Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors.
The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.
Canals
Meritxell
M
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Milligan
Graeme
G
D048588:Benzoxazines / Q000494:pharmacology; D002186:Cannabinoids / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000077285:Rimonabant
D048588:Benzoxazines; D002186:Cannabinoids; D008024:Ligands; D009025:Morpholines; D009281:Naphthalenes; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D048049:Extracellular Signal-Regulated MAP Kinases; D000077285:Rimonabant