Introduction

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Elimination of L-type calcium current (I_Ca-L) is commonly required during pharmacological or physiological studies assessing modulation of I_K in ventricular myocytes. Successful block of I_Ca-L has been achieved by use of either organic or inorganic calcium channel antagonists (Reuter et al., 1985; Kamp and Miller, 1987; Kamp et al., 1988). However, specificity of block of calcium channel antagonists relative to cardiac potassium currents is of great concern in order not to bias interpretation of records obtained.

Previous studies have suggested that calcium channel antagonists may not be as specific as generally thought (Hume, 1985; Nerbonne and Gurney, 1987; Richard et al., 1988; Fan and Hiraoka, 1991; Follmer et al., 1992). For example, cobalt modifies amplitude and kinetics of deactivating I_K tail current in guinea pig ventricular myocytes (Fan and Hiraoka, 1991). Moreover, in this species, cobalt positively shifts the voltage-dependent activation curve of I_K (Fan and Hiraoka, 1991). In cat ventricular myocytes, cadmium shifts to more positive potentials the voltage-dependent activation curve of I_K (Follmer et al., 1992). Contrasting results have been obtained in studies looking at the effects of organic I_Ca-L blockers on I_K. Among dihydropyridine derivatives, nisoldipine does not reduce I_K tail current after 1-sec depolarizing pulses in calf Purkinje fibers although nicardipine reduces I_K in frog atrial fibers at concentrations greater than 0.01 µM (Kass, 1982; Richard et al., 1988).

In guinea pig ventricular myocytes, I_K is composed of a rapidly activating, inwardly rectifying component (I_Kr) and a slowly activating, outwardly rectifying component (I_Ks) (Sanguinetti and Jurkiewicz, 1990a). I_Kr is the major repolarizing current during normal action potential duration and the target of most methanesulfonamide class III antiarrhythmic agents (Sanguinetti and Jurkiewicz, 1990a; Follmer and Colatsky, 1990; Wettwer et al., 1992). In contrast, I_Ks is selectively blocked by diuretics and has a more predominant role in repolarization during prolonged action potential duration or rapid heart rates during which I_Ks activation may accumulate (Courtney et al., 1992; Jurkiewicz and Sanguinetti, 1993; Turgeon et al., 1994; Daleau and Turgeon, 1994). Numerous studies assessing I_K properties in guinea pig ventricular myocytes have used either cadmium or nisoldipine to block I_Ca-L. Although these drugs became the gold standard blockers of I_Ca-L, no study carefully looked at their effects on I_K components in guinea pig ventricular myocytes.

Therefore, the objective of our study was to compare effects of cadmium and nisoldipine on the amplitude, kinetics and activation and deactivation current-voltage curves of I_Kr and I_Ks in guinea pig ventricular myocytes.

	Methods

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Patch-Clamp Experiments

Cell preparation and solutions. Experiments were performed on single ventricular myocytes obtained from adult guinea pig hearts by use of an enzymatic dissociation technique. All solutions used during the cell isolation procedure were oxygenated and maintained at 37°C. The hearts were mounted on a Langendorff apparatus and rinsed for 2 min with a calcium-free solution (solution A) containing (in mM): NaCl 132, KCl 4.8, MgCl₂ 1.2, HEPES 10, glucose 5, pH was adjusted to 7.35 with NaOH. Then, hearts were perfused with a low sodium-high potassium HEPES-buffered solution (solution B, in mM): NaCl 17, HEPES 10, KCl 5.4, K-glutamate 128, MgCl₂ 1) for a period of 2 min. At the end of this period, perfusion of solution B containing collagenase (final concentration 300 U/ml; Worthington Biochemical Corp., Freehold, NJ) and protease (0.7 U/ml; Sigma Chemical Co., St. Louis, MO) was started and continued until the system pressure dropped to 15 mm Hg (approximately 15 min). Hearts were finally reperfused with a solution made of 60% solution B and 40% solution A containing 0.5 mM CaCl₂. At this point, the ventricles were cut down and minced slightly to increase cell yield. After filtration through 200-µm nylon mesh, the dispersed cells were washed by centrifugation (200 rpm, 2 min), resuspended in solution A containing 1.8 mM CaCl₂ and maintained at 30°C before use.

Electrophysiological Measurements

A small aliquot of dissociated cells was placed in a 0.5-ml chamber mounted on the stage of an inverted microscope (model CK2, Olympus, Lake Success, NY). Cells were allowed to adhere to the coverslip at the bottom of the chamber and then superfused continuously with the external solution pre-warmed (30°C) by a Peltier device (Medical System Corp., Greenvale, NY). In our experiments, complete replacement of external solution contained in the chamber was achieved within 2 to 3 min when the superfusion rate was 2 ml/min.

All currents were recorded in the whole cell, voltage-clamp configuration of the patch-clamp technique using an Axopatch-1D amplifier (Axon Instruments Inc., Burlingame, CA). Voltage-clamp command pulses were generated by a 12-bit digital-to-analog converter (model TL/1, Axon Instruments Inc.) controlled by the pClamp software package (version 4.05b; Axon Instruments Inc.). Heat-polished patch-clamp pipette electrodes used (capillary glass from Radnoti, Starbore glass capillary tubing 1.2 mm O.D.) had a tip resistance of 3 to 5 M when filled with the pipette solution. Series resistance was compensated 50 to 80% to improve fidelity of whole cell voltage-clamp measurements.

Protocols

Rod-shape cells with clear cross striations, resting potential of at least 78 mV and stable I_K and I_K1 currents (as assessed during a baseline period of at least 4 min) were used. Effects of nisoldipine and cadmium on the I_Kr and I_Ks activating components of I_K were studied in cells held at 40 mV (to inactivate I_Na) and depolarized by pulses lasting either 250 msec (I_K250), 450 msec (I_K450) or 5000 msec (I_K5000). Test potentials of depolarizing pulses varied between 20 and +50 mV. I_K was measured from the peak magnitude of tail current obtained upon repolarization to 40 mV.

Data Storage and Analysis

Currents were filtered at either 2 KHz (I_K250 and I_K450 protocols) or 100 Hz (I_K5000 protocol) by a four-pole Bessel filter (3 dB/octave). Currents were sampled at 4 KHz (I_K250 and I_K450) and 400 Hz (I_K5000) by use of a 12-bit analog-to-digital converter (TL-1 DMA, Axon Instruments) and stored on hard disk for subsequent analysis. Data are presented as mean ± S.D. Statistically significant differences in I_K activating curves for I_K250 and I_K5000 in the presence of cadmium and nisoldipine were compared by a Student's paired t test. Best fit of data was established by comparison of ² analysis. Level of statistical significance was set at P < .05.

	Results

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Comparative effects of cadmium and nisoldipine on the delayed rectifier potassium current. Figure 1A illustrates outward currents elicited by a 5-sec pulse to various depolarizing potentials from a holding potential of 40 mV. Deactivating currents (tails) were recorded on repolarization to 40 mV. In these recordings, the same guinea pig ventricular myocyte was perfused with a low-calcium (0.1 mM) Tyrode solution containing either 0.1 mM cadmium (left panels) or nisoldipine 0.2 µM (right panels). When nisoldipine replaced cadmium in the perfusate, I_K5000 activating and tail currents elicited by depolarizing steps to voltages of more than 0 mV decreased although those elicited by lower depolarizing voltages increased. Figure 1B illustrates current-voltage relationships observed during the experiment shown in figure 1A for the deactivating currents. At a test potential of +50 mV, superfusion of the myocyte with the nisoldipine containing buffer reduced I_K5000 tail current by 90 pA (27%). In contrast, at a test potential of 20 mV, I_K5000 tail current was increased 25 pA (50%) by nisoldipine. A reversal of the effect was observed upon reperfusion of the myocyte with the cadmium containing buffer solution (fig. 1C).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A shows IK activating currents elicited by 5-sec voltage tests between 10 and +50 mV and tail currents recorded following repolarization to 40 mV, during application of either 0.1 mM cadmium or 0.2 µM nisoldipine. B compares the current-voltage relationship of IK tail current amplitude obtained in the presence of either 0.1 mM cadmium or 0.2 µM nisoldipine. Changes in IK tail amplitude caused by nisoldipine were fully reversible (C).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Current-voltage relationship of IK tail current obtained upon repolarization to 40 mV after test pulses of a duration of either 250 (A) or 5000 (B) msec. This relationship illustrates mean data of normalized current from 9 cells (* P < .05).

View this table: [in this window] [in a new window]   TABLE 1 Changes in the fast time constant (1) of IK5000 deactivating current induced by exposure of cells to cadmium or nisoldipine

Effects of cadmium on the delayed rectifier potassium current. In this series of experiments, control recordings were obtained during superfusion of cells with normal Tyrode solution (containing 1.8 mM calcium). Test pulse duration was either 250 msec (I_K250), 450 msec (I_K450) or 5000 msec (I_K5000). Under control conditions, a large inward L-type calcium current was elicited (fig. 3). When the external solution was replaced by a solution containing cadmium 0.1 mM (calcium concentration lowered to 0.1 mM to reproduce experimental conditions used during study of I_K), the L-type calcium current was eliminated. However, the amplitude of I_K tail currents was increased for all pulse durations tested at +10 mV and for I_K tail current elicited by 5 sec pulses to 10 mV (fig. 4). In contrast, the amplitudes of I_K tail current was reduced for the shorter pulses (i.e., 450 and 250 msec) after voltage steps to 10 mV. This effect was reproducibly observed in five cells tested (fig. 4, A and B). Amplitudes of I_K250 and I_K5000 tail current normalized by cell capacitance recorded after test pulses to voltages <0 mV during cadmium tended to be decreased compared to control conditions but were increased for voltage pulses  +20 mV (P < .05).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of cadmium on IK were assessed after a control period during which cells were bathed in a normal Tyrode solution. IK current was elicited by voltage pulses of three different durations (5000 msec  IK5000; 450 msec  IK450 and 250 msec  IK250) at two voltage levels (10 and +10 mV). In control conditions, test pulses elicited large inward calcium currents that activated and inactivated during IK activation. However, this current was totally eliminated by 0.1 mM cadmium.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Current-voltage relationship of IK tail current observed in five cells tested in the presence or absence of 0.1 mM cadmium (* P < .05). Current was normalized for cell capacitance and obtained on repolarization to 40 mV after 250- (A) or 5000- (B) msec depolarizing pulse durations.

View this table: [in this window] [in a new window]   TABLE 2 Changes in the fast time constant (1) of IK5000 deactivating current indced by cadmium

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Recordings of membrane currents obtained in cells previously exposed to E-4031 (5 µM) to eliminate IKr. Currents were elicited by 5000-millisecond depolarizing steps from 10 to +50 mV followed by repolarization to 30 mV. Recordings were obtained in the presence of a low calcium concentration (0.1 mM) at baseline (A) and during superfusion with 0.1 mM cadmium (B). Dashed arrows indicate zero current. To better discriminate between ICa inactivating and IKs activating currents, time-independent background current was subtracted from all recordings except for the activating current elicited by a test pulse to 10 mV. Plot of current-voltage relation of IK tail current measured after long pulses (5000 msec) to various test potentials at baseline () and in the presence of 0.1 mM cadmium () is illustrated in C.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent effect of cadmium on the current-voltage relationship of IK tail current recorded after 5-sec test pulses to stepwise increased potentials. Effects of three concentrations of cadmium, i.e., 20 µM (filled circles), 100 µM (open circles) and 500 µM (squares) on the same cell are presented.

Effects of nisoldipine on the delayed rectifier potassium current. Effects of nisoldipine 0.2 µM on I_K were determined and compared to recordings obtained using a normal Tyrode solution (containing 1.8 mM calcium) as the external solution. Traces in figure 7A show that this concentration of nisoldipine entirely blocks I_Ca-L but does not alter amplitude of I_K250 tail current. As well no changes were observed on I_K activating and tail currents during long pulses (5000 msec; fig. 7B). These effects were reproduced in five cells tested (fig. 8, A and B). Finally, no significant changes in I_K5000 deactivating constant (₁) were observed by the superfusion of cells with nisoldipine 0.2 µM (table 3).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   IK activating and tail currents elicited by pulse protocols that allow discrimination between effects on the rapid and slow components of IK are shown in control conditions (superfusion of a normal Tyrode solution containing 1.8 mM calcium) and in the presence of 0.2 µM nisoldipine. In A, cell potential was clamped at 0 mV during 250 msec and then at 40 mV to deactivate IK, conditions that favor the presence of the rapid component of IK. Isi was suppressed during superfusion of nisoldipine but IK tail was kept unchanged by the drug. Also, nisoldipine did not modified IK in conditions that favor the presence of the slow component of IK (5-sec test pulse to +50 mV) (B). In this case, no inward current was observed because the voltage of +50 mV is near or more than the reversal potential of Isi.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Current-voltage relationship of IK tail current observed in five cells tested in the presence or absence of 0.2 µM nisoldipine. Current was normalized for cell capacitance and obtained upon repolarization to 40 mV following 250- (A) or 5000- (B) msec depolarizing pulse durations.

View this table: [in this window] [in a new window]   TABLE 3 Changes in the fast time constant (1) of IK5000 deactivating current induced by nisoldipine

	Discussion

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Our experiments examined the effects of cadmium and nisoldipine on the two components of I_K, namely I_Kr and I_Ks. Studies were conducted at concentrations of cadmium and nisoldipine usually used to block I_Ca-L in experiments designed to characterize modulation of I_K (Arena and Kass, 1988; Balser et al., 1990; Sanguinetti and Jurkiewicz, 1990a; Daleau and Turgeon, 1994). Our findings have shown a decrease in I_K amplitude at low voltage steps which is consistent with an induction of a shift in I_Kr activation previously reported (Follmer et al., 1992). In addition, our results also provide evidence that the inorganic calcium channel blocker cadmium also shifts to positive voltages the activation curve of I_Ks and increases its amplitude. However, the organic dihydropyridine derivative calcium channel antagonist nisoldipine, had no effects on I_K activation curve and had minimal effects on deactivation kinetics. These results suggest that nisoldipine is a better tool than cadmium for use in study of I_K.

I_K recorded from guinea pig ventricular myocytes in the presence of 0.1 mM cadmium was decreased when elicited by voltage steps  0 mV but increased when elicited by voltage steps  20 mV compared to I_K obtained in the presence of nisoldipine or a 1.8 mM CaCl₂-containing Tyrode solution. Also, with E-4031 in the extracellular solution, cadmium increased I_K amplitude elicited at high voltage tested and positively shifts the I-V curve at low voltages. From these experiments, we cannot discriminate between a positive shift in I_Kr activation or a reduction in I_Kr amplitude. However, it has been described in cat ventricular myocytes, where I_K is mainly due to I_Kr, that cadmium added directly to the bath solution containing 3 µM nitrendipine, decreased I_K at low depolarizing potentials but increased peak I_K tail current and shifted the voltage dependence of activation to more positive potentials by about 15 mV (Follmer et al., 1992). On the basis of our results and the results of Follmer et al. (1992), we propose that cadmium shifts toward positive potentials I_Kr and I_Ks I-V curves and increases I_Ks amplitude elicited at voltages  +20 mV.

In contrast, experiments conducted with nisoldipine were not associated with alteration in I_K characteristics which confirms previous results (Kass, 1982) obtained in Purkinje fibers with 10 µM nisoldipine (i.e., 50 times the concentration usually used to block I_Ca). From these experiments, we concluded that cadmium is almost exclusively responsible for the difference observed in I_K characteristics recorded in the presence of either cadmium or nisoldipine. In guinea pig ventricular myocytes, lanthanum, an inorganic calcium current blocker, blocks I_Kr and shifts positively the voltage dependency of I_Ks activation curve (Sanguinetti and Jurkiewicz, 1990b). Cobalt, another inorganic salt, alters I_K properties similar to lanthanum, with a more pronounced reduction of I_K at negative potentials and a positive shift of I_K I-V curve in the whole voltage range of I_Ks activation (Fan and Hiraoka, 1991).

Deactivation kinetics of I_K were faster in the presence of cadmium compared to nisoldipine. Recordings obtained showed that the fast time constant (₁) of I_K deactivation was increased by about 180% when 0.1 mM cadmium was replaced by 0.2 µM nisoldipine. This effect was voltage independent. However, when cadmium was added to a normal Tyrode solution, ₁ was decreased to 65 to 70% of control value, consistent with the increase of ₁ observed when cadmium was replaced by nisoldipine in the bath solution. In contrast, no changes in ₁ were observed when nisoldipine was added to a normal Tyrode solution. Modification in the kinetics of I_K produced by cadmium suggests a direct interaction of the compound with negatively charged gating particles. Follmer et al. (1992) also observed a faster decay of I_K tail currents in the presence of cadmium in cat ventricular myocytes. In contrast, such an effect was absent in frog heart cells (Duchatelle-Gourdon et al., 1989). In our experiments, cadmium had minimum effects on the fast time constant of deactivation in the presence of E-4031 which is consistent with a predominant effect on I_Kr deactivation kinetics.

Finally we have assessed effects of different concentrations of cadmium on I_K, i.e., 20, 100 and 500 µM (fig. 6). Changes in I_K amplitude observed at lower depolarizing potentials were almost unaffected by cadmium concentration of more than 20 µM although increase in I_K observed at positive depolarizing potentials remains concentration dependent.

In conclusion, we have shown that cadmium shifts the activation curve and increases the amplitude of the slow component of the delayed rectifier potassium current in guinea pig ventricular myocytes. These effects are complementary to the previously demonstrated shift in I_Kr I-V curve caused by cadmium. These effects were observed at a concentration that overlaps with its effects on the L-type calcium current. Therefore, these effects must be considered in the interpretation of I_K-modulation studies where this cation is present. However, nisoldipine seems to be a more powerful and specific tool, at a concentration aimed to fully inhibit I_Ca in I_K studies.