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

J Pharmacol Exp Ther. 2009 Aug;330(2):567-74. doi: 10.1124/jpet.109.152710. Epub 2009 May 15.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / administration & dosage*
  • GTP-Binding Proteins / physiology*
  • Male
  • Mice
  • Mice, Knockout
  • Pertussis Toxin / pharmacology*
  • Piperidines / administration & dosage*
  • Pyrazoles / administration & dosage*
  • Receptor, Cannabinoid, CB1 / agonists
  • Receptor, Cannabinoid, CB1 / deficiency
  • Receptor, Cannabinoid, CB1 / physiology*
  • Receptors, Opioid, mu / agonists
  • Receptors, Opioid, mu / physiology*
  • Rimonabant
  • Signal Transduction / drug effects
  • Signal Transduction / physiology


  • Piperidines
  • Pyrazoles
  • Receptor, Cannabinoid, CB1
  • Receptors, Opioid, mu
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Pertussis Toxin
  • GTP-Binding Proteins
  • Rimonabant