CB1 receptor-independent actions of SR141716 on G-protein signaling: coapplication with the mu-opioid agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol unmasks novel, pertussis toxin-insensitive opioid signaling in mu-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 [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) assay, we have found that 10 microM SR141716 slightly but significantly decreases the basal [(35)S]GTPgammaS binding in membranes of the wild-type and CB(1) receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with micro-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB(1) receptor-independent. Although the specific MOR agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) saturably and concentration-dependently stimulated [(35)S]GTPgammaS binding, SR141716 (10 microM) 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 microM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 microM 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 (IC(50) = 5.7 microM). 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.
Cinar
Resat
R
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Szücs
Mária
M
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000008:administration & dosage*; D019204:GTP-Binding Proteins / Q000502:physiology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D037342:Pertussis Toxin / Q000494:pharmacology*; D010880:Piperidines / Q000008:administration & dosage*; D011720:Pyrazoles / Q000008:administration & dosage*; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000172:deficiency / Q000502:physiology*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000502:physiology*; D000077285:Rimonabant; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000077285:Rimonabant