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

Abstract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a cAMP-dependent ClϪ 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 ([Ca 2ϩ ] i ), we showed that, in addition to P2Y2R, Chinese hamster ovary (CHO) cells also express functional P2Y1R. P2Y1R were activated by 2-methylthioadenosine 5Ј-diphosphate Ͼ 2-methylthioadenosine-5Ј-triphosphate Ͼ ADP with an EC 50 of 30 nM, 0.2 M, and 0.8 M, respectively. Activation of P2Y1R increased [Ca 2ϩ ] i , which was prevented by the P2Y1R antagonists pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS) (10 M) and N6-methyl 2Ј-deoxyadenosine 3Ј,5Ј-bisphosphate (MRS2179) (10 M) and by pretreatment with P2Y1R antisense oligodeoxynucleotides. In CHO-K1 and CHO-KNUT (mock-transfected) cells lacking CFTR, both P2Y1R and P2Y2R caused [Ca 2ϩ ] 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 PTXinsensitive. In CHO-BQ1 cells, P2Y1R activation by ADP (100 M) failed to inhibit both forskolin (1 M)-induced CFTR activation, measured using iodide ( 125 I) efflux, and forskolin (0.1-10 M)-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.Cystic fibrosis (CF), one of the most common lethal autosomal recessive genetic disease, is due to mutations in CFTR (Riordan et al., 1989), a cAMP-regulated Cl Ϫ channel that is localized in apical membranes of epithelia (Cheng et al., 1991). However, the pleiotropic effects due to CFTR dysfunctions and observed on epithelial ion transport in CF largely exceed the impairment of the Cl Ϫ channel function and may be related to other regulatory properties of CFTR. Indeed, in addition to functioning as a cAMP-regulated Cl Ϫ channel, CFTR has been shown to regulate several ion channels by notably activating P2YR subsequent to ATP release (Schwiebert et al., 1995;Urbach and Harvey, 1999).Extracellular nucleotides are important signaling moleThis 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.aspet...

Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells

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Differential Effects of the Gβ5-RGS7 Complex on Muscarinic M3 Receptor–Induced Ca2+ Influx and Release

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