A3 adenosine and CB1 receptors activate a PKC-sensitive Cl- current in human nonpigmented ciliary epithelial cells via a gβγ-coupled MAPK signaling pathway

Chanjuan Shi, Anna Szczesniak, Lucy Mao, Christine Jollimore, Miguel Coca-Prados, Orlando Hung, Melanie E.M. Kelly

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

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 (ICl,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 β-adrenergic receptor kinase (ct-βARK), inhibited ICl,Aden. The phosphatidyl inositol 3-kinase (PI3K) inhibitor, wortmannin, had no effect on ICl,Aden; however, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, inhibited ICl,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 (ICl,Win). 5. Transfection of NPCE cells with the human CB 1 (hCB1) receptor, increased ICl,Win, consistent with increased receptor expression, and ICl,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. ICl,Win was inhibited by PTX preincubation, by transfection with ct-βARK 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 Gi/o/Gβγ signaling pathway, in a manner independent of PI3K but involving MAPK.

Original languageEnglish
Pages (from-to)475-486
Number of pages12
JournalBritish Journal of Pharmacology
Volume139
Issue number3
DOIs
Publication statusPublished - Jun 2003

ASJC Scopus Subject Areas

  • Pharmacology

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't

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