CHARACTERIZATION OF LIGAND-BINDING TO THE CANNABINOID RECEPTOR OF RAT-BRAIN MEMBRANES USING A NOVEL METHOD - APPLICATION TO ANANDAMIDE

Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [H-3]CP 55,940 and the Multiscreen Filtration Sys tem. Binding of [H-3]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 mu g of membrane protein/well, the K-D for [H-3]CP 55,940 is 461 pM and the B-max is 8 60 fmol/mg of protein. The apparent K-D of [H-3]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 pM at 100 mu g of membrane protein. Binding of [H-3] CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest rati o 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 cannabinoi d receptor, is 1.3 mu M in buffer alone and 143 nM in the presence of 0.15 mM phenylmethylsulfonyl fluoride. When [C-14]anandamide is incuba ted with rat forebrain membranes at room temperature, it is degraded t o arachidonic acid; the hydrolysis is inhibited by 0.15 mM phenylmethy lsulfonyl fluoride. These results support the hypothesis that anandami de is a high-affinity ligand of the cannabinoid receptor and that it i s rapidly degraded by membrane fractions.