Curve-fit values of MAEA-stimulated [35S]GTPγS and [3H]SR141716A binding in CB1-HEK and CB1-HEK-CRIP1a cells pretreated with vehicle, WIN55,212-2, or THC
Cells were pretreated for 4 hours with 10 μM WIN55,212-2, 6 μM THC, or vehicle. Varying concentrations of MAEA were incubated with membranes prepared from the indicated cell lines in the presence of 10 μM GDP and 0.1 nM [35S]GTPγS, as described in Materials and Methods. Varying concentrations of [3H]SR141716A were incubated with membranes prepared from the indicated cell lines, as described in Materials and Methods. Emax, EC50, Bmax, and KD values were derived from nonlinear regression analysis of the concentration-effect or saturation binding curves. Data are mean values ± S.E.M. (n = 4–5).
| Cell Line Pretreatment | |||
|---|---|---|---|
| Vehicle | WIN55,212-2 | THC | |
| MAEA-stimulated [35S]GTPγS binding | |||
| CB1-HEK | |||
| Emax (% Stim) | 128.5 ± 8.0 | 42.7 ± 6.9** | 100.5 ± 8.5 |
| EC50 (nM) | 308 ± 71 | 5412 ± 1870§ | 4609 ± 2137§ |
| CB1-HEK-CRIP1a | |||
| Emax (% Stim) | 112.6 ± 5.1 | 36.9 ± 4.5** | 104.3 ± 7.3 |
| EC50 (nM) | 423 ± 82 | 4112 ± 633§§ | 5175 ± 1316§§ |
| [3H]SR141716A binding | |||
| CB1-HEK | |||
| Bmax (pmol/mg) | 1.19 ± 0.11 | 0.56 ± 0.10** | 0.31 ± 0.04** |
| KD (nM) | 1.78 ± 0.23 | 1.37 ± 0.34 | 1.78 ± 0.59 |
| CB1-HEK-CRIP1a | |||
| Bmax (pmol/mg) | 0.97 ± 0.15 | 0.86 ± 0.14 | 0.62 ± 0.12 |
| KD (nM) | 1.72 ± 0.46 | 2.18 ± 0.69 | 2.40 ± 1.07 |
↵** P < 0.01, different from vehicle-treated cells of the same type, as determined by one-way ANOVA with posthoc Dunnett’s test; §P = 0.05, §§P < 0.01 different from vehicle-treated cells of the same type, as determined by one-way ANOVA of square root transformed data with posthoc Dunnett’s test.