Block Of Potassium Current Research Articles

SGK1 inhibition attenuates the action potential duration in reengineered heart cell models of drug-induced QT prolongation

Drug-induced QT prolongation (DI-QTP) is a clinical entity in which administration of a human ether-à-go-go-related gene/rapid delayed rectifier potassium current blocker such as dofetilide prolongs the cardiac action potential duration (APD) and the QT interval on the electrocardiogram. Inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) reduces the APD at 90% repolarization (APD90) in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from patients with congenital long QT syndrome. Here, we test the efficacy of 2 novel SGK1 inhibitors-SGK1-I1 and SGK1-I2-in iPSC-CM models of dofetilide-induced APD prolongation. Normal iPSC-CMs were treated with dofetilide to produce a DI-QTP iPSC-CM model. SGK1-I1's and SGK1-I2's therapeutic efficacy for shortening the dofetilide-induced APD90 prolongation was compared to mexiletine. The APD90 values were recorded 4hours after treatment using a voltage-sensing dye. The APD90 was prolonged in normal iPSC-CMs treated with dofetilide (673 ± 8 ms vs 436 ± 4 ms; P < .0001). While 10 mM mexiletine shortened the APD90 of dofetilide-treated iPSC-CMs from 673 ± 4 to 563 ± 8 ms (46% attenuation; P < .0001), 30 nM of SGK1-I1 shortened the APD90 from 673 ± 8 to 502 ± 7 ms (72% attenuation; P < .0001). Additionally, 300 nM SGK1-I2 shortened the APD90 of dofetilide-treated iPSC-CMs from 673 ± 8 to 460 ± 7 ms (90% attenuation; P < .0001). These novel SGK1-Is substantially attenuated the pathological APD prolongation in a human heart cell model of DI-QTP. These preclinical data support the development of this therapeutic strategy to counter and neutralize DI-QTP, thereby increasing the safety profile for patients receiving drugs with torsadogenic potential.

Simulation of ventricular rate control during atrial fibrillation using ionic channel blockers

BackgroundThe atrioventricular (AV) node is the only compartment that conducts an electrical impulse between the atria and the ventricles. The main role of the AV node is to facilitate efficient pumping by conducting excitation slowly between the two chambers as well as reduce the ventricular rate during atrial fibrillation (AF). MethodsUsing computer simulations, we investigated excitation conduction from the right atrium to the bundle of His during high-rate atrial excitation with or without partial blocking of the calcium or potassium ionic current. ResultsOur simulations revealed differences in rate reduction and repolarization effects between calcium and potassium current blocking and high degree of potassium current blocking required to reduce the ventricular rate during AF. ConclusionsOur simulation results explain why potassium current blockers are not recommended for controlling ventricular rate during AF.

Early afterdepolarizations promote transmural reentry in ischemic human ventricles with reduced repolarization reserve

AimsAcute ischemia is a major cause of sudden arrhythmic death, further promoted by potassium current blockers. Macro-reentry around the ischemic region and early afterdepolarizations (EADs) caused by electrotonic current have been suggested as potential mechanisms in animal and isolated cell studies. However, ventricular and human-specific arrhythmia mechanisms and their modulation by repolarization reserve remain unclear. The goal of this paper is to unravel multiscale mechanisms underlying the modulation of arrhythmic risk by potassium current (IKr) block in human ventricles with acute regional ischemia. Methods and resultsA human ventricular biophysically-detailed model, with acute regional ischemia is constructed by integrating experimental knowledge on the electrophysiological ionic alterations caused by coronary occlusion. Arrhythmic risk is evaluated by determining the vulnerable window (VW) for reentry following ectopy at the ischemic border zone. Macro-reentry around the ischemic region is the main reentrant mechanism in the ischemic human ventricle with increased repolarization reserve due to the ATP-sensitive potassium current (IK(ATP)) activation. Prolongation of refractoriness by 4% caused by 30% IKr reduction counteracts the establishment of macro-reentry and reduces the VW for reentry (by 23.5%). However, a further decrease in repolarization reserve (50% IKr reduction) is less anti-arrhythmic despite further prolongation of refractoriness. This is due to the establishment of transmural reentry enabled by electrotonically-triggered EADs in the ischemic border zone. EADs are produced by L-type calcium current (ICaL) reactivation due to prolonged low amplitude electrotonic current injected during the repolarization phase. ConclusionsElectrotonically-triggered EADs are identified as a potential mechanism facilitating intramural reentry in a regionally-ischemic human ventricles model with reduced repolarization reserve.

Reduced conduction failure of the main axon of polymodal nociceptive C-fibres contributes to painful diabetic neuropathy in rats

Painful diabetic neuropathy is a common complication of diabetes mellitus and can affect many aspects of life and severely limit patients' daily functions. Signals of painful diabetic neuropathy are believed to originate in the peripheral nervous system. However, its peripheral mechanism of hyperalgesia has remained elusive. Numerous studies have accumulated that polymodal nociceptive C-fibres play a crucial role in the generation and conduction of pain signals and sensitization of which following injury or inflammation leads to marked hyperalgesia. Traditionally, the number of nociceptive primary afferent firings is believed to be determined at the free nerve endings, while the extended main axon of unmyelinated C-fibres only involves the reliable and faithful propagation of firing series to the central terminals. We challenged this classic view by showing that conduction of action potential can fail to occur in response to repetitive activity when they travel down the main axon of polymodal nociceptive C-fibres. Quantitative analysis of conduction failure revealed that the degree of conduction failure displays a frequency-dependent manner. Local administration of low threshold, rapidly activating potassium current blocker, α-dendrotoxin (0.5 nM) and persistent sodium current blocker, low doses of tetrodotoxin (<100 nM) on the main axon of C-fibres can reciprocally regulate the degree of conduction failure, confirming that conduction failure did occur along the main axon of polymodal nociceptive C-fibres. Following streptozotocin-induced diabetes, a subset of polymodal nociceptive C-fibres exhibited high-firing-frequency to suprathreshold mechanical stimulation, which account for about one-third of the whole population of polymodal nociceptive C-fibres tested. These high-firing-frequency polymodal nociceptive C-fibres in rats with diabetes displayed a marked reduction of conduction failure. Delivery of low concentrations of tetrodotoxin and Nav1.8 selective blocker, A-803467 on the main axon of C-fibres was found to markedly enhance the conduction failure in a dose-dependent manner in diabetic rats. Upregulated expression of sodium channel subunits Nav1.7 and Nav1.8 in both small dorsal root ganglion neurons and peripheral C-fibres as well as enhanced transient and persistent sodium current and increased excitability in small dorsal root ganglion neurons from diabetic rats might underlie the reduced conduction failure in the diabetic high-firing-frequency polymodal nociceptive C-fibres. This study shed new light on the functional capability in the pain signals processing for the main axon of polymodal nociceptive C-fibres and revealed a novel mechanism underlying diabetic hyperalgesia.

A Comparative Study of the Effect of Ciguatoxins on Voltage-Dependent Na+ and K+ Channels in Cerebellar Neurons

Ciguatera is a global disease caused by the consumption of certain warm-water fish (ciguateric fish) that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. The effect of ciguatoxin standards and contaminated ciguatoxin samples was evaluated by electrophysiological recordings in cultured cerebellar neurons. The toxins affected both voltage-gated sodium (Nav) and potassium channels (Kv) although with different potencies. CTX 3C was the most active toxin blocking the peak inward sodium currents, followed by P-CTX 1B and 51-OH CTX 3C. In contrast, P-CTX 1B was more effective in blocking potassium currents. The analysis of six different samples of contaminated fish, in which a ciguatoxin analogue of mass 1040.6, not identical with the standard 51-OH CTX 3C, was the most prevalent compound, indicated an additive effect of the different ciguatoxins present in the samples. The results presented here constitute the first comparison of the potencies of three different purified ciguatoxins on sodium and potassium channels in the same neuronal preparation and indicate that electrophysiological recordings from cultured cerebellar neurons may provide a valuable tool to detect and quantify ciguatoxins in the very low nanomolar range.

Enhanced efficacy with MgSO4 due to an additive, alternative, or dual mode of action?

Pharmacological conversion of atrial fibrillation–atrial flutter (AF–AFl) is still an important therapeutic modality in daily patient care. Frequently used drugs are the class III anti-arrhythmics dofetilide, sotalol, and ibutilide. By blocking (specific) potassium currents, they will prolong the duration of the action potential (APD) providing less space for re-entry to manoeuvre. Their effectiveness is still not optimal and is also hampered by their pro-arrhythmic risk due to dose-dependent lengthening of the (ventricular) repolarization times. Their use is therefore restricted, including hospitalizations when therapy is started, as was the case in this single site (Hartford Hospital) retrospective investigation (2000–08) about the effectiveness of dofetilide to acutely convert AF–AFl.1 To protect their patients for dofetilide-induced torsade de pointes (TdP) arrhythmias, some ( n = 50) from the 160 eligible patients with AF–AFl received adjunctive magnesium sulfate (Mg). The evaluation demonstrated that a single or repeated bolus of Mg (mean ± 3 g MgSO4) did not only protect for drug-induced adverse effects (0/50 vs. 1/110 TdP occurrences), but also improved the efficacy of dofetilide to suppress/prevent AF–AFl: 38% without vs. 50% with Mg, P < 0.05. This important improvement with this combination (doubling of the odds), which was recently also shown for ibutilide and Mg by … *Corresponding author. Tel: +31 30 2538900, Fax: +31 30 2539036, Email: m.a.vos{at}umcutrecht.nl

The role of auditory nerve innervation and dendritic filtering in shaping onset responses in the ventral cochlear nucleus

Neurons in the ventral cochlear nucleus (VCN) that respond primarily at the onset of a pure tone stimulus show diversity in terms of peri-stimulus-time-histograms (PSTHs), rate-level functions, frequency tuning, and also their responses to broad band noise. A number of different mechanisms have been proposed as contributing to the onset characteristic: e.g. coincidence, depolarisation block, and low-threshold potassium currents. We show that a simple point neuron receiving convergent inputs from high-spontaneous rate auditory nerve (AN) fibers, with no special currents and no peri-stimulatory shifts in firing threshold, is sufficient to produce much of the diversity seen experimentally. Three sub-classes of onset PSTHs: onset-ideal (OI), onset-chopper (OC) and onset-locker (OL) are reproduced by variations in innervation patterns and dendritic filtering. The factors shaping responses were explored by systematically varying key parameters. An OI response in this model requires a narrow range of AN input best frequencies (BF) which only produce supra-threshold depolarizations during the stimulus onset. For OC and OL responses, receptive fields were wider. Considerable low pass filtering of AN inputs away from BF results in an OL, whilst relatively unfiltered inputs produce an OC response. Rate-level functions in response to pure tones can be sloping, or plateau. These can be also reproduced in the model by the manipulation of the AN innervation. The model supports the coincidence detection hypothesis, and suggests that differences in excitatory innervation and dendritic filtering constant are important factors to consider when accounting for the variation in response characteristics seen in VCN onset units.

Atrial Antifibrillatory Effects of Structurally Distinct IKurBlockers 3-[(Dimethylamino)methyl]-6-methoxy-2-methyl-4-phenylisoquinolin-1(2H)-one and 2-Phenyl-1,1-dipyridin-3-yl-2-pyrrolidin-1-yl-ethanol in Dogs with Underlying Heart Failure

Drug discovery efforts have focused recently on atrial-selective targets, including the Kv1.5 channel, which underlies the ultrarapid delayed rectifier current, I(Kur), to develop novel treatments for atrial fibrillation (AF). Two structurally distinct compounds, a triarylethanolamine TAEA and an isoquinolinone 3-[(dimethylamino)-methyl]-6-methoxy-2-methyl-4-phenylisoquinolin-1(2H)-one (ISQ-1), blocked I(Kur) in Chinese hamster ovary cells expressing human Kv1.5 with IC(50) values of 238 and 324 nM, respectively. In anesthetized dogs, i.v. infusions of TAEA and ISQ-1 elicited comparable 16% increases in atrial refractory period, with no effect on ventricular refractory period or QTc interval. Plasma concentrations at end infusion for TAEA and ISQ-1 were 58.5 +/- 23.6 and 330.3 +/- 43.5 nM, respectively. The abilities of TAEA and ISQ-1 to terminate AF, with comparison to the rapidly activating component of delayed rectifier potassium current blocker (+)-N-[1'-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4'-piperidin)-6-yl]methanesulfonamide] monohydrochloride (MK-499) and the class IC 1-[2-[2-hydroxy-3-(propylamino)-propoxy]phenyl]-3-phenyl-1-propanone (propafenone), were assessed in conscious dogs with heart failure and inducible AF (entry criterion). All test agents administered in i.v. bolus regimens terminated AF in at least half of animals tested; conversely no agent was universally effective. MK-499, ISQ-1, TAEA, and propafenone terminated AF in five of six, four of seven, four of six, and five of six animals at plasma concentrations of 32.6 +/- 18.7, 817 +/- 274, 714 +/- 622, and 816 +/- 240 nM, respectively. Directed cardiac electrophysiologic studies in anesthetized dogs using i.v. bolus (consistent with AF studies) plus infusion regimens with TAEA and ISQ-1 demonstrated significant increases in atrial refractory period (12-15%), A-H and P-A intervals, but no effects on ventricular refractory period, H-V, and HEG intervals. The demonstration of AF termination with TAEA and ISQ-1 in the dog heart failure model extends the profile of antiarrhythmic efficacy of Kv1.5 blockade.

Epicardial Activation of Left Ventricular Wall Prolongs QT Interval and Transmural Dispersion of Repolarization

Epicardial pacing of the left ventricle (LV) has been shown to prolong the QT interval and predispose to the development of torsade de pointes arrhythmias. The present study examines the cellular basis for QT prolongation and arrhythmogenesis after reversal of the direction of activation of the LV wall. A transmural ECG and transmembrane action potentials were simultaneously recorded from epicardial, M, and endocardial cells of arterially perfused canine LV wedge preparations. QT interval increased from 297.6+/-3.9 to 314.0+/-5.7 ms (n=12; P<0.001) and transmural dispersion of repolarization (TDR) increased from 35.5+/-5.2 to 70.3+/-6.2 ms (n=12; P<0.001) as pacing was shifted from endocardium to epicardium. Conduction time between M and epicardial cells increased from 12.1+/-1.2 to 24.2+/-1.5 ms (n=12; P<0.001). Amplification of TDR was further accentuated in the presence of rapidly activating delayed rectifier potassium current blockers (E-4031 and cisapride), increasing from 50.5+/-7.6 to 86.1+/-6.2 ms (n=8; P<0.01). Torsade de pointes arrhythmias could be induced during epicardial, but not endocardial, pacing of LV in the presence of rapidly activating delayed rectifier potassium current blockade. Reversal of the direction of activation of the LV wall, as occurs during biventricular pacing, leads to a prominent increase in QT and TDR as a result of earlier repolarization of epicardium and delayed activation and repolarization of the midmyocardial M cells. The increase in TDR creates the substrate for the development of torsade de pointes under long-QT conditions.

Endogenous rhythm generation in the pre-Bötzinger complex and ionic currents: modelling and in vitro studies.

The pre-Bötzinger complex is a small region in the mammalian brainstem involved in generation of the respiratory rhythm. As shown in vitro, this region, under certain conditions, can generate endogenous rhythmic bursting activity. Our investigation focused on the conditions that may induce this bursting behaviour. A computational model of a population of pacemaker neurons in the pre-Bötzinger complex was developed and analysed. Each neuron was modelled in the Hodgkin-Huxley style and included persistent sodium and delayed-rectifier potassium currents. We found that the firing behaviour of the model strongly depended on the expression of these currents. Specifically, bursting in the model could be induced by a suppression of delayed-rectifier potassium current (either directly or via an increase in extracellular potassium concentration, [K+]o) or by an augmentation of persistent sodium current. To test our modelling predictions, we recorded endogenous population activity of the pre-Bötzinger complex and activity of the hypoglossal (XII) nerve from in vitro transverse brainstem slices (700 micro m) of neonatal rats (P0-P4). Rhythmic activity was absent at 3 mm[K+]o but could be triggered by either the elevation of [K+]o to 5-7 mm or application of potassium current blockers (4-AP, 50-200 micro m, or TEA, 2 or 4 mm), or by blocking aerobic metabolism with NaCN (2 mm). This rhythmic activity could be abolished by the persistent sodium current blocker riluzole (25 or 50 micro m). These findings are discussed in the context of the role of endogenous bursting activity in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.

Sodium Channel-Blocking Properties of Spiradoline, a κ Receptor Agonist, are Responsible for Its Antiarrhythmic Action in the Rat

Spiradoline (U-62,066E), a selective kappa (kappa) receptor agonist, was examined for actions on the cardiovascular system and on myocardial ionic currents in rats. We initially characterized cardiac, hemodynamic, and antiarrhythmic actions of spiradoline in isolated perfused rat hearts and pentobarbital-anesthetized rats. Electrophysiologic studies in isolated myocytes were used to elucidate the mechanism for changes observed in vivo in the ECG, as well as for antiarrhythmic actions against electrical and ischemia-induced arrhythmias. In isolated rat hearts, spiradoline reduced heart rate and cardiac contractility and increased the PR interval and QRS width of the ECG in a concentration-dependent manner. In anesthetized rats, spiradoline dose-dependently reduced blood pressure and heart rate and prolonged the PR interval and QRS width. At slightly higher doses, it increased the QaT interval of the ECG. RSh, an index of sodium channel blockade in the rat, also was dose-dependently increased. Electrical stimulation of the left ventricle suggested that spiradoline may exert its antiarrhythmic action by blockade of myocardial sodium currents. The electrophysiologic actions of spiradoline on sodium currents, the transient outward (i(to)) and sustained plateau potassium (ik(sus)) currents were studied in isolated cardiac rat myocytes by whole-cell patch-clamp techniques. Spiradoline (15-500 microM) reduced peak sodium current in a rapid, reversible, and concentration-dependent manner; it also increased the rate of decay of I(to) and reduced the amplitude of Ik(sus). At a concentration of 150 microM, spiradoline produced a 24 +/- 2 mV hyperpolarizing shift in sodium current inactivation kinetics but did not alter activation processes. Spiradoline showed both tonic and frequency-dependent components of sodium current block. Thus spiradoline produced its antiarrhythmic actions via sodium channel blockade in myocardial tissue, although higher doses also block potassium currents. This combined ion channel-blocking property may be of added clinical benefit in the setting of myocardial ischemia.

Female gender as a risk factor for drug-induced cardiac arrhythmias: evaluation of clinical and experimental evidence.

One of the most pronounced gender-based differences in response to drugs is women's far greater risk of developing the life-threatening ventricular arrhythmia called torsades de pointes (TdP). A review of the literature and databases of the Food and Drug Administration reveals that a much higher percentage of women than men develop TdP arrhythmias after taking a variety of drugs, such as antihistamines (terfenadine, astemizole), antibiotics (erythromycin), antimalarials (halofantrine), antiarrhythmics (quinidine, d-sotalol), and miscellaneous other drugs. All of these drugs have in common the ability to block potassium currents, thereby prolonging cardiac repolarization and the QT interval on the ECG. The available experimental data support the hypothesis that gender differences in specific cardiac ion current densities are responsible, at least in part, for the greater susceptibility of females for developing TdP arrhythmias. In isolated perfused rabbit hearts (Langendorff technique), female rabbit hearts display greater baseline and drug-induced (quinidine and d-sotalol) changes in QT intervals than male hearts, and at least two different repolarizing potassium current densities (IKr and IKl) are found to be significantly lower in ventricular cardiomyocytes from female rabbits compared with those from males. Thus, it appears that as in humans, clear gender differences exist in the electrophysiologic characteristics governing cardiac repolarization in rabbits. This model and perhaps others should be examined as predictors of functional and pharmacologic differences between men and women. Understanding the potential mechanisms responsible for the greater risk of drug-induced arrhythmias in women could lead to screening methods for identification of individuals at risk for drug-induced arrhythmias or to the development of drugs with reduced risk of inducing arrhythmia.

Using the right drug: a treatment algorithm for regular supraventricular tachycardias.

Despite the recent advent of and the successful results from catheter ablation, pharmacological therapy is still used by most clinicians as the first line therapy in patients with regular supraventricular tachycardias. Before prescribing an antiarrhythmic agent, documentation of the arrhythmia using a 12-lead electrocardiogram (ECG) is necessary to identify the type of tachycardia. The ECG diagnosis is based on the presence and polarity of the P wave, the P to QRS relationship, the presence of QRS alternation and the effect of bundle branch block on tachycardia rate. Most regular supraventricular tachycardias use the atrioventricular node either passively, as in atrial tachycardias or flutter, or actively, as paroxysmal junctional tachycardias. The Sicilian Gambit approach attempted to introduce some rationale in the choice of an antiarrhythmic agent, taking into account tachycardia mechanism, by defining the critical components of the tachycardia and the vulnerable parameter, i.e. the component that may readily be affected by an appropriate antiarrhythmic agent. For this approach, an electrophysiological study is particularly useful. The most common regular paroxysmal supraventricular tachycardias include atrioventricular nodal re-entrant tachycardias and atrioventricular re-entrant tachycardias which use an overt or concealed accessory atrioventricular connection (Kent bundle) or atriofascicular connection (Mahaim). For acute termination of paroxysmal junctional tachycardia, intravenous adenosine is the drug of choice. For the prevention of the tachycardia attacks in atrioventricular nodal re-entrant tachycardia, the agents with a depressive effect on the antegrade slow pathway, such as calcium channel blockers or beta-blockers, are likely to be effective. If they fail, sodium channel blockers (propafenone or flecainide) may be indicated. In tachycardias involving accessory connections, agents that affect fast channel dependent tissue (propafenone, flecainide, cibenzoline, disopyramide or hydroquinidine) are effective. Potassium current blockers, such as sotalol or amiodarone, represent an alternative therapy. In atrial tachycardias, the use of propafenone, flecainide or sotalol constitute a logical choice. In drug-resistant cases, amiodarone is the most potent agent. Radiofrequency ablation of the slow atrioventricular nodal pathway, of an accessory connection or of an atrial focus, is indicated in drug-resistant or drug-intolerant patients and is increasingly offered as an alternative therapy.

Voltage-dependent calcium current with properties of T-type current in Sertoli cells from immature rat testis in primary cultures.

We have applied the whole-cell configuration of the patch-clamp technique to investigate calcium currents in Sertoli cells from immature rat testis in primary culture. Cesium-filled pipettes were used to block potassium currents. In the presence of 10 mM extracellular calcium (Ca+), depolarizations elicited transient inward currents in the range -70 to +10 mV with a maximal amplitude of -1.3 pA/pF at -30 mV. This component activated in the range -70 to -20 mV (V0.5 = -42 mV) and inactivated in the range -75 to -45 mV (V0.5 = 61 mV). Currents were not modified when barium (Ba2+) was substituted for Ca2+. They were suppressed by Ca(2+)-free solutions, insensitive to Bay K 8644 (an L-type channel opener), and inhibited by 1 mM cobalt, 10 microM nickel, 10 microM isradipine, and 1-10 microM omega conotoxine GVIA (four calcium-channel blockers). We conclude that calcium channels with the properties of T-type calcium channels of excitable cells are located in the membrane of immature Sertoli cells in primary cultures. Because these channels do not appear to be directly sensitive to FSH, their involvement in calcium movements remains to be determined.

Use-dependent effects of the class III antiarrhythmic agent NE-10064 (azimilide) on cardiac repolarization: block of delayed rectifier potassium and L-type calcium currents.

We studied the effects of NE-10064 (azimilide), a new antiarrhythmic agent reported to be a selective blocker of the slowly activating component of the delayed rectifier, IKs. In ferret papillary muscles, NE-10064 increased effective refractory period (ERP) and decreased isometric twitch tension in a concentration-dependent manner (0.3-30 microM). Increases in ERP showed reverse use-dependence, and were greater at 1 than at 3 Hz. In contrast, changes in tension were use dependent, with larger decreases observed at 3 than at 1 Hz. In guinea pig ventricular myocytes, NE-10064 (0.3-3 microM) significantly prolonged action potential duration (APD) at 1 Hz. At 3 Hz, NE-10064 (0.3-1 microM) increased APD only slightly, and at 10 microM decreased APD and the plateau potential. NE-10064 potently blocked the rapidly activating component of the delayed rectifier, IKr (IC50 0.4 microM), and inhibited IKs (IC50 3 microM) with nearly 10-fold less potency. NE-10064 (10 microM) did not block the inward rectifier potassium current (IKl). NE-10064 (10 microM) blocked the L-type calcium current (ICa) in a use-dependent manner; block was greater at 3 than at 1 Hz. We conclude that (a) NE-10064's block of potassium currents is relatively selective for IKr over IKs, (b) NE-10064 inhibits ICa in a use-dependent fashion, and (c) NE-10064's effects on ERP and tension in papillary muscle as well as APD and action potential plateau level in myocytes may be explained by its potassium and calcium channel blocking properties.

Embryotoxic effects of L-691,121, a class III antiarrhythmic agent, in rats.

L-691,121 is a class III antiarrhythmic agent which blocks potassium currents, leading to prolongation of cardiac potential and prevention of cardiac arrhythmia. In a developmental toxicity study in rats, there was a dose-dependent decrease in embryonic/fetal survival, and death of the entire litter was seen at an oral dose of 0.8 mg/kg per day. The critical period for embryolethality was determined as gestational days (GD) 10-13. In a study where females received 1 mg/kg on a critical day (GD 10 or 12) and were killed at 24-h intervals, a high embryonic mortality was seen at 72 h (GD 10 treatment) or 48 h (GD 12 treatment) after dosing. The surviving embryos had morphological abnormalities such as enlarged cardiac tube and pericardium, generalized edema, and hematoma. In order to investigate a possible mechanism for the embryolethality, GD 11 embryos were dissected from females at 4 h after dosing of 1 mg/kg and incubated for 5 h in vitro. The embryonic heart rates were decreased for the first 2 h after incubation but tended to recover to control levels thereafter. When GD 11 embryos were incubated for 4 h with the drug, there were decreases in the heart rates during the entire observation period. In a washout study where the embryos were transferred to drug-free medium after 1-h exposure, decreased heart rates recovered to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The roles of K+ conductance in expiratory pattern generation in anaesthetized cats.

1. The potassium current blockers caesium and tetraethylammonium were injected intracellularly by ionophoretic current into brainstem expiratory neurones of the ventral group. Neurones were identified by their spontaneous activity and by antidromic excitation from the spinal cord at the C2-C3 level. 2. The duration of action potentials increased and the early and late after-hyperpolarizations were completely suppressed. These effects on action potentials were reversible, recovered with an exponential time course within 3 min, and could be reproduced when blockers were applied repetitively into the same neurone. They were ascribed to blockade of potassium channels in the somatic membrane region. 3. Potassium channel blockers modified postsynaptic potentials: early-inspiratory hyperpolarizations were reversibly depressed while postinspiratory and expiratory depolarizations were irreversibly enhanced. The former effect was associated with a decrease of the neuronal input conductance. The latter effect was cumulative upon repetitive ionophoretic applications of potassium blockers. 4. The results demonstrate that potassium currents exert two different roles in expiratory pattern generation. Together with chloride currents, they contribute to the phasic early-inspiratory inhibition. They seem to be calcium-dependent and GABAB receptor-controlled currents which predominate near to the cell body. 5. Potassium currents also operate throughout the postinspiratory and late-expiratory periods. They seem to include persistent potassium currents which modulate the excitatory respiratory drive provided by the respiratory rhythm generator. We assume that these currents, widely distributed over the somatodendritic membrane area, are a target for neuromodulation by transmitters and intracellular second messengers.

Regulation of calcium channel current in A7r5 vascular smooth muscle cells by cyclic nucleotides.

In vascular smooth muscle (VSM) cells, the slow inward calcium current (ICa) may be regulated by phosphorylation of the calcium channel protein or of associated regulatory proteins. We investigated the role of several protein kinase systems in the regulation of ICa in cultured A7r5 cells, a clonal cell line derived from rat aorta. The perforated-patch voltage-clamp technique was used to record whole cell ICa. To isolate the ICa, the pipette contained high Cs+ and the bath contained 140 mM tetraethylammonium to block potassium currents. Ba2+ was used as the charge carrier. In control experiments, ICa was stable for at least 15 min. Compared with 23 +/- 3% in the time-control group (i.e., run-down; n = 10), 3 mM 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibited peak ICa by 53 +/- 3% (n = 9) within 15 min. Similarly, 3 mM 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP) inhibited ICa by 59 +/- 4 (n = 11). Application of 30 microM forskolin inhibited ICa by 58 +/- 9% (n = 6) within 5 min (compared with 4 +/- 3% for the 5-min time control). Forskolin also shifted the reversal potential to the left, suggesting a stimulation of an outward current. In the presence of the protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the same dose of forskolin had no effect (n = 7). The water-soluble analogue of forskolin (L-858051, 30 microM) decreased ICa by 72 +/- 11% (n = 9) and reduced the outward current component.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfhydryl alkylating agents induce calcium current in skeletal muscle fibers of a crustacean (Atya lanipes)

Voltage-clamp experiments using the three-microelectrode voltage clamp technique were performed on ventroabdominal flexor muscles of the crustacean Atya lanipes. Potassium and chloride currents were found to underlie the normal, passive response of the muscle. Blocking potassium currents with tetraethylammonium and replacing chloride ions with methanesulfonate did not unmask an inward current. By treating the muscle with the sulfhydryl-alkylating agent 4-cyclopentene-1,3-dione an inward current was detected. The current induced by the agent is carried by Ca2+, since it is abolished in Ca(2+)-free solutions. The induced Ca2+ current is detected at about -40 mV and reaches a mean maximum value of -78 microA/cm2 at ca. -10 mV. At this potential the time to peak is close to 15 msec. The induced Ca2+ current inactivated with 1-sec prepulses which did not elicit detectable Ca2+ current; the fitted hx curve had a midpoint of -38 mV and a steepness of 5.0 mV. Measurements of isometric tension were performed in small bundles of fibers, and the effects of the sulfhydryl-alkylating agents 4-cyclopentene-1,3-dione and N-ethylmaleimide were investigated. Tetanic tension was enhanced in a strictly Ca(2+)-dependent manner by 4-cyclopentene-1,3-dione. The amplitude of K+ contractures increased after treatment with N-ethylmaleimide. It is concluded that Ca2+ channels are made functional by the sulfhydryl-specific reagents and that the increase in tension is probably mediated by an increase in Ca2+ influx through the chemically induced Ca2+ channels.

Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects.

1. The effects of brief exposures to hypoxia on the membrane currents of isolated hippocampal CA1 neurons were studied with the use of the whole-cell variation of the patch-clamp technique. Neurons were acutely dissociated from immature (day 2-7) and mature (day 21-43) rats. 2. In the current-clamp mode, Na-cyanide (CN) hyperpolarized both mature and immature neurons. In the voltage-clamp mode, CN decreased the magnitude of the hyperpolarizing holding current in both age groups. 3. CN did not have a consistent effect on the voltage-dependent calcium and potassium currents of immature and mature CA1 neurons but decreased the voltage-dependent inward current of neurons at both ages. This effect was age dependent: the inward current of immature neurons decreased by only 10%, but that of mature neurons decreased by approximately 40%. 4. The decrease in the magnitude of the hyperpolarizing holding current and the depression of the voltage-dependent inward current of mature neurons were observed during brief exposure to N2 (PO2 = 0), indicating that the electroresponses observed with CN were the result of blocking oxidative respiration. 5. The hypoxia-sensitive inward current was blocked by tetrodotoxin (TTX) but was not blocked by cadmium or cesium + tetraethylammonium (TEA). Therefore this current was identified as the voltage-dependent, fast-inactivating sodium current (INa). 6. The isolated sodium current was studied with the use of cadmium to block calcium and TEA + cesium to block potassium currents. In mature neurons, CN left-shifted the steady-state inactivation curve for INa and slowed the deactivation kinetics of INa. CN caused little or no change in INa activation, fast inactivation, recovery from inactivation, or current-voltage (I-V) relationship. 7. We conclude that brief exposures to CN and hypoxia alter the intrinsic excitability of CA1 neurons by at least two mechanisms: 1) alterations in leakage currents and 2) alterations in the fast Na+ conductance that are maturationally dependent. We propose that the alterations in the Na+ conductance may play an adaptive role by reducing O2 demands and thus possibly delaying neuronal injury.