RT Journal Article
SR Electronic
T1 Pharmacological and Signaling Properties of Endogenous P2Y1 Receptors in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing Chinese Hamster Ovary Cells
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 533
OP 539
DO 10.1124/jpet.103.063396
VO 309
IS 2
A1 Marcet, Brice
A1 Chappe, Valérie
A1 Delmas, Patrick
A1 Verrier, Bernard
YR 2004
UL http://jpet.aspetjournals.org/content/309/2/533.abstract
AB 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. The American Society for Pharmacology and Experimental Therapeutics