title: Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells.
abstract: OBJECTIVE
  Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM).
  
  
  METHODS
  CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis.
  
  
  RESULTS
  WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 microM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Galpha(q/11) subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations>or=15 microM, and PTX-sensitive increases in ERK1/2 phosphorylation.
  
  
  CONCLUSIONS
  This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.
authors.affiliation: Department of Pharmacology and Laboratory for Retina and Optic Nerve Research, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: B T
authors.initials: BT
authors.lastname: McIntosh
authors.forename: B
authors.initials: B
authors.lastname: Hudson
authors.forename: S
authors.initials: S
authors.lastname: Yegorova
authors.forename: C A B
authors.initials: CA
authors.lastname: Jollimore
authors.forename: M E M
authors.initials: ME
authors.lastname: Kelly
mesh_terms: D001095:Arachidonic Acids / Q000494:pharmacology; D048588:Benzoxazines / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D063386:Cannabinoid Receptor Agonists; D063387:Cannabinoid Receptor Antagonists; D002460:Cell Line; D002478:Cells, Cultured; D003511:Cyclohexanols / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009025:Morpholines / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009281:Naphthalenes / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D043882:Receptors, Cannabinoid / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D001095:Arachidonic Acids; D048588:Benzoxazines; D063386:Cannabinoid Receptor Agonists; D063387:Cannabinoid Receptor Antagonists; D003511:Cyclohexanols; D009025:Morpholines; D009281:Naphthalenes; D010880:Piperidines; D011720:Pyrazoles; D043882:Receptors, Cannabinoid; C088155:methanandamide; C103505:AM 251; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases