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, 126 (3), 665-72

Agonist-inverse Agonist Characterization at CB1 and CB2 Cannabinoid Receptors of L759633, L759656, and AM630

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Agonist-inverse Agonist Characterization at CB1 and CB2 Cannabinoid Receptors of L759633, L759656, and AM630

R A Ross et al. Br J Pharmacol.

Abstract

We have tested our prediction that AM630 is a CB2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB2 receptor agonists. Binding assays with membranes from CHO cells stably transfected with human CB1 or CB2 receptors using [3H]-CP55940, confirmed the CB2-selectivity of L759633 and L759656 (CB2/CB1 affinity ratios = 163 and 414 respectively) and showed AM630 to have a Ki at CB2 receptors of 31.2 nM and a CB2/CB1 affinity ratio of 165. In CB2-transfected cells, L759633 and L759656 were potent inhibitors of forskolin-stimulated cyclic AMP production, with EC50 values of 8.1 and 3.1 nM respectively and CB1/CB2 EC50 ratios of > 1000 and > 3000 respectively. AM630 inhibited [35S]-GTPgammaS binding to CB2 receptor membranes (EC50 = 76.6 nM), enhanced forskolin-stimulated cyclic AMP production in CB2-transfected cells (5.2 fold by 1 microM), and antagonized the inhibition of forskolin-stimulated cyclic AMP production in this cell line induced by CP55940. In CB1-transfected cells, forskolin-stimulated cyclic AMP production was significantly inhibited by AM630 (22.6% at 1 microM and 45.9% at 10 microM) and by L759633 at 10 microM (48%) but not 1 microM. L759656 (10 microM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory effect of CP55940 on cyclic AMP production which was not statistically significant. We conclude that AM630 is a CB2-selective ligand that behaves as an inverse agonist at CB2 receptors and as a weak partial agonist at CB1 receptors. L759633 and L759656 are both potent CB2-selective agonists.

Figures

Figure 1
Figure 1
Structures of AM630, L759656 and L759633.
Figure 2
Figure 2
Inhibition by (a) CP55940 (n=9), (b) L759633 (n=3) and (c) L759656 (n=4) of forskolin-stimulated cyclic AMP production in CB2-transfected CHO cells. Each symbol represents mean percentage change in forskolin-stimulated cyclic AMP production±s.e.mean. Forskolin-stimulated cyclic AMP production in the absence of any cannabinoid has been normalized to 100%. Mean Emax values for CP55940, L759633 and L759656, with 95% confidence limits shown in brackets, were 21.9% (5.1 to 38.7), 0.5% (−25.3 to 26.3%) and 5.9% (−10.6 to 22.4%) respectively (GraphPad Prism).
Figure 3
Figure 3
The effect of AM630 on forskolin-stimulated cyclic AMP production in CB1 and CB2-transfected CHO cells (±s.e.mean; n=3 to 8). Each column represents mean percentage change in forskolin-stimulated cyclic AMP production±s.e.mean. Forskolin-stimulated cyclic AMP production in the absence of AM630 has been normalized to 100%. Asterisks indicate values significantly less than 100% (*P<0.05; **P<0.01; ***P<0.001; one-sample t-test). The mean EC50 value of AM630 for enhancement of forskolin-stimulated cyclic AMP production in the CB2-transfected cells with its 95% confidence limits shown in brackets is 230.4 nM (48.4 and 1096 nM) (GraphPad Prism).
Figure 4
Figure 4
The effect of 1 μM AM630 on CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB1 and CB2-transfected CHO cells. Each symbol represents mean percentage change in forskolin-stimulated cyclic AMP production±s.e.mean (n=3). Forskolin-stimulated cyclic AMP production in the absence of AM630 and CP55940 has been normalized to 100%. Mean EC50 values of CP55940 in the CB1-transfected cells with their 95% confidence limits shown in brackets are 0.5 nM (0.2 and 1.2 nM) in the presence of AM630 and 0.2 nM (0.1 and 0.4 nM) in its absence (GraphPad Prism).
Figure 5
Figure 5
The effect of 100 nM AM630 or 50 nM SR144528 on CP55940-induced inhibition of forskolin-stimulated cyclic AMP production in CB2-transfected CHO cells. Each symbol represents mean percentage change in forskolin-stimulated cyclic AMP production±s.e.mean (n=3 or 4). Forskolin-stimulated cyclic AMP production in the absence of AM630, SR144528 and CP55940 has been normalized to 100%. The mean dextral shift in the log concentration-response curve of CP55940 produced by AM630 is 21.3 and its 95% confidence limits are 3.0 and 649.4 (symmetrical (2+2) dose parallel line assay). It did not deviate significantly from parallelism.
Figure 6
Figure 6
Effects of AM630 and SR144528 on specific binding of [35S]-GTPγS to CB2-transfected CHO cell membranes. Each symbol represents mean percentage decrease in binding±s.e.mean (n=3 to 5). The mean EC50 values of AM630 and SR144528 for inhibition of [35S]-GTPγS binding with their 95% confidence limits shown in brackets are 76.6 nM (16.5 and 356.2 nM) and 10.4 nM (3.6 and 29.7 nM) respectively. Corresponding values for the maximum degree of inhibition produced are 47.2±5.8% and 48.5±2.9% respectively (mean Emax values±s.e.mean; non-linear regression analysis; GraphPad Prism).
Figure 7
Figure 7
Effects of AM630 on inhibition of forskolin-stimulated cyclic AMP production by 10 nM CP55940 in CB1- and CB2-transfected CHO cells (±s.e.mean; n=4 to 6). Each column represents mean percentage change in forskolin-stimulated cyclic AMP production. Forskolin-stimulated cyclic AMP production in the absence of AM630 and CP55940 has been normalized to 100%. Asterisks indicate significant differences from forskolin-stimulated cyclic AMP production in the presence of CP55940 and absence of AM630 (*P<0.05; **P<0.01; Kruskall-Wallis test followed by Dunn's multiple comparison test).

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