Abstract
The CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) has been shown by many investigators to inhibit basal G-protein activity, i.e., to display inverse agonism at high concentrations. However, it is not clear whether this effect is cannabinoid CB1 receptor-mediated. Using the ligand-stimulated [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) assay, we have found that 10 μM SR141716 slightly but significantly decreases the basal [35S]GTPγS binding in membranes of the wild-type and CB1 receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with μ-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB1 receptor-independent. Although the specific MOR agonist Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) saturably and concentration-dependently stimulated [35S]GTPγS binding, SR141716 (10 μM) inhibited the basal by 25% and competitively inhibited DAMGO stimulation in the mouse cortex. In MOR-CHO membranes, DAMGO caused a 501 ± 29% stimulation of the basal activity, which was inhibited to 456 ± 22% by 10 μM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 μM each) resulted in a 169 ± 22% stimulation of the basal activity (that was completely inhibited by the prototypic opioid antagonist naloxone) because of pertussis toxin (PTX) treatment to uncouple MORs from Gi/Go proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 μM). These results suggest the emergence of novel, PTX-insensitive G-protein signaling that is blocked by naloxone when MORs are activated by the combination of DAMGO and SR141716.
Footnotes
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This work was supported by the National Office for Research and Technology Szeged Neurobiological Knowledge Center [Grant 08/2004].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.152710.
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; [35S]GTPγS, guanosine-5′-O-(3-[35S]thio)triphosphate; BSA, bovine serum albumin; CB1, type 1 cannabinoid receptor; CB2, type 2 cannabinoid receptor; CHO, Chinese hamster ovary; DAMGO, Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol; GTP-γ-S-Li4, guanosine 5′-[γ-thio]triphosphate tetralithium salt; KO, knockout; MOR, μ-opioid receptor; PTX, pertussis toxin; Win55,212-2, R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolo[1,2,3-de]-1,4 benzoxazin-yl]-(1-naphthalenyl) methanone mesylate; SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; O-2050, (6aR, 10aR)-3-(1-methane-sulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran; wt, wild type; ANOVA, analysis of variance.
- Received February 25, 2009.
- Accepted May 14, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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