Purinoceptor-coupled Cl- channels in mouse heart: a novel, alternative pathway for CFTR regulation.
1. P2-purinoceptors couple extracellular ATP to the activation of a Cl- current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart. 2. Extracellular adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; 100 microM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPgammaS while a PKC activator, phorbol 12, 13-dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPgammaS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl- channels. 3. Isoprenaline increased ICl,ATP pre-activated by ATPgammaS or PDBu, while isoprenaline or forskolin alone failed to activate any Cl- current in these myocytes. Adenosine 3',5'-cyclic monophosphothionate, a PKA inhibitor, prevented ATPgammaS or PDBu activation of ICl,ATP. Thus, ICl,ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. 4. Glibenclamide (50 microM) significantly blocked ICl,ATP activated by ATPgammaS or by the CFTR channel activator, levamisole. 5. The slope conductance of the unitary ICl,ATP in cell-attached patches was 11.8 +/- 0.3 pS, resembling the known properties of CFTR Cl- channels in cardiac myocytes. 6. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 7. We conclude that ICl,ATP in mouse heart is due to activation of CFTR Cl- channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.
Duan
D
D
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557-0046, USA.
Ye
L
L
Britton
F
F
Miller
L J
LJ
Yamazaki
J
J
Horowitz
B
B
Hume
J R
JR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction
D017931:DNA Primers; D018048:Receptors, Purinergic P2; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C022571:adenosine 5'-O-(3-thiotriphosphate); D015240:Phorbol 12,13-Dibutyrate; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D007545:Isoproterenol