Effects of the Diuretic Agent Indapamide on Na⁺, Transient Outward, and Delayed Rectifier Currents in Canine Atrial Myocytes

From the Department of Medicine (Z.W., S.N.), University of Montreal; the Research Center (Y.L., L.Y., Z.W., S.N.), Montreal Heart Institute; and the Department of Pharmacology and Therapeutics (L.Y., S.N.), McGill University, Montreal, Quebec, Canada.

Correspondence to Stanley Nattel, MD, Research Center, Montreal Heart Institute, 5000 Belanger Street East, Montreal, Quebec H1T 1C8, Canada. E-mail nattel{at}icm.umontreal.ca

	Abstract

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Abstract—The diuretic agent indapamide has been reported to block the slow component of the delayed rectifier K⁺ current (I_Ks) without altering the rapid component (I_Kr) or the inward rectifier current and has been used as a pharmacological probe for I_Ks; however, the effects of indapamide on Na⁺ (I_Na), L-type Ca²⁺ (I_Ca), and transient outward K⁺ (I_to) currents have not been determined. We applied tight-seal, whole-cell, patch-clamp techniques to assess the effects of indapamide on I_Na, I_to, I_Ca, and I_Ks in canine atrial myocytes. Indapamide inhibited I_Na, I_to, and I_Ks in a concentration-dependent and reversible way, without altering I_Ca. Block increased with depolarization, with the 50% blocking concentration (EC₅₀) decreasing from 129±26 µmol/L (at -60 mV) to 79±17 µmol/L (at -10 mV) for I_Na, from 174±19 µmol/L (at +10 mV) to 98±7 µmol/L (at +60 mV) for I_to, and from 148±28 µmol/L (at +10 mV) to 86±18 µmol/L (at +60 mV) for I_Ks. Significant inhibition was seen at concentrations as low as 10 µmol/L for all 3 currents. In addition, indapamide effectively inhibited the ultrarapid delayed rectifier current in a voltage-independent way, with an EC₅₀ of 138±7 µmol/L at +10 mV. Standard microelectrode experiments showed the effects of indapamide on the action potential to be consistent with the ionic actions seen. We conclude that in addition to its well-recognized I_Ks-blocking action, indapamide also inhibits I_Na and I_to effectively and with similar potency. Thus, indapamide is not a reliable pharmacological probe with which to study the specific effects of I_Ks blockade, and I_Na and I_to block may contribute to the potential profile of cardiac actions of the compound.

Key Words: ion channel blocker • ECG • cardiac antiarrhythmic drug • heart electrophysiology

	Introduction

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The delayed rectifier K⁺ current (I_K) plays a critical role in the repolarization of the cardiac action potential.¹ Two components of I_K have been recognized since the 1960s.² Study of the physiological role of these components was simplified by the demonstration that the rapid, inwardly rectifying component (I_Kr) is selectively blocked by a variety of class III antiarrhythmic agents, including D-sotalol and E-4031.^{3 4} More recently, our understanding of I_Kr and the slower component, I_Ks, was substantially enhanced by the elucidation of their underlying molecular bases, channels encoded by the human ether-a-go-go–related gene (HERG)⁵ and channels resulting from the coassembly of subunits encoded by minK and K_vLQT1, respectively.^{6 7}

Numerous studies have used selective I_Kr-blocking drugs to evaluate the physiological role of I_Kr, and the results of selective pharmacological blockade of I_Kr are well established. On the other hand, the role of I_Ks has been more difficult to determine because of a lack of specific blockers. Several years ago, the diuretic agent indapamide was shown to be a selective blocker of I_Ks.⁸ I_Ks blockade has been suggested to be a potential contributor to the torsades de pointes–potentiating properties of indapamide, along with diuretic-induced hypokalemia.⁸ Furthermore, indapamide has been shown to have antiarrhythmic properties against ventricular arrhythmias caused by ischemia and reperfusion in guinea pig hearts⁹ and to potentiate the effects of D-sotalol on ventricular repolarization in dogs.¹⁰ These results have been interpreted as effects resulting from the I_Ks-selective blocking actions of indapamide.^{9 10} A limitation to our understanding of these findings and of the usefulness of indapamide as a pharmacological probe is the incomplete information available about the channel-blocking profile of the drug. Indapamide block of I_K in guinea pig ventricle strongly suggests block of I_Ks with minimal or no inhibition of I_Kr, and the drug does not alter inward rectifier K⁺ current.⁸ However, indapamide effects on other important currents, such as the Na⁺ current (I_Na), the transient outward K⁺ current (I_to), and L-type Ca²⁺ current (I_Ca), were not determined. The present study was designed to determine whether indapamide inhibits I_Na, I_to, or I_Ca, and if so, how the magnitude of these effects compares with changes in I_Ks at the same drug concentration.

	Materials and Methods

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Myocyte Isolation
Adult mongrel dogs of either sex (18.4 to 26.8 kg) were anesthetized with pentobarbital sodium (30 mg/kg IV), and their hearts were quickly removed and immersed in Tyrode's solution (for composition, see Solutions and Drugs) at room temperature. All solutions were equilibrated with 100% O₂. The right coronary artery was cannulated, and the right atrium was dissected free and perfused with Ca²⁺-containing Tyrode's solution at 37°C for 10 minutes until the effluent was clear of blood. Any leaks from arterial branches were ligated with silk thread to ensure adequate perfusion. The tissue was then perfused at 12 mL/min with Ca²⁺-free Tyrode's solution for 20 minutes, followed by 40 minutes of perfusion with the same solution containing collagenase (100 U/mL CLS II collagenase, Worthington Biochemical) and 1% BSA (Sigma Chemical Co). Tissue samples (2 to 3 mm in diameter) were removed every 5 minutes, beginning 40 minutes after the onset of exposure to collagenase. Samples were minced into small chunks (1.5 mm³), and cells were obtained by trituration with a Pasteur pipette. Cells were kept at room temperature in a high-K⁺ storage solution (see Solutions and Drugs) before being used.

Solutions and Drugs
The standard Tyrode's solution for cell isolation and patch-clamp studies contained the following (mmol/L): NaCl 136, KCl 5.4, MgCl₂ 1.0, NaH₂PO₄ 0.33, CaCl₂ 2.0, dextrose 10, and HEPES 10 (pH adjusted to 7.4 with NaOH). The high-K⁺ storage solution contained (mmol/L) KCl 20, KH₂PO₄ 10, dextrose 25, mannitol 40, potassium glutamate 70, ß-hydroxybutyric acid 10, taurine 20, and EGTA 10, along with 1% albumin (pH adjusted to 7.4 with KOH). The standard Tyrode's solution was used to record I_to, with a holding potential (HP) of -50 mV used to inactivate I_Na; when a more negative HP was used to study I_to, NaCl was replaced with Tris-HCl. The extracellular solution used to record I_Ks contained (mmol/L) NaCl 136, KCl 5.4, MgCl₂ 1.0, NaH₂PO₄ 0.33, dextrose 10, and HEPES 10. The extracellular solution used to record I_Ca contained (mmol/L) tetraethylammonium chloride (TEA) 136, KCl 5.4, MgCl₂ 1.0, NaH₂PO₄ 0.33, CaCl₂ 2.0, dextrose 10, and HEPES 10. The extracellular solution used to record I_Na contained (mmol/L) CsCl 132.5, NaCl 5.0, MgCl₂ 1.0, CaCl₂ 1.0, dextrose 11, and HEPES 20. The pipette solution used to study K⁺ currents contained (mmol/L) potassium aspartate 110, KCl 20, MgCl₂ 1.0, HEPES 5.0, EGTA 5.0, Mg₂-ATP 5.0, GTP 0.1, and Na₂-phosphocreatine 5.0. The pipette solution used to record I_Ca contained (mmol/L) CsCl 20.0, cesium aspartate 110.0, HEPES 10.0, EGTA 10, MgCl₂ 1.0, Mg₂-ATP 5.0, phosphocreatine 5.0, and GTP (lithium salt) 0.1. The pipette solution used to record I_Na contained (mmol/L) CsF 135, NaCl 5.0, HEPES 5.0, EGTA 10, and Mg₂-ATP 5.0. The pH levels of internal and external solutions were adjusted to 7.2 and 7.35, respectively, with the use of KOH (pipette solution) and NaOH (external solution) for K⁺ currents. CsOH (both pipette and external solutions) was used for studies of I_Ca and I_Na. CdCl₂ (0.2 mmol/L) was added to the superfusate to block I_Ca for experiments studying I_to, and 0.1 mmol/L CdCl₂ was used for I_Na recording. 4-Aminopyridine (4-AP, 2 mmol/L) and ryanodine (2 µmol/L) were added to the superfusate for I_Ca recording to block I_to, the canine ultrarapid delayed rectifier current (I_Kur.d), and the Ca²⁺-dependent Cl^- current. 4-AP (2 mmol/L), nifedipine (10 µmol/L), dofetilide (1 µmol/L), and atropine (0.2 µmol/L) were added to block I_to, I_Kur.d, I_Ca, I_Kr, and any acetylcholine-dependent K⁺ current, respectively, for I_Ks recording. In selected experiments we also used TEA, made up as a 2 mol/L stock solution with pH adjusted to 7.0 with HCl. All chemicals and drugs were obtained from Sigma.

Voltage-Clamp Technique
Only quiescent rod-shaped cells lacking membrane deformities and showing clear cross striations were studied. A small aliquot of the solution containing the isolated cells was placed in a 1-mL chamber mounted on the stage of an inverted microscope. Five minutes was allowed for cell adhesion to the bottom of the chamber, and then the cells were superfused at 3 mL/min with the standard extracellular solution. The bath temperature was kept constant to study I_Na (17°C), I_Ca (23°C), and I_to and I_Ks (36°C) by a Peltier-effect device. In order to minimize rundown of I_Ca during drug infusion, a multiple-capillary device was positioned next to the cell, and the perfusate was changed within 300 ms with the use of a solenoid switch.

The whole-cell patch-clamp technique was used to record ionic currents in the voltage-clamp mode. Borosilicate glass electrodes (outer diameter, 1.0 mm) were used, with tip resistances of 2.5 to 5 M for studies of I_to, I_Ks, and I_Ca and 1.0 to 1.5 M for I_Na, when filled with the appropriate internal solution. Currents were recorded with an Axopatch 1-D amplifier (Axon Instruments). Command pulses were generated by a 12-bit digital-to-analog converter controlled by pCLAMP software (Axon Instruments). Recordings were low pass–filtered at 2 kHz for K⁺ currents and I_Ca and at 5 kHz for I_Na, and series resistance (R_S) was compensated. Recordings were digitized at twice the filter frequency (model TM 125, Scientific Solutions) and stored on the hard disk of an IBM-compatible computer. Time-dependent currents without apparent inactivation (delayed rectifiers) were measured on the basis of time-dependent step or tail current amplitude. Inactivating current (I_to, I_Na, and I_Ca) amplitudes were measured on the basis of differences between peak current and steady-state current at the end of a depolarizing pulse.

Junction potentials were zeroed before formation of the membrane-pipette seal in Tyrode's solution. Mean seal resistance averaged 21.8±4.3 G (n=21 cells). Several minutes after seal formation, the membrane was ruptured by gentle suction to establish the whole-cell configuration for voltage clamping. R_S was electrically compensated to minimize the duration of the capacitive surge on the current recording and the (current-induced) voltage drop across the pipette (access resistance). R_S along the clamp circuit was estimated by dividing the time constant obtained by fitting the decay of the capacitive transient by the calculated membrane capacitance (C_m, the time integral of the capacitive surge measured in response to 5-mV hyperpolarizing steps from an HP of -60 mV divided by the voltage step).

Before R_S compensation, the decay of the capacitive surge for cells used to study I_Na had time constants of 340±37 µs (C_m, 50.3±3.4 pF; n=12). Time constants averaged 471±54 µs (C_m, 64.5±7.8 pF; n=16) for cells used to study I_to and 484±64 µs (C_m, 64.1±6.6 pF; n=10) for cells used to study I_Ks. Precompensation R_S values were 6.8±0.4, 7.3±0.5, and 7.6±0.6 M for cells used to study I_Na, I_to, and I_Ks, respectively. After compensation, the time constants were reduced to 134±14, 158±16, and 189±15 µs, and R_S values were reduced to 1.8±0.1, 2.8±0.2, and 3.5±0.2 M for electrodes used to study I_Na, I_to and I_Ks, respectively. Cells with significant leak current were rejected, and leakage compensation was not applied.

Standard Microelectrode Experiments
In order to evaluate the effects of indapamide on the action potential under conditions as close to physiological as possible, we applied standard fine-tipped microelectrode techniques to canine right atrial preparations (2x2 cm) perfused at 18 mL/min via the right coronary artery. A heated-water sleeve and insulated bath were used to maintain the preparation at 37°C. The extracellular solution for perfusion contained (mmol/L) NaCl 120, KCl 4, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, CaCl₂ 1.25, and dextrose 5.5, aerated with 95% O₂/5% CO₂. Floating glass microelectrodes mounted on AgCl-coated silver wire and filled with 3 mol/L KCl (tip resistance, 15 to 20 M) were coupled to a World Precision Instruments KS-700 amplifier and used to record action potentials. The preparations were stimulated continuously with 2-ms twice-threshold pulses at 1 Hz. Action potentials were recorded under control conditions and then after the addition of 300 µmol/L indapamide to the intracellular solution. Because the preparations contracted vigorously, it was difficult to maintain stable impalement of the same cell under both control and drug conditions. We therefore recorded multiple action potentials before and after drug exposure in each preparation.

Data Analysis
Group data are expressed as the mean±SEM. Statistical comparisons were performed with a paired t test (for comparisons between group means when only 2 groups were compared) and ANOVA with Bonferroni-adjusted t tests for multiple comparisons. A 2-tailed P<0.05 was taken to indicate statistical significance. Nonlinear curve-fitting was performed with the use of the CLAMPfit routine in pCLAMP or Sigmaplot software (Jandel Scientific).

	Results

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Effects of Indapamide on I_Ks
Experiments to study I_Ks were performed in the presence of 1 µmol/L dofetilide to prevent contamination by I_Kr. Currents were elicited by 3-s depolarizing pulses at 0.1 Hz from -50 mV to the voltages indicated, followed by 1-s repolarizations to -40 mV to observe tail currents (inset of Figure 1). Indapamide produced concentration-dependent reversible inhibition of I_Ks as illustrated by results from a representative cell in Figure 1A. Step and tail currents were almost completely eliminated by the largest concentration tested (1000 µmol/L). Figure 1B shows the current-voltage relation for I_Ks under control conditions and in the presence of indapamide in 5 cells in which data were obtained under control conditions and after each concentration of the drug at all voltages. Indapamide progressively reduced I_Ks at all voltages at which appreciable current was detected.

View larger version (23K): [in this window] [in a new window]   Figure 1. IKs inhibition by indapamide (IP) (1 µmol/L dofetilide was present in the bath to block IKr). A, Currents from a representative cell, recorded with 3-s depolarizing pulses (0.1 Hz) from -50 to +60 mV, followed by a 1-s repolarization to -40 mV. Results were obtained in the same cell under control conditions, in the presence of 10, 100, 300, and 1000 µmol/L IP, and after washout (). B, Current-voltage relations for IKs under control conditions and in the presence of 4 concentrations of IP, as obtained with the voltage protocol (3-s steps at 0.1 Hz followed by 1-s repolarizations to -40 mV) shown in the inset at the top of the figure. Results are mean±SEM for 5 cells studied under all conditions. C, Voltage dependence of IKs activation (mean±SEM of 5 cells). Results were obtained by dividing tail currents (Itail) at each step potential by Itail after a pulse to +60 mV. Curves are best-fit Boltzmann relations to mean data.

The voltage dependence of I_Ks activation was determined by normalizing tail currents for each step voltage to the tail current after a pulse to +60 mV. The results were fitted by a Boltzmann function of the form I/I_max=1/{1+exp[(V_1/2-V)/k]}, where I is tail current at step voltage V, I_max is tail current after a step to +60 mV, V_1/2 is half-activation voltage, and k is a slope factor. Mean data for 5 cells under control conditions and in the presence of 100 and 300 µmol/L indapamide are shown in Figure 1C, along with best-fit Boltzmann functions to mean data. Overall, V_1/2 averaged 24.6±6.7, 21.9±4.7, and 23.1±6.3 mV for control and 100 and 300 µmol/L indapamide, and the slope factors were 13.3±0.9, 12.6±3.1, and 12.2±1.9 mV, respectively (P=NS for V_1/2 and k in the presence of the drug versus control).

Envelope-of-tails tests were performed before and after indapamide, with the protocol illustrated in Figure 2A. Original recordings under control conditions and in the presence of 100 µmol/L indapamide are shown. Figure 2B shows mean±SEM tail/step current ratios under control conditions and at 3 drug concentrations. As expected for I_K in the presence of 1 µmol/L dofetilide, the ratio is constant and unchanged by test pulse (TP) duration under all conditions.

View larger version (20K): [in this window] [in a new window]   Figure 2. Envelope of tails for IKs (data obtained in presence of 1 µmol/L dofetilide). IP indicates indapamide. A, Results from a representative cell obtained with the protocol at the top (1 Hz). B, Tail/step current ratio (It/Is) for IKs (mean±SEM for 5 cells studied under all conditions).

Drug Effects on I_Na
Figure 3A shows I_Na recordings obtained during 40-ms depolarizations applied at 0.1 Hz from an HP of -140 to -40 mV under control conditions and then in the presence of progressively increasing indapamide concentrations. In 7 cells, I_Na averaged 2.56±0.33 nA under control conditions compared with 2.45±0.32, 2.15±0.28, 1.35±0.21, and 1.12±0.21 pA in the presence of 10 (P<0.01 versus control), 100 (P<0.01), 300 (P<0.01), and 1000 (P<0.001) µmol/L indapamide, respectively. In 6 cells in which washout data were obtained, 97.8±2.1% reversal of drug effect was noted. Figure 3B shows mean data for I_Na as a function of TP voltage in 5 cells studied under control conditions and in the presence of 300 µmol/L indapamide. The drug produced significant and substantial reductions in I_Na at all voltages between -50 and -10 mV. As shown by the triangles in Figure 3B, the percentage reduction in I_Na caused by the drug increased at more positive voltages, and the effect of the drug showed significant (P<0.05) voltage dependence.

View larger version (25K): [in this window] [in a new window]   Figure 3. INa inhibition by indapamide (IP). A, Original recordings from a representative cell obtained with 40-ms pulses to -40 mV (0.1 Hz) under control conditions, in the presence of progressively increasing drug concentrations, and after washout. B, Current-voltage relation for INa (mean±SEM for 5 cells) under control conditions () and in the presence of 300 µmol/L IP (). indicates percent reduction of INa caused by the drug at each voltage (P<0.05 for voltage dependence of INa inhibition). **P<0.01 vs control.

Figure 4 shows the results of experiments to assess the use dependence of drug block of I_Na and effects on voltage-dependent inactivation. On the onset of 50-ms pulses to -40 mV, I_Na showed slight decreases under control conditions. In the presence of indapamide (300 µmol/L), a use-dependent onset of block was observed, as shown by the mean±SEM data for 5 cells studied before and after the drug at 1 Hz (Figure 4A) and 2 Hz (Figure 4B). The time constants for onset of block averaged 0.084±0.02 and 0.057±0.01 pulse^-1 at 1 and 2 Hz, respectively. To study shifts in the voltage dependence of I_Na inactivation, 2-s prepulses were applied to voltages between -130 and -30 mV and were followed by a 50-ms TP to -40 mV. Indapamide (1000 µmol/L) shifted the inactivation V_1/2 from a mean of -96.8±0.9 mV to -105.7±1.1 mV (P<0.01) in 5 cells (Figure 4C). Washout was attempted in all cells and was incomplete, with V_1/2 averaging -103.1±1.0 mV after 12 minutes of washout, likely reflecting a time-dependent negative inactivation shift, which opposed the reversal of drug effect on washout. The drug also significantly altered the slope of the inactivation curve, with k averaging 4.3±0.2 mV under control conditions, 5.9±0.3 mV (P<0.001) in the presence of indapamide, and 4.4±0.1 mV after washout (P=NS versus control).

View larger version (19K): [in this window] [in a new window]   Figure 4. Use dependence and voltage-dependent inactivation of INa under control conditions and in presence of indapamide (IP, 300 µmol/L) in 5 cells. A and B, Current as function of pulse number after the onset of 50-ms pulses from -140 to -40 mV at 1 Hz (A) and 2 Hz (B). The onset of block was monoexponential, with rate constants of 0.084±0.02 and 0.057±0.01 pulse-1 at 1 and 2 Hz, respectively. C, Voltage dependence of INa inactivation in 5 cells, studied with 2-s prepulses to the voltages indicated, followed by 50-ms TPs to -40 mV (0.1 Hz). Results are mean±SEM, and best-fit Boltzmann relations are shown.

Effects of Indapamide on I_to
The effects of indapamide on I_to are illustrated in Figure 5. Figure 5A shows representative currents recorded on 60-ms pulses from -50 to +60 mV (0.1 Hz) in one cell under control conditions, in the presence of 10, 100, 300, and 1000 µmol/L indapamide, and after washout of the drug at the highest concentration. The current progressively decreased as drug concentration increased. Mean data for the effects of indapamide on I_to in 7 cells are shown in Figure 5B. Significant changes were noted at concentrations as low as 10 µmol/L. Figure 5C shows effects of indapamide on the voltage dependence of I_to inactivation, evaluated with the use of 1000-ms prepulses to a variety of voltages, followed by a 200-ms TP to +60 mV. Mean data for 4 cells are shown along with the best-fit Boltzmann distribution curves in Figure 5C. Mean values for V_1/2 and k under control conditions were -30.9±0.5 and 6.6±0.7 mV, respectively. In the presence of 100 and 1000 µmol/L indapamide, V_1/2 averaged -37.9±1.8 (P<0.05 versus control) and -45.9±2.1 (P<0.01 versus control), and k averaged 7.5±0.7 (P<0.05 versus control) and 9.9±0.9 mV (P<0.01 versus control), respectively.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effects of indapamide (IP) on Ito. A, Representative recordings with the protocol shown (0.1 Hz) from one cell studied under control conditions, in the presence of 4 concentrations of IP, and after washout (W/O). B, Mean±SEM current-voltage relations for Ito obtained in 8 cells studied under all conditions indicated with protocol in inset (0.1 Hz). *P<0.05 and **P<0.01 vs control. C, Voltage dependence of Ito inactivation in 4 cells studied with the use of 1-s prepulses from an HP of -80 mV followed by 200-ms TPs to +60 mV. Results are mean±SEM, and best-fit Boltzmann relations to mean data are shown.

Figure 5 shows drug effects on both I_to and a sustained pedestal component of current. We have previously shown that the pedestal component is carried by a highly 4AP- and TEA-sensitive ultrarapid delayed rectifier current, which we have called I_Kur.d (for ultrarapid delayed rectifier, dog).¹¹ We performed additional experiments to verify the effects of indapamide on I_to in the absence of I_Kur.d and on I_Kur.d itself. Figure 6A shows currents recorded in the same fashion as in Figure 5A, but with TEA added to the superfusate for all recordings at a concentration (10 mmol/L) that fully inhibits I_Kur.d.¹¹ As in Figure 5A, I_to is inhibited in a concentration-dependent and reversible way; however, in the absence of I_Kur.d, only the transient component is affected (Figure 6A)—the sustained current inhibition seen in Figure 5A is absent. Mean I_to density-voltage relations from 3 cells studied in the presence of 10 mmol/L TEA before and after each indapamide concentration are shown in Figure 6B. The concentration-dependent effects on I_to are quite similar to those noted in a separate group of 7 cells the absence of TEA (Figure 5B).

View larger version (18K): [in this window] [in a new window]   Figure 6. A. Original recordings of Ito in the absence and presence of indapamide (IP) at the concentrations indicated by the legend at the bottom and after washout (W/O). The same protocols and solutions were applied as in Figure 5A, except that TEA (10 mmol/L) was present in all extracellular solutions to inhibit IKur.d. B, Mean±SEM data for IP inhibition of Ito obtained in 3 cells at all concentrations, with 10 mmol/L TEA in the superfusate to inhibit IKur.d. *P<0.05 and **P<0.01 vs control.

We used 100-ms prepulses to +40 mV 10 ms before TPs, as shown in Figure 7A, in order to inactivate I_to and record selectively I_Kur.d.¹¹ Indapamide produced concentration-dependent and reversible inhibition of I_Kur.d, as illustrated in Figure 7A and illustrated by the mean data from 6 cells shown in Figure 7B. Figure 7C shows the percentage change in I_Kur.d as a function of TP. No significant voltage dependence was noted for the effects of indapamide on I_Kur.d.

View larger version (28K): [in this window] [in a new window]   Figure 7. A, IKur.d recordings obtained with the protocol shown in the inset. IKur.d was separated from nonspecific background (time-independent) current with the use of 1000 µmol/L indapamide (IP), which fully inhibited all time-dependent currents. B, Mean±SEM IKur.d step current as a function of TP voltage recorded from 6 cells as shown in panel A. *P<0.05 and **P<0.01 vs control. C, Percentage change in IKur.d as a function of TP.

Effects of Indapamide on I_Ca
Figure 8 shows examples of I_Ca before and after exposure to 1000 µmol/L indapamide. In the cell shown, I_Ca was not altered. Similar results were obtained in a total of 7 cells. For example, I_Ca at +10 mV averaged 339±45 pA under control conditions and 331±41 pA in the presence of indapamide in these 7 cells.

View larger version (21K): [in this window] [in a new window]   Figure 8. ICa recordings obtained with the protocol shown (0.1 Hz) from a representative cell before (A) and after (B) 1000 µmol/L indapamide (IP). Similar results were obtained in 7 cells.

Concentration Dependence of I_Ks, I_Na, I_to, and I_Kur.d Inhibition
Figure 9 shows the concentration-dependent inhibition of I_Ks, I_Na, I_to, and I_Kur.d by indapamide on voltage steps to +50, -40, +60, and +50 mV, respectively, with inhibition of each current fitted by an equation of the form E=E_max · Cⁿ/(EC₅₀ⁿ+Cⁿ), where E is the effect observed at concentration C, EC₅₀ is the concentration for half-maximal inhibition, and n is the Hill coefficient. At the voltages indicated, EC₅₀ values averaged 103±21 µmol/L for I_Na, 98±7 µmol/L for I_to, 87±20 µmol/L for I_Ks, and 128±11 µmol/L for I_Kur.d and were not significantly different among currents.

View larger version (12K): [in this window] [in a new window]   Figure 9. Concentration-response curve for indapamide inhibition of INa (at -40 mV, n=7), Ito (at +60 mV, n=7), IKs (at +50 mV, n=7), and IKur.d (at +50 mV, n=6). Results are mean±SEM. Curves shown are best nonlinear curve fits by the equation E=Emax/{1+(EC50/[D])n}, where Emax is the maximal effect, E is the effect at a concentration [D], EC50 is the concentration for half-maximal action, and n is the Hill coefficient.

Figure 10 shows an analysis of the voltage dependence of drug potency. The EC₅₀ was determined as illustrated in Figure 9 at each TP for which there was measurable current. Significant voltage dependence of the EC₅₀ was noted for I_Na (P<0.05), I_to (P<0.01), and I_Ks (P<0.05). In contrast, the EC₅₀ for I_Kur.d inhibition was not significantly voltage dependent.

View larger version (22K): [in this window] [in a new window]   Figure 10. EC50 (mean±SEM) for inhibition of INa (A), Ito (B), IKs step current (C), and IKur.d (D) as a function of TP. Results for INa were obtained from 5 cells with the protocol illustrated in Figure 3A, results for Ito were from 3 cells studied as shown in Figure 6A, results for IKs were from 5 cells studied as shown in Figure 1A, and results for IKur.d were from 6 cells studied as shown in Figure 7A. The EC50 decreased at more positive voltages for INa, IKs, and Ito (P<0.05 for voltage dependence of effects on INa and IKs and P<0.01 for Ito).

Effects on Action Potentials
Action potentials were recorded from 18 cells before and 18 cells after exposure to indapamide in 3 preparations. Examples of typical recordings are shown in Figure 11. Figure 11A shows a control action potential, and an action potential after exposure to indapamide is shown in Figure 11B. Indapamide reduced the overshoot, decreased the amplitude of phase 1 repolarization, and raised the plateau level. For direct comparison, the same action potentials are superimposed in Figure 11C. Overall, indapamide did not significantly alter resting potential, which averaged -78±1 mV before and -76±1 mV after the drug, but reduced action potential amplitude from 102±1 to 94±1 mV (P<0.001). Consistent with the effect of the drug on I_to, it strongly delayed early repolarization; eg, action potential duration (APD) to 20% repolarization averaged 43±2 ms before and 83±2 ms after the drug (P<0.001), a 97±7% increase. APD to 50% repolarization was also substantially increased, by 41±5% (from 91±3 to 129±2 ms, P<0.001), and APD to 90% repolarization was also significantly increased (from 193±4 to 229±3 ms, P<0.001), albeit to a much lesser extent (by 17±3%).

View larger version (8K): [in this window] [in a new window]   Figure 11. Typical action potentials recorded at 1 Hz before (A) and after (B) indapamide (IP). To facilitate visual comparison, the same action potentials are superimposed in panel C.

	Discussion

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Since the demonstration by Turgeon et al⁸ that indapamide blocks I_Ks effectively with no measurable effect on I_Kr, the drug has been used as a tool to block I_Ks.^{9 10 12} In the present study, we show that indapamide inhibits I_Na and I_to with a potency similar to that of its I_Ks-blocking action.

Comparison With Previous Studies of Indapamide
Turgeon et al⁸ noted that the EC₅₀ for indapamide block of tail currents depends on activating pulse potential, with smaller values at more positive step potentials at which I_Ks is more important than I_Kr. In the present study, we found that the blocking potency of I_Ks depends on step potential under conditions that prevent any contamination from I_Kr. The EC₅₀s that we measured for I_Ks inhibition, in the range of 100 to 150 µmol/L, are very similar to those reported by Turgeon et al for comparable voltages. Furthermore, we found qualitatively similar voltage dependence of the indapamide EC₅₀ for blocking I_Ks, I_to, and I_Na. Indapamide shifted the inactivation voltage dependence of both I_to and I_Na and produced use-dependent I_Na block, suggesting state-dependent blocking mechanisms. Because the goal of the present study was to determine whether indapamide blocks currents other than I_Ks in order to clarify its specificity of action, and not to evaluate in detail the mechanisms by which it blocks them, we did not perform additional experiments to study in greater detail state-dependent blocking actions. We were unable to identify other studies of indapamide on cardiac I_Na or I_to in the literature.

Potential Significance
There is a need for selective blockers with which to study the role of I_Ks in cardiac repolarization and in the control of arrhythmia occurrence. Since the demonstration that indapamide strongly inhibits I_Ks without blocking I_Kr or inward rectifier K⁺ current,⁸ investigators have used the drug as a probe for I_Ks. Indapamide has been shown to suppress ventricular arrhythmias resulting from cardiac ischemia followed by reperfusion⁹ and to modulate the electrophysiological effects of the I_Kr blocker D-,L-sotalol in dogs.¹⁰ These actions have been interpreted in terms of the physiological role of I_Ks; however, our results suggest that the effects on I_Na or I_to could have been involved in the actions of the drug and that great caution is needed in interpreting the electrophysiological results of indapamide administration. Two more recently described blockers of I_Ks may be more specific tools, the experimental compounds chromanol 293B^{13 14} and L-735821.¹⁵ Although the latter compounds have some ancillary effects (293B blocks I_to at concentrations 20 times those that inhibit I_Ks¹⁴ and L-735821 shifts the voltage dependence of I_Ks¹⁵ ), they appear to be much more selective in blocking I_Ks than indapamide.

Diuretic agents are known to potentiate the risk of acquired long-QT syndromes with associated ventricular tachyarrhythmias in patients taking action potential–prolonging drugs.^{16 17 18 19} In addition to the widely recognized potential importance of diuretic-induced electrolyte abnormalities, such as hypokalemia and hypomagnesemia, the demonstration that compounds such as indapamide⁸ and triamterene²⁰ inhibit I_K raised the possibility of direct electrophysiological mechanisms of cardiotoxicity by these agents. Our studies indicate that in addition to affecting I_K, indapamide can alter cardiac electrophysiology by blocking I_Na and I_to and that the latter actions are as likely to be manifest at a given drug concentration as I_K block. Therefore, a broader spectrum of ion channel blockade may contribute to the direct cardiac effects of indapamide.

Potential Limitations
We did not evaluate state-dependent drug actions in any detail. Although we obtained evidence in terms of voltage- and use-dependent properties pointing to state-dependent blocking properties of indapamide, our goal was not to define mechanisms of channel block by the drug but to evaluate further its specificity. Substantial additional work would be necessary to define in detail the mechanisms by which indapamide interacts with each of the channels we studied. Furthermore, such questions might be better addressed by studying channels encoded by specific cDNAs expressed in model systems, thus avoiding contamination from other currents and permitting the study of molecular mechanisms with site-directed mutagenesis.^{21 22}

We selected the temperature for voltage-clamp studies in order to achieve adequate voltage-clamp and current recording. Consequently, K⁺ current was recorded at 36°C; I_Ca, at 23°C; and I_Na, at 17°C. Ion current properties are known to be temperature dependent, and drug-induced channel blocking actions may also vary with temperature. This needs to be considered in interpreting our results. This limitation does not apply to our recordings of K⁺ currents, which were all obtained at the same temperature and which showed the effects of indapamide on I_to and I_Kur.d to be of the same potency as the effects on I_Ks. Action potential recordings from multicellular preparations at 37°C showed drug effects inconsistent with pure I_Ks inhibition and consistent with inhibition of I_to (decreased phase 1 amplitude and delayed early repolarization) and I_Na (decreased action potential amplitude with unchanged resting potential).

Time-dependent hyperpolarizing shifts in the voltage dependence of I_Na inactivation are common in tight-seal voltage-clamp studies of I_Na. They probably occurred to some extent in our experiments, as evidenced by the incomplete washout of drug effects on the half-inactivation voltage of I_Na. We dealt with this issue by using a very negative HP (-140 mV), at which inactivation appeared to be negligible under both control and drug conditions (Figure 4C). With the use of this HP, complete reversibility of even very large drug effects on I_Na was noted on drug washout (see Figure 3A), indicating that the concentration-dependent inhibitory effect of indapamide on I_Na that we observed was not an artifact of time-dependent shifts in I_Na availability. At the same time, we cannot exclude a quantitative contribution of time-dependent I_Na availability voltage shifts to the extent of tonic block.

	Acknowledgments

 
This study was supported by the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. Dr Wang is a Research Scholar of the Heart and Stroke Foundation of Canada. The authors thank Nathalie Talbot for excellent technical assistance, France Thériault for secretarial help, and Jianlin Feng for useful discussion and suggestions.

Received December 11, 1997; accepted May 14, 1998.

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