L-759633

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]‐GTPγS binding to CB2 receptor membranes (EC50=76.6 nM), enhanced forskolin‐stimulated cyclic AMP production in CB2‐transfected cells (5.2 fold by 1 μM), 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 μM and 45.9% at 10 μM) and by L759633 at 10 μM (48%) but not 1 μM. L759656 (10 μM) 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.British Journal of Pharmacology (1999) 126, 665–672; doi:10.1038/sj.bjp.0702351

Activation of CB2 has been demonstrated to induce directed immune cell migration. However, the ability of CB2 to act as a chemoattractant receptor in macrophages remains largely unexplored. Using a real-time chemotaxis assay and a panel of chemically diverse and widely used CB2 agonists, we set out to examine whether CB2 modulates primary murine macrophage chemotaxis. We report that of 12 agonists tested, only JWH133, HU308, L-759,656 and L-759,633 acted as macrophage chemoattractants. Surprisingly, neither pharmacological inhibition nor genetic ablation of CB2 had any effect on CB2 agonist-induced macrophage chemotaxis. As chemotaxis was pertussis toxin sensitive in both WT and CB2-/- macrophages, we concluded that a non-CB1/CB2, Gi/o-coupled GPCR must be responsible for CB2 agonist-induced macrophage migration. The obvious candidate receptors GPR18 and GPR55 could not mediate JWH133 or HU308-induced cytoskeletal rearrangement or JWH133-induced β-arrestin recruitment in cells transfected with either receptor, demonstrating that neither are the unidentified GPCR. Taken together our results conclusively demonstrate that CB2 is not a chemoattractant receptor for murine macrophages. Furthermore we show for the first time that JWH133, HU308, L-759,656 and L-759,633 have off-target effects of functional consequence in primary cells and we believe that our findings have wide ranging implications for the entire cannabinoid field.