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 [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 micrograms of membrane protein/well, the KD for [3H]CP 55,940 is 461 pM and the Bmax is 860 fmol/mg of protein. The apparent KD of [3H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 pM at 100 micrograms 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 Ki of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 microM in buffer alone and 143 nM in the presence of 0.15 mM 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 mM 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.
Hillard
C J
CJ
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226.
Edgemond
W S
WS
Campbell
W B
WB
D000818:Animals; D001095:Arachidonic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002186:Cannabinoids / Q000378:metabolism; D003511:Cyclohexanols / Q000378:metabolism; D063388:Endocannabinoids; D005374:Filtration / Q000379:methods; D008024:Ligands; D008297:Male; D008566:Membranes / Q000378:metabolism; D009448:Neurochemistry / Q000379:methods*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D051381:Rats; D017207:Rats, Sprague-Dawley; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000378:metabolism*; D013997:Time Factors
D001095:Arachidonic Acids; D002186:Cannabinoids; D003511:Cyclohexanols; D063388:Endocannabinoids; D008024:Ligands; D053284:Polyunsaturated Alkamides; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D010664:Phenylmethylsulfonyl Fluoride; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; C078814:anandamide