Forskolin-stimulated cyclic AMP production in the absence of AM630 has been normalized to 100%
Forskolin-stimulated cyclic AMP production in the absence of AM630 has been normalized to 100%. Tris, 10?mM MgCl2, 100?mM NaCl and 0.2?mM EDTA at pH 7.4. Incubations were carried out at 30C for 90?min in a total volume of 500?l. The reaction was terminated by the addition of 4?ml of ice-cold wash buffer (50?mM Tris and 1?mg?ml?1 BSA, pH 7.4) followed by rapid filtration under vacuum through Whatman GF/B glass-fibre filters (pre-soaked in wash buffer) using a 12-tube Brandel cell harvester. The tubes were washed three times with 4?ml of wash buffer. Filters were oven dried, placed in 5?ml of scintillation fluid and bound radioactivity was determined by liquid scintillation counting. Basal binding of [35S]-GTPS was determined in the presence of 20?M GDP and absence of cannabinoid. Non-specific binding was determined in the presence of 10?M GTPS. Analysis of data Values have been expressed as means and variability as s.e.mean or as 95% confidence limits. Mean values have been compared using the Kruskall-Wallis test followed by Dunn’s multiple comparison test. A value <0.05 was considered to be significant. Effects of test compounds on forskolin-stimulated cyclic AMP production have been expressed in percentage terms. This was calculated from the equation [100(f?b)]/(f?b) where f?, f and b are values of cyclic AMP production (pmol?ml?1), f? in the presence of forskolin and the test compound, f in the presence of forskolin only and b in the absence of both forskolin and the test compound. Drug-induced inhibition of specific [35S]-GTPS binding was expressed as the percentage decrease below the basal level of [35S]-GTPS binding using the equation [100(d?d)]/d where d and d WIN 55,212-2 mesylate are d.p.m. in the presence and absence of the drug respectively. Values for EC50, IC50 and maximal effects (Emax) and the 95% confidence limits of these values have been calculated by non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, U.S.A.). The ability of AM630 to antagonize CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB2 transfected cells is expressed in terms of the concentration ratio. This has been defined as the concentration of CP55940 that produces a particular degree of inhibition in the presence of AM630 at a concentration, B, divided by the concentration of CP55940 that produces an identical degree of inhibition in the absence of AM630. Since AM630 behaved as an inverse agonist at CB2 receptors (see Results), it was considered inappropriate to insert concentration ratio values into the Schild equation in order to obtain a KB value of AM630 at these receptors. Concentration ratio values and their 95% confidence limits have been determined by symmetrical (2+2) dose parallel line assays (Colquhoun, 1971), using responses to pairs of agonist concentrations located on the steepest part of each log concentration-response curve. This method was also used to establish whether log concentration-response curves of CP55940 constructed in the presence and absence of AM630 deviated significantly from parallelism. Drugs CP55940 (?)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol was supplied by Pfizer, WIN55212-2 (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone by Research Biochemicals International, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and 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 by Sanofi Recherche and L759633 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6,9-trimethyl-6a,7,10,10a?-tetrahydro?-6H-benzo[c]chromene] and L759656 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6-dimethyl-9-methylene?-?6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene] by Merck Frosst. AM630 (6-iodopravadoline) was synthesized in the laboratory of Dr A. Makriyannis. [3H]-CP55940 (126?Ci?mmol?1) and [3H]-WIN55212-2 (45?Ci mmol?1) were supplied by NEN Life Science Products. Cannabinoids were stored as 1?mg?ml?1 stock solutions in ethanol and diluted in assay buffer. At the highest concentrations used, ethanol by itself had no detectable effect on specific binding of [3H]-CP55940, [3H]-WIN55212-2 or [35S]-GTPS or on forskolin-stimulated cyclic AMP production (data not shown). Results Cannabinoid binding experiments The radioligand binding data shown in Table 1 confirm that CP55940 is a high-affinity non-selective ligand for cannabinoid receptors. The data also confirm L759656 and L759633 to be markedly CB2-selective, with much lower Ki values in CB2 transfected cell membranes than in membranes of CB1 transfected cells. AM630 is also CB2-selective with a CB1/CB2 Ki ratio of 165 in transfected CHO cell membranes. Further experiments showed AM630 to be no less effective in displacing [3H]-CP55940 than [3H]-WIN55212-2 from CB2 receptors on CHO cell membranes (Table 1). SR144528 was also equally effective in displacing [3H]-CP55940 and [3H]-WIN55212-2 from CB2 receptors (Table 1). Effects of CP55940, L759656, L759633 and AM630 on cyclic AMP production Cyclic AMP concentrations in the absence and presence of 2?M forskolin were 5.11.8 and 46.313.1?pmol?ml?1 respectively in CB1-transfected cells (n=6) and 5.62.6 and 52.811.7?pmol?ml?1 respectively Rabbit polyclonal to PDE3A in CB2-transfected cells (n=6). CP55940 was highly potent in inhibiting forskolin-stimulated cyclic AMP production in both CB1- and CB2-transfected cells (Table 2 and Figure 2). The CB1/CB2 EC50 ratio of 0.9 reflects the non-selective nature of this agonist. In contrast, L759633 and L759656 showed greater potency against forskolin-stimulated cyclic AMP production in CB2-transfected cells than in CB1-transfected cells (Table 2 and Figure 2). Indeed,.Thus it would seem that, depending on the CB1 receptor-containing preparation used, AM630 can behave as a CB1 receptor agonist, antagonist or inverse agonist. buffer) using a 12-tube Brandel cell harvester. The tubes were washed three times with 4?ml of wash buffer. Filters were oven dried, placed in 5?ml of scintillation fluid and bound radioactivity was determined by liquid scintillation counting. Basal binding of [35S]-GTPS was determined in the presence of 20?M GDP and absence of cannabinoid. Non-specific binding was determined in the presence of 10?M GTPS. Analysis of data Values have been expressed as means and variability as s.e.mean or as 95% confidence limits. Mean values have been compared using the Kruskall-Wallis test followed by Dunn’s multiple comparison test. A value <0.05 was considered to be significant. Effects of test compounds on forskolin-stimulated cyclic AMP production have been expressed in percentage terms. This was calculated from the equation [100(f?b)]/(f?b) where f?, f and b are values of cyclic AMP production (pmol?ml?1), f? in the presence of forskolin and the test compound, f in the presence of forskolin only and b in the absence of both forskolin and the test compound. Drug-induced inhibition of specific [35S]-GTPS binding was expressed as the percentage decrease below the basal level of [35S]-GTPS binding using the equation [100(d?d)]/d where d and d are d.p.m. in the presence and absence of the drug respectively. Values for EC50, IC50 and maximal effects (Emax) and the 95% confidence limits of these values have been calculated by non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, U.S.A.). The ability of AM630 to antagonize CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB2 transfected cells is expressed in terms of the concentration ratio. This has been defined as the concentration of CP55940 that produces a particular degree of inhibition in the presence of AM630 at a concentration, B, divided by the concentration of CP55940 that produces an identical degree of inhibition in the absence of AM630. Since AM630 behaved as an inverse agonist at CB2 receptors (see Results), it was considered inappropriate to insert concentration ratio values into the Schild equation in order to obtain a KB value of AM630 at these receptors. Concentration ratio values and their 95% confidence limits have been determined by symmetrical (2+2) dose parallel line assays (Colquhoun, 1971), using responses to pairs of agonist concentrations located on the steepest part of each log concentration-response curve. This method was also used to establish whether log concentration-response curves of CP55940 constructed in the presence and absence of AM630 deviated significantly from parallelism. Drugs CP55940 (?)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol was supplied by Pfizer, WIN55212-2 (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone by Research Biochemicals International, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and 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 by Sanofi Recherche and L759633 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6,9-trimethyl-6a,7,10,10a?-tetrahydro?-6H-benzo[c]chromene] and L759656 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6-dimethyl-9-methylene?-?6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene] by Merck Frosst. AM630 (6-iodopravadoline) was synthesized in the laboratory of Dr A. Makriyannis. [3H]-CP55940 (126?Ci?mmol?1) and [3H]-WIN55212-2 (45?Ci mmol?1) were supplied by NEN Life Science Products. Cannabinoids were stored as 1?mg?ml?1 stock solutions in ethanol and diluted in assay buffer. At the highest concentrations used, ethanol by itself had no detectable effect on specific binding of [3H]-CP55940, [3H]-WIN55212-2 or [35S]-GTPS or on forskolin-stimulated cyclic AMP production (data not shown). Results Cannabinoid binding experiments The radioligand binding data shown in Table 1 confirm that CP55940 is a high-affinity non-selective ligand for cannabinoid receptors. The data also confirm L759656 and L759633 to be markedly CB2-selective, with much lower Ki values in CB2 transfected cell membranes than in membranes of CB1 transfected cells. AM630 is also CB2-selective with a CB1/CB2 Ki ratio of 165 in transfected CHO cell membranes. Further experiments showed AM630 to be no less effective in displacing [3H]-CP55940 than [3H]-WIN55212-2 from CB2 receptors on CHO cell membranes (Table 1). SR144528 was also equally effective in displacing [3H]-CP55940 and [3H]-WIN55212-2 from CB2 receptors (Table 1). Effects of CP55940,.Possibly, AM630 is antagonizing a CB2 receptor agonist that is being spontaneously released by this WIN 55,212-2 mesylate cell line. Tris, 10?mM MgCl2, 100?mM NaCl and 0.2?mM EDTA at pH 7.4. Incubations were carried out at 30C for 90?min in a total volume of 500?l. The reaction was terminated by the addition of 4?ml of ice-cold wash buffer (50?mM Tris and 1?mg?ml?1 BSA, pH 7.4) followed by rapid filtration under vacuum through Whatman GF/B glass-fibre filters (pre-soaked in wash buffer) using a 12-tube Brandel cell harvester. The tubes were washed three times with 4?ml of wash buffer. Filters were oven dried, placed in 5?ml of scintillation fluid and bound radioactivity was determined by liquid scintillation counting. Basal binding of [35S]-GTPS was determined in the presence of 20?M GDP and absence of cannabinoid. Non-specific binding was determined in the presence of 10?M GTPS. Analysis of data Values have been expressed as means and variability as s.e.mean or as 95% confidence limits. Mean values have been compared using the Kruskall-Wallis test followed by Dunn’s multiple comparison test. A value <0.05 was considered to be significant. Effects of test compounds on forskolin-stimulated cyclic AMP production have been expressed in percentage terms. This was calculated from your equation [100(f?b)]/(f?b) where f?, f and b are values of cyclic AMP production (pmol?ml?1), f? in the presence of forskolin and the test compound, f in the presence of forskolin only and b in the absence of both forskolin and the test compound. Drug-induced inhibition of specific [35S]-GTPS binding was expressed as the percentage decrease below the basal level of [35S]-GTPS binding using the equation [100(d?d)]/d where d and d are d.p.m. in the presence and absence of the drug respectively. Values for EC50, IC50 and maximal effects (Emax) and the 95% confidence limits of these values have been calculated by non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, U.S.A.). The ability of AM630 to antagonize CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB2 transfected cells is expressed in terms of the concentration ratio. This has been defined as the concentration of CP55940 that produces a particular degree of inhibition in the presence of AM630 at a concentration, B, divided from the concentration of CP55940 that produces an identical degree of inhibition in the absence of AM630. Since AM630 behaved as an inverse agonist at CB2 receptors (see Results), it was considered inappropriate to insert concentration ratio values into the Schild equation in order to obtain a KB value of AM630 at these receptors. Concentration ratio values and their 95% confidence limits have been determined by symmetrical (2+2) dose parallel line assays (Colquhoun, 1971), using responses to pairs of agonist concentrations located on the steepest part of each log concentration-response curve. This method was also used to establish whether log concentration-response curves of CP55940 constructed in the presence and absence of AM630 deviated significantly from parallelism. Drugs CP55940 (?)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol was supplied by Pfizer, WIN55212-2 (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone by Research Biochemicals International, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and 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 by Sanofi Recherche and L759633 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6,9-trimethyl-6a,7,10,10a?-tetrahydro?-6H-benzo[c]chromene] and L759656 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6-dimethyl-9-methylene?-?6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene] by Merck Frosst. AM630 (6-iodopravadoline) was synthesized in the laboratory of Dr A. Makriyannis. [3H]-CP55940 (126?Ci?mmol?1) and [3H]-WIN55212-2 (45?Ci mmol?1) were supplied by NEN Life Science Products. Cannabinoids were stored as 1?mg?ml?1 stock solutions in ethanol and diluted in assay buffer. At the highest concentrations used, ethanol by itself had no detectable effect on specific binding of [3H]-CP55940, [3H]-WIN55212-2 or [35S]-GTPS or on forskolin-stimulated cyclic AMP production (data not shown). Results Cannabinoid binding experiments The radioligand binding data shown in Table 1 confirm WIN 55,212-2 mesylate that CP55940 is a high-affinity non-selective ligand for cannabinoid receptors. The data also confirm L759656 and L759633 to be markedly CB2-selective, with much lower Ki values in CB2 transfected cell membranes than in membranes of CB1 transfected cells. AM630 is also CB2-selective with a CB1/CB2 Ki ratio of 165 in transfected CHO cell membranes. Further experiments showed AM630 to be no less effective in.and R.A.R.) and by grants DA3801 (to A.M.) and DA9158 (to A.M. glass-fibre filters (pre-soaked in wash buffer) using a 12-tube Brandel cell harvester. The tubes were washed three times with 4?ml of wash buffer. Filters were oven dried, placed in 5?ml of scintillation fluid and bound radioactivity was determined by liquid scintillation counting. Basal binding of [35S]-GTPS was determined in the presence of 20?M GDP and absence of cannabinoid. Non-specific binding was determined in the presence of 10?M GTPS. Analysis of data Values have been expressed as means and variability as s.e.mean or as 95% confidence limits. Mean values have been compared using the Kruskall-Wallis test followed by Dunn’s multiple comparison test. A value <0.05 was considered to be significant. Effects of test compounds on forskolin-stimulated cyclic AMP production have been expressed in percentage terms. This was calculated from the equation [100(f?b)]/(f?b) where f?, f and b are values of cyclic AMP production (pmol?ml?1), f? in the presence of forskolin and the test compound, f in the presence of forskolin only and b in the absence of both forskolin and the test compound. Drug-induced inhibition of specific [35S]-GTPS binding was expressed as the percentage decrease below the basal level of [35S]-GTPS binding using the equation [100(d?d)]/d where d and d are d.p.m. in the presence and absence of the drug respectively. Values for EC50, IC50 and maximal effects (Emax) and the 95% confidence limits of these values have been calculated by non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, U.S.A.). The ability of AM630 to antagonize CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB2 transfected cells is expressed in terms of the concentration ratio. This has been defined as the concentration of CP55940 that produces a particular degree of inhibition in the presence of AM630 at a concentration, B, divided by the concentration of CP55940 that produces an identical degree of inhibition in the absence of AM630. Since AM630 behaved as an inverse agonist at CB2 receptors (see Results), it was considered inappropriate to insert concentration ratio values into the Schild equation in order to obtain a KB value of AM630 at these receptors. Concentration ratio values and their 95% confidence limits have been determined by symmetrical (2+2) dose parallel line assays (Colquhoun, 1971), using responses to pairs of agonist concentrations located on the steepest part of each log concentration-response curve. This method was also used to establish whether log concentration-response curves of CP55940 constructed in the presence and absence of AM630 deviated significantly from parallelism. Drugs CP55940 (?)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol was supplied by Pfizer, WIN55212-2 (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone by Research Biochemicals International, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and 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 by Sanofi Recherche and L759633 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6,9-trimethyl-6a,7,10,10a?-tetrahydro?-6H-benzo[c]chromene] and L759656 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6-dimethyl-9-methylene?-?6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene] by Merck Frosst. AM630 (6-iodopravadoline) was synthesized in the laboratory of Dr A. Makriyannis. [3H]-CP55940 (126?Ci?mmol?1) and [3H]-WIN55212-2 (45?Ci mmol?1) were supplied by NEN Life Science Products. Cannabinoids were stored as 1?mg?ml?1 stock solutions in ethanol and diluted in assay buffer. At the highest concentrations used, ethanol by itself had no detectable effect on specific binding of [3H]-CP55940, [3H]-WIN55212-2 or [35S]-GTPS or on forskolin-stimulated cyclic AMP production (data not shown). Results Cannabinoid binding experiments The radioligand binding data shown in Table 1 confirm that CP55940 is a high-affinity non-selective ligand for cannabinoid receptors. The data also confirm L759656 and L759633 to be markedly CB2-selective, with much lower Ki values in CB2 transfected cell membranes than in membranes of CB1 transfected cells. AM630 is also CB2-selective with a CB1/CB2 Ki ratio of 165 in transfected CHO cell membranes. Further experiments showed AM630 to be no less effective in displacing [3H]-CP55940 than [3H]-WIN55212-2 from CB2 receptors on CHO cell membranes (Table 1). SR144528 was also equally effective in displacing [3H]-CP55940 and [3H]-WIN55212-2 from CB2 receptors (Table 1). Effects of CP55940, L759656, L759633 and AM630 on cyclic AMP production Cyclic AMP concentrations in the absence and presence of 2?M forskolin were 5.11.8 and 46.313.1?pmol?ml?1 respectively in CB1-transfected cells (n=6) and 5.62.6 and 52.811.7?pmol?ml?1 respectively in CB2-transfected cells (n=6). CP55940 was highly potent in inhibiting forskolin-stimulated cyclic AMP production in both CB1- and CB2-transfected cells (Table 2 and Figure 2). The CB1/CB2 EC50 ratio of 0.9 reflects the non-selective nature of this agonist. In contrast, L759633 and L759656 showed greater potency against forskolin-stimulated cyclic AMP production in CB2-transfected.Our results also confirm previous reports that CP55940 is a cannabinoid receptor agonist that acts with more or less equal potency at CB1 and CB2 receptors (Felder et al., 1995). dried, placed in 5?ml of scintillation fluid and bound radioactivity was determined by liquid scintillation counting. Basal binding of [35S]-GTPS WIN 55,212-2 mesylate was determined in the presence of 20?M GDP and absence of cannabinoid. Non-specific binding was determined in the presence of 10?M GTPS. Analysis of data Values have been expressed as means and variability as s.e.mean or as 95% confidence limits. Mean values have been compared using the Kruskall-Wallis test followed by Dunn’s multiple comparison test. A value <0.05 was considered to be significant. Effects of test compounds on forskolin-stimulated cyclic AMP production have been expressed in percentage terms. This was calculated from the equation [100(f?b)]/(f?b) where f?, f and b are values of cyclic AMP production (pmol?ml?1), f? in the presence of forskolin and the test compound, f in the presence of forskolin only and b in the absence of both forskolin and the test compound. Drug-induced inhibition of specific [35S]-GTPS binding was expressed as the percentage decrease below the basal level of [35S]-GTPS binding using the equation [100(d?d)]/d where d and d are d.p.m. in the presence and absence of the drug respectively. Values for EC50, IC50 and maximal effects (Emax) and the 95% confidence limits of these values have been calculated by non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, U.S.A.). The ability of AM630 to antagonize CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB2 transfected cells is expressed in terms of the concentration ratio. This has been defined as the concentration of CP55940 that produces a particular degree of inhibition in the presence of AM630 at a concentration, B, divided by the concentration of CP55940 that produces an identical degree of inhibition in the absence of AM630. Since AM630 behaved as an inverse agonist at CB2 receptors (see Results), it was considered inappropriate to insert concentration ratio values into the Schild equation in order to obtain a KB value of AM630 at these receptors. Concentration ratio values and their 95% confidence limits have been determined by symmetrical (2+2) dose parallel line assays (Colquhoun, 1971), using responses to pairs of agonist concentrations located on the steepest part of each log concentration-response curve. This method was also used to establish whether log concentration-response curves of CP55940 constructed in the presence and absence of AM630 deviated significantly from parallelism. Drugs CP55940 (?)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol was supplied by Pfizer, WIN55212-2 (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone by Research Biochemicals International, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and 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 by Sanofi Recherche and L759633 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6,9-trimethyl-6a,7,10,10a?-tetrahydro?-6H-benzo[c]chromene] and L759656 [(6aR,10aR)-3-(1,1-dimethyl-heptyl)-1-methoxy-6,6-dimethyl-9-methylene?-?6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene] by Merck Frosst. AM630 (6-iodopravadoline) was synthesized in the laboratory of Dr A. Makriyannis. [3H]-CP55940 (126?Ci?mmol?1) and [3H]-WIN55212-2 (45?Ci mmol?1) were supplied by NEN Life Science Products. Cannabinoids were stored as 1?mg?ml?1 stock solutions in ethanol and diluted in assay buffer. At the highest concentrations used, ethanol by itself had no detectable effect on specific binding of [3H]-CP55940, [3H]-WIN55212-2 or [35S]-GTPS or on forskolin-stimulated cyclic AMP production (data not shown). Results Cannabinoid binding experiments The radioligand binding data shown in Table 1 confirm that CP55940 is a high-affinity non-selective ligand for cannabinoid receptors. The data also confirm L759656 and L759633 to be markedly CB2-selective, with much lower Ki values in CB2 transfected cell membranes than in membranes of CB1 transfected cells. AM630 is also CB2-selective with a CB1/CB2 Ki ratio of 165 in transfected CHO cell membranes. Further experiments showed AM630 to be no less effective in displacing [3H]-CP55940 than [3H]-WIN55212-2 from CB2 receptors on CHO cell membranes (Table 1). SR144528 was also equally effective in displacing [3H]-CP55940 and [3H]-WIN55212-2 from CB2 receptors (Table 1). Effects of CP55940, L759656, L759633 and AM630 on cyclic AMP production Cyclic AMP concentrations in the absence and presence of 2?M forskolin were 5.11.8 and 46.313.1?pmol?ml?1 respectively in CB1-transfected cells (n=6) and 5.62.6 and 52.811.7?pmol?ml?1 respectively in CB2-transfected cells (n=6). CP55940 was highly.