Characterization of Ligand Binding to the Cannabinoid Receptor of Rat Brain Membranes Using a Novel Method: Application to Anandamide

Abstract: Ligand binding to the cannabinoid receptor of brain membranes has been characterized using ³H]CP 55,940 and the Multiscreen Filtration System. Binding of ³H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 µg of membrane protein/well, the K _D for ³H]CP 55,940 is 461 p M and the B _max is 860 fmol/mg of protein. The apparent K _D of ³H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 p M at 100 µg of membrane protein. Binding of ³H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The K _i of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 µ M in buffer alone and 143 n M in the presence of 0.15 m M phenylmethylsulfonyl fluoride. When ¹⁴C]anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 m M phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high-affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions.