title: 9-Anthracene carboxylic acid is more suitable than DIDS for characterization of calcium-activated chloride current during canine ventricular action potential.
abstract: Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance as channel malfunctions can lead to sudden cardiac death by inducing arrhythmias. Therefore, researchers frequently use inhibitors to selectively block a certain ion channel like 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for calcium-activated chloride current (ICl(Ca)). This study aims to explore which blocker is preferable to study ICl(Ca). Whole-cell voltage-clamp technique was used to record ICa,L, IKs, IKr and IK1, while action potentials were measured using sharp microelectrodes. DIDS- (0.2 mM) and 9-AC-sensitive (0.5 mM) currents were identical in voltage-clamp conditions, regardless of intracellular Ca(2+) buffering. DIDS-sensitive current amplitude was larger with the increase of stimulation rate and correlated well with the rate-induced increase of calcium transients. Both drugs increased action potential duration (APD) to the same extent, but the elevation of the plateau potential was more pronounced with 9-AC at fast stimulation rates. On the contrary, 9-AC did not influence either the AP amplitude or the maximal rate of depolarization (V max), but DIDS caused marked reduction of V max. Both inhibitors reduced the magnitude of phase-1, but, at slow stimulation rates, this effect of DIDS was larger. All of these actions on APs were reversible upon washout of the drugs. Increasing concentrations of 9-AC between 0.1 and 0.5 mM in a cumulative manner gradually reduced phase-1 and increased APD. 9-AC at 1 mM had no additional actions upon perfusion after 0.5 mM. The half-effective concentration of 9-AC was approximately 160 μM with a Hill coefficient of 2. The amplitudes of ICa,L, IKs, IKr and IK1 were not changed by 0.5 mM 9-AC. These results suggest that DIDS is equally useful to study ICl(Ca) during voltage-clamp but 9-AC is superior in AP measurements for studying the physiological role of ICl(Ca) due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (ICa,L, IKr, IKs, IK1); however, ICa,L tracings can be contaminated with ICl(Ca) when measured in voltage-clamp condition.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, 4012, Debrecen, Nagyerdei krt 98, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Váczi
authors.forename: Bence
authors.initials: B
authors.lastname: Hegyi
authors.forename: Ferenc
authors.initials: F
authors.lastname: Ruzsnavszky
authors.forename: Kornél
authors.initials: K
authors.lastname: Kistamás
authors.forename: Balázs
authors.initials: B
authors.lastname: Horváth
authors.forename: Tamás
authors.initials: T
authors.lastname: Bányász
authors.forename: Péter P
authors.initials: PP
authors.lastname: Nánási
authors.forename: Norbert
authors.initials: N
authors.lastname: Szentandrássy
authors.forename: János
authors.initials: J
authors.lastname: Magyar
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000200:Action Potentials / Q000187:drug effects*; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology; D020746:Calcium Channels, L-Type / Q000502:physiology; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000502:physiology*; D051668:Delayed Rectifier Potassium Channels / Q000502:physiology; D004285:Dogs; D005260:Female; D006352:Heart Ventricles / Q000187:drug effects*; D066298:In Vitro Techniques; D008297:Male; D024661:Potassium Channels, Inwardly Rectifying / Q000502:physiology; D016276:Ventricular Function / Q000187:drug effects
chemical_list: D000873:Anthracenes; D020746:Calcium Channels, L-Type; D018118:Chloride Channels; D051668:Delayed Rectifier Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C010030:9-anthroic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium