Abstract

The purpose of these studies was to support the hypothesis that an undiscovered cannabinoid receptor exists in brain. [³⁵S]GTPγS 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 CB₁receptors, including CP55940, HU-210, and Δ⁹-tetrahydrocannabinol, failed to stimulate [³⁵S]GTPγS binding in CB₁ ^−/−membranes. In CB₁ ^−/− membranes, SR141716A affected both basal and anandamide- or WIN55212-2-induced stimulation of [³⁵S]GTPγS binding only at concentrations greater than 1 μM. In CB₁ ^+/+ membranes, SR141716A inhibited only 84% of anandamide and 67% of WIN55212-2 stimulated [³⁵S]GTPγS binding with an affinity appropriate for mediation by CB₁ receptors (K _B ≈ 0.5 nM). The remaining stimulation seemed to be inhibited with lower potency (IC₅₀ ≈ 5 μM) 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 μ 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 [³H]WIN55212-2, but not [³H]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.