title: Characterization of anandamide-stimulated cannabinoid receptor signaling in human ULTR myometrial smooth muscle cells.
abstract: Accumulating evidence highlights the importance of the endocannabinoid anandamide (AEA) as a key mediator in reproductive physiology. Current data suggest potential roles for AEA in gametogenesis, fertilization, and parturition. AEA exerts its actions through two G protein-coupled receptors, termed cannabinoid receptor 1 (CB1), and 2 (CB2), and the ligand-gated transient receptor potential vanilloid receptor type 1 (TRPV1) ion channel. At present, the cellular mechanism(s) and consequences of AEA signaling in reproductive tissues, especially the myometrium, are poorly understood. Here, we examine the expression of CB1, CB2, and TRPV1 in the human myometrial smooth muscle cell-line (ULTR) and characterize intracellular signaling after stimulation with AEA. Radioligand binding analysis revealed a total CB receptor expression of 76 +/- 24 fmol/mg protein, with both quantitative PCR and competition binding studies indicating a negligible CB2 component. AEA caused Galpha(i/o)-dependent inhibition of adenylate cyclase to reduce intracellular cAMP levels. In addition, AEA caused a 2.5- to 3.5-fold increase in ERK activation, which was ablated by inhibition of Galpha(i/o), phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca(2+)/calmodulin-dependent protein kinase or protein kinase C activities. TRPV1 channel activation with capsaicin failed to activate ERK. Consistent with these findings, the selective agonists, arachidonyl-2-chloroethylamide (CB1) and L759656 (CB2), and selective antagonists AM251 (CB1) and JTE907 (CB2), provided pharmacological evidence that the ERK signaling pathway is activated through endogenously expressed CB1. These findings provide an insight into myometrial AEA signaling, highlighting a potential role for endocannabinoids in the regulation of gene expression in myometrial smooth muscle cells.
authors.affiliation: Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brighton
authors.forename: John
authors.initials: J
authors.lastname: McDonald
authors.forename: Anthony H
authors.initials: AH
authors.lastname: Taylor
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
authors.forename: David G
authors.initials: DG
authors.lastname: Lambert
authors.forename: Justin C
authors.initials: JC
authors.lastname: Konje
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
mesh_terms: D001095:Arachidonic Acids / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival; D063388:Endocannabinoids; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008954:Models, Biological; D009130:Muscle, Smooth / Q000166:cytology* / Q000187:drug effects; D009215:Myometrium / Q000166:cytology* / Q000187:drug effects; D016133:Polymerase Chain Reaction; D053284:Polyunsaturated Alkamides / Q000494:pharmacology*; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D048049:Extracellular Signal-Regulated MAP Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C078814:anandamide