Figure 8.

Binding and Functional Analysis of Terpenes at the CB1. Fitted curve values reported as the mean with the 95% confidence interval. (A) CB1-CHO membranes were subjected to competition binding assay with terpenes and WIN55,212-2 against ³H-CP55,940. Data represents the mean ± SEM of specific binding (n = 5 independent experiments). IC₅₀ values: WIN55,212-2 = 90.4 nM (36–206); Geraniol = 44.2 μM (11.1–193). Other terpenes did not provide competition curves that could be fit. (B) CB1-CHO cells were treated with varying concentrations of terpene or WIN55,212-2 for 30 min. The ability to inhibit forskolin-stimulated cAMP accumulation was measured accordingly (see Methods). Data represents the mean ± SEM of % of forskolin-stimulated cAMP (n = 4 independent experiments). IC₅₀ value: WIN55,212-2 = 591 nM (289–1,180). Terpenes did not provide full inhibition curves. (C) CB1-CHO-DX cells were treated with varying concentrations of compounds and βarrestin2 recruitment was assessed after 1.5 h of treatment (see Methods). Data represents the mean ± SEM of max WIN55,212-2 recruitment (n = 3 independent experiments). EC₅₀ value: WIN55,212-2 = 1,600 nM (1,110–2,310). (D) CB1-CHO-DX cells were pretreated with 500 μM terpene for 5 min, followed by varying concentrations of WIN55,212-2 for 1.5 h (see Methods). Data represents the mean ± SEM of max WIN55,212-2 recruitment (n = 3 independent experiments). EC₅₀ values: WIN55,212-2 alone = 254 nM (194–331); WIN + Geraniol = 3,080 nM (2180–4350); WIN + α-Humulene = 471 nM (295–743); WIN + Linalool = 679 nM (480–956); WIN + β-Pinene = 302 nM (147–621); WIN + β-Caryophyllene = 283 nM (169–470).