Purinoceptor‐coupled Cl⁻ channels in mouse heart: a novel, alternative pathway for CFTR regulation

Abstract

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. Extracellular adenosine‐5′‐O‐(3‐thiotriphosphate) (ATPγS; 100 μM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPγS while a PKC activator, phorbol 12,13‐dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPγS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl− channels. Isoprenaline increased ICl,ATP pre‐activated by ATPγS 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 ATPγS 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. Glibenclamide (50 μM) significantly blocked ICl,ATP activated by ATPγS or by the CFTR channel activator, levamisole. 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. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 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.

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

“…For this purpose, we used guinea pig ventricular cells, because these cells, unlike mouse cells (5,16), develop I Cl,CFTR in response to ␤-adrenergic stimulation (9,11,12). In the experiment shown in Fig.…”

A Novel Anionic Inward Rectifier in Native Cardiac Myocytes

“…10,11 Functional data suggest that I Cl.PKC and I Cl.ATP may also be attributed to activation of CFTR Cl Ϫ channels. [12][13][14][15][16] Although the molecular entity responsible for I Cl.Ca remains to be determined, it may be encoded by a member of the new CLCA gene family. 17 With physiological Cl Ϫ gradients, all of these Cl Ϫ currents activate predominantly at depolarized voltages, exhibit outward rectification, and thus contribute significantly to shortening of action potential duration; although activation of I Cl.Ca under conditions of intracellular Ca 2ϩ overload may, in addition, contribute to the development of oscillatory delayed afterdepolarizations.…”

“…16,31 To prevent contamination from Ca 2ϩ and K ϩ currents, and the cationic inward rectifiers I K1 and I f , nisoldipine (1 mol/L), 4-aminopyridine (4-AP, 2 mmol/L), Ba 2ϩ (2 mmol/L), and Cs ϩ (10 mmol/L) were present continuously in the extracellular bath solutions and cations in the intracellular pipette solution and, in some experiments, in the bath solution were replaced by the large impermeant cation, N-methyl-D-glucamine (NMDG). Hypotonic (230 mOsm/kg H 2 O, measured by freezing-point depression) bath solutions contained (mmol/L) NaCl 100, MgCl 2 between the bath and a Ag/AgCl reference electrode immersed in pipette solution was used to minimize changes in liquid junction potential, and junction potentials were zeroed before formation of the membrane-pipette seal.…”

Effects of bisindolylmaleimide PKC inhibitors on p90RSK activity in vitro and in adult ventricular myocytes

“…The use of these inhibitors, particularly GF109203X, in myocardial tissue and cells has implicated PKC-mediated signalling events in the regulation of physiological functions, such as contraction (Pi & Walker, 2000;Szokodi et al, 2002;von Lewinski et al, 2003) and protein synthesis (Ponicke et al, 1999), as well as a variety of pathophysiological processes, such as myocyte hypertrophy (Mullan et al, 1997;Ponicke et al, 1999;Ruf et al, 2002) and ischaemic cell death (Kitakaze et al, 1996;Chen et al, 1999;Inagaki et al, 2000;Fryer et al, 2001). In addition, GF109203X and Ro31-8220 have been used to investigate the roles of PKC in the regulation of sarcolemmal ion-transporting proteins, such as K þ (Hu et al, 1996;Wang et al, 2001a), Ca 2 þ (Woo & Lee, 1999;Hu et al, 2000) and Cl À (Middleton & Harvey, 1998;Duan et al, 1999) channels and the Na þ /K þ pump (Jo et al, 2000). These inhibitors have also been utilised by this and other laboratories to explore the involvement of PKC isoforms in the stimulation of the sarcolemmal Na þ /H þ exchanger (NHE1) by diverse stimuli, such as adrenergic (Puce´at et al, 1993;Snabaitis et al, 2000), thrombin (Yasutake et al, 1996), angiotensin (Gunasegaram et al, 1999) and opioid (Bian et al, 2000) receptor agonists, anaesthetic agents (Kanaya et al, 2001) and oxidative stress (Snabaitis et al, 2002).…”