Effects of diltiazem and nifedipine on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Abstract

1. It is unknown whether the widely used L-type Ca(2+) channel antagonists diltiazem and nifedipine would block the repolarization K(+) currents, transient outward current (I(to1)) and ultra-rapid delayed rectifier K(+) current (I(Kur)), in human atrium. The present study was to determine the effects of diltiazem and nifedipine on I(to1) and I(Kur) in human atrial myocytes with whole-cell patch-clamp technique. 2. It was found that diltiazem substantially inhibited I(to1) in a concentration-dependent manner, with an IC(50) of 29.2+/-2.4 microM, and nifedipine showed a similar effect (IC(50)=26.8+/-2.1 muM). The two drugs had no effect on voltage-dependent kinetics of the current; however, they accelerated I(to1) inactivation significantly, suggesting an open channel block. 3. In addition, diltiazem and nifedipine suppressed I(Kur) in a concentration-dependent manner (at +50 mV, IC(50)=11.2+/-0.9 and 8.2+/-0.8 microM, respectively). These results indicate that the Ca(2+) channel blockers diltiazem and nifedipine substantially inhibit I(to1) and I(Kur) in human atrial myocytes.

Figure 1

Diltiazem effect on I_to1. (A) Representative voltage-dependent I_to1 (capacitance compensated) recorded in an atrial myocyte with the voltage steps protocol shown in the inset at 0.2 Hz under control conditions (a), in the presence of 5 and 50 μM diltiazem (b, c). I_to1 was substantially inhibited by the application of diltiazem for 6 min, and the effect was recovered by the drug washout for 8 min (d). (B) Time-dependent effect of 50 μM diltiazem on I_to1 elicited by the voltage step shown in the left inset delivered every 10 s in a typical experiment. I_to1 measured was peak to ‘quasi'-steady-state level. The original I_to1 traces at corresponding time points are shown in the right inset of the panel.

Figure 2

Effects of verapamil and diltiazem on I_to1. (A) I_to1 traces recorded with the voltage protocol as shown in the inset of (c) in a representative myocytes during control (a), in the presence of 10 μM verapamil (Verap.) for 6 min (b), co-presence of verapamil and 50 μM diltiazem (Dilt.) for 6 min (c), and washout of diltiazem for 8 min. Verapamil actually induced an increase of measured I_to1 by selectively inhibiting I_Kur. (B) I–V relationships of I_to1 in the presence of 10 μM verapamil (control), co-presence of verapamil and 5, 10, 50, 100, and 200 μM diltiazem (6 min for each concentration), and after the drug washout for 10 min. Diltiazem inhibited I_to1 in a concentration-dependent manner (P<0.05 or 0.01 vs control, and the effect was reversed by 90% after the drug washout. The statistical significance was analyzed by repeated-measures ANOVA. (C) Concentration–response relationship for diltiazem inhibition of I_to1. Symbols are mean data at +50 mV (the error bars are smaller than the size of the data symbol), and solid line is the best-fit Hill equation, IC₅₀=29.2±2.4 μM, Hill co-efficient=0.98±0.08 (n=6), and E_max=65.2%.

Figure 3

Effects of diltiazem on time-dependent kinetics of I_to1. (a) I_to1 traces recorded from a representative cell upon a 300-ms voltage step to +50 from −50 mV in the presence of 10 μM verapamil (control) and co-presence of verapamil and 50 μM diltiazem. Raw data (points) of I_to1 under control conditions were fitted to a monoexponential function (solid lines, superimposed with raw data) with time constants shown. After the application of 50 μM diltiazem, the data were fitted only by a biexponential equation, with fast and slow time constants (τ₁ and τ₂) shown. (b) Mean values of time constants at +50 mV under control conditions, in the presence of 1, 5, 10, and 50 μM diltiazem. The time constant was reduced by the application of 1 and 5 μM diltiazem (n=7, ^*P<0.05, ^**P<0.01 vs control). Diltiazem at concentrations higher than 10 μM had τ₁ and τ₂. The τ₁ decreased with increasing diltiazem concentration (^##P<0.01 vs 10 μM diltiazem), and the τ₂ did not show significant difference. (c) Time to peak of I_to1 activation at 0 to +60 mV under control conditions and in the presence of 50 μM diltiazem. Diltiazem significantly reduced the time to peak of I_to1 (n=7, ^**P<0.01 vs control). The statistical significance was analyzed by repeated-measures ANOVA.

Figure 4

Effects of diltiazem on voltage-dependence and restoration of I_to1. (a) Representative current traces and protocol (inset) used to evaluate voltage-dependent inactivation I_to1 recorded in the presence of 10 μM verapamil. (b) Voltage-dependent variables for I_to1 activation (Act.) and inactivation (Inact.) were fitted to the Boltzmann distribution: y=1/{1+exp[(V_m−V_0.5)/S]}, where V_m is membrane potential, V_0.5 is the midpoint, and S is slope. For activation, V_0.5 and S were 16.9±1.4 and −11.2±0.3 mV for control, and 18.4±0.9 and −11.9±0.4 mV for 50 μM diltiazem (Dilt.) treatment (n=7, P=NS). For inactivation, V_0.5 and S were –29.2±1.1 and 7.5±0.6 mV under control conditions, and −30.4±1.4 and 7.6±0.8 mV in the presence of 50 μM diltiazem (n=6, P=NS). (c) Representative current traces recorded in a typical experiment in the presence of 10 μM verapamil by 300-ms paired pulses to +50 mV after a 30-ms step of −40 mV (to inactivate I_Na) from −80 mV with varying P1 and P2 interval (inset), which are used for assessing time-dependent recovery of I_to1 from inactivation. (d) Mean data for time course of recovery of I_to1 from inactivation in the absence and presence of 50 μM diltiazem in six cells. Data were best fit to monoexponential function. No change in recovery time constant of I_to1 was observed after the application of diltiazem (n=6, P=NS).

Figure 5

Effect of diltiazem on I_Kur. (A) Representative voltage-dependent I_Kur (capacitance compensated) recorded at 0.2 Hz in a typical experiment with a 100-ms prepulse to +40 mV to inactivate I_to1, followed by 150-ms test pulses to between –40 and +50 from –50 mV after a 10-ms interval, then to –30 mV (as shown in the inset of (c) under control conditions (a), in the presence of 5 and 50 μM diltiazem (b, c). I_Kur was substantially suppressed by the application of diltiazem, and the effect was significantly recovered by washout of the drug for 6 min (d). (B) Time-dependent effect of 10 μM diltiazem on I_Kur elicited by 150-ms voltage step to +50 from −50 mV (as shown in the left inset) delivered every 10 s. The original I_Kur races at corresponding time points are shown in the right inset.

Figure 6

Concentration-dependent effect of diltiazem on I_Kur. (a) I–V relationships of I_Kur under control conditions, in the presence of 1, 5, 10, 50, and 100 μM diltiazem (6 min for each concentration), and after the drug washout for 10 min. Diltiazem inhibited I_Kur in a concentration-dependent manner, and the effect was reversed by 95% (at +50 mV) after the drug washout. (b) Percent reduction of I_Kur at 0 to +50 mV by diltiazem with multiple concentrations. Diltiazem significantly inhibited I_Kur at concentrations from 1 μM (P<0.05 vs control) to 5, 10, 50, and 100 μM (n=8, P<0.01 vs control). Significant voltage dependence was observed for the drug effect at 10–100 μM, and stronger effect was observed at potentials positive to +10 and +50 mV (P<0.05 or 0.01 vs 0 mV). The statistical significance was analyzed by repeated-measures ANOVA. (c) Concentration–response relationships of I_Kur block by diltiazem at +50 mV. The symbols are mean values of inhibitory effect in cells exposed to different concentrations (0.1–200 μM) of diltiazem. Solid lines were best fit to Hill equation. Mean IC₅₀ was 11.2±0.9 μM, Hill co-efficient was 0.96±0.07, and E_max was 64% (n=7).

Figure 7

Inhibition of I_to1 by nifedipine. (A) Time-dependent effect of 30 μM nifedipine on I_to1 elicited by voltage step to +50 from −50 mV (as shown in the left inset) delivered every 10 s. Nifedipine reversibly suppressed I_to1. The original I_to1 traces at corresponding time points are shown in the right inset. (B) Voltage-dependent I_to1 traces recorded with the voltage protocol as shown in the inset in a typical experiment under control conditions (a), in the presence of 5 and 50 μM nifedipine (b, c) for 5 min. I_to1 was significantly inhibited by nifedipine, and the effect recovered upon the drug washout for 6 min.

Figure 8

Effect of nifedipine on I_to1 under conditions of inhibiting I_Kur with 10 μM verapamil. (A) I_to1 traces recorded with the same voltage protocol as shown in the inset in a representative myocyte during control (a, 10 μM verapamil), in the co-presence of verapamil and 5 (b) and 50 (c) μM nifedipine (Nif.) for 6 min, and washout of nifedipine for 10 min (d). (B) I–V relationships of I_to1 under control conditions (10 μM verapamil), in the co-presence of verapamil and 5, 10, 50, 100 μM nifedipine (6 min for each concentration), and after the drug washout for 10 min. Nifedipine (5–100 μM) significantly inhibited I_to1 at voltages from 0 to +60 mV (n=7, P<0.05 or 0.01). The statistical significance was analyzed by repeated-measures ANOVA. (C) Concentration-dependent response of I_to1 to nifedipine. IC₅₀ at +50 mV was 26.8±2.1 μM with a Hill co-efficient of 0.97±0.02 (n=6), and E_max was 85.1%.

Figure 9

Effects of nifedipine on inactivation and time to peak of I_to1. (a) I_to1 traces recorded in a typical experiment upon a 300-ms voltage step to +50 from −50 mV in the presence of 10 μM verapamil (control), and co-presence of verapamil and 50 μM nifedipine. Raw data (points) of I_to1 under control conditions were well fit to a mono-exponential function (solid lines, superimposed with raw data) with time constant shown. The data after application of 50 μM nifedipine were only fit to a biexponential equation with fast and slow time constants (τ₁ and τ₂) shown. (b) Mean values of I_to1 inactivation time constants under control conditions, and in the presence of 1 (n=7, P<0.05 vs control), 5, 10 and 50 μM nifedipine (^#P<0.05, ^##P<0.01 vs 5 μM nifedipine). (c) Time to peak of I_to1 as a function of test potentials under control conditions and in the presence of 50 μM nifedipine. The time to peak of I_to1 was significantly reduced at 0 to +60 mV by the application of diltiazem (n=6, ^**P<0.01 vs control). The statistical significance was analyzed by repeated-measures ANOVA.

Figure 10

Effect of nifedipine on I_Kur. (A) Time-dependent effect of 30 μM nifedipine on I_Kur elicited by voltage protocol shown in the left inset delivered every 10 s in a typical experiment. Nifedipine reversibly suppressed I_Kur. (B) Voltage-dependent I_Kur recorded in a representative myocyte by the voltage protocol shown in the inset under control conditions (a), in the presence of 5 and 50 μM nifedipine for 5 min (b, c), and after wash out of the drug for 6 min (d). Nifedipine substantially suppressed I_Kur, and the effect recovered upon the drug washout.

Figure 11

Nifedipine effect on voltage-dependent I_Kur. (a) I–V relationships of I_Kur under control conditions, in the presence of 1, 5, 10, 50, and 100 μM nifedipine (6 min for each concentration), and after the drug washout for 10 min. Nifedipine inhibited I_Kur in a concentration-dependent manner, and the effect was reversed by 95% (at +50 mV) by washout of the drug for 10 min. (b) Percent inhibition of I_Kur at voltages from 0 to +50 mV by nifedipine with multiple concentrations. Nifedipine significantly inhibited I_Kur at concentrations from 1 to 5, 10, 50, and 100 μM (n=8, P<0.05 or 0.01 vs control), and no voltage-dependent effect was observed for the drug action. The statistical significance was analyzed by the repeated measures ANOVA. (c) Concentration-dependent inhibition of I_Kur by nifedipine at +50 mV. The symbols are mean values of inhibitory effect in seven cells exposed to different concentrations of nifedipine. Solid lines were fit to Hill equation. Mean IC₅₀ was 8.2±0.8 μM, Hill co-efficient was 1.2±0.2, and E_max was 76%.