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Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5'-O-(3-[35S]thio)-triphosphate binding assay in rat cerebellar membranes.
J Pharmacol Exp Ther. 1998 May; 285(2):553-60.JP

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]GTP gamma S) 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 GTP gamma S 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 GTP gamma S 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]GTP gamma S 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, (-)-delta-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 (-)-delta-tetrahydrocannabinol, cannabinol and anandamide, which produced no concentration-dependent stimulation of [35S]GTP gamma S 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]GTP gamma S binding assay than has previously been reported.

Authors+Show Affiliations

Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9580597

Citation

Griffin, G, et al. "Evaluation of Cannabinoid Receptor Agonists and Antagonists Using the guanosine-5'-O-(3-[35S]thio)-triphosphate Binding Assay in Rat Cerebellar Membranes." The Journal of Pharmacology and Experimental Therapeutics, vol. 285, no. 2, 1998, pp. 553-60.
Griffin G, Atkinson PJ, Showalter VM, et al. Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5'-O-(3-[35S]thio)-triphosphate binding assay in rat cerebellar membranes. J Pharmacol Exp Ther. 1998;285(2):553-60.
Griffin, G., Atkinson, P. J., Showalter, V. M., Martin, B. R., & Abood, M. E. (1998). Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5'-O-(3-[35S]thio)-triphosphate binding assay in rat cerebellar membranes. The Journal of Pharmacology and Experimental Therapeutics, 285(2), 553-60.
Griffin G, et al. Evaluation of Cannabinoid Receptor Agonists and Antagonists Using the guanosine-5'-O-(3-[35S]thio)-triphosphate Binding Assay in Rat Cerebellar Membranes. J Pharmacol Exp Ther. 1998;285(2):553-60. PubMed PMID: 9580597.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5'-O-(3-[35S]thio)-triphosphate binding assay in rat cerebellar membranes. AU - Griffin,G, AU - Atkinson,P J, AU - Showalter,V M, AU - Martin,B R, AU - Abood,M E, PY - 1998/5/15/pubmed PY - 1998/5/15/medline PY - 1998/5/15/entrez SP - 553 EP - 60 JF - The Journal of pharmacology and experimental therapeutics JO - J Pharmacol Exp Ther VL - 285 IS - 2 N2 - 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]GTP gamma S) 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 GTP gamma S 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 GTP gamma S 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]GTP gamma S 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, (-)-delta-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 (-)-delta-tetrahydrocannabinol, cannabinol and anandamide, which produced no concentration-dependent stimulation of [35S]GTP gamma S 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]GTP gamma S binding assay than has previously been reported. SN - 0022-3565 UR - https://www.unboundmedicine.com/medline/citation/9580597/Evaluation_of_cannabinoid_receptor_agonists_and_antagonists_using_the_guanosine_5'_O__3_[35S]thio__triphosphate_binding_assay_in_rat_cerebellar_membranes_ L2 - https://jpet.aspetjournals.org/cgi/pmidlookup?view=long&pmid=9580597 DB - PRIME DP - Unbound Medicine ER -