Evidence that the plant cannabinoid Delta9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist

Abstract

Delta9-tetrahydrocannabivarin (THCV) displaced [(3)H]CP55940 from specific binding sites on mouse brain and CHO-hCB(2) cell membranes (K(i)=75.4 and 62.8 nM, respectively).THCV (1 microM) also antagonized CP55940-induced stimulation of [(35)S]GTPgammaS binding to these membranes (apparent K(B)=93.1 and 10.1 nM, respectively). In the mouse vas deferens, the ability of Delta9-tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent K(B)-value (96.7 nM) approximating the apparent K(B)-values for its antagonism of CP55940- and R-(+)-WIN55212-induced stimulation of [(35)S]GTPgammaS binding to mouse brain membranes. THCV also antagonized R-(+)-WIN55212, anandamide, methanandamide and CP55940 in the vas deferens, but with lower apparent K(B)-values (1.5, 1.2, 4.6 and 10.3 nM, respectively).THCV (100 nM) did not oppose clonidine, capsaicin or (-)-7-hydroxy-cannabidiol-dimethylheptyl-induced inhibition of electrically evoked contractions of the vas deferens. Contractile responses of the vas deferens to phenylephrine hydrochloride or beta,gamma-methylene-ATP were not reduced by 1microM THCV or R-(+)-WIN55212, suggesting that THCV interacts with R-(+)-WIN55212 at prejunctional sites. At 32 microM, THCV did reduce contractile responses to phenylephrine hydrochloride and beta,gamma-methylene-ATP, and above 3 microM it inhibited electrically evoked contractions of the vas deferens in an SR141716A-independent manner. In conclusion, THCV behaves as a competitive CB(1) and CB(2) receptor antagonist. In the vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R-(+)-WIN55212 in this tissue than in brain membranes. The bases of these agonist- and tissue-dependent effects remain to be established.

Figure 1

Structures of THC and THCV.

Figure 2

Displacement of [³H]CP55940 by THCV from specific binding sites in (a) mouse whole brain membranes and (b) CHO-hCB₂ cell membranes. Each symbol represents the mean percent displacement±s.e.m. Mean K_i-values for this displacement with 95% confidence limits shown in brackets were calculated by the Cheng–Prusoff equation to be (a) 75.4 nM (53.4 and 106.3 nM; n=4–8) in mouse whole brain membranes and (b) 62.8 nM (52.5 and 75.3 nM; n=6) in CHO-hCB₂ cell membranes.

Figure 3

The effect of 1 μM THCV on the mean log concentration–response curve of CP55940 for stimulation of [³⁵S]GTPγS binding (a) to mouse whole brain membranes and (b) to CHO-hCB₂ cell membranes. Each symbol represents the mean percentage increase in [³⁵S]GTPγS binding±s.e.m. (n=6). Mean apparent K_B-values of THCV for its antagonism of CP55940 have been calculated from these data and these values are listed in Table 1.

Figure 4

The mean log concentration–response curve of THCV in the mouse isolated vas deferens. Each symbol represents the mean value±s.e.m. for inhibition of electrically evoked contractions expressed as a percentage of the amplitude of the twitch response measured immediately before the first addition to the organ bath of THCV. Tissues were exposed to THCV concentrations ranging either from 3.2 nM to 10 μM (n=10) or from 1 to 100 μM (n=10).

Figure 5

Upper panel: the effect of pretreatment with THCV on mean increases in tension of the mouse isolated vas deferens induced by β,γ-methylene ATP in the presence of DMSO (○) or 32 μM THCV (•). For the construction of log concentration–response curves, β,γ-methylene ATP was first added 30 min after DMSO or THCV (n=7 or 8). Lower panels: the effect of pretreatment with R-(+)-WIN55212 or THCV on mean increases in tension of the mouse isolated vas deferens induced by (a) 32 μM phenylephrine or (b) 3.2 μM phenylephrine. Additions of phenylephrine were made 30 min after DMSO (open columns), THCV or R-(+)-WIN55212 (n=8). In all panels, mean increases in tension are expressed in grams±s.e.m. The asterisks indicate significant differences between responses to β,γ-methylene ATP (unpaired t-test) or to phenylephrine (ANOVA followed by Dunnett's test) in the absence of added cannabinoids, and corresponding responses in the presence of R-(+)-WIN55212 or THCV (^*P<0.05; ^***P<0.001).

Figure 6

Upper panel: the effect of pretreatment with THCV on the mean log concentration–response curve of R-(+)-WIN55212 in the mouse isolated vas deferens. Each symbol represents the mean value±s.e.m. for inhibition of electrically evoked contractions expressed as a percentage of the amplitude of the twitch response measured immediately before the first addition of R-(+)-WIN55212 to the organ bath. THCV or DMSO was added 30 min before the first addition of R-(+)-WIN55212, further additions of which were made at 15-min intervals. Each log concentration–response curve was constructed cumulatively without washout (n=6–9). Lower panel: Schild plot for antagonism of R-(+)-WIN55212 by 10–1000 nM THCV, in which values for log x−1 were calculated from the data shown in the upper panel (x=concentration ratio). The slope of this plot is 0.99±0.14 and this does not differ significantly from unity (P>0.05; one-sample t-test). The mean apparent K_B-value of THCV for its antagonism of R-(+)-WIN55212 has been calculated from this slope by Schild analysis and is listed in Table 1.

Figure 7

The effect of pretreatment with THCV on the mean log concentration–response curves of anandamide (n=7 or 8) and clonidine (n=8) in the mouse isolated vas deferens. Each symbol represents the mean value±s.e.m. for inhibition of electrically evoked contractions expressed as a percentage of the amplitude of the twitch response measured immediately before the first addition of twitch inhibitor to the organ bath. THCV or DMSO was added 30 min before the first addition of anandamide or clonidine, further additions of which were made at 15- and 5-min intervals, respectively. Each log concentration–response curve was constructed cumulatively without washout.