Inhibitory effect of amiodarone on Na(+)/Ca(2+) exchange current in guinea-pig cardiac myocytes

Abstract

The effect of amiodarone on Na(+)/Ca(2+) exchange current (I(NCX)) was examined in single guinea-pig ventricular myocytes using the whole-cell voltage clamp technique. I(NCX) was recorded by ramp pulses from the holding potential of -60 mV in the presence of 140 mM Na(+) and 2 mM Ca(2+) in the external solution, and 20 mM Na(+) and 398 nM free Ca(2+) (19 mM Ca(2+) and 30 mM BAPTA) in the internal solution. External application of amiodarone suppressed I(NCX) in a concentration-dependent manner. The IC(50) value was 3.3 microM with a Hill coefficient of 1. Intracellular application of trypsin via the micropipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the cytoplasmic side of the molecule. This inhibitory effect of amiodarone on the Na(+)/Ca(2+) exchanger may contribute to the cardioprotective action of the drug.

Figure 1

(A) Chart recording of membrane current. The horizontal bars above the current indicate when 3 μM amiodarone and 100 μM KB-R7943 were applied externally. (B) I-V curves obtained at the corresponding time points indicated in A. a is control, b in the presence of amiodarone and c in the presence of KB-R7943. (C) Difference I-V curves between a and c (a-c) and between b and c (b-c) in B.

Figure 2

Difference I-V curves at different concentrations of amiodarone. (A) 1 μM, (B) 3 μM, (C) 10 μM, (D) 30 μM amiodarone. Each I-V curve was after subtraction of the current recorded in the presence of 100 μM KB-R7943. Control is before amiodarone application. The duration of application of amiodarone was 10∼20 min.

Figure 3

Concentration-inhibition curves of amiodarone. The average data obtained at 50 mV for the outward I_NCX and at −100 mV for the inward I_NCX were fitted. The IC₅₀ values of amiodarone were 3.3 and 3.6 μM for the outward and inward I_NCX, respectively. The Hill coefficient was 1 for the both components.

Figure 4

(A) Chart recording of the current observed when the pipette solution contained trypsin (2.5 μg ml⁻¹). The horizontal bars above the current indicate where trypsin, amiodarone 10 μM and KB-R7943 100 μM were applied. (B) I-V curves obtained at the corresponding labels in (A). a is control and b in presence of amiodarone and c in the presence of KB-R7943. (C) Difference I-V curves between a and c (a-c) and between b and c (b-c) in B.

Figure 5

Comparison of the inhibitory effect of 10 μM amiodarone on I_NCX in the absence (left) and presence of trypsin (right) in the pipette solution. The data of trypsin were obtained by the experiments shown in Figure 4.