Animals

MF1 mice were purchased from Harlan UK Ltd (Blackthorn, UK), whereas C57BL/6 CB1 receptor knockout mice and the wild-type (WT) littermates were obtained from NIH (Rockville, MD, USA). Mice were maintained on a 12/12h light/dark cycle with free access to food and water.

CHO cells

CHO cells stably transfected with cDNA encoding human cannabinoid CB2 receptors were maintained in Dulbecco’s modified Eagles’s medium (DMEM) nutrient mixture F-12 HAM, supplemented with 2mm l-glutamine, 10% fetal bovine serum (FBS), 0.6% penicillin–streptomycin, hygromycin B (300μgml−1) and geneticin (600μgml−1). The CHO cells stably transfected with cDNA-encoding human cannabinoid CB1 receptors (Bmax=2980±802fmolmg−1protein) were maintained in DMEM F-12 supplemented with 10% FBS, geneticin (400μgml−1) and zeocin (250μgml−1). The native CHO cells were maintained in DMEM nutrient mixture F-12 HAM, which was supplemented with 2mm l-glutamine, 5% FBS and 2% penicillin–streptomycin. All cells were maintained at 37°C and 5% CO2 in their respective media and were passaged twice a week using non-enzymatic cell dissociation solution.

Membrane preparation

Binding assays with [3H]CP55940 and with [35S]GTPγS were performed with mouse whole-brain membranes, prepared as described by Thomas et al. (2004), or with native CHO cell membranes, or with membranes from CHO cells transfected with either human CB1 or CB2 receptors (Ross et al., 1999). The buffers used in the preparation of [35S]GTPγS-binding assay brain membranes were additionally supplemented with 100mm NaCl2.

The CHO cells were removed from flasks by scraping and then frozen as a pellet at −20°C until required. The CB1-CHO cells were additionally FBS-starved for 24h before their removal from flasks. Before use in a radioligand-binding assay, cells were defrosted, diluted in 50mm Tris buffer (radioligand displacement assay) or GTPγS-binding buffer ([35S]GTPγS-binding assay) and homogenized with a 1ml hand-held homogenizer. Protein assays were performed using a Bio-Rad Dc kit (Bio-Rad, Hercules, CA, USA).

Radioligand displacement assay

The assays were carried out with [3H]CP55940, 1mgml−1 bovine serum albumin (BSA) and 50mmTris buffer, total assay volume 500μl, using the filtration procedure described previously (Ross et al., 1999; Thomas et al., 2005). Binding was initiated by the addition of either the brain membranes (33μg protein per tube) or the hCB2-CHO cells (25μg protein per tube) and all assays were performed at 37°C for 60min. Specific binding was defined as the difference between the binding that occurred in the presence and the absence of 1μm unlabelled CP55940. The concentration of [3H]CP55940 used in the displacement assays was 0.7nm. All drugs were stored as a stock solution of 10mm in dimethyl sulphoxide (DMSO), the vehicle concentration in all assay tubes being 0.1% DMSO. The binding parameters for [3H]CP55940, determined by fitting data from saturation-binding experiments to a one-site saturation plot using GraphPad Prism, were 2336±878fmolmg−1 protein (Bmax) and 2.31±1.33nm (Kd) in mouse brain membranes and 72418±4279fmolmg−1 protein (Bmax) and 1.04±0.14nm (Kd) in hCB2-CHO cells (Thomas et al., 2005).

[35S]GTPγS-binding assay

The method used for measuring agonist-stimulated [35S]GTPγS-binding to CB1 and to human CB2receptors was as described previously by Thomas et al. (2005). The GTPγS-binding buffer contained 50mm Tris-HCl, 50mm Tris base, 5mm MgCl2, 1mm EDTA, 100mm NaCl, 1mmdithiothreitol and 0.1% BSA. Briefly, the assay conditions for experiments conducted in mouse brain membranes included 30μm GDP, 10μgml−1 protein and 0.1nm [35S]GTPγS, in a final volume of 500μl. The corresponding assay conditions for experiments conducted in hCB2-CHO cell membranes were 320μm GDP, 10μgml−1 protein and 0.7nm [35S]GTPγS, in a final volume of 250μl. Experiments conducted in native CHO cells were performed under identical conditions to those used for experiments with hCB2-CHO cell membranes, whereas the conditions used for the CB1-CHO cells were similar to those used for experiments conducted with mouse brain membranes. The only difference from the mouse brain experimental conditions was that the NaCl in the GTPγS buffer was omitted. Additionally, CB1-CHO cells were 24h FBS-starved, unlike the CB2-CHO and native CHO cells. Non-specific binding was measured in the presence of 30μmGTPγS and the drugs were incubated in the assay for 60min at 30°C. Additionally, mouse brain membranes were preincubated for 30min at 30°C with 0.5Uml−1 adenosine deaminase (200Umg−1) to remove endogenous adenosine. All drugs, with the exception of morphine, were stored as a stock solution of 1 or 10mm in DMSO. The vehicle concentration in experiments conducted using one of these drugs was 0.1% DMSO or 0.11% DMSO in the presence of an antagonist. In experiments with morphine, which was stored as a stock solution of 10mm in distilled water, we used a vehicle concentration of 0.01% DMSO.

Analysis of data

Values have been expressed as means and variability as s.e.m. or as 95% confidence intervals (CI). The concentration of a drug that produced a 50% displacement of [3H]CP55940 from specific binding sites (IC50) was calculated using GraphPad Prism 4. Its dissociation constant (Ki) was calculated using the equation of Cheng and Prusoff (1973). Net ligand-stimulated [35S]GTPγS-binding values were calculated by subtracting basal binding values (obtained in the absence of ligand) from ligand-stimulated values (obtained in the presence of ligand) as detailed elsewhere (Ross et al., 1999). These values were compared with the level of basal binding by performing a one-sample t-test (GraphPad Prism). A P-value <0.05 was considered to be significant. Values for EC50, for maximal effect (Emax) and for the s.e.m. or 95% confidence limits of these values have been calculated by nonlinear regression analysis using the equation for a sigmoid concentration–response curve (GraphPad Prism).

The apparent dissociation constant (KB) values for antagonism of agonists by cannabidiol, rimonabant, SR144528 or O-2654 have been calculated by Schild analysis from the concentration ratio, defined as the concentration of an agonist that elicits a response of a particular size in the presence of a competitive reversible antagonist at a concentration, B, divided by the concentration of the same agonist that produces an identical response in the absence of the antagonist. The methods used to determine concentration ratios and apparent KB values and to establish whether log concentration–response plots deviated significantly from parallelism are detailed elsewhere (Pertwee et al., 2002). Mean values have been compared using Student’s two-tailed t-test for unpaired data or one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test (GraphPad Prism). A P-value <0.05 was considered to be significant.

Our experiments were further analysed to determine the effect of cannabidiol or SR144528 on CP55940-stimulated [35S]GTPγS binding to hCB2-CHO cell membranes or to determine the effect of cannabidiol or rimonabant on CP55940-stimulated [35S]GTPγS binding to mouse brain membranes. Thus, we subtracted the inhibitory effect that cannabidiol, SR144528 or rimonabant induced on the basal level of [35S]GTPγS binding, determined in the absence of any other compound, from the effect obtained in the presence of CP55940 and re-calculated the apparent KBvalues in the same manner as described above.

Materials

Cannabidiol was supplied by GW Pharmaceuticals (Porton Down, Wiltshire, UK), THC by the National Institute on Drug Abuse (Bethesda, MD, USA) and O-2654 by Dr Raj Razdan (Organix Inc., MA, USA). Rimonabant and SR144528 were obtained from Sanofi-Aventis (Montpellier, France). CP55940, R-(+)-WIN55212 and morphine were from Tocris (Bristol, UK) and BSA, TRIZMA hydrochloride, TRIZMA base, l-glutamine, penicillin–streptomycin, non-enzymatic cell dissociation solution, guanosine 5′-diphosphate (GDP) and 8-cyclopentyl-1,3-dipropylxantine (DPCPX) from Sigma-Aldrich (St Louis, MO, USA). [3H]CP55940 (160Cimmol−1) was obtained from APBiotech (Little Chalfont, UK) and [35S]GTPγS (1250Cimmol−1) from PerkinElmer (Boston, MA, USA). GTPγS, adenosine deaminase and hygromycin B from Roche Diagnostic (Indianapolis, IN, USA) and the geneticin from Gibco (Paisley, UK).

Experiments with mouse brain membranes

Initially, we compared the abilities of rimonabant and cannabidiol to antagonize CP55940-induced stimulation of [35S]GTPγS binding to mouse brain membranes. At 1μm, cannabidiol shared the ability of 10nm rimonabant to produce a rightward shift in the log concentration–response curve of CP55940 (Figure 1a and b). Both the apparent KB values of rimonabant (0.09nm) and cannabidiol (79nm) for the antagonism of CP55940-induced stimulation of [35S]GTPγS binding to mouse brain were well below their corresponding CB1 Ki values (2.2nm and 4.9μm, respectively, see also Table 1). Similar apparent KB values were obtained for the antagonism of R-(+)-WIN55212-mediated [35S]GTPγS binding by either 1μm cannabidiol or 10nm rimonabant to mouse brain membranes (Figure 2). The apparent KB values of cannabidiol or rimonabant, with 95% CI in parantheses, were 138nm (87 and 225nm) and 0.3nm (0.16 and 0.52nm), respectively, for this antagonism of R-(+)-WIN55212 in mouse brain membranes.

Figure 1

[35S]GTPγS binding to mouse brain membranes. The effect of (a) 1μm cannabidiol (n=4–5), (b) 10nm rimonabant (n=4) or (c) 1μm O-2654 (n=5) on the mean log concentration–response curve of …

Figure 2

[35S]GTPγS binding to mouse brain membranes. The effect of (a) 1μm cannabidiol (n=6) or (b) 10nm rimonabant (n=5) on the mean log concentration–response curve of R-(+)-WIN55212 for stimulation of [35S]GTP …

Table 1

The mean apparent KB values for antagonism of CP55940-induced [35S]GTPγS binding to membranes and the Ki values for the displacement of [3H]CP55940 from membranes

Next, we confirmed that under our laboratory assay conditions, it was possible to detect not only stimulation of [35S]GTPγS binding to mouse brain membranes by the established CB1 receptor agonists, CP55940 and R-(+)-WIN55212, but also inhibition of such binding by a CB1 receptor inverse agonist, rimonabant (Figure 3, Table 2). We then went on to investigate the effect of cannabidiol by itself on [35S]GTPγS binding to mouse brain membranes. Although this compound had no detectable effect at 1 or 100nm, it produced significant inhibition at 1 and 10μm (Figure 3a). The inhibitory effect produced by 1μm cannabidiol did not deviate significantly from that of 1μmrimonabant, whereas the inhibitory effect of 10μm cannabidiol greatly exceeded that of 10μmrimonabant (P<0.05; ANOVA followed by Bonferroni’s multiple comparison test; n=6 and 8).

Figure 3

[35S]GTPγS binding to mouse brain membranes. The effect of (a) CP55940 (n=9), R-(+)-WIN55212 (n=7–8), rimonabant (n=7–8) and cannabidiol (n=6) or (b) rimonabant (n=7–8) and O-2654 (n=6–10) on the level of …

Table 2

The mean Emax values±s.e.m. and the mean EC50 values, with 95% CI in parentheses for the efficacy of tested cannabinoids in mouse brain or hCB2-CHO cell membranes

Our next experiments were performed with the putative neutral CB1 receptor antagonist, O-2654 (Thomas et al., 2004), to establish how it compared with cannabidiol as a modulator of [35S]GTPγS binding to mouse brain membranes. In contrast to CP55940, R-(+)-WIN55212, cannabidiol or rimonabant, O-2654 neither inhibited nor enhanced [35S]GTPγS binding between 0.1nm and 1μm to mouse brain membranes (Figure 3b). Unexpectedly, at a concentration of 10μm, O-2654 significantly inhibited the binding of [35S]GTPγS to mouse brain membranes by an amount that did not differ significantly from the inhibition produced by 10μm rimonabant (P>0.05; ANOVA followed by Bonferroni’s multiple comparison test; n=8). We also found that O-2654 shared the ability of cannabidiol to antagonize CP55940 (Figure 1c). However, unlike cannabidiol and rimonabant, O-2654 is only slightly more potent as a CB1 receptor antagonist (apparent KB=51nm; Table 1) than as a CB1 receptor ligand (Ki=114nm) (Thomas et al., 2004). TheKi values of rimonabant, cannabidiol and O-2654 and their mean apparent KB values for antagonism of CP55940-induced stimulation of [35S]GTPγS binding to mouse brain membranes are shown in Table 1.

Because cannabidiol produced an inverse effect at 10μm in mouse brain membranes, which was so much greater than that produced by rimonabant, we investigated whether this effect of cannabidiol is CB1 receptor-mediated. This issue was addressed by establishing whether membranes prepared from the brains of mice whose CB1 receptors had been genetically deleted (CB1−/− C57BL/6 mice) responded differently, in the [35S]GTPγS assay, to cannabidiol from brain membranes obtained from their WT littermates.

In these experiments, 10μm cannabidiol was no less effective as an inhibitor of [35S]GTPγS binding to CB1−/− than to WT C57BL/6 mouse brain membranes (Figure 4a). It is noteworthy, however, that cannabidiol was less potent at inhibiting [35S]GTPγS binding to WT C57BL/6 mouse brain membranes than to the MF1 mouse brain membranes that were used in all our other experiments. Thus, 1μm cannabidiol produced detectable inhibition only in the MF1 mouse brain membranes (Figures 3a and ​and4a).4a). Rimonabant was also less potent in this assay when it was conducted with WT C57BL/6 rather than MF1 mouse brain membranes. Thus, like cannabidiol, 1μm rimonabant inhibited [35S]GTPγS binding only to the MF1 mouse brain membranes (Figures 3and ​and4b).4b). Neither 1 nor 10μm rimonabant inhibited [35S]GTPγS binding to CB1−/− mouse brain membranes (Figure 4b). This finding was unexpected, as Breivogel et al. (2001) have reported that rimonabant can inhibit [35S]GTPγS binding to brain membranes obtained from C57BL/6 CB1−/−mice.

Figure 4

[35S]GTPγS binding to mouse brain membranes. The effect of (a) cannabidiol (n=5–6) or (b) rimonabant (n=6) on the level of [35S]GTPγS binding to mouse whole-brain membranes prepared from either C57BL/6 mice whose CB1 receptors …

Cannabidiol and rimonabant exhibited lower inhibitory potency in the [35S]GTPγS-binding assay when this was performed with brain membranes obtained from C57BL/6 mice rather than from MF1 mice. Consequently, as we had already found O-2654 to possess at least 10 times less inhibitory potency than either cannabidiol or rimonabant in this assay when it was performed with MF1 mouse brain membranes, and as O-2654 inhibited [35S]GTPγS binding to these membranes at 10μm but not 1μm, we did not investigate the effect of O-2654 on [35S]GTPγS binding to C57BL/6 mouse brain membranes.

Because the results from our experiments with CB1−/− mouse brain membranes suggest that cannabidiol can inhibit [35S]GTPγS binding through a CB1 receptor-independent mechanism, we performed experiments with membranes prepared either from CHO cells transfected with human CB1 receptors or from untransfected CHO cell membranes. We found, however, that although binding of [35S]GTPγS to the hCB1-CHO cell membranes was stimulated by cannabidiol at concentrations between 1 and 1000nm and inhibited by cannabidiol at 10μm (Figure 5), none of these concentrations of cannabidiol affected [35S]GTPγS binding to the membranes obtained from untransfected CHO cells (data not shown).

Figure 5

[35S]GTPγS binding to hCB1-CHO cell membranes. The effect of cannabidiol (n=5–15) on the level of [35S]GTPγS binding to CB1 transfected CHO cell membranes. Each symbol represents the mean percentage increase in [35S]GTPγ …

We also investigated whether cannabidiol would antagonize ligand-induced activation of a non-cannabinoid G protein-coupled receptor. More specifically, we addressed the question of whether cannabidiol can block the activation of opioid receptors by morphine as, like the CB1 receptor, opioid receptors are thought to signal primarily through Gi/o proteins (Corbett et al., 2006). We selected morphine for these experiments as it is expected to target all the opioid receptor subtypes that are thought to be present in mouse brain membranes (Mignat et al., 1995). We found that 1μmcannabidiol (n=4–6) did not significantly antagonize morphine-induced enhancement of [35S]GTPγS binding to mouse brain membranes (data not shown).

Experiments with cannabidiol and SR144528 using human CB2-CHO cell membranes

Having established the effect of cannabidiol on CB1 receptor-containing systems (mouse brain and CB1-CHO cell membranes), we compared the abilities of SR144528 and cannabidiol to displace [3H]CP55940 from hCB2-CHO cell membranes. The results (Figure 6) provided the CB2 Ki values for SR144528 and cannabidiol shown in Table 1. We next investigated whether cannabidiol shares the ability of SR144528 to antagonize CP55940-induced stimulation of [35S]GTPγS binding to hCB2-CHO cell membranes. At a concentration of 1μm, cannabidiol produced a downward as well as a rightward shift of the log concentration–response curve of CP55940 (Figure 7a) and its apparent KB value was calculated to be 64.5 times less than its CB2 Ki value (Table 1). Similar results were obtained from experiments with SR144528 performed under the same assay conditions (Figure 7b). Thus, SR144528 induced a downward as well as rightward displacement of the CP55940 log concentration–response curve and exhibited an apparent KB value that was 15 times less than its CB2 Ki value (Table 1).

Figure 6

Displacement of [3H]CP55940 from hCB2-CHO cell membranes. The ability of SR144528, cannabidiol or O-2654 to displace [3H]CP55940 from specific binding sites in hCB2-CHO cell membranes. Each symbol represents the mean percent displacement±s.e.m. …

Figure 7

[35S]GTPγS binding to membranes from hCB2-CHO cell membranes. The effect of (a) 1μm cannabidiol (n=4–5), (b) 100nm SR144528 (n=5–6) or (c) 1μm O-2654 (n=3–5) on the mean log concentration–response …

We also investigated whether cannabidiol shares the ability of SR144528 to behave as a CB2receptor inverse agonist, as measured by inhibition of [35S]GTPγS binding to hCB2-CHO cell membranes. Cannabidiol was indeed found to produce an inhibitory effect on [35S]GTPγS binding, as was SR144528 (Figure 8a). The Emax of cannabidiol did not deviate significantly from that of SR144528, whereas its pEC50 (6.3±0.7) was markedly greater than that of SR144528 (9.1±0.3). A summary of these results can be found in Table 2.

Figure 8

[35S]GTPγS binding to membranes from hCB2-CHO cell membranes. The effect of (a) CP55940 (n=6–12), SR144528 (n=5) and cannabidiol (n=8) or (b) SR144528 (n=5) and O-2654 (n=6) on the level of [35S]GTPγS binding to CB2transfected …

Experiments with O-2654 using human CB2-CHO cell membranes

As O-2654 attenuated CP55940 responses in mouse brain membranes in a manner that is consistent with it being a neutral CB1 receptor antagonist, it was of interest to determine whether O-2654 also behaves as a neutral antagonist at the CB2 receptor. Accordingly, we first tested how well O-2654 displaces [3H]CP55940 from the hCB2-CHO cell membranes (Figure 6), the results obtained indicating that O-2654 binds 2.4 times more readily to the CB2 receptor than to the CB1receptor (Table 1). We then addressed the question of whether O-2654 resembles cannabidiol (and SR144528) in antagonizing CP55940-induced inhibition of [35S]GTPγS binding more potently than it displaces [3H]CP55940 from hCB2-CHO cell membranes. Our experiments showed that this was not so, as the apparent KB value of O-2654 for antagonism of CP55940 (Figure 7c) was not markedly different from its CB2 Ki value (Table 1). O-2654 appeared to induce downward as well as rightward displacements of the CP55940 log concentration–response curve in the hCB2-CHO cell membranes in these experiments (Figure 7c). However, in contrast to our findings with cannabidiol and SR144528, this downward displacement was not statistically significant. Thus, the 95% CI for the bottom of the CP55940 log concentration–response curves in the absence or presence of 1μm O-2654 overlapped. Although, we also discovered that when added by itself, O-2654 exhibits cannabidiol-like potency and efficacy as an inhibitor of [35S]GTPγS binding to hCB2-CHO cell membranes (Table 2 and Figure 8b), this should not be taken as evidence that O-2654 is a CB2 receptor inverse agonist. Thus, the potency and efficacy of O-2654 as an inhibitor of [35S]GTPγS binding remained the same irrespective of whether the bioassay was performed with hCB2-CHO cell membranes or with membranes from cells that had not been transfected with CB2receptors (n=6; Figure 9). In contrast, CP55940 (n=6), cannabidiol (n=8), or SR144528 (n=8) did not modulate [35S]GTPγS binding to membranes prepared from CHO cells that had not been transfected with CB2 receptors (data not shown).

Figure 9

[35S]GTPγS binding to membranes from untransfected cells or cells transfected with human CB2 receptors. The effect of O-2654 on the level of [35S]GTPγS binding to untransfected CHO cell membranes (n=6) or CB2-transfected CHO cell membranes…

Finally, it is unlikely that cannabidiol and SR144528 each appear to be more potent as an antagonist of CP55940-induced stimulation of [35S]GTPγS binding to hCB2-CHO cell membranes than as a displacer of [3H]CP55940 from CB2 receptors because the buffers used in the [35S]GTPγS and [3H]CP55940-binding assays were different. Thus, the ability of SR144528 (n=5; data not shown) to displace [3H]CP55940 from hCB2-CHO cell membranes was unaffected when GTPγS buffer was used in this bioassay instead of the standard Tris/BSA buffer. We have also reported similar findings previously with Δ9-tetrahydrocannabivarin (Thomas et al., 2005).

A summary of the CB2 Ki values of cannabidiol, SR144528 and O-2654 and of the mean apparentKB values of these compounds for antagonism of CP55940-induced stimulation of [35S]GTPγS binding to hCB2-CHO cell membranes can be found in Table 1.

This investigation was supported by grants from GW Pharmaceuticals, the BBSRC and the National Institute on Drug Abuse.

CI

confidence intervals

CP55940

(–)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol

Δ9-THC

Δ9-tetrahydrocannabinol

DMEM

Dubbecco’s modified Eagle’s medium

DMSO

dimethyl sulphoxide

DPCPX

8-cyclopentyl-1,3-dipropylxanthine

FBS

fetal bovine serum

hCB1-CHO

Chinese hamster ovary cells stably transfected with human CB1 receptors

hCB2-CHO

Chinese hamster ovary cells stably transfected with human CB2 receptors

R-(+)-WIN55212

(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone

SR144528

N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide

• Bisogno T, Hanus L, De Petrocellis L, Tchilibon S, Ponde DE, Brandi I, et al. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br J Pharmacol. 2001;134:845–852. [PMC free article] [PubMed]
• Bouaboula M, Desnoyer N, Carayon P, Combes T, Casellas P. Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation. Mol Pharmacol. 1999;55:473–480. [PubMed]
• Breivogel CS, Griffin G, Di Marzo V, Martin BR. Evidence for a new G protein-coupled cannabinoid receptor in mouse brain. Mol Pharmacol. 2001;60:155–163. [PubMed]
• Carrier EJ, Auchampach JA, Hillard CJ. Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc Nat Acad Sci USA.2006;103:7895–7900. [PMC free article] [PubMed]
• Cheng YC, Prusoff WH. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099–3108. [PubMed]
• Corbett AD, Henderson G, McKnight AT, Paterson SJ. 75 years of opioid research: the exciting but vain quest for the Holy Grail. Br J Pharmacol. 2006;147:S153–S162.[PMC free article] [PubMed]
• ElSohly MA, Slade D. Chemical constituents of marijuana: the complex mixture of natural cannabinoids. Life Sci. 2005;78:539–548. [PubMed]
• Kathmann M, Flau K, Redmer A, Trankle C, Schlicker E. Cannabidiol is an allosteric modulator at mu- and delta-opioid receptors. Naunyn-Schmiedeberg’s Arch Pharmacol.2006;372:354–361. [PubMed]
• Leff P. The two-state model of receptor activation. Trends Pharmacol Sci. 1995;16:89–97.[PubMed]
• Lunn CA, Fine JS, Rojas-Triana A, Jackson JV, Fan X, Kung TT, et al. A novel cannabinoid peripheral cannabinoid receptor-selective inverse agonist blocks leukocyte recruitment in vivo. J Pharmacol Exp Ther. 2006;316:780–788. [PubMed]
• Mignat C, Wille U, Ziegler A. Affinity profiles of morphine, codeine, dihydrocodeine and their glucuronides at opioid receptor subtypes. Life Sci. 1995;56:793–799. [PubMed]
• Pertwee RG. Pharmacology of cannabinoid receptor ligands. Curr Med Chem. 1999;6:635–664. [PubMed]
• Pertwee RG. The pharmacology and therapeutic potential of cannabidiol Cannabinoids 2004. Kluwer Academic/Plenum Publishers: New York; 32–83.83In: Di Marzo, V. (eds)
• Pertwee RG. Inverse agonism and neutral antagonism at cannabinoid CB1 receptors. Life Sci.2005;76:1307–1324. [PubMed]
• Pertwee RG, Griffin G, Lainton JAH, Huffman JW. Pharmacological characterization of three novel cannabinoid receptor agonists in the mouse isolated vas deferens. Eur J Pharmacol.1995;284:241–247. [PubMed]
• Pertwee RG, Ross RA, Craib SJ, Thomas A. Cannabidiol antagonizes cannabinoid receptor agonists and noradrenaline in the mouse vas deferens. Eur J Pharmacol. 2002;456:99–106. [PubMed]
• Pertwee RG, Thomas A, Stevenson LA, Maor Y, Mechoulam R. Evidence that (−)-7-hydroxy-4′-dimethylheptyl-cannabidiol activates a non-CB1, non-CB2, non-TRPV1 target in the mouse vas deferens. Neuropharmacology. 2005;48:1139–1146. [PubMed]
• Petitet F, Jeantaud B, Reibaud M, Imperato A, Dubroeucq MC. Complex pharmacology of natural cannabinoids: evidence for partial agonist activity of delta-9-tetrahydrocannabinol and antagonist activity of cannabidiol on rat brain cannabinoid receptors. Life Sci.1998;63:PL1–PL6. [PubMed]
• Portier M, Rinaldi-Carmona M, Pecceu F, Combes T, Poinot-Chazel C, Calandra B, et al. SR144528, an antagonist for the peripheral cannabinoid receptor that behaves as an inverse agonist. J Pharmacol Exp Ther. 1999;288:582–589. [PubMed]
• Rhee MH, Kim SK. SR144528 as inverse agonist of CB2 cannabinoid receptor. J Vet Sci.2002;3:179–184. [PubMed]
• Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A, et al. Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656 and AM630. Br J Pharmacol. 1999;126:665–672. [PMC free article] [PubMed]
• Savinainen JR, Saario SM, Niemi R, Jarvinen T, Laitinen JT. An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1and cannabinoid CB1 receptors. Br J Pharmacol. 2003;140:1451–1459. [PMC free article][PubMed]
• Showalter VM, Compton DR, Martin BR, Abood ME. Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther. 1996;278:989–999. [PubMed]
• Thomas A, Baillie GL, Phillips AM, Ross RA, Pertwee RG. Cannabidiol is an inverse agonist and exhibits unexpectedly high potency as an antagonist at mouse brain CB1 and human CB2 receptors Symposium on the Cannabinoids 2006. Burlington, Vermont, International Cannabinoid Research Society; p 8.
• Thomas A, Ross RA, Saha B, Mahadevan A, Razdan RK, Pertwee RG. 6′-azidohex-2′-yne-cannabidiol: a potential neutral, competitive cannabinoid CB1 receptor antagonist. Eur J Pharmacol. 2004;487:213–221. [PubMed]
• Thomas A, Stevenson LA, Wease KN, Price MR, Baillie G, Ross RA, et al. Evidence that the plant cannabinoid Δ9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist. Br J Pharmacol. 2005;146:917–926. [PMC free article] [PubMed]
• Thomas B, Gilliam AF, Burch DF, Roche MJ, Seltzman HH. Comparative receptor binding analyses of cannabinoid agonists and antagonists. J Pharmacol Exp Ther. 1998;285:285–292. [PubMed]