Download Effect of disopyramide on potassium currents in rabbit ventricular

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
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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).
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