Characterization of ligand binding to the cannabinoid receptor of rat brain membranes using a novel method: application to anandamide. J Neurochem 1995 Feb;64(2):677-83
Date
02/01/1995Pubmed ID
7830060DOI
10.1046/j.1471-4159.1995.64020677.xScopus ID
2-s2.0-0028813933 (requires institutional sign-in at Scopus site) 75 CitationsAbstract
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.
Author List
Hillard CJ, Edgemond WS, Campbell WBAuthors
William B. Campbell PhD Professor in the Pharmacology and Toxicology department at Medical College of WisconsinCecilia J. Hillard PhD Associate Dean, Center Director, Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AnimalsArachidonic Acids
Brain
Cannabinoids
Cyclohexanols
Endocannabinoids
Filtration
Ligands
Male
Membranes
Neurochemistry
Phenylmethylsulfonyl Fluoride
Polyunsaturated Alkamides
Rats
Rats, Sprague-Dawley
Receptors, Cannabinoid
Receptors, Drug
Time Factors
