Inhibitory effect of aprindine on Na+/Ca2+ exchange current in guinea-pig cardiac ventricular myocytes

Abstract

1. Using the whole-cell voltage clamp technique, the effect of aprindine on Na+/Ca2+ exchange current (I(NCX)) was examined in guinea-pig single cardiac ventricular myocytes and CCL39 fibroblasts expressing a dog cardiac Na+/Ca2+ exchanger (NCX1). 2. I(NCX) was recorded by ramp pulses from the holding potential of -60 mV with the external solution containing 140 mM Na+ and 1 mM Ca2+, and the pipette solution containing 20 mM Na+, 20 mM BAPTA and 13 mM Ca2+ (433 nM free Ca2+). 3. External application of aprindine suppressed I(NCX) in a concentration-dependent manner. The IC50 values of outward (measured at 50 mV) and inward (measured at -100 mV) I(NCX) components were 48.8 and 51.8 microM with Hill coefficients of 1.3 and 1, respectively. 4. Intracellular application of trypsin via the pipette solution did not change the blocking effect of aprindine, suggesting that aprindine does not affect the exchanger from the cytoplasmic side. 5. Aprindine inhibited I(NCX) of a mutant NCX1 with a deletion of amino acids 247 - 671 in the large intracellular domain between the transmembrane segments 5 and 6 in a similar manner to that of the wild-type, suggesting that the site of aprindine inhibition is not in the large intracellular domain of NCX1. 6. A kinetic study indicated that aprindine was cooperatively competitive with KB-R7943, another inhibitor of NCX and that aprindine was a competitive inhibitor with respect to external Ca2+. 7. We conclude that aprindine may modestly inhibit I(NCX) in a therapeutic range of concentrations (around 2.5 approximately 6.9 microM) possibly at an external or intra-membranous site of the exchanger.

Figure 1

Effect of 300 μM aprindine on I_NCX. (A) Typical chart recording of membrane current. The bars above the current indicate when 300 μM aprindine and 100 μM KB-R7943 were applied externally. (B) I – V curves obtained at the time points corresponding to the labels in (A). (a) is the control and (b) in the presence of aprindine and (c) in the presence of KB-R7943. (C) Difference I – V curves between a and b (a-b) and between b and c (b-c) in (B).

Figure 2

Aprindine concentration – inhibition curve. Average I_NCX values measured at +50 mV were fitted to a logistic equation. The IC₅₀ of aprindine was 48.8 μM and the Hill coefficient was 1.3. Each point indicates mean±s.e. (number of cells).

Figure 3

Effect of trypsin on inhibition by aprindine. (A) Typical chart recording of current. The pipette solution contained 2.5 mg ml⁻¹ trypsin. BDM, a trypsin-sensitive inhibitor of I_NCX, did not inhibit I_NCX, indicating the presence of trypsin in the cell. (B) I – V curves obtained at the points a∼d in (A). (C) Summarized data of the inhibitory effect of 300 μM aprindine on I_NCX in the absence (left) and presence of trypsin (right) in the pipette solution. The values are means±s.e. ^*P<0.05 based on unpaired t-test.

Figure 4

Effects of aprindine on I_NCX of wild-type (WT) NCX1 and a mutant with a deletion of amino acids 247 – 671 (Δ247 – 671) expressed in CCL39 cells. (A and B) I – V curves of control (c), in the presence of 100 μM aprindine (a) and 100 μM KB-R7943 (k). (C) Summary of the results of A and B. Per cent inhibition of WT, Δ247 – 671 and cardiac myocytes I_NCX by aprindine. (D) Comparison of I_NCX densities between WT and Δ247 – 671.

Figure 5

(A) Dixon plot of reciprocal or normalized I_NCX versus aprindine concentration under control conditions and in the presence of fixed concentrations of KB-R7943. The three fitted lines intersect at a point, indicating that aprindine and KB-R7943 are co-operative pure competitive inhibitors. (B) Hanes – Woolf plot for determining the mode of aprindine inhibition of I_NCX with respect to external Ca²⁺. Parallel fitted lines indicate that aprindine is a competitive inhibitor with respect to external Ca²⁺.