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Circulation. 1995;91:2834-2843

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(Circulation. 1995;91:2834-2843.)
© 1995 American Heart Association, Inc.

Articles

Anticholinergic Effects of Class III Antiarrhythmic Drugs in Guinea Pig Atrial Cells

Different Molecular Mechanisms

Katsumi Mori, MD; Yukio Hara, PhD; Toshihiro Saito, MD, PhD; Yoshiaki Masuda, MD, PhD; Haruaki Nakaya, MD, PhD

From the Department of Pharmacology and the Third Department of Internal Medicine, School of Medicine, Chiba University, Chiba, Japan.

Correspondence to Haruaki Nakaya, MD, PhD, Department of Pharmacology, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260, Japan.


*    Abstract
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 *Abstract
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Background It is well known that vagal stimulation increases the vulnerability to atrial fibrillation via muscarinic receptor–mediated shortening of refractory period. Recently it has been reported that some class III antiarrhythmic drugs effectively terminate or prevent atrial flutter and fibrillation by prolonging atrial effective refractory period. However, effects of class III antiarrhythmic drugs on the muscarinic acetylcholine receptor–operated K+ current (IK.ACh), which is important for the repolarization phase of the action potential in atrial cells, have not been thoroughly examined.

Methods and Results Effects of three class III antiarrhythmic drugs, d,l-sotalol, E-4031, and MS-551, on the carbachol (1 µmol/L)–induced action potential shortening and outward K+ current were examined in guinea pig atrial cells by conventional microelectrode and patch clamp techniques. In isolated left atria, d,l-sotalol (100 µmol/L), E-4031 (3 µmol/L), and MS-551 (30 µmol/L) partially reversed the carbachol-induced action potential shortening. In isolated single atrial cells, IK.ACh was activated by extracellular application of carbachol (1 µmol/L) or adenosine (10 µmol/L) or by intracellular loading of GTP{gamma}S (100 µmol/L). Sotalol (3 to 1000 µmol/L), E-4031 (1 to 100 µmol/L), and MS-551 (1 to 100 µmol/L) inhibited the carbachol-induced IK.ACh in a concentration-dependent manner, and their IC50 (half-maximal inhibition) values were 35.5, 7.8, and 11.4 µmol/L, respectively. However, the GTP{gamma}S-induced and adenosine-induced IK.ACh were inhibited by high concentrations of E-4031 and MS-551 but not by sotalol.

Conclusions Sotalol may inhibit IK.ACh by the blockade of the atrial muscarinic receptors, whereas E-4031 and MS-551 may inhibit the current not only by blocking the muscarinic receptors but also by depressing the function of the K+ channel itself and/or G proteins. These drugs may potentially be useful for the prevention and termination of atrial flutter and fibrillation through their inhibitory action on IK.ACh.


Key Words: potassium • antiarrhythmia agents • receptors, muscarinic • atrium


*    Introduction
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 *Introduction
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Class III antiarrhythmic drugs are currently receiving renewed interest as antifibrillatory therapies to reduce the likelihood of sudden cardiac death.1 2 Their antifibrillatory activity has been ascribed to their prolonging effects on action potential duration (APD) resulting from blockade of the voltage-gated K+ channels. It has been demonstrated that in ventricular cells, most class III antiarrhythmic drugs inhibit the delayed rectifier K+ current (IK) and some of them inhibit the inward rectifier K+ current (IK1) and/or the transient outward current (Ito).3 4 5 6 7 8 However, effects of class III antiarrhythmic drugs on the ligand-operated K+ currents have not been thoroughly examined.

Recently, experimental and clinical reports have indicated that class III antiarrhythmic drugs may be useful for the treatment of atrial flutter and fibrillation.9 10 11 12 13 14 However, effects of class III antiarrhythmic drugs on the membrane current system of atrial cells are less well defined. It is acknowledged that the muscarinic acetylcholine (ACh) receptor–operated K+ current (IK.ACh) plays an important role in the repolarization of the action potential as well as the maintenance of the resting potential in atrial cells.15 This ligand-operated K+ current is regulated by the receptor–GTP-binding protein pathway.16 17 It has been reported that some class I and IV antiarrhythmic drugs inhibit IK.ACh by blocking muscarinic ACh receptors or by blocking the K+ channel itself and/or GTP-binding proteins.18 19 20 21 However, effects of class III antiarrhythmic drugs on IK.ACh have not been thoroughly examined. If they suppress IK.ACh, it may partly explain the efficacy of the class III antiarrhythmic drugs for the prevention and treatment of atrial flutter and fibrillation, which can be experimentally elicited by vagal stimulation.22 23 Therefore, the present study was conducted to examine effects of three class III antiarrhythmic drugs, sotalol, E-4031, and MS-551, on the action potential and IK.ACh in guinea pig atrial cells.


*    Methods
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Action Potential Study
All experiments were performed under the regulations of the Animal Research Committee of the School of Medicine, Chiba University. Guinea pigs weighing 250 to 300 g were killed by a blow to the head, and their hearts were rapidly removed. The hearts were immersed in an oxygenated Tyrode's solution, and left atria were dissected. The preparations were pinned to the bottom of a tissue chamber and continuously superfused with the modified Tyrode's solution equilibrated with 95% O2/5% CO2. The composition of the solution was (in mmol/L) NaCl 125, KCl 4, NaH2PO4 1.8, MgCl2 0.5, CaCl2 2.7, NaHCO3 25, and glucose 5.5. The bath temperature was kept constant at 36.0±1.0°C.

Transmembrane potentials were recorded by standard microelectrode techniques, as previously described.24 The preparation was electrically stimulated at 1.0 Hz with pulses of 1-ms duration at twice the diastolic threshold with a bipolar electrode. Stimuli were delivered from an electronic stimulator (Nihon Kohden S-7272B). Transmembrane action potentials were recorded with glass microelectrodes filled with 3 mol/L KCl, which had a tip resistance of 10 to 30 M{Omega}. The microelectrode was connected to the input stage of a high-impedance amplifier with capacitance neutralization (Nihon Kohden MZ-4). The amplified signals were displayed on a dual-beam oscilloscope (Nihon Kohden VC-10) and photographed by a Polaroid camera.

After an equilibration period of 1 to 2 hours, a stable impalement was obtained and control recordings were made. The preparations were then exposed to solutions containing various concentrations of class III antiarrhythmic drugs. In some experiments, effects of class III antiarrhythmic drugs on the muscarinic receptor–mediated action potential shortening were examined in atrial preparations. First, the preparations were exposed to 1 µmol/L carbachol, a muscarinic agonist that is resistant to hydrolysis by cholinesterase. The cholinergic agonist produced a marked shortening of the action potential in atrial preparations. Ten minutes after the introduction of 1 µmol/L carbachol, d,l-sotalol (100 µmol/L), E-4031 (3 µmol/L), or MS-551 (30 µmol/L) was added to the superfusate, and the antagonizing effects of the class III antiarrhythmic drugs on the muscarinic receptor–mediated action potential shortening were observed. Preliminary experiments revealed that a 30-minute superfusion period was sufficient for action potential changes to reach a steady state. The recording of the transmembrane potential was repeated at the end of the drug-superfusion period.

Patch-Clamp Study
Single atrial cells of the guinea pig heart were isolated by an enzymatic dissociation method, as previously described.25 Briefly, the heart was removed from the open-chest guinea pigs anesthetized with pentobarbital sodium and mounted on a modified Langendorff perfusion system for retrograde perfusion of the coronary circulation with a normal HEPES-Tyrode's solution. The perfused medium was then changed to a nominally Ca2+-free Tyrode's solution and then to a solution containing 0.02% wt/vol collagenase (Wako). After digestion, the heart was perfused with a high-K+/low-Cl-solution (KB solution).26 Atrial tissue was cut into small pieces in the KB solution, and the cell suspension was stored in a refrigerator (4°C) for later use. The composition of the normal HEPES-Tyrode's solution was (in mmol/L) NaCl 143, KCl 5.4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, glucose 5.5, and HEPES-NaOH buffer (pH 7.4) 5.0. The composition of KB solution was (in mmol/L) KOH 70, L-glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, MgCl2 3, glucose 10, EGTA 1.0, and HEPES-KOH buffer (pH 7.4) 10.

Whole-cell membrane currents were recorded by the patch-clamp method.27 Single atrial cells were placed in a recording chamber (1-mL volume) attached to an inverted microscope (Olympus IMT-2) and superfused with the HEPES-Tyrode's solution at a rate of 10 mL · min-1. The temperature of the external solution was kept constant at 36.0±1.0°C. Glass patch pipettes with a diameter of 1.5 mm were filled with an internal solution. The composition of the pipette solution was (in mmol/L) potassium aspartate 110, KCl 20, MgCl2 1.0, potassium ATP 5.0, potassium phosphocreatine 5.0, EGTA 10, and HEPES-KOH buffer (pH 7.4) 5.0. GTP (sodium salt; 100 µmol/L) or GTP{gamma}S (tetralithium salt; 100 µmol/L) was also added to the pipette solution. The free Ca2+ concentration in the pipette solution was adjusted to pCa 8 according to the calculation by Fabiato and Fabiato28 with the correction of Tsien and Rink.29 The resistance of the patch pipette filled with the internal solution was 2 to 3 M{Omega}. After the gigaohm seal between the tip of the electrode and the cell membrane was established, the membrane patch was disrupted by more negative pressure applied to make the whole-cell voltage-clamp mode. The electrode was connected to a patch-clamp amplifier (Nihon Kohden CEZ-2300). Recordings were filtered at 1-kHz bandwidth, and series resistance was compensated. Command pulses were generated by a 12-bit digital-to-analog converter controlled by PCLAMP software (Axon Instruments, Inc). Current signals were digitized and stored on the hard disk of an IBM-compatible computer.

The tight-seal, whole-cell voltage-clamp technique was used. A liquid junctional potential of -8 mV was corrected. The cells were held at -50 mV to examine effects of these drugs on the muscarinic ACh receptor–regulated K+ channel. The IK.ACh was activated by the extracellular application of 1 µmol/L carbachol or 10 µmol/L adenosine in the GTP-loaded cells or by the intracellular loading of a nonhydrolyzable GTP analogue, GTP{gamma}S. In some experiments, 1 µmol/L ACh instead of 1 µmol/L carbachol was used to activate IK.ACh. Effects of various concentrations of d,l-sotalol, E-4031, and MS-551 on IK.ACh activated in three different ways were examined. In part of the experiments, effects of d-sotalol on the carbachol-induced IK.ACh were also examined.

To calculate percent inhibition of IK.ACh by various drugs, the difference between the steady-state current in solution containing either 1 µmol/L carbachol or 10 µmol/L adenosine and the current level in the absence of any agonist was taken as 100% in the GTP-loaded cells. In the GTP{gamma}S-loaded cells, the difference between the persistent outward current in the absence of agonist and the initial current level just after the break of the patch membrane in the pipette was taken as 100%.

Mechanical Function Study
To determine whether three class III antiarrhythmic drugs competitively interact with atrial muscarinic receptors, further mechanical experiments were conducted using isolated guinea pig atrial preparations. Left atria were mounted vertically in a 20-mL water-jacketed bath containing the modified Tyrode's solution bubbled with 95% O2/5% CO2 at 36±1°C. The lower end of the left atrium was fixed on a hook, and the other end was connected to a force transducer (Nihon Kohden TB-651T). Isometric tensions developed in the preparations were recorded on a chart recorder (NEC San-ei 8K21) through a preamplifier (Nihon Kohden SEN-6104). The resting tension applied to the preparation was adjusted to 0.5 g. The atria were electrically driven by rectangular pulses 0.5 Hz in frequency, 5 ms in duration, and twice the threshold voltage. The pulses were delivered from an electronic stimulator (Nihon Kohden S-7272B). The preparations were allowed to equilibrate for at least 60 minutes before the experiments.

The concentration-response curves for the negative inotropic effect of methacholine, another muscarinic agonist, were determined in a cumulative manner by increasing its concentration in steps of 0.5 log units. At the completion of the concentration-response curve, the preparations were washed out thoroughly. After the force of contraction returned to predrug level, a class III antiarrhythmic drug was applied. After the incubation time of 30 minutes, the concentration-response curve for methacholine was obtained in the presence of a drug. We confirmed that repetitive application of methacholine to the preparation did not affect the concentration-response curves for the negative inotropic effect of methacholine. The concentrations of methacholine producing half-maximal response in the absence and presence of various concentrations of the test drugs were obtained from log-probit plots of the individual response versus concentration. Affinity measurements were estimated by Schild analysis,30 fitting linear regression by least-squares and verifying parallelism before calculating dose ratios.

Drugs
The compounds used were as follows: d,l-sotalol and d-sotalol (Bristol-Myers Squibb Co), E-4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulfonamide dihydrochloride dihydrate) (Eisai Co, Ltd), MS-551 (1,3-dimethyl-6-{2-N-(2-hydroxyethyl)-3-(4-nitrophenyl) propylamino] ethylamino}-2,4-(1H,3H)-pyrimidinedione hydrochloride) (Mitsui Pharmaceuticals), carbachol chloride (Wako), methacholine chloride (Wako), ACh chloride (Daiichi), and adenosine (Wako). All drugs were dissolved in distilled water.

Statistics
All values are presented in terms of mean±SEM. Student's t test was used for statistical analysis of the paired observations. Two-way ANOVA was also used in comparing the effects and concentration-response curves of the three drugs. One-way ANOVA was used to test the differences among the groups. A value of P<.05 was considered significant. The IC50 values (the concentrations required to produce 50% of the maximal inhibitory effect) were obtained with a Macintosh computer (Apple Computer, Inc) and the SIGMA PLOT program (Jandel Corp). The slope of the Schild plot analyzed with respect to difference from unity and 95% confidence limit of the slope were calculated according to Brown and Hollander.31


*    Results
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Effects of Class III Antiarrhythmic Drugs on Action Potentials in Left Atria
Fig 1ADown illustrates representative changes in action potential configuration produced by d,l-sotalol, E-4031, and MS-551 in isolated left atria stimulated at 1.0 Hz. These drugs increased APD without affecting action potential amplitude (APA), resting membrane potential (RMP), and the maximum upstroke velocity of phase 0 depolarization (|$$max). Changes in APD at 90% repolarization level (APD90) after these class III antiarrhythmic drugs are summarized in Fig 1BDown. The baseline values of APD90, RMP, APA, and |$$ were 70.0±3.0 ms, -87.9±0.9 mV, 120.7±1.1 mV, and 264.4±21.3 V · s-1, respectively (n=15). There were no significant differences in any of the baseline values of action potential parameters between subgroups. Sotalol, E-4031, and MS-551 prolonged APD90 in a concentration-dependent manner. E-4031 was most potent and sotalol was least potent in prolonging APD among the three drugs on a molar basis. None of these drugs significantly affected |$$.



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  		Figure 1. Effects of d,l-sotalol, E-4031, and MS-551 on action potentials in guinea pig left atria driven at 1.0 Hz. A, Superimposed records of action potentials obtained before and after exposure to class III antiarrhythmic drug. C, S, E, and M indicate control action potentials, those after 100 µmol/L d,l-sotalol, 3 µmol/L E-4031, and 30 µmol/L MS-551, respectively. B, Concentration-response curves for increases in action potential duration at 90% repolarization level ({Delta}APD90) with d,l-sotalol, E-4031, and MS-551 in isolated left atria. Increases in APD90 are indicated on the ordinate, and the concentrations of drugs are on the abscissa. Values are expressed as mean±SEM of five experiments in each group.

Effects of Class III Antiarrhythmic Drugs on Muscarinic ACh Receptor–Mediated Action Potential Shortening in Left Atria
Carbachol, a cholinergic agonist, at a concentration of 1 µmol/L markedly shortened APD, as shown in Fig 2ADown. In left atrial preparations, 1 µmol/L carbachol decreased APD90 from 69.3±2.1 to 19.6±1.9 ms (n=19). Addition of 100 µmol/L d,l-sotalol, 3 µmol/L E-4031, or 30 µmol/L MS-551 partially reversed the carbachol-induced action potential shortening, as shown in Fig 2ADown. At these concentrations, three class III antiarrhythmic drugs produced a submaximal prolongation of APD in untreated atrial preparations, as already mentioned (Fig 1BUp). Sotalol, E-4031, and MS-551 partially but significantly reversed the muscarinic receptor–mediated action potential shortening to 60.8±2.1% (n=5), 40.1±1.8% (n=8), and 55.0±3.6% (n=6) of control, respectively (Fig 2BDown).



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  		Figure 2. Effects of d,l-sotalol, E-4031, and MS-551 on the muscarinic receptor–mediated action potential shortening in guinea pig left atria. A, Superimposed records obtained before and after carbachol and carbachol plus class III antiarrhythmic drug. C, CCh, S, E, and M indicate control, 1 µmol/L carbachol, 100 µmol/L d,l-sotalol, 3 µmol/L E-4031, and 30 µmol/L MS-551, respectively. B, Influences of class III antiarrhythmic drugs on the carbachol-induced action potential shortening. Action potential duration at 90% repolarization level (APD90) in the control condition (C) was taken as 100% (solid bars). APD90, expressed as percent of the control, after 1 µmol/L carbachol and carbachol plus class III antiarrhythmic drug is indicated by open and hatched bars, respectively. Values are expressed as mean±SEM of five to eight experiments in each group. *Significant difference (P<.05) between APD90 after carbachol alone and that after the addition of class III antiarrhythmic drug. Reversal of APD90 after d,l-sotalol and MS-551 was significantly greater than that after E-4031 when compared at these concentrations.

Effects of Class III Antiarrhythmic Drugs on Muscarinic ACh Receptor–Regulated K+ Channel Current in the GTP-Loaded Atrial Cells
Effects of sotalol, E-4031, and MS-551 on the carbachol-induced K+ channel current in the GTP-loaded cells were examined. On application of 1 µmol/L carbachol to the bath solution, an outward K+ current was rapidly activated at a holding potential of -50 mV. After the activation, the carbachol-induced K+ current gradually declined despite the continuous presence of carbachol, possibly because of a desensitization.32 33 After the current had almost reached a steady level, d,l-sotalol, E-4031, or MS-551 was added to the bath solution. Sotalol, E-4031, and MS-551 depressed the carbachol-induced K+ current effectively in a concentration-dependent manner (Fig 3Down). On washout of the class III antiarrhythmic drugs, the outward current reappeared. Similar inhibition of the carbachol-induced IK.ACh was also observed with d-sotalol, and the concentration-response curves for the inhibitory effects of d-sotalol and d,l-sotalol were almost superimposable (data not shown). The IC50 values of d,l-sotalol, E-4031, and MS-551 for depressing the carbachol-induced current were 35.5, 7.8, and 11.4 µmol/L, respectively (see Fig 6Down). These class III antiarrhythmic drugs also inhibited the IK.ACh induced by extracellular application of 1 µmol/L ACh in a concentration-dependent manner (data not shown).



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  		Figure 3. Tracings showing effects of class III antiarrhythmic drugs on the muscarinic ACh receptor–regulated K+ channel current (IK.ACh) in guinea pig atrial cells. IK.ACh was activated by the extracellular application of carbachol in the GTP (100 µmol/L)– loaded atrial cells (right inset). The holding potential was -50 mV. Application of carbachol, d,l-sotalol, E-4031, and MS-551 is shown by the bars above each original current trace.



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  		Figure 6. Concentration-response curves for the inhibitory effects of class III antiarrhythmic drugs on the muscarinic K+ channel current in isolated atrial cells. The muscarinic K+ channel current was activated by extracellular application of carbachol, adenosine, or intracellular loading of GTP{gamma}S. Percent inhibition of the outward current is indicated on the ordinates, and the concentrations of class III antiarrhythmic drugs are on the abscissas. Values are expressed as mean±SEM of 4 to 10 experiments in each point.

Effects of Class III Antiarrhythmic Drugs on the Muscarinic K+ Channel Current Activated by Intracellular GTP{gamma}S-Loading in Atrial Cells
It has been demonstrated that pertussis toxin–sensitive GTP-binding proteins couple muscarinic receptors with a specific K+ channel in atrial cells.17 Intracellular application of the nonhydrolyzable GTP analogues can directly activate the GTP-binding proteins and evoke antagonist-resistant, persistent activation of the muscarinic K+ channels.16 In the GTP{gamma}S (100 µmol/L)– loaded cells, the K+ current was activated gradually, even in the absence of any agonists. We examined effects of d, l-sotalol, E-4031, and MS-551 on the muscarinic K+ channel uncoupled from the membrane receptors in the GTP{gamma}S-loaded cells (Fig 4Down). The GTP{gamma}S-induced K+ current was inhibited by E-4031 and MS-551 less effectively than the carbachol-induced K+ current. In other words, high concentrations of these drugs were needed to inhibit the GTP{gamma}S-induced current (Fig 4Down). The IC50 values of E-4031 and MS-551 for depressing the GTP{gamma}S-induced current were 133.9 and 76.7 µmol/L, respectively (see Fig 6Up). In contrast with E-4031 and MS-551, d,l-sotalol failed to affect the GTP{gamma}S-induced current in concentrations up to 1000 µmol/L, as shown in Figs 4Down and 6Up. These findings indicate that E-4031 and MS-551, but not sotalol, directly inhibit the muscarinic K+ channel itself and/or G proteins.



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  		Figure 4. Tracings showing effects of class III antiarrhythmic drugs on the muscarinic K+ channel current activated by intracellular loading of GTP{gamma}S (100 µmol/L) in atrial cells. Note that an outward current increased gradually even in the absence of any agonist in the bath solution. The cells were held at -50 mV. Extracellular application of d,l-sotalol, E-4031, or MS-551 and intracellular loading of GTP{gamma}S are shown by the bars above each original current trace.

Effects of Class III Antiarrhythmic Drugs on Adenosine Receptor–Regulated K+ Channel Current in the GTP-Loaded Atrial Cells
To confirm the hypothesis mentioned above, effects of class III antiarrhythmic drugs on the K+ channel current induced by adenosine receptor stimulation in GTP-loaded cells were examined. Although carbachol and adenosine act on different membrane receptors, ie, muscarinic-ACh and A1-adenosine receptors, these agonists induce the muscarinic K+ channel current through the activation of pertussis toxin–sensitive GTP-binding protein in atrial cells.34 On extracellular application of 10 µmol/L adenosine, an outward current through the K+ channel was induced by activation of A1-adenosine receptors in GTP-loaded atrial cells (Fig 5Down). The adenosine-induced outward K+ current also decreased gradually in the continuous presence of adenosine. After the current had almost reached a steady level, d,l-sotalol, E-4031, or MS-551 was applied to the bath solution. Although both E-4031 and MS-551 inhibited the adenosine-induced K+ current, the magnitude of the inhibition of the current was smaller than that of the carbachol-induced current compared at the same concentrations (Figs 5Down and 6Up). The IC50 values of E-4031 and MS-551 for depressing the adenosine-induced current were 172.8 and 122.0 µmol/L, respectively. These IC50 values were similar to those of the two drugs for depressing the GTP{gamma}S-induced current. The concentration-response curves of E-4031 and MS-551 for the inhibitory action on these K+ currents were almost superimposable, as shown in Fig 6Up. In contrast, d,l-sotalol failed to depress the adenosine-induced current in concentrations up to 1000 µmol/L (Figs 5Down and 6Up). These findings support the proposition that the muscarinic K+ channel itself and/or G proteins can be inhibited by E-4031 and MS-551 but not by sotalol.



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  		Figure 5. Tracings showing effects of class III antiarrhythmic drugs on the adenosine receptor–regulated K+ channel current in guinea pig atrial cells. The K+ current was activated by extracellular application of 10 µmol/L adenosine in the GTP (100 µmol/L)–loaded atrial cells (right inset). The holding potential was -50 mV. Application of adenosine and d,l-sotalol, E-4031, and MS-551 is shown by the bars above each original current trace.

Effects of Class III Antiarrhythmic Drugs on the Negative Inotropic Response to Methacholine in Left Atria
Since the results of the patch-clamp experiments indicated that class III antiarrhythmic drugs might interact with atrial muscarinic receptors, further pharmacological experiments using guinea pig left atrial preparations were conducted. In this series of experiments, we used methacholine, a muscarinic agonist having properties slightly different from those of carbachol.35 In electrically driven left atria, methacholine produced a negative inotropic response in a concentration-dependent manner. The maximal negative inotropic response produced by methacholine in the absence of any class III antiarrhythmic drugs (92.4±1.4% decrease from control) was not statistically different from the maximal response in the presence of one of the drugs. Sotalol, E-4031, and MS-551 shifted the concentration-response curves for the negative inotropic effect of methacholine to the right in a parallel manner (Fig 7Down). The slope of d,l-sotalol obtained from the Schild plot was 1.03, which was almost unity, indicating that sotalol shows a purely competitive interaction with atrial muscarinic receptors. The pA2 value of d,l-sotalol obtained from this functional study was 4.31. Conversely, the slope values of the Schild plot with E-4031 and MS-551 were slightly larger than unity (1.14 and 1.28, respectively), although the slope value of E-4031 was not significantly different from unity. These results reflect that these two drugs, especially MS-551, produced a greater rightward shift of the concentration-response curve of methacholine in high concentrations, possibly because of their additional inhibitory action on the K+ channel itself and/or G proteins. The pA2 values of E-4031 and MS-551, tentatively determined from these functional studies, were 4.79 and 4.60, respectively.



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  		Figure 7. Curves showing effects of d,l-sotalol, E-4031, and MS-551 on the negative inotropic effect of methacholine in electrically driven guinea pig left atria. Percent of the maximal negative inotropic response to methacholine is indicated on the ordinates, and concentration of the cholinergic agonist is on the abscissas. S, E, and M indicate d,l-sotalol, E-4031, and MS-551, respectively. Note that three drugs produced rightward shifts of the concentration-response curves for the negative inotropic effect of methacholine in a concentration-dependent manner. A Schild plot of each class III antiarrhythmic drug is shown in the lower panels. DR indicates dose ratio. Logarithmic values of DR-1 are indicated on the ordinates of the Schild plots. The slope values of d,l-sotalol, E-4031, and MS-551 were 1.03, 1.14, and 1.28, respectively. Values are expressed as mean±SEM of 5 to 15 experiments.


*    Discussion
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*Discussion
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K+ Channels as Targets for Class III Antiarrhythmic Drugs
Class III antiarrhythmic agents are defined as drugs that act primarily by prolonging the APD. These drugs are believed to block cardiac K+ channels and thereby reduce the repolarizing currents. More than six different potassium channels have been described in the heart.36 Of these, most currently available class III antiarrhythmic drugs have been shown to block the outward currents through voltage-gated K+ channels, such as the delayed rectifier K+ current (IK), the transient outward current (Ito), and the inward rectifier K+ current (IK1).1 3 8 The IK has been further divided into two components on the basis of various electrophysiological properties.37 38 Both E-4031 and sotalol have been demonstrated to inhibit the rapidly activating component (IKr) without affecting the slowly activating component (IKs) in guinea pig ventricular cells.5 We recently reported that MS-551 also inhibits IK of rabbit ventricular cells, which is saturated around +20 mV and resembles the IKr of guinea pig ventricular cells.7 This drug also inhibited Ito and IK1 in rabbit ventricular cells.7 The class III antiarrhythmic drug tedisamil reportedly depresses Ito in rat ventricular myocytes.4 It was recently reported that RP 58866 and its active enantiomer RP 62719 are highly selective blockers of IK1.39 Thus, most class III antiarrhythmic drugs preferentially act on IK. However, some class III antiarrhythmic drugs selectively or nonselectively inhibit Ito and/or IK1.

Effects of Class III Antiarrhythmic Drugs on IK.ACh
It has been reported that some class I and IV antiarrhythmic drugs inhibit IK.ACh in guinea pig atrial cells.18 19 20 21 Two mechanisms by which these antiarrhythmic drugs inhibit IK.ACh have been proposed: some drugs block the muscarinic receptors and others inhibit the muscarinic K+ channel itself and/or GTP-binding proteins. Disopyramide and pilsicanide belong to the former group, whereas quinidine, flecainide, propafenone, and cibenzoline belong to the latter group.18 20 21 Verapamil, a calcium channel blocker, inhibits not only the muscarinic receptors but also the K+ channel itself and/or G proteins.19 However, effects of class III antiarrhythmic drugs on IK.ACh were not examined. While this paper was in preparation, Escande et al40 reported that RP 58866, a selective IK1 channel blocker, at a concentration of 10 µmol/L slightly depressed IK.ACh. However, the magnitude of the IK.ACh inhibition was very small, and they did not analyze the underlying molecular mechanisms. The present study has clearly demonstrated that three class III antiarrhythmic drugs, sotalol, E-4031, and MS-551, inhibit IK.ACh by different molecular mechanisms. Sotalol effectively inhibited the carbachol-induced IK.ACh, although it failed to affect the outward K+ current induced by intracellular loading of GTP{gamma}S. Since nonhydrolyzable GTP analogues such as GTP{gamma}S bring about antagonist-resistant and persistent activation of IK.ACh,16 these findings indicate that d,l-sotalol may depress the muscarinic K+ channel current by blocking the muscarinic ACh receptors (Fig 8Down). This concept was supported by the findings that sotalol failed to affect the adenosine-induced K+ current in guinea pig atrial cells. The inhibition of the carbachol-induced IK.ACh was observed not only with d,l-sotalol but also with d-sotalol, indicating that d-sotalol can also interact with the muscarinic ACh receptors. In contrast, all the currents induced by carbachol, adenosine, and GTP{gamma}S were inhibited by E-4031 and MS-551, although higher concentrations of these drugs were needed to inhibit the GTP{gamma}S-induced and adenosine-induced currents. These findings suggest that MS-551 and E-4031 block the muscarinic receptors and inhibit the function of the muscarinic K+ channel itself and/or GTP-binding proteins in high concentrations (Fig 8Down).



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  		Figure 8. Schematic representation of the mechanisms by which class III antiarrhythmic drugs inhibit the muscarinic K+ channel current (IK.ACh). IK.ACh can be activated by extracellular application of carbachol, adenosine, or intracellular loading of GTP{gamma}S through the activation of GTP-binding protein (G). Sotalol inhibits IK.ACh by blocking M2 receptors, whereas MS-551 and E-4031 inhibit the current by blocking M2 receptors and depressing the function of K+ channel itself and/or G proteins.

It may be argued that carbachol may not be an ideal muscarinic agonist. However, these class III antiarrhythmic drugs also inhibited the ACh-induced outward current in a concentration-dependent manner. Furthermore, the concentration-response curves for the negative inotropic effect of methacholine, another muscarinic agonist, were shifted to the right in a parallel manner by these class III antiarrhythmic drugs. The slope of the Schild plot with sotalol was almost unity, indicating that sotalol shows a competitive interaction with atrial muscarinic receptors. However, the slope values of the other two drugs, especially of MS-551, were greater than unity. These results might stem from the direct inhibitory action on the K+ channel itself and/or GTP-binding proteins in high concentrations. The concentration-response curves for the inhibitory action of MS-551 on the carbachol-induced current and the GTP{gamma}S-induced current in atrial cells were relatively close, as shown in Fig 6Up. Therefore, the direct inhibitory action of MS-551 might affect the slope value of the Schild plot more seriously. In addition, preliminary radioligand binding experiments in our laboratory showed that these class III antiarrhythmic drugs competitively displaced [3H]N-methylscopolamine binding to muscarinic receptors of guinea pig left atrial membranes (H. Uemura, Y. Hara, M. Endou, K. Mori, H. Nakaya, unpublished observations). Therefore, it can be concluded that these three class III antiarrhythmic drugs in common interact with atrial muscarinic receptors. One may ask whether the concentrations of class III antiarrhythmic drugs that inhibit IK.ACh are comparable to those that prolong APD in ventricular cells. The concentration-response curves for the APD-prolonging effects of E-4031, MS-551, and d,l-sotalol obtained from guinea pig ventricular muscles were similar to those in left atria (H. Nakaya, N. Tohse, M. Kanno, unpublished data). When the concentration-response curves of the three drugs in Fig 6Up were compared with those in Fig 1Up, it would appear that sotalol most potently and E-4031 least potently inhibit IK.ACh in concentrations that produce class III effects in vivo. It was reported that in humans, single and repeated oral administration of 320 mg sotalol resulted in plasma concentrations of 2100 to 3300 µg/L at 3 hours after dosing.41 After intravenous administration of 2 mg/kg sotalol, the plasma concentration was shown to reach a similar level.42 The plasma concentrations of sotalol were well correlated with QTc prolongation, reduction in premature ventricular contractions, and hemodynamic effects in vivo.41 43 44 These plasma concentrations correspond to {approx}10 µmol/L. This concentration of sotalol inhibited the carbachol (1 µmol/L)–induced IK.ACh by only 17.8% but was not far from the apparent dissociation constant for atrial muscarinic receptors (48.6 µmol/L) calculated from its pA2 value. Indeed, intravenous administration of a larger dose of sotalol (loading dose 6 mg/kg plus maintenance dose 3 mg · kg-1 · h-1), which resulted in plasma levels of 12 300 to 9900 µg/L (30 to 40 µmol/L), very effectively terminated atrial fibrillation and prevented its induction in dogs.11 Therefore, high doses of these class III antiarrhythmic drugs may partly exert their inhibitory action on IK.ACh in vivo.

Atrial Fibrillation/Flutter and Class III Antiarrhythmic Drugs
Several experimental models simulating atrial flutter have been developed over the years. They include atrial injury by intercaval crush,9 45 the use of Y-shaped right atrial incision,10 46 and atrial enlargement due to tricuspid insufficiency.47 It has been reported that stimulation of atrial muscarinic receptors by vagal stimulation or ACh infusion can readily induce atrial flutter and fibrillation during rapid atrial pacing in anesthetized dogs.11 22 23 The induction of atrial flutter and fibrillation is assumed to stem from muscarinic receptor–mediated shortening of APDs and refractory periods. Efficacy against experimental flutter and fibrillation has been demonstrated for class III antiarrhythmic drugs, including sotalol9 11 and E-4031.45 48 49 Wang et al11 recently demonstrated that a high dose of d,l-sotalol effectively suppressed the shortening of atrial effective refractory period and the wavelength for reentry during vagal stimulation, thereby terminating and preventing cholinergic atrial fibrillation. Therefore, the inhibition of IK.ACh by sotalol observed in this study may play a role in exerting the antiarrhythmic action in experimental atrial fibrillation. Several clinical reports12 13 14 also showed that sotalol was useful in the termination and prevention of atrial flutter and fibrillation. Enhancement of vagal tone can result in the clinical occurrence of atrial fibrillation,50 although the contribution of increased vagal tone may be variable. Therefore, the anticholinergic effect of sotalol in atrial cells may play a role in the establishment of the antiarrhythmic effect against atrial fibrillation.

Possible but Less Likely Anticholinergic Effects in Extracardiac Tissues
It is recognized that class I antiarrhythmic drugs with anticholinergic activity cause untoward effects such as dry mouth, constipation, and urinary retention.51 Since these class III antiarrhythmic drugs also interacted with cardiac muscarinic receptors in this study, one may expect that these drugs may exert such extracardiac side effects. However, anticholinergic side effects have not been commonly experienced with sotalol in clinical settings.52 At the present time, we do not have a clear understanding of this apparent discrepancy, but we suggest the following explanation. It is known that M3 (glandular-type muscarinic) receptors are functionally important in peripheral tissues such as gastrointestinal tract, urinary bladder, and salivary glands, whereas M2 (cardiac-type muscarinic) receptors are important in the heart.53 There is a class I antiarrhythmic drug showing a high affinity for cardiac-type M2 receptors but a low affinity for glandular type M3 receptors, which results in less anticholinergic effect in peripheral tissues.54 Our preliminary experiments revealed that d,l-sotalol antagonized the negative inotropic response to carbachol in guinea pig left atria several times more potently than the contractile response to carbachol in guinea pig ileum (H. Uemura, Y. Hara, M. Endou, K. Mori, H. Nakaya, unpublished observations). These results suggest that sotalol may have a lower affinity for glandular M3 receptors than for cardiac M2 receptors. Functional and radioligand binding studies are now under way to evaluate anticholinergic effects of these class III antiarrhythmic drugs in peripheral tissues.

Conclusions
This study has demonstrated for the first time the molecular mechanisms by which class III antiarrhythmic drugs inhibit IK.ACh. Sotalol inhibits IK.ACh by blocking the muscarinic receptors in atrial cells. In contrast, E-4031 and MS-551 inhibit the K+ current by blocking the muscarinic receptors and depressing the function of the K+ channel itself and/or G proteins. These effects of class III antiarrhythmic drugs antagonize the muscarinic receptor–mediated shortening of APD and refractory period in atrial cells. The inhibition of IK.ACh may play a role in the termination or prevention of atrial flutter and fibrillation by class III antiarrhythmic drugs.


*    Acknowledgments
 
The authors thank M. Tamagawa and I. Sakurada for their excellent technical assistance and I. Sakashita for her secretarial assistance.

Received August 17, 1994; revision received November 21, 1994; accepted December 18, 1994.


*    References
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