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Characterization of Anandamide-Stimulated Cannabinoid Receptor Signaling in Human ULTR Myometrial Smooth Muscle Cells

Endocannabinoid Research Group (P.J.B., A.H.T., J.C.K., J.M.W.), Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom; Department of Cardiovascular Sciences (J.M., D.G.L.), Pharmacology and Therapeutics Group, Division of Anesthesia, Critical Care and Pain Management, University of Leicester, Leicester LE1 5WW, United Kingdom; and Department of Cell Physiology and Pharmacology (R.A.J.C., J.M.W.), University of Leicester, Leicester LE1 9HN, United Kingdom

Address all correspondence and requests for reprints to: Jonathon M. Willets, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom. E-mail: jmw23{at}le.ac.uk.

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 G_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 G_i/o, phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca²⁺/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.