title: A3 adenosine and CB1 receptors activate a PKC-sensitive Cl- current in human nonpigmented ciliary epithelial cells via a G beta gamma-coupled MAPK signaling pathway.
abstract: (1) We examined A3 adenosine and CB1 cannabinoid receptor-coupled signaling pathways regulating Cl(-) current in a human nonpigmented ciliary epithelial (NPCE) cell line. (2) Whole-cell patch-clamp recordings demonstrated that the A3 receptor agonist, IB-MECA, activates an outwardly rectifying Cl(-)current (I(Cl,Aden)) in NPCE cells, which was inhibited by the adenosine receptor antagonist, CGS-15943 or by the protein kinase C (PKC) activator, phorbol 12,13 dibutyrate (PDBu). (3) Treatment of NPCE cells with pertussis-toxin (PTX), or transfection with the COOH-terminus of beta-adrenergic receptor kinase (ct-betaARK), inhibited I(Cl,Aden). The phosphatidyl inositol 3-kinase (PI3K) inhibitor, wortmannin, had no effect on I(Cl,Aden); however, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, inhibited I(Cl,Aden). (4) Reverse transcription-polymerase chain reaction experiments and immunocytochemistry confirmed mRNA and protein expression for the CB1 receptor in NPCE cells, and the CB1 receptor agonist, Win 55,212-2, activated a PDBu-sensitive Cl(-) current (I(Cl,Win)). (5) Transfection of NPCE cells with the human CB1 (hCB1) receptor, increased I(Cl,Win), consistent with increased receptor expression, and I(Cl,Win) in hCB1 receptor-transfected cells was decreased after application of a CB1 receptor inverse agonist, SR 141716. (6) Constitutive activity for CB1 receptors was not significant in NPCE cells as transfection with hCB1 receptors did not increase basal Cl(-) current, nor was basal current inhibited by SR 141716. (7) I(Cl,Win) was inhibited by PTX preincubation, by transfection with ct-betaARK and by the MEK inhibitor, PD98059, but unaffected by the PI3K inhibitor, wortmannin. (8) We conclude that both A3 and CB1 receptors activate a PKC-sensitive Cl(-) current in human NPCE cells via a G(i/o)/Gbetagamma signaling pathway, in a manner independent of PI3K but involving MAPK.
authors.affiliation: Laboratory for Retina and Optic Nerve Research, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7.
authors.forename: Chanjuan
authors.initials: C
authors.lastname: Shi
authors.forename: Anna
authors.initials: A
authors.lastname: Szczesniak
authors.forename: Lucy
authors.initials: L
authors.lastname: Mao
authors.forename: Christine
authors.initials: C
authors.lastname: Jollimore
authors.forename: Miguel
authors.initials: M
authors.lastname: Coca-Prados
authors.forename: Orlando
authors.initials: O
authors.lastname: Hung
authors.forename: Melanie E M
authors.initials: ME
authors.lastname: Kelly
mesh_terms: D002461:Cell Line, Transformed; D018118:Chloride Channels / Q000378:metabolism*; D002923:Cilia / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000502:physiology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D043684:Receptor, Adenosine A3 / Q000502:physiology*; D043884:Receptor, Cannabinoid, CB1 / Q000502:physiology*
chemical_list: D018118:Chloride Channels; D004791:Enzyme Inhibitors; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D043684:Receptor, Adenosine A3; D043884:Receptor, Cannabinoid, CB1; D011493:Protein Kinase C