Introduction

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Cannabinoid drugs are thought to produce their unique pharmacological profile of effects through activation of specific membrane receptors (Devane et al., 1988; Munro et al., 1993). These receptors, termed CB₁ and CB₂, couple to guanine nucleotide binding proteins (G proteins) as described for a large variety of other receptors (Matsuda et al., 1990; Munro et al., 1993; Kenakin, 1996). Cannabinoid receptors are found throughout both the central nervous system and the periphery (Herkenham et al., 1991; Munro et al., 1993). Within the central nervous system, cannabinoid receptors are found to be localized in many brain areas, with the regions of densest receptor localization including the cerebellum, hippocampus, cortex and the basal ganglia (Herkenham et al., 1991). This distribution may be related to the pharmacological effects of administered cannabinoid drugs (Pertwee, 1993) and is very similar irrespective of species (Herkenham et al., 1991; Jansen et al., 1992; Glass et al., 1997).

Several functional assays are presently used to characterize cannabinoid compounds including whole animal tests, such as inhibition of locomotor activity and the development of hypothermia; inhibition of electrically stimulated contractions of isolated smooth muscle preparations; and inhibition of forskolin-stimulated cAMP accumulation in tissues and cell lines. In addition, the effects of cannabinoids on ion channel conductance and on the activity of mitogen-activated protein kinase have been used (for review see: Martin et al., 1995; Bouaboula et al., 1995).

Recently, a method for measuring cannabinoid-stimulated [³⁵S]GTPS binding in brain membranes has been described (Sim et al., 1995). This model measures the first step in functional activation of the receptor as a result of agonist binding, therefore allowing for the delineation of agonist/antagonist activity regardless of the second messenger system involved. Following activation of the receptor by an agonist, the affinity of the G protein alpha subunit increases with respect to GTP vs. GDP. As a consequence, GDP is displaced from the G protein and GTP or GTPS binds. If a radioactive label, such as [³⁵S], is attached to the GTPS molecule, then the formation of the G protein/[³⁵S]GTPS complex may be directly measured using liquid scintillation spectrophotometry (Weiland and Jakobs, 1994).

The aim of this study was to investigate the effects of a range of structurally diverse cannabinoid receptor ligands on [³⁵S]GTPS binding. Further to this was an attempt to correlate a compound's ability to stimulate GTPS binding with previously reported receptor affinity data and also with in vivo potency data.

	Experimental Procedures

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Materials. Male Sprague-Dawley rats (150-250 g) were obtained from Harlan (Dublin, VA). GDP and GTPS were purchased from Boehringer Mannheim (Indianapolis, IN). [³⁵S]GTPS (1000-1200 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Other reagent grade chemicals were purchased from Sigma (St. Louis, MO). THC and cannabinol were obtained from NIDA. SR141716A, CP 55,940 and CP 55,244 were generously provided by Pfizer (Groton, CT), and WIN 55212-2 was purchased from Research Biochemicals (Natick, MA). Anandamide, fluoromethanandamide and O-1064 were synthesized by Dr. Raj Razdan (Organix, Woburn, MA). HU-210 was generously provided by Prof. Raphael Mechoulam (Hebrew University, Jerusalem, Israel). Deoxy-HU-210, JWH-030 and JWH-073 were synthesized by Dr. John Huffman (Clemson University, Clemson, SC). All compounds were stored as 1 mg/ml solutions in ethanol.

Membrane preparation. Cerebella were dissected on ice from 3 fresh male Sprague-Dawley rat brains. The pooled tissue was suspended in centrifugation buffer (50 mM Tris HCl, 1 mM EGTA, 3 mM MgCl₂; pH 7.4) and homogenized using a Kontes Potter-Elvehjem glass-Teflon grinding system (Fisher Scientific, Sprinfield, NJ). The homogenate was centrifuged at 48,000 × g for 20 min at 4°C. The pellet was then resuspended in assay buffer (50 mM Tris HCl, 9 mM MgCl₂, 0.2 mM EGTA, 150 mM NaCl; pH 7.4), homogenized, and centrifuged as previously. The final P2 pellet was then resuspended in assay buffer, homogenized, and diluted to a concentration of ~ 2 µg/µl with assay buffer. The protein concentration was determined by the method of Bradford (1976). Aliquots were then stored at 80°C.

[³⁵S]GTPS binding. The methods for measuring agonist-stimulated [³⁵S]GTPS binding were adapted from those of Sim et al. (1995). Rat cerebellar membranes (10 µg) were incubated in assay buffer containing .1% fatty acid free bovine serum albumin with GDP 1-100 µM, [³⁵S]GTPS 0.05 nM and cannabinoid compounds/ethanol control in siliconized glass tubes. The total assay volume was 0.5 ml, which was incubated at 30°C for 30 min. An incubation time of 60 min was used in experiments with HU-210 as a time course experiment demonstrated this to be the optimal time for maximal stimulation of [³⁵S]GTPS binding (data not shown). Experiments with anandamide also included 50 µM PMSF. The reaction was terminated by addition of 2 ml ice-cold wash buffer (50 mM Tris HCl, 5 mM MgCl₂; pH 7.4) followed by rapid filtration under vacuum through Whatman GF/C glass-fiber filters using a 12-well sampling manifold. The tubes were washed once with 2 ml of ice-cold wash buffer, and the filters were washed twice with 4 ml of ice-cold wash buffer. Filters were placed into 7 ml plastic scintillation vials (RPI Corp., Mount Prospect, IL). Bound radioactivity was determined by liquid scintillation spectrophotometry after extraction in 5 ml BudgetSolve scintillation fluid. Non-specific binding was determined using 10 µM GTPS. Basal binding was assayed in the absence of agonist and in the presence of GDP. The stimulation by agonist was defined as a percentage increase above basal levels (i.e., {[dpm (agonist)  dpm (no agonist)]/dpm (no agonist)}× 100).

Data analysis. Data are reported as mean ± S.E.M. of three to eight experiments, performed in triplicate. Nonlinear regression analysis of concentration-response data was performed using Prism 2.0 software for the Macintosh (GraphPAD Software, San Diego, CA) to calculate E_max and EC₅₀ values. One-way ANOVA using Dunnett's post-hoc (P < .05) was used for statistical analysis. The equilibrium dissociation constant (K_d) for the interaction of the antagonist and the receptor has been calculated from the equation (dose ratio  1) = [B]  K_d, where [B]is the concentration of the antagonist used in the experiment (Pertwee et al., 1995a).

	Results

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Effects of GDP on agonist-stimulated [³⁵S]GTPS binding. Preliminary experiments were directed at optimizing the assay conditions for WIN 55212-2 stimulation of [³⁵S]GTPS binding. To optimize this assay, the conditions should ideally overcome spontaneous agonist-independent guanine nucleotide exchange at the G protein, as has been demonstrated to occur with other G protein-coupled receptors (Kenakin, 1996), and also to maximize the ability of an agonist to induce GDP dissociation and [³⁵S]GTPS association with the G protein following receptor activation (Weiland and Jakobs, 1994). The combination of these factors may be expected to result in both a decrease in basal and an increase in agonist-stimulated [³⁵S]GTPS binding. Therefore, the influences of GDP, sodium and magnesium ions on WIN 55212-2-stimulated [³⁵S]GTPS binding were investigated.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Effect of GDP on basal and WIN 55212-2 (10 µM) stimulated [35S]GTPS binding. A, Data represent percentage total specific [35S]GTPS binding in the presence () and absence () of WIN 55212-2 (10 µM). B, Data represent percentage stimulation over basal binding induced by WIN 55212-2 (10 µM) (). Results are presented as mean ± S.E. for n = 4 experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of 10 µM () and 100 µM () GDP on the concentration-response curves of WIN 55212-2 (A), CP 55,940 (B) and THC (C). Data represent percentage stimulation over basal binding. Results are presented as mean ± S.E. for n = 4 experiments.

View this table: [in this window] [in a new window]   TABLE 1 Effect of GDP concentration on the ability of three cannabinoid receptor agonists to stimulate [35S]GTPS binding in rat cerebellar membranes

View this table: [in this window] [in a new window]   TABLE 2 Ability of various cannabinoid receptor ligands to stimulate [35S]GTPS binding in rat cerebellar membranes at a GDP concentration of 100 µM

Effects of various cannabinoid receptor ligands on [³⁵S]GTPS binding. To examine the effects of a number of structurally diverse cannabinoid ligands on[³⁵S]GTPS binding, membranes were incubated with varying concentrations of ligands from each of the four classes of cannabinoids; (1) classic cannabinoids (THC; HU-210; deoxy-HU-210; cannabinol), (2) nonclassic cannabinoids (CP 55,940; CP 55,244), (3) aminoalkylindoles (WIN 55212-2; JWH-030; JWH-073) and (4) eicosanoids (anandamide; fluoromethanandamide; O-1064). HU-210 and CP 55,244 were used as they have previously been shown, in several assay systems, to be high potency cannabinoid agonists (Howlett, 1995). Deoxy-HU-210 was used as this has a much higher affinity for CB₂ receptors than it does for CB₁ (Huffman et al., 1996). JWH-030 and JWH-073 were used as these indole analogues have been shown to have affinity for both CB₁ and CB₂ receptors and have not previously been tested in any functional assays (Showalter et al., 1996; B.R. Martin, unpublished results). Fluoromethanandamide and O-1064 were used as high affinity, metabolically stable anandamide analogues (Adams et al., 1995). The structures for each of these compounds are shown in figure 3. To keep conditions for the comparison of each compound identical, the GDP concentration was kept constant (100 µM). The results of these experiments are shown in table 2. CP 55,244 was the most potent of the compounds, and also produced the highest percentage stimulation of binding, with EC₅₀ and E_max values of 0.47 (0.22-0.96) nM and 165% (148-183%), respectively. WIN 55212-2, deoxy-HU-210 and HU-210 all had E_max values that did not differ significantly from CP 55,244 and therefore may also be classified as full agonists. CP 55,940, fluoromethanandamide, O-1064, JWH-030 and JWH-073 all stimulated [³⁵S]GTPS binding, but with a significantly lesser maximal effect, and may therefore be classed as partial agonists. The maximal percentage stimulations of these partial agonists ranged from 114% (97-131%) with CP 55,940 to 29% (19-40%) with JWH-073. THC, anandamide and cannabinol did not stimulate [³⁵S]GTPS binding under these conditions. The ability of THC to antagonize CP 55,940-induced stimulation of [³⁵S]GTPS was also tested. THC was found to produce a slight, but nonsignificant rightward shift of the concentration-response curve of CP 55,940 (results not shown). Following the observation that THC stimulated [³⁵S]GTPS binding with a GDP concentration of 10 µM, but not at 100 µM, anandamide was also tested at this lower GDP concentration. However, it was found that anandamide did not stimulate [³⁵S]GTPS binding at this GDP concentration. Furthermore, lowering the sodium and magnesium ion concentrations did not affect anandamide's ability to stimulate GTPS binding. PMSF, at a concentration of 50 µM, was included in the assay buffer for experiments using anandamide and, at this concentration, was found not to effect [³⁵S]GTPS binding. To examine the possibility that tissue metabolism accounted for the lack of stimulatory effect of anandamide on GTPS binding, a radioligand displacement curve was constructed. It was found that in rat cerebellum, anandamide, in the presence of 50 µM PMSF, displaced 1 nM [³H]CP 55,940 with a K_i = 145.4 nM (results not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Structures of compounds used in this study.

Effect of the selective CB₁ antagonist, SR141716A, on cannabinoid-stimulated [³⁵S]GTPS binding. Since the discovery of the CB₁ selective antagonist in 1994 (Rinaldi-Carmona et al., 1994), this compound has been used in a wide variety of assays to assess the role of the CB₁ receptor in the production of a cannabimimetic effect (Rinaldi-Carmona et al., 1994; Pertwee et al., 1995a; Compton et al., 1996). To determine the role of the CB₁ receptor in the production of cannabinoid-stimulated [³⁵S]GTPS binding, concentration-response curves of a number of the more potent and efficacious agonists used in this study were compared in the presence and absence of SR141716A.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of SR141716A at concentrations of 1 nM (), 3 nM () and 10 nM () on the log concentration response curve of CP 55,940 (). Data represent percentage stimulation over basal binding. Results are presented as mean ± S.E. for n = 4 experiments.

	Discussion

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Effects of GDP on agonist-stimulated [³⁵S]GTPS binding. WIN 55212-2 was chosen for the initial optimization experiments as it has been used as a standard cannabinoid receptor agonist, acting as a potent full agonist, in many different assays (Howlett, 1995; Martin et al., 1995). It also displays a high affinity for both subtypes of cannabinoid receptor (Showalter et al., 1996) and has been demonstrated to stimulate [³⁵S]GTPS binding in rat cerebellar membranes (Selley et al., 1996). Optimal WIN 55212-2-stimulated [³⁵S]GTPS binding occurred with the conditions described in the results section and were used for all subsequent experiments. A significantly greater maximal percentage stimulation was observed at 100 µM GDP than at 10 µM. The E_max and the potency of WIN 55212-2 obtained under these conditions correspond with previous studies that used varying assay conditions with both rat cerebellar membranes and membrane preparations from other brain areas (Selley et al., 1996; Hosohata et al., 1997). When similar experiments were conducted using CP 55,940, an identical trend was observed. However, THC, at the higher GDP concentration (100 µM), produced no significant stimulation of binding whereas, at a GDP concentration of 10 µM, a clear concentration-dependent stimulation of [³⁵S]GTPS binding was observed. This apparently contradictory set of results may be explained by considering the role of GDP in this assay. As previously discussed in the results section, GDP is included in the assay to promote G protein inactivation. High efficacy agonists, such as CP 55,940 and WIN 55212-2, are thought to be more efficient at overcoming this `GDP block' than agonists of lower efficacy, such as THC (Selley et al., 1996). This difference may be as a result of a better ability of high efficacy compounds to induce and/or stabilize changes in receptor conformation. Therefore, by reducing the "GDP block," THC may now be capable of stimulating [³⁵S]GTPS binding. It has also been suggested that excess GDP may cause a decrease in the catalytic rate of G protein activation, to which high efficacy agonists may be less susceptible (Emmerson et al., 1996). The results presented here are consistent with these possibilities. This observation has previously been demonstrated in other studies (Selley et al., 1996; Breivogel et al., 1997a).

Effects of various cannabinoid receptor ligands on [³⁵S]GTPS binding. The pharmacologies of the cannabinoid receptor ligands tested in this study, in the presence of 100 µM GDP, demonstrate a wide range of activities. The compounds ranged from the highly potent and efficacious through to compounds displaying little or no concentration-dependent stimulation of [³⁵S]GTPS binding. Of the range of compounds tested; CP 55,244, HU-210, deoxy-HU-210 and WIN 55212-2 may be regarded as full agonists; CP 55,940, fluoromethanandamide, O-1064, JWH-030 and JWH-073 displayed varying degrees of partial agonism and THC, anandamide and cannabinol were not active (although THC was active at 10 µM GDP). This disparity of activities, and the finding that several compounds, known to act as full agonists in other functional assays, have little or no activity in this assay, requires further examination.

Effect of the selective CB₁ antagonist, SR141716A, on cannabinoid-stimulated [³⁵S]GTPS binding. The results of this study demonstrate that in rat cerebellar membranes, cannabinoid-induced stimulation of [³⁵S]GTPS binding is mediated by specific cannabinoid receptors. The ability of SR141716A to antagonize cannabimimetic effects has been demonstrated in detail in many other assays. In isolated smooth muscle preparations, for example, SR141716A has been reported to antagonize electrically evoked contractions with K_d values ranging from ~1 through 10 nM (Rinaldi-Carmona et al., 1994; Pertwee et al., 1995a). Similarly, Selley et al. (1996) demonstrated that SR141716A, at a concentration of 200 nM, antagonized WIN 55212-2-induced stimulation of [³⁵S]GTPS binding in rat cerebellar membranes. Although the authors did not quote a K_d value, it was estimated to be no greater than 2 nM. The K_d values of SR141716A calculated in the presence of five cannabinoid receptor agonists correlate closely with each other, irrespective of the agonist used, suggesting that cannabinoid agonist-induced stimulation of [³⁵S]GTPS binding is mediated by a single receptor site, CB₁. The agreement of the K_d values found in this study with those previously reported using other experimental models is further evidence of the validity of this assay for the study of cannabinoid receptor antagonists.