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CELLULAR AND MOLECULAR

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

Institut de Neurosciences Physiologiques et Cognitives, Centre National de la Recherche Scientifique-INPC, Marseille, France (B.M., B.V.); McGill University, Department of Physiology, Montreal, Canada (V.C.); and Intégration des Informations Sensorielles, Centre National de la Recherche Scientifique-UMR 6150, Faculté de Médecine, l'Institut Fédératif de Recherche Jean Roche, Marseille, France (P.D.)

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 G_q/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 G_q/11-proteins. In contrast, in CFTR-expressing CHO cells (CHO-BQ1), the P2Y1R response was completely PTX-sensitive, indicating that P2Y1R couples to G_i/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.

Address correspondence to: Dr. Brice Marcet, INPC-CNRS, 31 Chemin Joseph Aiguier 13402 Marseille cedex 20, France. E-mail: marcet{at}dpm.cnrs-mrs.fr