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

Br J Pharmacol. 2003 Jun;139(3):475-86. doi: 10.1038/sj.bjp.0705266.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Transformed
  • Chloride Channels / metabolism*
  • Cilia / drug effects
  • Cilia / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Activation / physiology
  • Enzyme Inhibitors / pharmacology
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • GTP-Binding Protein beta Subunits / physiology*
  • GTP-Binding Protein gamma Subunits / physiology*
  • Humans
  • MAP Kinase Signaling System / drug effects
  • MAP Kinase Signaling System / physiology
  • Protein Kinase C / metabolism*
  • Receptor, Adenosine A3 / physiology*
  • Receptor, Cannabinoid, CB1 / physiology*

Substances

  • Chloride Channels
  • Enzyme Inhibitors
  • G-protein Beta gamma
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Receptor, Adenosine A3
  • Receptor, Cannabinoid, CB1
  • Protein Kinase C