Evidence for a new G protein-coupled cannabinoid receptor in mouse brain

The purpose of these studies was to support the hypothesis that an undiscovered cannabinoid receptor exists in brain. [S-35]GTP gammaS binding was stimulated by anandamide and WIN55212-2 in brain membranes from both CB1+/+ and CB1-/- mice. In contrast, a wide variety of other compounds that are known to activate CB1 receptors, including CP55940, HU-210, and Delta (9)-tetrahydrocannabinol, failed to stimulate [S-35]GTP gammaS binding in CB1-/- membranes. In CB1-/- membranes, SR141716A affected both basal and anandamide- or WIN55212-2-induced stimulation of [S-35]GTP gammaS binding only at concentrations greater than 1 muM. In CB1+/+ membranes, SR141716A inhibited only 84% of anandamide and 67% of WIN55212-2 stimulated [S-35]GTP gammaS binding with an affinity appropriate for mediation by CB1 receptors (K-B approximate to 0.5 nM). The remaining stimulation seemed to be inhibited with lower potency (IC50 approximate to 5 muM) similar to that seen in CB1-/- membranes or in the absence of agonist. Further experiments determined that the effects of anandamide and WIN55212-2 were not additive, but that the effect of mu opioid, adenosine A1, and cannabinoid ligands were additive. Finally, assays of different central nervous system (CNS) regions demonstrated significant activity of cannabinoids in CB1-/- membranes from brain stem, cortex, hippocampus, diencephalon, midbrain, and spinal cord, but not basal ganglia or cerebellum. Moreover, some of these same CNS regions also showed significant binding of [H-3]WIN55212-2, but not [H-3]CP55940. Thus anandamide and WIN55212-2 seemed to be active in CB1-/- mouse brain membranes via a common G protein-coupled receptor with a distinct CNS distribution, implying the existence of an unknown cannabinoid receptor subtype in brain.