Pharmacological and signaling properties of endogenous P2Y1 receptors in cystic fibrosis transmembrane conductance regulator-expressing Chinese hamster ovary cells.
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 ([Ca(2+)](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 EC(50) of 30 nM, 0.2 microM, and 0.8 microM, respectively. Activation of P2Y1R increased [Ca(2+)](i), which was prevented by the P2Y1R antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) and N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS2179) (10 microM) and by pretreatment with P2Y1R antisense oligodeoxynucleotides. In CHO-K1 and CHO-KNUT (mock-transfected) cells lacking CFTR, both P2Y1R and P2Y2R caused [Ca(2+)](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 microM) failed to inhibit both forskolin (1 microM)-induced CFTR activation, measured using iodide ((125)I) efflux, and forskolin (0.1-10 microM)-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.
Marcet
Brice
B
Institut de Neurosciences Physiologiques et Cognitives, Centre National de la Recherche Scientifique-INPC, Marseille, France. marcet@dpm.cnrs-mrs.fr
Chappe
Valérie
V
Delmas
Patrick
P
Verrier
Bernard
B
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004347:Drug Interactions; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1
D020319:Oligodeoxyribonucleotides, Antisense; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D002118:Calcium