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A3 adenosine and CB1 receptors activate a PKC‐sensitive Cl − current in human nonpigmented ciliary epithelial cells via a Gβ γ ‐coupled MAPK signaling pathway

2003; Wiley; Volume: 139; Issue: 3 Linguagem: Inglês

10.1038/sj.bjp.0705266

1476-5381

Chanjuan Shi, Anna Szcześniak, Lucy Mao, Christine A. B. Jollimore, Miguel Coca‐Prados, Orlando Hung, Michael E. Kelly,

Ion channel regulation and function

We examined A3 adenosine and CB1 cannabinoid receptor‐coupled signaling pathways regulating Cl − current in a human nonpigmented ciliary epithelial (NPCE) cell line. 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). Treatment of NPCE cells with pertussis‐toxin (PTX), or transfection with the COOH‐terminus of β ‐adrenergic receptor kinase (ct‐ β ARK), 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 . 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 ). 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. 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. I Cl,Win was inhibited by PTX preincubation, by transfection with ct‐ β ARK and by the MEK inhibitor, PD98059, but unaffected by the PI3K inhibitor, wortmannin. We conclude that both A3 and CB1 receptors activate a PKC‐sensitive Cl − current in human NPCE cells via a G i/o /G βγ signaling pathway, in a manner independent of PI3K but involving MAPK. British Journal of Pharmacology (2003) 139 , 475–486. doi: 10.1038/sj.bjp.0705266