title: Identification of N-arachidonoyl dopamine as a highly biased ligand at cannabinoid CB1 receptors.
abstract: OBJECTIVE
  N-arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro.
  
  
  METHODS
  Human or rat cannabinoid CB1 receptors were expressed in AtT20, CHO or HEK 293 cells. NADA displacement of radiolabelled cannabinoids, and CB1 receptor mediated activation of K channels or ERK phosphorylation, release of intracellular calcium ([Ca]i ) and modulation of adenylyl cyclase were measured in addition to NADA effects on CB1 receptor trafficking.
  
  
  RESULTS
  At concentrations up to 30 μM, NADA failed to activate any signalling pathways via CB1 receptors, with the exception of mobilization of [Ca]i . The elevations of [Ca]i were insensitive to pertussis toxin, and reduced or abolished by blockers of Gq /11 -dependent processes including U73122, thapsigargin and a peptide antagonist of Gq /11 activation. Prolonged NADA incubation produced modest loss of cell surface CB1 receptors. The prototypical cannabinoid agonist CP55940 signalled as expected in all assays.
  
  
  CONCLUSIONS
  NADA is an ineffective agonist at most canonical cannabinoid receptor signalling pathways, but did promote mobilization of [Ca]i via Gq -dependent processes and some CB1 receptor trafficking. This signalling profile is distinct from that of any known cannabinoid, and suggests that NADA may have a unique spectrum of effects in vivo. Our results also indicate that it may be possible to identify highly biased CB1 receptor ligands displaying a subset of the pharmacological or therapeutic effects usually attributed to CB1 ligands.
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, NSW, 2109, Australia.
authors.forename: William J
authors.initials: WJ
authors.lastname: Redmond
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, and Centre for Brain Research, University of Auckland, Auckland, New Zealand.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Cawston
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, and Centre for Brain Research, University of Auckland, Auckland, New Zealand.
authors.forename: Natasha L
authors.initials: NL
authors.lastname: Grimsey
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, NSW, 2109, Australia.
authors.forename: Jordyn
authors.initials: J
authors.lastname: Stuart
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, NSW, 2109, Australia.
authors.forename: Amelia R
authors.initials: AR
authors.lastname: Edington
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, and Centre for Brain Research, University of Auckland, Auckland, New Zealand.
authors.forename: Michelle
authors.identifier: http://orcid.org/0000-0002-5997-6898
authors.initials: M
authors.lastname: Glass
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, NSW, 2109, Australia.
authors.forename: Mark
authors.identifier: http://orcid.org/0000-0003-2538-2001
authors.initials: M
authors.lastname: Connor
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002186:Cannabinoids / Q000494:pharmacology; D002460:Cell Line; D003511:Cyclohexanols / Q000494:pharmacology; D004298:Dopamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008024:Ligands; D010766:Phosphorylation; D015221:Potassium Channels / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D001095:Arachidonic Acids; D002186:Cannabinoids; D003511:Cyclohexanols; D008024:Ligands; D015221:Potassium Channels; D043884:Receptor, Cannabinoid, CB1; C474941:arachidonyl dopamine; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D048049:Extracellular Signal-Regulated MAP Kinases; D000262:Adenylyl Cyclases; D002118:Calcium; D004298:Dopamine