Bepridil differentially inhibits two delayed rectifier K(+) currents, I(Kr) and I(Ks), in guinea-pig ventricular myocytes - PubMed (original) (raw)

Bepridil differentially inhibits two delayed rectifier K(+) currents, I(Kr) and I(Ks), in guinea-pig ventricular myocytes

J C Wang et al. Br J Pharmacol. 1999 Dec.

Abstract

1. We investigated the effects of bepridil on the two components of the delayed rectifier K(+) current, i.e., the rapidly activating (I(Kr)) and the slowly activating (I(Ks)) currents using tight-seal whole-cell patch-clamp techniques in guinea-pig ventricular myocytes, under blockade of L-type Ca(2+) current with nitrendipine (5 microM) or D600 (1 microM). 2. Bepridil decreased I(Ks) under blockade of I(Kr) with E4031 (5 microM), in a concentration-dependent manner. The concentration-dependent inhibition of I(Ks) by bepridil was fitted by a curve, assuming one-to-one interactions between the channel and the drug molecule. The concentration of half-maximal inhibition (IC(50)) was found to be 6.2 microM. 3. The effect of bepridil on I(Kr) was assessed using an envelope-of-tails test. In the control condition, a ratio of the tail current to the time-dependent current measured during depolarization was large (>1) at shorter pulses (<200 ms), and it decreased to a steady state value of approximately 0.4 with increases in the pulse duration. Bepridil at a concentration of 2 microM did not decrease this ratio at shorter pulses. 4. In a short-pulse (duration=50 ms) experiment that largely activates I(Kr), the drug was found to block I(Kr) in a cooperative manner (Hill coefficient=3.03) and the IC(50) was 13.2 microM. 5. These results suggest that bepridil at a clinical therapeutic concentration ( approximately 2 microM) selectively blocks I(Ks) but does not inhibit I(Kr). This may relate to the characteristic frequency-dependent effects of bepridil on the action potential duration (APD), e.g., the non-reverse use-dependent prolongation of APD.

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Figures

Figure 1

Figure 1

(A,B) Effects of bepridil on the outward current of a guinea-pig ventricular myocyte. Current families in the absence (A) and presence (B) of 10 μ

M

bepridil. (C) Current-voltage relationship of bepridil-sensitive time-dependent current. The ordinate shows the difference in the time-dependent current at the end of the depolarizing pulses (abscissa), measured between corresponding traces in panels A and B. The lack of an apparent inward-going rectification in the I–V curve suggests that bepridil blocked the slowly activating component of the delayed-rectifier K+ current, i.e., IKs. The L-type Ca2+ current was blocked by 1 μ

M

D600. Temperature, 35°C.

Figure 2

Figure 2

(A) Effects of 10 μ

M

bepridil on the slowly activating delayed rectifier K+ current, IKs, which was recorded after blockade of the rapidly activating component of the current, IKr, with 5 μ

M

E4031. (B) Concentration-dependent effect of bepridil on IKs. The per cent inhibition of the tail current evoked after depolarization to +60 mV (duration of 1 s) was plotted against the drug concentrations tested. The data were fitted by the following equation, by assuming one-to-one interaction between the channel and the drug molecule: Fractional inhibition (per cent inhibition of IKs)=Imax *C/ (C+Kd), where Imax is the maximal inhibition and Kd is a dissociation constant. Imax and Kd were calculated to be 100% and 6.2 μ

M

.

Figure 3

Figure 3

Voltage-independent effects of bepridil on IKs. The experiments were conducted in the presence of 5 μ

M

E4031. Each data point and vertical bar indicate the mean and s.e.mean from three experiments.

Figure 4

Figure 4

(A) Envelope-of-tails test in the absence (a) and presence of 2 μ

M

bepridil (b). Depolarizing pulses to +50 mV from −40 mV were applied by increasing the pulse duration. (B) The ratio of the tail current (IKtail) to the time-dependent current during the pulse (IKdepo) was plotted against the pulse duration. Bepridil shifted the curve upward, preserving a large value in the IKtail/IKdepo ratio at the short pulse duration of ⩽200 ms. (C) The IKtail/IKdepo ratio of the bepridil-sensitive current, which was obtained by subtracting the current in the presence of bepridil from the current in the absence of bepridil. The absence of a large IKtail/IKdepo ratio at shorter pulse durations suggests that bepridil at 2 μ

M

did not block IKr, but did block IKs (cf. Figure 7C).

Figure 5

Figure 5

(A) Envelope-of-tails test in the absence and presence of 10 μ

M

bepridil. (B) Envelope-of-tails test of the current blocked by 10 μ

M

bepridil. A slight increase in the IKtail/IKdepo ratio at shorter pulses suggests that a higher concentration of bepridil (∼10 μ

M

) blocks both IKr and IKs.

Figure 6

Figure 6

Effects of bepridil on IKr tail current. (A) Typical examples of the effect of bepridil at 2 and 10 μ

M

on IKr evoked by short depolarizing pulses (duration=50 ms; see inset). (B) The concentration-dependent inhibition of bepridil on IKr, examined using a short-pulse protocol. The data were fitted by a Hill equation: Fractional inhibition (per cent inhibition of IKr)={K·Ch/(1+K·Ch)}·100, where C, K, and h are the drug concentration, dissociation constant, and Hill coefficient, respectively. Best fit was obtained when K=0.0004 and h=3.03. The figures in parentheses indicate the number of cells tested.

Figure 7

Figure 7

A scheme explaining the bepridil-induced changes in the profile of an envelope-of-tails test. The ordinate unit in panel D is arbitrary. See text for details.

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