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Naunyn-Schmiedeberg’s Arch Pharmacol (1998) 357 : 268–275 © Springer-Verlag 1998 O R I G I N A L A RT I C L E Lászlo Virág · András Varró · Julius Gy. Papp Effect of disopyramide on potassium currents in rabbit ventricular myocytes Received: 1 September 1997 / Accepted: 10 November 1997 Abstract The effects of disopyramide (1–30 µM) on the 4-aminopyridine sensitive transient outward current (Ito), on the rapid component of the delayed rectifier potassium current (IKr) and on the inward rectifier potassium current (Ik1) were studied in single rabbit ventricular myocytes at 35° C by applying the whole-cell configuration of the patch clamp technique. Disopyramide signifiantly decreased the amplitude of Ito (from 1510 ± 122 pA at control to 1015 ± 21 pA after 30 µM disopyramide at +50 mV; n = 5). This effect was not voltage- or use-dependent. Disopyramide (10 µM) influenced neither the recovery from inactivation of Ito nor the steady-state inactivation curve. The drug dose dependently decreased the time constant of the fast component of the decay of Ito (τf = 6.41 ± 0.25 ms, n = 24 for control; and 2.20 ± 0.38 ms, n = 5 after 30 µM disopyramide at +50 mV). The fractional block caused by 30 µM disopyramide as a function of time was well fitted by a single exponential function with time constant of 1.48 ± 0.18 ms (n = 5), most likely reflecting the binding kinetics of the drug to the open channel. The offset kinetics of the drug was estimated by using a double-pulse protocol and its time constant was 3.9 ± 0.5 ms. Disopyramide (30 µM) did not influence significantly the onset of inactivation measured at –20 mV. The estimated EC50 value for the Ito block by disopyramide was 14.1 µM. Our results are consistent with an open-channel block of Ito by disopyramide, however, a weak, drug-induced increase of the rate of inactivation and a moderate tonic block cannot be excluded. The amplitude of the outward tail current attributed to IKr was depressed dose dependently by disopyramide (after clamping the cells back to the holding potential from +30 mV, 139.5 ± 10.9 pA for control, and 30.7 ± 3.2 pA in the presence of 10 µM disopyramide; n = 11). The estimated EC50 was 1.8 µM. Ito is thus less sensitive to disopyramide L. Virág · A. Varró · J. G. Papp (Y) Department of Pharmacology, Albert Szent-Györgyi Medical University, Dóm tér 12, P.O. Box 115, H-6701 Szeged, Hungary than IKr. Ik1 was not influenced significantly by disopyramide, even when applied in the highest tested concentration (30 µM). It is concluded that in rabbit ventricular myocytes disopyramide blocks not only IKr, but also Ito, both of which may play an important role in the well established repolarization lengthening and antiarrhythmic effects of the drug. Key words Disopyramide · Ito · IK · Ik1 · Patch clamp Introduction The CAST (1989) and SWORD (Waldo et al. 1996) studies have focussed attention on the possible proarrhythmic potency of drugs that reduce conduction velocity (class I drugs) or prolong the action potential (class III drugs). To develop new agents with less proarrhythmic potency, it is very important to understand better the mechanisms of action of antiarrhythmic drugs presently used in clinical practice. The patch-clamp technique has yielded abundant information about the effects of newer antiarrhythmic drugs on cardiac transmembrane ionic currents, but relatively few such studies have been carried out with older antiarrhythmic agents. Therefore, in this study we have analysed the effects of a widely used antiarrhythmic drug, disopyramide, on some important transmembrane potassium currents underlying cardiac repolarization. Disopyramide, an agent with Vaughan-Williams class Ia action, has been used for the treatment of ventricular and supraventricular arrhythmias. Its well-established action includes use-dependent depression of the fast sodium current (INa) underlying the suppression of excitability and conduction velocity (Yatani and Akaike 1985; Gruber and Carmeliet 1989; Hiraoka et al. 1989; Sunami et al. 1991). Another important effect of disopyramide inherent with the Ia antiarrhythmic action is the less-well explored lengthening of repolarization in ventricular muscle (Sekiya and Vaughan-Williams 1963; Kus and Sasyniuk 1975). Repolarization of the action potential in cardiac cells can 269 be due to changes in the magnitude or kinetics of several transmembrane ionic currents. One of the mechanisms by which antiarrhythmic drugs increase action potential duration (APD) is the depression of the delayed rectifier potassium current (IK). Another important current influencing repolarization is the inward rectifier potassium current (Ik1). APD can also be lengthened by increasing inward currents such as the L-type calcium current (ICa) or INa (Buggisch et al. 1985). Disopyramide however influences both ICa and INa oppositely; i.e. by depressing these currents (Yatani and Akaike 1985; Kodama et al. 1986; Coraboeuf et al. 1988; Kotake et al. 1988; Sunami et al. 1991). The ATP-sensitive potassium current (IK,ATP), which is activated in ischaemic/hypoxic conditions and leads to arrhythmogenic shortening of the APD, was also reported to be suppressed by disopyramide (Horie et al. 1992; Wu et al. 1992; de Lorenzi et al. 1995). However, very little information is available about the action of disopyramide on the transient outward current (Ito), the rapid component of the delayed rectifier (IKr) and the inward rectifier potassium current (Ik1) (Coraboeuf et al. 1988; Carmeliet 1993; Martin et al. 1994). Thus the exact mechanism of the effect of disopyramide on repolarization has not been fully elucidated as yet. The present experiments were therefore carried out to gain deeper insight in the mechanisms of disopyramide-evoked repolarization lengthening by measuring the interaction of the drug with the Ito, IKr and Ik1 channels. (mM): NaCl 144, NaH2PO4 0.33, KCl 4.0, CaCl2 1.8, MgCl2 0.53, Glucose 5.5, HEPES 5.0, pH 7.4. Superfusion was maintained by gravity flow. Micropipettes were fabricated from borosilicate glass capillaries (Clark) using a computer-controlled horizontal puller (Mecanex) and had a resistance of 1.5–2.5 MΩ when filled with a pipette solution containing (in mM) KCl 140, MgCl2 4, K2ATP 5, HEPES 10, EGTA 1. The pH of the solution was adjusted to 7.2 by KOH. The external solution in all experiments contained 0.25 mM CdCl2 to block ICa completely. The membrane currents were recorded with an Axopatch-1D amplifier (Axon Instruments, Burlinghdale Calif., USA) using the whole-cell configuration of the patch-clamp technique. After establishing high- (1–10 GΩ)-resistance seals by gentle suction, the cell membrane beneath the tip of the electrode was disrupted by further suction or by applying 1.5-V electrical pulses for 1–5 ms. The cell capacitance was measured by applying a 10-mV hyperpolarizing voltage step from the holding potential of –10 mV. The capacity (103 ± 3 pF, n = 57) was calculated by integration of the capacitive transient divided by the amplitude of the voltage step (10 mV). The series resistance was typically 4–8 MΩ before compensation (usually 50–80% depending on the voltage protocols). Those experiments in which the series resistance was high or substantially increased during the measurements were discarded from the analysis. The membrane currents were digitized using a 333 kHz, analogue-to-digital converter (Digidata 1200, Axon) under software control (PClamp 6.0, Axon). The results were analysed using software programs purchased from Axon (PClamp 6.0). Experiments were carried out at 37° C. Statistical analysis was performed using Student's t-test for paired data. The results were considered to be significant at P < 0.05 level. Numerical data are expressed as means ± SE. Results Effect of disopyramide on Ito Methods Preparation of myocytes. Single ventricular myocytes were obtained by enzymatic dissociation of rabbit hearts. The animals (1–2 kg) were sacrificed by cervical dislocation after receiving 400 IU/kg heparin i.v. The chest was opened and the heart quickly removed and placed into cold (4° C) solution of the following composition (mM): NaCl 135, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, HEPES 10, NaHCO3 4.4, glucose 10, CaCl2 1.8, (pH 7.2). The heart was mounted on a modified, 60-cm high Langendorff column and perfused with the oxygenated and prewarmed (37° C) solution described above. After washing the blood out (3–5 min), the heart was perfused with nominally Ca-free solution with a perfusion pump (flow rate approximately 24 ml/min) for 4 min followed by 12–15 min perfusion (12 ml/min) with the same solution containing 0.5 mg/ml Collagenase (Sigma type I) and 0.04 mg/ml Pronase E (Sigma) with 0.1% albumin. In the 5th min of the enzyme perfusion the [Ca2+] was elevated by 200 µM. After removing the heart from the cannula, the right ventricular free wall was placed into enzyme-free solution containing 1.8 mM CaCl2 and 1% albumin and equilibrated at 37° C for 10 min whereafter the tissue was cut into small fragments. After gentle agitation, the cells were separated from the chunks by filtering through nylon mesh. Sedimentation was used for harvesting cells; as soon as most myocytes reached the bottom of the vessel the supernatant was removed and replaced by Tyrode solution containing 1.8 mM CaCl2. This procedure was repeated 2 times. The cells were stored at room temperature in HEPES-buffered Tyrode solution. Experimental techniques. One drop of cell suspension was placed in a transparent recording chamber mounted on the stage of an inverted microscope (TMS Nikon, Tokyo, Japan) and the individual myocytes allowed to settle to the bottom of the recording chamber for at least 5 min before superfusion. HEPES-buffered Tyrode solution was used as normal superfusate. This solution contained There is general agreement that Ito consists of two components (Coraboeuf and Carmeliet 1982; Escande et al. 1987; Hiraoka and Kawano 1989). The first component of Ito is sensitive to the K+ channel blocker 4-aminopyridine. The second component is most likely a Ca2+-sensitive Cl– current (Zygmunt and Gibbons 1991). It therefore depends on Ca2+ release from the sarcoplasmic reticulum and is abolished by agents that block Ca2+ release, such as ryanodine and caffeine. In our experiments the Ca2+-dependent component was absent because the cells were dialysed by the pipette solution containing 1 mM EGTA. It is difficult to separate IK from Ito completely. It therefore cannot be ruled out that this current contaminated the measurements of Ito. However, the amplitude of IK is small compared with that of Ito. Also, the inactivation kinetics of Ito are considerably faster than the activation of IK, even considering that in the rabbit heart only the rapid component of the delayed rectifier is present. Although both activation and inactivation of INa are faster than those of Ito, it is theoretically possible that changes of INa may influence the measurement of Ito. With less negative holding potentials, INa could have been inactivated but in this case the amplitude of Itowould have been greatly reduced because of partial inactivation. Because continuous application of TTX throughout the measurements to eliminate INa would have greatly increased the cost of the study, we tested the effect of 50 µM TTX on Ito in five separate experiments. Application of TTX did not significantly alter the amplitude of the current in the voltage range of –10 to 270 +50 mV (not shown) suggesting that the possible influence of INa is negligible. Figure 1 shows the effect of 30 µM disopyramide on Ito recorded in single rabbit ventricular myocytes. The current was activated by 400-ms depolarizing voltage pulses from the holding potential of –90 mV to test potentials ranging from 0 to +60 mV with a pulse frequency of 0.33 Hz. The amplitude of Ito was measured as the difference between the peak of Ito and the sustained current at the end of the Fig. 1 A, B The effect of 30 µM disopyramide on transient outward current (Ito) in rabbit ventricular myocytes. The current was activated by 400-ms depolarizing voltage pulses from holding potential of –90 mV to test potentials ranging from 0 to 50 mV with a pulse frequency of 0.33 Hz. A Original current traces in control conditions (left) and in the presence of 30 µM disopyramide (right) recorded at 0, 10, 20, 30, 40, 50 mV test potentials. B Effect of 30 µM disopyramide on current amplitude at different test potentials (open circles: control, closed circles: 30 µM disopyramide). * P < 0.05, n = 5 Fig. 2 A Effect of disopyramide on the inactivation kinetics of Ito. Upper panel, original current traces, lower panel, concentration/response curve for effect of disopyramide on the fast inactivation time constant (open circle, pooled control, n = 24; closed circles drug n = 5–9, * P < 0.05). B Dose-Concentration/ response curves for the inhibition of Ito by disopyramide, calculated from the charge movement through the channels (n = 4–8). The current was activated by a train of voltage pulses from the holding potential of –90 mV to 50 mV with a pulse frequency of 0.33 Hz pulse. Disopyramide significantly decreased the amplitude of the current (1510 ± 122 pA control, 1015 ± 21 pA after 30 µM disopyramide at +50 mV; n = 5). Original current traces obtained in a representative experiment in control conditions and after application of 30 µM disopyramide are shown in Fig. 1 A. Figure 1 B shows the effect of the drug on the current/voltage relationship. The drug effect was not significantly voltage-dependent. Superimposed traces of a control response and those recorded in the presence of 30 µM disopyramide show in Fig. 2 A (upper panel) that the decay of the current was accelerated by the drug. The effect of disopyramide on the decay of Ito was studied by applying 300-ms depolarizing voltage pulses from the holding potential of –90 mV to +50 mV in every 3 s. The decay of current was well fitted by a double exponential function. The time constant for the initial fast component decreased as a function of increasing disopyramide concentrations (Fig. 2 A). The time constant for the slow component was 30–100 ms and showed no apparent dependence on drug concentration in the 1–30 µM range. The acceleration of the decay kinetics of the current is an important drug effect, as reflected in the dose/response curve in Fig. 2 B, which shows the total charge movement through the channels as a functin of disopyramide concentration. The total charge movement was calculated by integrating the current traces from the peak to the end of the pulse taking the steady-state current as baseline. The estimated EC50 value was 14.1 µM. Figure 3 A illustrates the results of experiments in which the possible use-dependent effect of 10 µM disopyramide on Ito was tested. After at least 1 min resting at the holding potential of –90 mV, a series of 400-ms depolarizing pulses to +50 mV were applied at 1 Hz. Disopyramide (10 µM) did not depress Ito use dependently. The recovery of Ito from inactivation (Fig. 3 B) was well fitted by a single exponential curve. Disopyramide (10 µM) did not influence the reactivation process (the time constant was 1080 ± 259 ms for control and 1112 ± 239 ms in the presence of 10 µM disopyramide, n = 5) suggesting that the 271 Fig. 3 A Lack of use-dependent effect of 10 µM disopyramide on Ito. After at least 1 min rest, a series of 400-ms depolarizing voltage pulses to +50 mV from holding potential of –90 mV were applied at a frequency of 1 Hz (see inset). The illustration shows current amplitude as a function of pulse number (open circles, control conditions; closed circles 10 µM disopyramide, * P < 0.05, n = 6). B Recovery of Ito from inactivation under control conditions (open circles) and after application of 10 µM disopyramide (closed circles, n = 5). The double-pulse protocol used (see inset) consisted of two identical 400-ms depolarizing pulses (P1, P2) to +50 mV from the holding potential of –90 mV. The P1-P2 interval was 0–10 s. The normalized current (P2/P1) was plotted as a function of P1-P2 interval. C Voltage dependence of the steady-state inactivation of Itounder control conditions (open circles) and in the presence of 10 µM disopyramide (closed circles, n = 6). Prepulses (500 ms long) to potentials ranging from –70 mV to 0 mV were applied before 400-ms depolarizing test pulses to +50 mV. The holding potential was –90 mV. D Onset of inactivation of Ito. Prepulses to –20 mV with a duration of 0–100 ms were applied before the 400ms test pulses to +50 mV. The holding potential was –90 mV. Control conditions (open circles), 30 µM disopyramide (closed circles, n = 5) offset kinetics of the drug are faster than the recovery of Ito from inactivation. The voltage dependence of the steady-state inactivation in the presence of 10 µM disopyramide was evaluated using a conventional prepulse protocol (see inset in Fig. 3 C). The results in Fig. 3 C show that 10 µM disopyramide produced no detectable shift in the steady-state inactivation curve, suggesting that the drug does not bind preferentially to the inactivated state of the channel, or at least does not influence the voltage dependence of the function of inactivation gate. The increased rate of decay of the current in the presence of disopyramide may result either from the blockade of open channels or the modification of inactivation gating i.e. increasing of the rate of inactivation. To test the latter possibility, the onset of inactivation was studied under control conditions and in the presence of 30 µM disopyramide (Fig. 3D). The voltage protocol is shown in the inset of Fig. 3D; the test pulse to +50 mV was pre- ceded by a conditioning pulse to –20 mV lasting for 0– 100 ms. The holding potential was –90 mV. Since at this conditioning potential an overwhelming portion of the channels can be expected to be in the inactivated state and very few channels in the open state, any change in the kinetics in the presence of the drug would be due to changing the inactivation gating rather than to open-channel block. The decline of the current amplitude with increasing duration of the conditioning pulse was well fitted by a single exponential function with a time constant of 25.5 ± 3.6 ms for control and 18.7 ± 2.0 ms in the presence of the drug (n.s., n = 5). This finding suggests that the inactivation gating at –20 mV is not influenced by disopyramide. Figure 4 A shows the fractional block by 30 µM disopyramide as a function of time after clamping a cell to +50 mV from the holding potential of –90 mV. This relationship was well fitted by a single exponential function with time constant of 1.48 ± 0.18 ms (n = 5) which reflects the binding kinetics of disopyramide to the open channel. The offset kinetics of the drug were estimated by using a double-pulse protocol (see inset, Fig. 4 B). In control conditions the amplitude of the current, normalized to the current amplitude activated by the prepulse, was just slightly changed when the interpulse interval increased representing the recovery of Ito from inactivation which is too slow compared with the present time scale. However, in the presence of 30 µM disopyramide the normalized current amplitude/interpulse interval relationship was well fitted by a single exponential function which reflects the rate of unbinding of the drug from the channel (Fig. 4 B). The time constant was 3.9 ± 0.5 ms (n = 3). Effect of disopyramide on the rapid component of delayed rectifier potassium current It is known that, in rabbit ventricular myocytes, only the rapid component of IK (IKr) exists (Giles and Imaizumi 272 Fig. 4 A Fractional block (drug-induced current reduction divided by control current) of Ito as a function of time after clamping the cell to the test potential of +50 mV in a representative experiment. The cell was clamped to +50 mV at time t = 0 ms. The inset shows the original current records from which the individual values of fractional block were calculated. This relationship was fitted by a single exponential function. Data obtained during the 1st ms were omitted from the fitting process because of distortion by capacitive transients (see inset). B Double-pulse protocol used for estimating the offset kinetics of disopyramide (see inset). The first pulse (P1) was a 5-ms depolarizing voltage pulse from the holding potential of –90 mV to +50 mV. The duration of the test pulse (P2) was 400 ms. The current amplitude, normalized to the current amplitude activated P1, is shown as a function of the P1-P2 interval under control conditions (open circles) and in the presence of 30 µM disopyramide (closed circles). * P < 0.05, n = 3 1988; Carmeliet 1992). The effect of disopyramide on this current was therefore measured by applying 3-s depolarizing voltage pulses from the holding potential of –40 mV to various test potentials ranging from –10 to 30 mV in 10 mV increments at a pulse frequency of 0.2 Hz (see inset, Fig. 5 B). On clamping the cells back to –40 mV, an outward tail current was observed, which was attributed to IKr. In cells exposed to disopyramide superfusion for 5–10 min significant reduction of the tail current was observed (after clamping the cells back to the holding potential from +30 mV, 139.5 ± 10.9 pA for control, 30.7 ± 3.2 pA in the presence of 10 µM disopyramide; n = 11). Original current traces obtained in a representative experiment in control conditions and after application of 10 µM disopyramide are shown in Fig. 5 A. Figure 5 B displays the peak tail current amplitude at –40 mV as a function of test potential in the absence and in the presence of 10 µM disopyramide. In Fig. 6 the dose/response relationship of the disopyramide evoked IKr block is displayed. It is important to note that the estimated EC50 value for IKr was 1.8 µM, considerably lower than that for Ito. Effect of disopyramide on the inward rectifier potassium current The possible effect of disopyramide on Ik1 was studied by measuring the steady state current level at the end of the 400-ms voltage pulse in the voltage range of –120 to 0 mV with a pulse frequency of 0.33 Hz. The holding potential was –90 mV (see inset, Fig. 7). As Fig. 7 shows, disopyramide superfusion, even at a relatively high concentration (30 µM), did not influence significantly the steady state current/voltage relation (–1077 ± 88 pA for control, –1057 ± 113 pA 30 µM disopyramide at –100 mV; n = 7) suggesting the lack of effect of disopyramide on Ik1. Fig. 5 A, B Effect of 10 µM disopyramide on the outward tail current, attributed to the rapid component of the delayed rectifier K+ current (IKr), in rabbit ventricular myocytes. Outward tail current was observed after clamping the cells back to –40 mV after application of 3-s depolarizing voltage pulse from the holding potential of –40 mV to various test potentials ranging from –10 to +30 mV with a pulse frequency of 0.2 Hz. A Original current traces under control conditions (left) and in the presence of 10 µM disopyramide (right) recorded at 0, +10, +20, +30 mV test potentials. B Effect of 10 µM disopyramide on the amplitude of the peak outward tail current at different test potentials (open circles control, closed circles 10 µM disopyramide). * P < 0.05, n = 8 273 Fig. 6 Concentration/response curve for the inhibition of IKr evoked by disopyramide, calculated from the amplitude of the peak outward tail current (n = 3–8) Fig. 7 Steady-state current/voltage relationship for the inward rectifier potassium current (open squares control, closed squares 30 µM disopyramide, n = 7). The steady-state current was measured at the end of 400-ms pulses in the voltage range of –120 to 0 mV. The holding potential was –90 mV Discussion In mammalian ventricular myocytes the most important K currents responsible for the repolarization are believed to be Ito, IK and Ik1. However, there are considerable species dependencies of these currents. Ito is a very important repolarizing current in the atrium and is responsible for the early repolarizing phase in the ventricle in various mammalian species including man, and it may also influence the plateau phase of the action potential. This current, which is a rapidly activating and inactivating, voltagegated transmembrane current in response to depolarization, is large in rabbit ventricular muscle (Giles and Imaizumi 1988; Hiraoka and Kawano 1989), but it is negligible or absent in guinea-pig ventricular myocytes (Josephson et al. 1984). The non-inactivating and relatively slowly activating, voltage-gated IK, which is considered as the most important current initiating the repolarization in ventricle, has two components in guinea-pig; a rapidly activating one with small amplitude (IKr) and a slowly activating one with large amplitude (IKs) (Sanguinetti and Jurkiewicz 1990; Colatsky et al. 1990). In rabbit ventricular cells IKs is small or absent and only IKr exists (Giles and Imaizumi 1988; Carmeliet 1992). The Ik1 plays a major role in maintaining the resting membrane potential and its amplitude is decreasing upon depolarization, but at potentials more negative than –40 mV it carries a repolarizing current contributing to the final repolarization (Kass et al. 1990; Martin and Chinn 1992; Shimoni et al. 1992). In this study the effects of disopyramide, an antiarrhythmic drug known to lengthen cardiac repolarization, on the above transmembrane K currents were studied in rabbit ventricular myocytes. Our main findings are as follows: (1) disopyramide decreases the amplitude of Ito in the 3–30 µM concentration range with an estimated EC50 of 14.1 µM; (2) the amplitude of IKr is markedly depressed by disopyramide with an estimated EC50 of 1.8 µM; (3) Ito is much less sensitive to disopyramide than IKr and (4) Ik1 is not influenced by disopyramide, even at the highest tested concentration (30 µM). In human ventricular myocytes large transient outward current are recorded, but IK is hardly detectable (Wettwer et al. 1993, 1994; Beuckelmann et al. 1993; Näbauer et al. 1993). Therefore, the main repolarizing current systems measured in our study in the rabbit are quite similar to those found by others in human ventricular myocytes. Consequently, the effect of disopyramide described in clinical practice may involve the depression of transient outward and the rapid component of the delayed rectifier potassium currents as observed in this study. The effect of disopyramide on Ito has not yet been extensively studied. In the only investigation reported so far (Coraboeuf et al. 1988) disopyramide, at concentrations as high as 60 µM, significantly depressed the amplitude of Ito in sheep Purkinje fibres measured by the two microelectrode, voltage-clamp technique. In the present study, in addition to confirming this previous finding in ventricular myocytes, we also showed that disopyramide, at therapeutically relevant concentrations (Koch-Weser 1979), decreased the amount of current. The block of Ito by disopyramide is not use-dependent and without any apparent effect on the recovery from inactivation and the steady-state inactivation of the current. These facts imply that the occupation of the binding site by disopyramide does not influence the operation of the inactivation gate. In this study, current decay increased with increasing disopyramide concentration. One possible explanation of this phenomenon is an increased rate of inactivation, though this seems unlikely because disopyramide did not affect significantly the onset of inactivation at –20 mV supposing that the inactivation process is weakly voltage dependent. The acceleration of the onset of inactivation in the presence of disopyramide proved to be not significant statistically, but a weak, drug-induced increase in the rate of inactivation, at least at higher drug concentrations, cannot be excluded. This assumption seems to be supported by the findings presented in Fig. 4 B. The figure clearly shows that in under control conditions about 30% of the channels are inactivated during the first 5-ms depolarizing pulse (first open circle, Fig. 4 B). After application of the drug this value is higher (last closed circle), though it is 274 not significantly different from the control value, indicating that the inactivation process might be faster in the presence of the drug. Therefore, the observed increase in the rate of decay of the current in the presence of disopyramide can be due to drug binding to the open state of the channel, thus causing open-channel block, however, the peak amplitude of the current is also decreased by the drug. Although decline of the current amplitude might also be due to the increased rate of current decay, minor binding of disopyramide to the closed state of the channel, i.e. a weak tonic block by the drug, cannot be ruled out. Similar properties have been noted for Ito block by another I/A antiarrhythmic drug, quinidine (Imaizumi and Giles 1987; Wang et al. 1995). These authors also observed an acceleration in the current decay, which was interpreted as being consistent with open-channel block. However, quinidine slowed the recovery of Ito from inactivation and caused use-dependent inhibition of the current. The lack of change in Ito reactivation kinetics in the case of disopyramide suggests that the unblocking kinetics of quinidine is slower than that of disopyramide. The first study reporting that disopyramide blocks IK was performed in the sinus node preparation using the two microelectrode voltage-clamp technique (Kotake et al. 1985; Kotake et al. 1988). Later, Hiraoka et al. (1989) have reported that disopyramide (11 µM) depresses IK in guinea-pig ventricular myocytes, and Carmeliet (1993) has extended these observations by showing that disopyramide blocks the rapid component of IK (IKr) use dependently in rabbit ventricular myocytes. Our results, in addition to confirming these previous findings in rabbit ventricular myocytes, provide evidence that this effect is present at relatively low concentrations; the estimated EC50 of 1.8 µM calculated from the dose/response relation is considerably lower than the corresponding value for Ito block. A similar effect on IK has also been reported with some other class I drugs, such as quinidine (Furukawa et al. 1989; Balser et al. 1991; Hiraoka et al. 1986), flecainide (Follmer and Colatsky 1990; Slawsky and Castle 1994) and propafenone (Slawsky and Castle 1994; Delpon et al. 1995). The effect of disopyramide on Ik1 seems rather controversial. Coraboeuf et al. (1988) have reported reduction of the instantaneous background current in the presence of disopyramide in sheep Purkinje fibres but the concentration of the drug (60 µM) was several times higher than the upper limit of the therapeutic range. Martin et al. (1994) have found no significant effect of disopyramide (1–20 µM) on the open probability of inward rectifier potassium channels in cell-attached patches of rabbit ventricular cells at room temperature. Our experiments with disopyramide support the results of the latter study, since disopyramide did not influence Ik1, even in relatively high concentrations (30 µM), thereby suggesting the lack of effect of the drug on the inward rectifier potassium current. In summary, it is concluded that, like some other class I antiarrhythmic drugs, disopyramide blocks both Ito and IKr. This may play a significant role in the prolongation of repolarization by disopyramide in ventricular and atrial tissues. This effect of disopyramide is very likely to be involved in the suppression of reentry-type ventricular tachycardia and atrial fibrillation in clinical settings. Acknowledgements This work was supported by grants from the Hungarian National Research Foundation (OTKA F 6328 and OTKA T 016651). References Balser JR, Bennett PB, Hondeghem LM, Roden DM (1991) Suppression of time-dependent outward current in guinea-pig ventricular myocytes. Action of quinidine and amiodarone. 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