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Research ArticleCELLULAR AND MOLECULAR

Pharmacological and Signaling Properties of Endogenous P2Y1 Receptors in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing Chinese Hamster Ovary Cells

Brice Marcet, Valérie Chappe, Patrick Delmas and Bernard Verrier
Journal of Pharmacology and Experimental Therapeutics May 2004, 309 (2) 533-539; DOI: https://doi.org/10.1124/jpet.103.063396
Brice Marcet
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Valérie Chappe
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Patrick Delmas
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Bernard Verrier
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Abstract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel that is defective in CF disease. CFTR activity has been shown to be regulated by the Gq/phospholipase C-linked P2Y2 subtype of P2Y nucleotide receptors (P2YR) in various systems. Here, we tested whether other P2YR may exert a regulation on CFTR activity and whether CFTR may in turn exert a regulation on P2YR signaling. Using reverse transcriptase-polymerase chain reactions, antisense oligodeoxynucleotide knockdown, and measurements of intracellular calcium concentration ([Ca2+]i), we showed that, in addition to P2Y2R, Chinese hamster ovary (CHO) cells also express functional P2Y1R. P2Y1R were activated by 2-methylthioadenosine 5′-diphosphate > 2-methylthioadenosine-5′-triphosphate > ADP with an EC50 of 30 nM, 0.2 μM, and 0.8 μM, respectively. Activation of P2Y1R increased [Ca2+]i, which was prevented by the P2Y1R antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (10 μM) and N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (MRS2179) (10 μM) and by pretreatment with P2Y1R antisense oligodeoxynucleotides. In CHO-K1 and CHO-KNUT (mock-transfected) cells lacking CFTR, both P2Y1R and P2Y2R caused [Ca2+]i mobilization via pertussis toxin (PTX)-insensitive Gq/11-proteins. In contrast, in CFTR-expressing CHO cells (CHO-BQ1), the P2Y1R response was completely PTX-sensitive, indicating that P2Y1R couples to Gi/o-proteins, whereas the P2Y2R response remained PTX-insensitive. In CHO-BQ1 cells, P2Y1R activation by ADP (100 μM) failed to inhibit both forskolin (1 μM)-induced CFTR activation, measured using iodide (125I) efflux, and forskolin (0.1-10 μM)-evoked cAMP increase. Together, our results indicate that, in contrast to P2Y2R, P2Y1R does not modulate CFTR activity in CHO cells and that CFTR expression may alter the G-protein-coupling selectivity of P2Y1R.

Footnotes

  • This work was supported by institutional grants from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), and by the French association Vaincre la Mucoviscidose. B.M. was supported by a doctoral fellowship from Vaincre la Mucoviscidose.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • DOI: 10.1124/jpet.103.063396.

  • ABBREVIATIONS: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator(s); P2YR, P2Y nucleotide receptor(s); CHO, Chinese hamster ovary; PTX, pertussis toxin; PLCβ, phospholipase Cβ; 2-MeSATP, 2-methylthioadenosine 5′-triphosphate; 2-MeSADP, 2-methylthioadenosine 5′-diphosphate; PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid; FSK, forskolin; RT-PCR, reverse transcriptase-polymerase chain reaction(s); PTO-ODN, phosphorothioate oligodeoxynucleotide; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; MRS2179, N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate; NHERF, Na+/H+ exchanger regulatory factor; PDZ, postsynaptic density 95/disc-large/ZO-1; AC, adenylate cyclase(s).

    • Received November 21, 2003.
    • Accepted January 21, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 309 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 309, Issue 2
1 May 2004
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Research ArticleCELLULAR AND MOLECULAR

Pharmacological and Signaling Properties of Endogenous P2Y1 Receptors in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing Chinese Hamster Ovary Cells

Brice Marcet, Valérie Chappe, Patrick Delmas and Bernard Verrier
Journal of Pharmacology and Experimental Therapeutics May 1, 2004, 309 (2) 533-539; DOI: https://doi.org/10.1124/jpet.103.063396

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Research ArticleCELLULAR AND MOLECULAR

Pharmacological and Signaling Properties of Endogenous P2Y1 Receptors in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing Chinese Hamster Ovary Cells

Brice Marcet, Valérie Chappe, Patrick Delmas and Bernard Verrier
Journal of Pharmacology and Experimental Therapeutics May 1, 2004, 309 (2) 533-539; DOI: https://doi.org/10.1124/jpet.103.063396
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