Effects of Delta9-tetrahydrocannabivarin on [35S]GTPgammaS binding in mouse brain cerebellum and piriform cortex membranes

Abstract

Background and purpose: We have recently shown that the phytocannabinoid Delta9-tetrahydrocannabivarin (Delta9-THCV) and the CB1 receptor antagonist AM251 increase inhibitory neurotransmission in mouse cerebellum and also exhibit anticonvulsant activity in a rat piriform cortical (PC) model of epilepsy. Possible mechanisms underlying cannabinoid actions in the CNS include CB1 receptor antagonism (by displacing endocannabinergic tone) or inverse agonism at constitutively active CB1 receptors. Here, we investigate the mode of cannabinoid action in [35S]GTPgammaS binding assays.

Experimental approach: Effects of Delta9-THCV and AM251 were tested either alone or against WIN55,212-2-induced increases in [35S]GTPgammaS binding in mouse cerebellar and PC membranes. Effects on non-CB receptor expressing CHO-D2 cell membranes were also investigated.

Key results: Delta9-THCV and AM251 both acted as potent antagonists of WIN55,212-2-induced increases in [35S]GTPgammaS binding in cerebellar and PC membranes (Delta9-THCV: pA2=7.62 and 7.44 respectively; AM251: pA2=9.93 and 9.88 respectively). At micromolar concentrations, Delta9-THCV or AM251 alone caused significant decreases in [35S]GTPgammaS binding; Delta9-THCV caused larger decreases than AM251. When applied alone in CHO-D2 membranes, Delta9-THCV and AM251 also caused concentration-related decreases in G protein activity.

Conclusions and implications: Delta9-THCV and AM251 act as CB1 receptors antagonists in the cerebellum and PC, with AM251 being more potent than Delta9-THCV in both brain regions. Individually, Delta9-THCV or AM251 exhibited similar potency at CB1 receptors in the cerebellum and the PC. At micromolar concentrations, Delta9-THCV and AM251 caused a non-CB receptor-mediated depression of basal [35S]GTPgammaS binding.

Figure 1

Log concentration–response curves for WIN55,212-2 (10 pM–10 μM) effects on percentage stimulation of [³⁵S]GTPγS binding in the presence of AM251 (31.6 pM–1 nM) in (a) cerebellar (n=4 separate experiments) and (b) PC (n=3 separate experiments) membranes. Plot symbols show mean percentage increase in [³⁵S]GTPγS binding±s.e.mean (n=minimum three trials per agonist concentration used). Note the progressive rightward shifts in WIN55,212-2 concentration–response curves in both cerebellar and PC membranes induced by increasing concentrations of AM251, consistent with an antagonistic effect. Schild plots were subsequently constructed for antagonism of WIN55,212-2 by AM251 in (c) cerebellar and (d) PC membranes, yielding slope and pA₂ values given in Table 1. Slopes of unity (dotted lines) are shown for reference. AM251, N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide; PC, piriform cortex; [³⁵S]GTPγS, [³⁵S]guanosine-5′-O-(3-thiotriphosphate); WIN55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate.

Figure 2

Log concentration–response curves for WIN55,212-2 (10 pM–10 μM) effects on percentage stimulation of [³⁵S]GTPγS binding in the presence of Δ⁹-THCV (100 nM–5 μM) in (a) cerebellar and (b) PC membranes (both n=3). Plot symbols show mean percentage increase in [³⁵S]GTPγS binding±s.e.mean (n=minimum three trials per agonist concentration used). Note the progressive rightward shifts in WIN55,212-2 concentration–response curves in both cerebellar and PC membranes induced by increasing concentrations of Δ⁹-THCV, consistent with an antagonistic effect. Schild plots were subsequently constructed for antagonism of WIN55,212-2 by Δ⁹-THCV in (c) cerebellar and (d) PC membranes, yielding slope and pA₂ values given in Table 1. Slopes of unity (dotted lines) are shown for reference. PC, piriform cortex; WIN55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; Δ⁹-THCV, Δ⁹-tetrahydrocannabivarin.

Figure 3

Log concentration–response curves for WIN55,212-2 (10 pM–10 μM), AM251 and Δ⁹-THCV (10 pM–100 μM for both) effects on percentage stimulation of [³⁵S]GTPγS binding in (a) cerebellar (WIN55,212-2, n=8; AM251, n=6 and Δ⁹-THCV, n=7) and (b) PC (WIN55,212-2, n=6; AM251, n=7 and Δ⁹-THCV, n=6) membranes. AM251, N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide; PC, piriform cortex; [³⁵S]GTPγS, [³⁵S]guanosine-5′-O-(3-thiotriphosphate); WIN55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; Δ⁹-THCV, Δ⁹-tetrahydrocannabivarin.

Figure 4

Bar charts summarizing effects of (a) AM251 and (b) Δ⁹-THCV (10–100 μMfor both) on percentage stimulation of [³⁵S]GTPγS binding in cerebellar, PC and CHO-D₂ cell membranes in the absence or presence of the selective adenosine A₁ antagonist, DPCPX (1 μM). AM251-induced depression of [³⁵S]GTPγS binding was significantly less in CHO-D₂ membranes vs PC and cerebellar membranes at all concentrations. Δ⁹-THCV-induced depression of [³⁵S]GTPγS binding was significantly less in CHO-D₂ membranes vs PC (10 and 50 μM) and cerebellar (50 μM only) membranes. At 100 μM Δ⁹-THCV, there were no significant differences in binding between CHO-D₂, cerebellar and PC (P>0.05 for both) membranes. AM251-induced decreases in [³⁵S]GTPγS binding were significantly attenuated by DPCPX in PC (all concentrations) and cerebellar (only at 100 μM) membranes. DPCPX had no significant effects on Δ⁹-THCV responses in each membrane preparation (b; P>0.2). Significance levels were tested using non-parametric Mann–Whitney U-tests and are shown as ^*P<0.05, ^**P<0.01 and ^***P<0.001. Minimum n=3 for each experiment in each membrane preparation. AM251, N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; PC, piriform cortex; Δ⁹-THCV, Δ⁹-tetrahydrocannabivarin.

Figure 5

Log concentration–response curves for (a) AM251 (1 nM–10 μM) and Δ⁹-THCV (1 nM–100 μM) on percentage stimulation of [³⁵S]GTPγS binding in CHO-D₂ cell membranes (both, n=3 separate experiments). (b) Agonist log concentration–response curves for dopamine (100 pM–100 μM), WIN55,212-2 (100 pM–10 μM) and NECA (100 pM–100 μM) effects on percentage stimulation of [³⁵S]GTPγS binding in CHO-D₂ cell membranes (all, n=3 separate experiments). Dopamine had an agonist concentration–response relationship at D_2short receptors (EC₅₀ value=164 nM; n=3); following linear regression, curves for WIN55,212-2 and NECA were found not to differ significantly from zero, indicating a lack of CB and adenosine receptor-mediated effects respectively. AM251, N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide; [³⁵S]GTPγS, [³⁵S]guanosine-5′-O-(3-thiotriphosphate); WIN55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; Δ⁹-THCV, Δ⁹-tetrahydrocannabivarin.