CB1 Receptor-Independent Actions of SR141716 on G-Protein Signaling: Coapplication with the μ-Opioid Agonist Tyr-D-AlaGly-(NMe)Phe-Gly-ol Unmasks Novel, Pertussis Toxin-Insensitive Opioid Signaling in μ-Opioid Receptor-Chinese Hamster Ovary Cells

The CB 1 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 CB 1 receptor-mediated. Using the ligand-stimulated [S-35]guanosine 5'-3-O(thio)triphosphate(GTP gamma S) assay, we have found that 10 mu M SR141716 slightly but significantly decreases the basal [S-35] GTP gamma S 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 mu-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)PheGly-ol (DAMGO) saturably and concentration-dependently stimulated [S-35]GTP gamma S binding, SR141716 (10 mu M) 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 mu M SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 mu M 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 G(i)/G(o) proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 mu M). 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.