PT - JOURNAL ARTICLE AU - Resat Cinar AU - Mária Szücs TI - CB<sub>1</sub> Receptor-Independent Actions of SR141716 on G-Protein Signaling: Coapplication with the μ-Opioid Agonist Tyr-<span class="sc">d</span>-Ala-Gly-(NMe)Phe-Gly-ol Unmasks Novel, Pertussis Toxin-Insensitive Opioid Signaling in μ-Opioid Receptor-Chinese Hamster Ovary Cells AID - 10.1124/jpet.109.152710 DP - 2009 Aug 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 567--574 VI - 330 IP - 2 4099 - http://jpet.aspetjournals.org/content/330/2/567.short 4100 - http://jpet.aspetjournals.org/content/330/2/567.full SO - J Pharmacol Exp Ther2009 Aug 01; 330 AB - 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. The American Society for Pharmacology and Experimental Therapeutics