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OtherANALGESIA AND DRUGS OF ABUSE

Evaluation of Cannabinoid Receptor Agonists and Antagonists Using the Guanosine-5′-O-(3-[35S]thio)-triphosphate Binding Assay in Rat Cerebellar Membranes

Graeme Griffin, Peter J. Atkinson, Vincent M. Showalter, Billy R. Martin and Mary E. Abood
Journal of Pharmacology and Experimental Therapeutics May 1998, 285 (2) 553-560;
Graeme Griffin
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Peter J. Atkinson
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Vincent M. Showalter
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Billy R. Martin
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Mary E. Abood
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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
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Journal of Pharmacology and Experimental Therapeutics
Vol. 285, Issue 2
1 May 1998
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OtherANALGESIA AND DRUGS OF ABUSE

Evaluation of Cannabinoid Receptor Agonists and Antagonists Using the Guanosine-5′-O-(3-[35S]thio)-triphosphate Binding Assay in Rat Cerebellar Membranes

Graeme Griffin, Peter J. Atkinson, Vincent M. Showalter, Billy R. Martin and Mary E. Abood
Journal of Pharmacology and Experimental Therapeutics May 1, 1998, 285 (2) 553-560;

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OtherANALGESIA AND DRUGS OF ABUSE

Evaluation of Cannabinoid Receptor Agonists and Antagonists Using the Guanosine-5′-O-(3-[35S]thio)-triphosphate Binding Assay in Rat Cerebellar Membranes

Graeme Griffin, Peter J. Atkinson, Vincent M. Showalter, Billy R. Martin and Mary E. Abood
Journal of Pharmacology and Experimental Therapeutics May 1, 1998, 285 (2) 553-560;
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