title: Characterization of the endocannabinoid system, CB(1)  receptor signalling and desensitization in human myometrium.
abstract: OBJECTIVE
  The endocannabinoid plays vital roles in several aspects of reproduction, including gametogenesis, fertilization and parturition. However, little is known regarding the presence or role of the endocannabinoid system in myometrial function. Here the presence of the endocannabinoid system and signalling properties of cannabinoid receptors were characterized.
  
  
  METHODS
  Components of the endocannabinoid system were identified using qRT-PCR, immunohistochemical, immunoblotting and radioligand binding experiments. Cannabinoid receptor signalling pathways were characterized using standard MAPK and second messenger assays.
  
  
  RESULTS
  Primary myometrium expresses the endocannabinoid synthesizing enzyme N-acyl-phosphatidyl ethanolamine-specific phospholipase D, endocannabinoid degrading enzyme fatty acid amide hydrolase and cannabinoid CB(1) , but not CB(2) receptors or transient receptor potential vanilloid-type-1 channels. The CB(1) receptor ligand anandamide caused a Gα(i/o) -dependent inhibition of adenylate cyclase reducing intracellular cAMP levels, and Gα(i/o) , phosphoinositide-3-kinase, Src-kinase-dependent ERK activation. CB(1) receptor-generated signals declined following continual anandamide stimulation, possibly due to ligand metabolism since free anandamide concentrations declined during the experiment from 2.5 µM initially, to 500 nM after >30 min. However, identical loss of CB(1) receptor responsiveness occurred in the presence of the metabolically stable derivative methanandamide. Moreover, RNAi-mediated depletion of arrestin3 (a negative regulator of receptor signalling) prevented loss of CB(1) receptor activity, enhancing and prolonging ERK signals.
  
  
  CONCLUSIONS
  The myometrium has the capacity to synthesize, respond to and degrade endocannabinoids. Furthermore, reduced CB(1) receptor responsiveness occurs as a consequence of receptor desensitization, not agonist depletion and we identify a key role for arrestin3 in this process.
authors.affiliation: Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester Royal Infirmary, Leicester, UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brighton
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Marczylo
authors.forename: Shashi
authors.initials: S
authors.lastname: Rana
authors.forename: Justin C
authors.initials: JC
authors.lastname: Konje
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
mesh_terms: D000328:Adult; D001095:Arachidonic Acids / Q000494:pharmacology; D019390:Arrestins / Q000235:genetics / Q000502:physiology; D001667:Binding, Competitive; D015153:Blotting, Western; D063385:Cannabinoid Receptor Modulators / Q000378:metabolism*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D063388:Endocannabinoids; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008875:Middle Aged; D009215:Myometrium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D053284:Polyunsaturated Alkamides / Q000494:pharmacology; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000494:pharmacology; D011869:Radioligand Assay; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D001095:Arachidonic Acids; D019390:Arrestins; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D008024:Ligands; D053284:Polyunsaturated Alkamides; D034741:RNA, Small Interfering; D043884:Receptor, Cannabinoid, CB1; C082333:arrestin3; C088155:methanandamide; C078814:anandamide