Characterization of anandamide and WIN55212-2-stimulated [35S]GTPγS binding and inhibition of agonist-stimulated [35S]GTPγS binding by SR141716A
| Agonist | SR141716A | Parameter | CB1+/+ | CB1−/− | ||
|---|---|---|---|---|---|---|
| Anandamide | WIN55212-2 | Anandamide | WIN55212-2 | |||
| μM | ||||||
| 0.1–30 | EC50(μM) | 1.4 ± 0.3 | 0.17 ± 0.08 | 3.6 ± 2.0 | 1.8 ± 0.7 | |
| 0.1–30 | Emax (fmol/mg) | 150 ± 7 | 180 ± 11 | 41 ± 3 | 64 ± 7 | |
| 10 | 0.1–30,000 | High K B (nM) | 0.26 ± 0.06 | 0.40 ± 0.09 | none | none |
| 10 | 0.1–30,000 | Low IC50 (μM) | 3.0 ± 1.1 | 13 ± 5 | 3.3 ± 2.1 | 11 ± 2 |
| 10 | 0.1–30,000 | % High-potency | 84 ± 6% | 66 ± 3% | ||
| 10 | 0.1–30,000 | K B for −SR (nM) | 0.42 ± 0.18 | 0.71 ± 0.21 | ||
| 10 | 0.1–30,000 | % Blocked −SR | 78 ± 1% | 67 ± 5% | ||
The first two rows were data obtained using various concentrations of either anandamide or WIN55212-2 alone. The next three rows are data obtained with 10 μM anandamide or 10 μM WIN55212-2 in the presence of various concentrations of SR141716A (see “SR + 10 μM WIN” and “SR + 10 μM anand.” in Fig. 2). “HighK B” and “Low IC50” refer to the two sites observed for inhibition of agonist-stimulated [35S]GTPγS binding in CB1 +/+ membranes. “High K B” is the affinity of the high potency site at which SR141716A inhibited agonist stimulation in CB1 +/+ membranes and was calculated from the high-affinity IC50 value as described under Materials and Methods. “Low IC50” is concentration of SR141716A that seem to inhibit half of the remaining stimulation by agonist in CB1 +/+ and was the only site observed in CB1 −/− membranes. “% High potency” is the percentage of agonist-stimulated binding inhibited by SR141716A at the high K B value. The last two rows were obtained by subtracting the effects of SR141716A alone from the data obtained for agonists plus SR141716A for each experiment before nonlinear fitting of the data. “K B for −SR” is the affinity value exhibited by SR141716A, and “% Blocked −SR” is the percentage of agonist-stimulated binding inhibited by SR141716A (see “SR + 10 μM WIN −SR alone” and “SR + 10 μM anand. − SR alone” in Fig. 2). Data are mean values ± S.E. obtained by nonlinear fitting from at least four experiments.