Evidence that the plant cannabinoid cannabigerol is a highly potent alpha2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist

Abstract

Background and purpose: Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Delta(9)-tetrahydrocannabinol, cannabidiol and Delta(9)-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor.

Experimental approach: The [(35)S]GTPgammaS binding assay, performed with mouse brain membranes, was used to test the ability of cannabigerol to produce G protein-coupled receptor activation or blockade. Its ability to displace [(3)H]CP55940 from mouse CB(1) and human CB(2) cannabinoid receptors and to inhibit electrically evoked contractions of the mouse isolated vas deferens was also investigated.

Key results: In the brain membrane experiments, cannabigerol behaved as a potent alpha(2)-adrenoceptor agonist (EC(50)= 0.2 nM) and antagonized the 5-HT(1A) receptor agonist, R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (apparent K(B)= 51.9 nM). At 10 microM, it also behaved as a CB(1) receptor competitive antagonist. Additionally, cannabigerol inhibited evoked contractions of the vas deferens in a manner that appeared to be alpha(2)-adrenoceptor-mediated (EC(50)= 72.8 nM) and displayed significant affinity for mouse CB(1) and human CB(2) receptors.

Conclusions and implications: This investigation has provided the first evidence that cannabigerol can activate alpha(2)-adrenoceptors, bind to cannabinoid CB(1) and CB(2) receptors and block CB(1) and 5-HT(1A) receptors. It will now be important to investigate why cannabigerol produced signs of agonism more potently in the [(35)S]GTPgammaS binding assay than in the vas deferens and also whether it can inhibit noradrenaline uptake in this isolated tissue and in the brain.

Figure 1

The chemical structure of cannabigerol.

Figure 2

The effect of cannabigerol on [³⁵S]GTPγS binding to whole brain membranes obtained from MF1 mice (n= 7 to 20). Each symbol represents the mean percentage change in binding ± SEM. Asterisks denote values that are significantly different from zero (**P < 0.01; ***P < 0.001; one-sample t-test).

Figure 4

Upper panels: mean increases in tension of the MF1 mouse isolated vas deferens induced by β,γ-methylene ATP in the presence of DMSO (circles) or cannabigerol (triangles). For the construction of log concentration-response curves, β,γ-methylene ATP was first added 30 min (A) after DMSO or 100 nM cannabigerol (n= 6 or 8) or (B) after DMSO or 1 µM cannabigerol (n= 8). The asterisk indicates a significant difference between the contractile response to 10 µM β,γ-methylene ATP in the absence of cannabigerol and the corresponding response in the presence of this cannabinoid (P < 0.05; unpaired t-test). Lower panel: mean increases in tension of the mouse isolated vas deferens induced by 32 µM phenylephrine in the absence or presence of 1 µM cannabigerol. The two mean values are not significantly different (P > 0.05; unpaired t-test). Additions of phenylephrine were made 30 min after DMSO (open columns) or cannabigerol (n= 8). In all panels, mean increases in tension are expressed in grams ± SEM. DMSO, dimethyl sulphoxide.

Figure 3

Mean log concentration-response curves of (A) cannabigerol (n= 13), (B) clonidine (n= 7) (C) dexmedetomidine (n= 5) and (D) maprotiline (n= 7) in the MF1 mouse isolated vas deferens constructed in the presence of yohimbine or its vehicle. Each symbol represents the mean value ± SEM for inhibition of electrically evoked contractions expressed as a percentage of the amplitude of the twitch response measured immediately before the first addition of cannabigerol, clonidine or maprotiline to the organ bath. Yohimbine or its vehicle was added 30 min before this first addition and all further additions were made at 5 or 15 min intervals (Methods). Each log concentration response curve was constructed cumulatively. The mean apparent K_B value of yohimbine with its 95% confidence limits shown in brackets is 10.1 nM (3.0 and 33.7 nM) against cannabigerol, 14.0 nM (4.9 and 40.6 nM) against clonidine, 8.7 nM (4.3 and 17.8 nM) against dexmedetomidine and 8.2 nM (1.5 and 46.0 nM) against maprotiline. In the absence of yohimbine, electrically evoked contractions were inhibited by cannabigerol, clonidine, dexmedetomidine and maprotiline with mean EC₅₀ values of 72.8 nM (23.4 and 227 nM), 6.3 nM (2.0 and 19.7 nM), 0.24 nM (0.14 and 0.40 nM) and 24.9 nM (5.6 and 111.2 nM) respectively. The 95% confidence limits of these mean values are shown in brackets. The corresponding E_max values are 51.7% (44.3 and 59.1%), 63.0% (54.1 and 71.8%), 85.8% (81.6 and 90.0%) and 58.2% (47.1 and 69.3%) respectively.

Figure 5

Mean log concentration-response curves of (A) dexmedetomidine (n= 4) and (B) cannabigerol (n= 12) constructed in the absence or presence of 100 nM yohimbine. Each symbol represents the mean percentage change in binding of [³⁵S]GTPγS to MF1 mouse whole brain membranes ± SEM. Mean EC₅₀ values of dexmedetomidine and cannabigerol in the absence of yohimbine with 95% confidence limits shown in brackets are 4.3 nM (0.7 and 25.5 nM) and 0.13 nM (0.004 and 4.4 nM) respectively. The corresponding mean E_max values are 26.5% (17.1 and 36.0%) and 14.8% (5.9 and 23.7%) respectively. The right-ward shifts produced by yohimbine in the log concentration response curves of dexmedetomidine and cannabigerol are significant and do not deviate significantly from parallelism (P > 0.05). The mean apparent K_B value of yohimbine for this antagonism, with its 95% confidence limits shown in brackets is 3.9 nM (1.0 and 15.1 nM) against dexmedetomidine and 1.8 nM (0.04 and 90.5 nM) against cannabigerol.

Figure 6

Displacement of [³H]CP55940 by cannabigerol from specific binding sites on (A) MF1 mouse whole brain membranes (n= 4) and (B) CHO-hCB₂ cell membranes (n= 8). Each symbol represents the mean percent displacement ± SEM. Mean K_i values with 95% confidence limits shown in brackets are (A) 381 nM (231 and 627 nM) for displacement from brain membranes and (B) 2.6 µM (1.4 and 4.7 µM) for displacement from CHO-hCB₂ cell membranes. CHO, Chinese hamster ovary.

Figure 7

Upper panels: the effect of 10 µM cannabigerol (CBG) on the mean log concentration-response curve of (A) anandamide and (B) CP55940 for stimulation of [³⁵S]GTPγS binding to mouse brain membranes. Lower panels: the effect of 10 µM cannabigerol (CBG) on the mean log concentration-response curve of (C) anandamide and (D) CP55940 for stimulation of [³⁵S]GTPγS binding after subtraction of the inhibitory effect induced by 10 µM cannabigerol on [³⁵S]GTPγS binding in the absence of any other compound (30.1 ± 3.8%; n= 17; Figure 2). This value was subtracted from all values of percent stimulation of [³⁵S]GTPγS binding by anandamide or CP55490 determined in the presence of cannabigerol. Experiments were performed with MF1 mouse whole brain membranes and stimulation of [³⁵S]GTPγS binding is expressed as mean percent stimulation ± SEM (n= 5 or 6). The right-ward shifts produced by cannabigerol in the log concentration response curves of anandamide and CP55940 do not deviate significantly from parallelism (P > 0.05). DMSO, dimethyl sulphoxide.

Figure 8

Mean log concentration-response curves of R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) constructed in the presence of (A) DMSO or 100 nM WAY100635 (n= 7), (B) DMSO or 1 µM cannabigerol (n= 10) or (C) DMSO or 10 µM cannabigerol (n= 12). Each symbol represents the mean percentage change in binding of [³⁵S]GTPγS to MF1 mouse whole brain membranes ± SEM. Neither the right-ward shift produced by WAY100635 in the log concentration response curve of 8-OH-DPAT nor that produced by 1 or 10 µM cannabigerol deviates significantly from parallelism (P > 0.05). Mean apparent K_B values with 95% confidence limits shown in brackets are (A) 1.0 nM (0.5 and 2.3 nM) for WAY100635, (B) 19.6 nM (6.9 and 55.8 nM) for 1 µM cannabigerol and (C) 28.2 nM (7.7 and 102.9 nM) for 10 µM cannabigerol. Panel (D): Schild plot for antagonism of 8-OH-DPAT by 100 nM, 316 nM, 1 µM, 3.16 µM and 10 µM cannabigerol (n= 5 to 12) with the 99% confidence band shown by dotted lines. The mean slope of this best-fit line with 95% confidence limits shown in brackets is 1.1 (1.0 and 1.2) and so does not differ significantly from unity. The mean apparent K_B value of cannabigerol calculated from this Schild plot, with 95% confidence limits shown in brackets, is 51.9 nM (37.6 and 68.2 nM). DMSO, dimethyl sulphoxide.