Regulation of extracellular UTP-activated Cl- current by P2Y-PLC-PKC signaling and ATP hydrolysis in mouse ventricular myocytes.
The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTPo)-induced chloride (Cl-) currents (I(Cl.UTP)) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTPo (0.1 to 100 microM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl- solutions, an anion selectivity of Cl- > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR(inh)-172, 10 microM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 microM), these properties matching those of CFTR Cl- channels. The potency order of nucleotides for an activation of the Cl- current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 microM), a P2Y receptor antagonist, strongly inhibited the UTPo -activation of the Cl- current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 microM), another P2Y receptor antagonist, induced little inhibition of I(Cl.UTP). The activation of I(Cl.UTP) was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPbetaS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTPo failed to activate the Cl- current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I(Cl.UTP) was persistently activated with a mixture of ATPgammaS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I(Cl.UTP) is due to the activation of CFTR Cl- channels through Gq/11-coupled P2Y2 receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.
Yamamoto
Shintaro
S
Department of Physiology, Saga University Faculty of Medicine, Saga, 849-8501 Japan. yamamot3@cc.saga-u.ac.jp
Ichishima
Kunihiko
K
Ehara
Tsuguhisa
T
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology; D006868:Hydrolysis; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism* / Q000494:pharmacology
D002712:Chlorides; C546386:P2ry2 protein, mouse; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D014544:Uridine Triphosphate