Abstract
Cannabinoid receptors are members of the superfamily of G protein-coupled receptors. Their activation has previously been shown to stimulate guanosine 5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding in a range of brain regions using both membrane preparations and autoradiography. This study evaluates the activities of structurally diverse cannabinoid receptor ligands in the GTPγS binding assay, comparing the relationship between receptor binding and activation and also examining efficacy differences between compounds. Using rat cerebellar membrane preparations, the effects of GDP concentration on GTPγS binding and the activities of a range of cannabinoid receptor ligands, including the CB1 selective antagonist SR141716A, were investigated. GDP concentration was found to have differing effects on cannabinoid-stimulated [35S]GTPγS binding depending on the nature of the agonist used. The stimulation produced by high efficacy compounds, such as CP 55,940 and WIN 55212–2, was increased by raising the GDP concentration, but that of a low efficacy agonist, (−)-Δ-tetrahydrocannabinol, was decreased. Of the cannabinoid compounds tested, a wide range of potencies (EC50) and levels of maximal stimulation (Emax) were observed. These ranged from CP 55,244 (Emax of 165, 148–183%, and an EC50 of 0.47, 0.22–0.96, nM) through (−)-Δ-tetrahydrocannabinol, cannabinol and anandamide, which produced no concentration-dependent stimulation of [35S]GTPγS binding under the same conditions. SR141716A competitively antagonized all the agonists against which it was tested, providing equilibrium dissociation constants (Kd values) in the sub-nanomolar range (0.06–0.40 nM), implicating a CB1 receptor mediated response. These results provide a more detailed characterization of the cannabinoid-stimulated [35S]GTPγS binding assay than has previously been reported.
Footnotes
-
Send reprint requests to: Dr. Mary Abood, Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980524, Richmond, VA 23298.
-
↵1 This work was supported in part by National Institute on Drug Abuse Grants DA-09978, DA-05274 and DA-09789 and the Council for Tobacco Research Grant CTR-4482.
- Abbreviations:
- [35S]GTPγS
- guanosine-5′-O-(3-[35S]thio)-triphosphate
- PMSF
- phenylmethylsulfonyl fluoride
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- THC
- (−)-Δ9-tetrahydrocannabinol
- CP 55
- 940, −)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol
- SR141716A
- N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride
- WIN55212–2
- (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone
- JWH-073
- 1-(1-butyl)-3-(1-naphthoyl)-indole
- JWH-030
- 3-naphthoyl-N-pentylpyrrole
- O-1064
- 2,16,16-trimethyl-all-cis-5,8,11,14-docosatetraenoyl-2′-fluoroethanolamide
- HU-210
- (−)-11-OH-Δ8-THC-dimethylheptyl
- ANOVA
- analysis of variance
- CB1
- central cannabinoid receptor
- CB2
- peripheral cannabinoid receptor
- Received September 8, 1997.
- Accepted January 20, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|