB are presented in Table 2.