IDENTIFICATION AND CHARACTERIZATION OF A NOVEL SYNTHETIC CANNABINOID CP-55,940 BINDER IN RAT-BRAIN CYTOSOL

We have detected the presence of a specific [H-3] CP 55,940 binder in the cytosol of rat cerebral cortex. Competition studies showed that on ly cold CP 55,940 and to a lesser extent Delta(9)THC was able to compe te with [H-3] CP 55,940; little competition was observed with either D elta(8)THC or anandamide. Scatchard analysis of the data indicate the presence of two distinct binding components having affinity constants (Kd) of 0.97 +/- 0.03 nM, 5.83 +/- 0.08 nM, and B-max of 3.31 +/- 0.06 pmol/mg protein, 22.2 +/- 1.2 pmol/mg protein respectively. The cytos olic CP 55,940 binder was heat stable up to 30 degrees C. Besides the brain cytosol, lesser amounts of binding were also detected in the spl een, and testis. Liver, kidney and muscle cytosol preparations were fo und to be devoid of this binder. Unlike the previously characterized b rain membrane cannabinoid receptor, this binder was found to be salt, sulfhydryl blocking reagents and nucleotide resistant. Interestingly, dithiothreitol (DTT), a protein-disulfide group reducing agent, inhibi ted the binding of [H-3] CP-55,940 to the receptor and approximately 8 0% binding inhibition was obtained at a 5 mM concentration. Western bl ot analysis using anti-receptor antibody reveal the presence of a 95-1 10, 50 and 38 kDa band in the brain, spleen and testis cytosolic prepa rations. In conclusion, we have identified the presence of a novel CP 55,940 binder in rat cerebral cortex cytosol possessing biochemical pr operties distinct from those previously observed using rat cerebral co rtex membrane cannabinoid receptor.