The effect of amiodarone on Na+/Ca2+ exchange current (INCX) was examined in single guinea‐pig ventricular myocytes using the whole‐cell voltage clamp technique.
INCX was recorded by ramp pulses from the holding potential of −60 mV in the presence of 140 mM Na+ and 2 mM Ca2+ in the external solution, and 20 mM Na+ and 398 nM free Ca2+ (19 mM Ca2+ and 30 mM BAPTA) in the internal solution.
External application of amiodarone suppressed INCX in a concentration‐dependent manner. The IC50 value was 3.3 μM 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+/Ca2+ exchanger may contribute to the cardioprotective action of the drug.
British Journal of Pharmacology (2000) 131, 80–84; doi:10.1038/sj.bjp.0703527
- Na+/Ca2+ exchange current
- I‐V curve
- current‐voltage relation curve
- 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulphonate
Amiodarone is the most promising drug in the treatment of life‐threatening supraventricular and ventricular tachyarrhythmias, and consequently the prevention of sudden cardiac death (reviews by Singh, 1994; Kodama et al., 1997; 1999). Amiodarone is a class III antiarrhythmic drug, but it also has class I, II and IV antiarrhythmic drug properties in the Vaughan Williams classification. In electrophysiological experiments on single cardiac myocytes, acute effects of amiodarone have been reported to block Na+ channels (Follmer et al., 1987), L‐type Ca2+ channels (Nishimura et al., 1989), two voltage‐gated K+ channels, i.e., the delayed rectifier K+ current (Balser et al., 1991) and the inward rectifier K+ current (Sato et al., 1994), and three ligand‐gated K+ channels i.e., the muscarinic acetylcholine receptor‐operated K+ current (Watanabe et al., 1996), the ATP‐sensitive K+ current (Takizawa & Nakaya, 1997) and the Na+‐activated K+ current (Mori et al., 1996). However, effects of amiodarone on Na+/Ca2+ exchange current (INCX) have not been examined.
Na+/Ca2+ exchange is a major mechanism for extruding Ca2+ in cardiac myocytes (Blaustein & Lederer, 1999). The driving force of Na+/Ca2+ exchange is the Na+ concentration gradient. Therefore an increase in intracellular Na+ concentration caused by digitalis, for example, impairs Ca2+ efflux by this transporter, accumulates Ca2+ under the membrane and subsequently leads to enhanced force of contraction of cardiac myocytes. Aomine & Fukui (1993) found that amiodarone decreased ouabain‐induced developed tension in ventricular papillary muscles from guinea‐pig, and predicted that amiodarone would inhibit Na+/Ca2+ exchange. INCX can be recorded using the whole‐cell patch clamp technique by loading Na+ and Ca2+ in both internal and external solutions (Kimura et al., 1986; 1999). Recently a relatively selective Na+/Ca2+ exchange inhibitor, KB‐R7943, has been developed (Watano et al,. 1996; Iwamoto et al., 1996; Kimura et al., 1999), which enables identifying the exchange current as a KB‐R7943 sensitive current. In the present study, we examined the effect of amiodarone on INCX in single guinea‐pig ventricular cells studied under whole‐cell voltage clamp conditions.
All experiments were performed under the regulation of the Animal Research Committee of the School of Medicine, Fukushima Medical University. Single ventricular cells were isolated from the guinea‐pig heart by the following method. Guinea‐pigs weighing 250–400 g were anaesthetized by intraperitoneal injection of pentobarbital. The chest was opened under artificial ventilation, the aorta was cannulated in situ, and the heart was mounted on a Langendorff perfusion system. After washing out the blood with Tyrode solution, the perfusate was changed to nominally Ca2+‐free Tyrode solution and then to one containing 0.01% w v−1 collagenase (Wako, Osaka, Japan) and 0.002% w v−1 alkaline protease (Nagase, Tokyo, Japan). After digestion for about 15–20 min, the enzymes were washed out by perfusing a high K+, low Cl− solution (modified KB solution; Isenberg & Klockner, 1982). The ventricular tissue was cut into the modified KB solution and gently shaken to isolate the cells. The cell suspension was stored in a refrigerator (4°C) for later use.
Whole‐cell membrane currents were recorded by the patch‐clamp method. Single cardiac ventricular cells were placed in a recording chamber (1 ml volume) attached to an inverted microscope (Nikon, Tokyo, Japan) and superfused with the Tyrode solution at a rate of 5 ml min−1. The temperature of the external solution was kept constant at 36±0.5°C. The Tyrode solution had the following composition (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, glucose 5.5 and HEPES–NaOH 5 (pH 7.4). The composition of the modified KB solution was (in mM): KOH 70, l‐glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, MgCl2 3, glucose 10, EGTA 0.2 and HEPES–KOH buffer 10 (pH 7.2). Patch pipettes were made from glass capillaries with a diameter of 1.5 mm by a vertical microelectrode puller (pp‐83, Narishige, Tokyo, Japan). The pipette resistance was 2∼3 MΩ, when filled with the pipette solution. The composition of the pipette solution was (in mM): NaCl 20, BAPTA 30, CaCl2 15–19 (free Ca2+ concentration 235–398 nM), CsCl2 120, MgCl2 3, Aspartic acid 50, MgATP 5, HEPES 10 (pH 7.2 with CsOH). The extracellular solution contained (in mM): NaCl 140, CaCl2 2, MgCl2 1, ouabain 0.02, nifedipine 0.01, ryanodine 0.01, HEPES–CsOH 5 (pH 7.2). The electrode was connected to a patch‐clamp amplifier (TM‐1000, Act ME, Tokyo, Japan). Recording signals were filtered at 2.5 kHz bandwidth, and the series resistance was compensated. Current signals were stored on‐line and analysed by a computer (PC‐9801RX, NEC, Tokyo, Japan) with the handmade software called RAM5.
The current‐voltage (I‐V) relationship was obtained by voltage clamp ramp pulses as described previously (Kimura et al., 1987; Watano et al., 1996). The holding potential was set at −60 mV. A double‐ramp protocol (positive and negative slopes) was employed. The membrane was initially depolarized from −60 to +60 mV, then hyperpolarized from +60 to −110 mV and then depolarized back to −60 mV at a constant rate of 640 mV s−1. The descending limb (from +60 to −110 mV) was plotted as the I‐V relationship without capacitance compensation. The Ca2+ current (ICa), K+ currents, Na+–K+ pump current and Ca2+ release channels of the sarcoplasmic reticulum were blocked by nifedipine, Cs+, ouabain and ryanodine, respectively.
Amiodarone, ouabain, ryanodine and nifedipine were purchased from Sigma Chemical Co., St Louis, U.S.A. KB‐R7943 (2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulphonate) was a gift from Kanebo Co. Ltd. (Osaka, Japan). Amiodarone was first dissolved in ethanol and was dissolved further in Na+‐external solution containing bovine serum albumin (1.0%) as described by Polster & Broekhuysen (1976). Ethanol concentration in the Na+‐external solution was less than 0.1%. Nifedipine and KB‐R7943 were initially dissolved in dimethylsulphoxide (DMSO) as stock solutions and added to the extracellular solution. The final concentration of DMSO was 0.1%, which did not affect the Na+/Ca2+ exchange current. Trypsin (2.5 μg ml−1) (Difco Laboratories, Detroit, MI, U.S.A.) was directly dissolved in the pipette solution. All the chemicals used were the highest grade available.
All the values were presented as mean±s.e.mean (number of experiments). Student's t‐test and analysis of variance were used for statistical analyses. A P value of less than 0.05 was considered significant. The concentration–response data were fitted and IC50 and Hill coefficient values were obtained by Delta Graph Professional (Polaroid Computing, Tokyo, Japan) on a Macintosh computer (Apple Computer, Mariani Avenue Cupertino, CA, U.S.A.). Per cent inhibition of the outward INCX at various concentrations of amiodarone was fitted by the following logistic equation:
Effects of amiodarone on INCX
INCX was induced by 2 mM Ca2+ and 140 mM Na+ in the external solution and 20 mM Na+ and 398 nM free Ca2+ in the pipette solution. Under these ionic conditions, the reversal potential of the exchange current at a 3Na:1Ca stoichiometry was calculated to be −73 mV. After establishing the whole‐cell clamp mode, the external solution was changed from Tyrode solution to the control external solution while monitoring the increase in current until it reached a steady state. As shown in Figure 1A, when the current became stable, the control external solution was switched to one containing amiodarone. Amiodarone at 3 μM immediately suppressed the fast Na+ current and then slowly suppressed the Na+/Ca2+ exchange current. Suppression of the latter current was slow, taking more than 5 min before steady state was attained. Figure 1B illustrates the current‐voltage (I‐V) relationships obtained in control conditions (a) and after exposure to amiodarone (b). The I‐V relationship recorded in the presence of amiodarone intersected with the control I‐V curve at about −60 mV. After the effect of amiodarone reached a steady state, a high concentration (100 μM) of KB‐R7943, a potent exchange current inhibitor, was applied to completely block INCX. Similar to the current recorded in the presence of amiodarone alone, the I‐V curve obtained in the presence of KB‐R7943 (c) also intersected with the control I‐V curve at about −60 mV (Figure 1B), suggesting that the amiodarone‐sensitive current was INCX. Figure 1C illustrates the net I‐V curves of INCX obtained by subtracting the I‐V curve obtained with KB‐R7943 (c) from those before (a) and after (b) amiodarone application, respectively.
We next tested different concentrations of amiodarone. Figure 2 illustrates representative net I‐V curve obtained by subtracting the current in the presence of KB‐R7943 (100 μM) from the current obtained under control conditions and following exposure to amiodarone. Each concentration of amiodarone was tested in a different cell. I‐V curves obtained under control conditions and in the presence of amiodarone were superimposed in each panel. Amiodarone inhibited INCX in a dose‐dependent manner. All the I‐V curves crossed the voltage axis at around −60 mV, indicating that the amiodarone‐sensitive currents were mainly produced by INCX. The current magnitude was measured at two different potentials; i.e. at +50 mV for the outward exchange current component and at −100 mV for the inward current component, and the per cent inhibition was calculated assuming that 100 μM KB‐R7943 completely inhibited INCX. The concentration–response curves of amiodarone were plotted in Figure 3. Sigmoidal fitting of the curves yielded the IC50 values of amiodarone at 3.3 and 3.6 μM with the Hill coefficient of 1 for the outward (n=27) and inward (n=27) components, respectively. These results indicate that amiodarone inhibited both direction of the exchange current equipotently.
Effect of trypsin on the amiodarone inhibition of INCX
We examined whether amiodarone inhibited INCX from the inner side of the membrane by perfusing trypsin through the pipette solution. Trypsin (2.5 μg ml−1) was included in the pipette solution and 10 μM amiodarone was applied in the external solution. As shown in Figure 4, amiodarone inhibited INCX by 37% in this cell. KB‐R7943 at 100 μM still substantially blocked INCX even after the trypsin treatment. As summarized in Figure 5, amiodarone blocked INCX by 28±3% (n=4) after trypsin treatment, while it blocked INCX by 75±4% (n=5) without trypsin. Therefore, the blocking effect of amiodarone was significantly attenuated by trypsin. This result suggests that amiodarone affects the Na+/Ca2+ exchanger from the intracellular side of the membrane.
Acute application of amiodarone has been reported to inhibit various ionic channels including Na+‐, Ca2+‐ and K+ channels (Kodama et al., 1997; 1999). In the present study, we found that amiodarone exerts an additional action by inhibiting INCX in a concentration‐dependent manner in guinea‐pig cardiac ventricular cells. The IC50 value was 3.3∼3.6 μM and the Hill coefficient was 1.
The site of action of amiodarone is most likely on the cytoplasmic side of the exchanger, possibly in the internal loop, because intracellular treatment of a proteolytic enzyme, trypsin via the pipette solution attenuated the blocking effect of amiodarone. The irreversible and slow time‐course of the suppression before reaching a steady state also supports this view. The Na+/Ca2+ exchanger has a long intracellular loop containing various regulatory sites (Nicoll et al., 1990; 1999; Philipson et al., 1996; Iwamoto et al., 1999). ‘De‐regulation’ of the exchanger by cytosolic trypsin or α‐chymotrypsin treatment has been already demonstrated to affect activation by intracellular Ca2+ and ATP, inactivation by internal Na+ (Hilgemann 1990; Matsuoka & Hilgemann, 1994), inhibition by H+ (Doering & Lederer 1993), and inhibition by the calmodulin inhibitor, W‐7 (Kimura, 1993). The site of action of amiodarone may be close to or overlap with the regulatory sites of internal Ca2+, Na+ and H+ in the exchanger molecule. An equally plausible explanation, however, could be that a modification by trypsin of an internal site(s) on the exchanger protein or a regulatory protein alters the conformation of the protein in such a way that an amiodarone binding site within the membrane becomes partially occluded.
The following is a list of the ionic currents affected by amiodarone and the concentration range over which these effects are observed. INa was blocked by amiodarone (0.1∼7.3 μM) use‐ and voltage‐dependently in dog cardiac Purkinje myocytes and cat ventricular cells (Follmer et al., 1987). Amiodarone allosterically inhibits batrachotoxin‐binding to the Na+ channel, indicating that it binds preferentially to inactivated Na+ channels (Sheldon et al., 1989). L‐type Ca+ channels were also blocked by amiodarone with an estimated dissociation constant of 5.8 μM in the resting state and 0.36 μM in the inactivated state (Nishimura et al., 1989). Amiodarone affects most types of K+ channels present in cardiac ventricular cells. Amiodarone at 10 μM halved IK tail current magnitude, but had no effect on Ito in rabbit ventricular cells (Kamiya et al., 1995; Varro et al., 1996). Ito of newborn rat ventricular myocytes was, however, inhibited with an IC50 4.9 μM. Amiodarone at 10∼20 μM suppressed IK1 by 12–14% in guinea‐pig ventricular myocytes (Sato et al., 1994). The ligand‐gated K+ currents are also susceptible. Amiodarone inhibited IK.Na with an IC50 of ∼1 μM in inside‐out patches excised from guinea‐pig ventricular myocytes (Mori et al., 1996). Amiodarone inhibited the carbachol‐induced, adenosine‐induced and GTPγS‐induced IK.Ach of guinea‐pig atrial cells (IC50∼2 μM) (Watanabe et al., 1996). Amiodarone inhibited single KATP channel current of guinea‐pig ventricular myocytes by inside‐out patch with the IC50 value of 0.3 μM (Takizawa & Nakaya, 1997).
Acute and chronic clinical administration of amiodarone results in plasma levels in the range of 0.06–6.5 μg ml−1, which corresponds to concentrations of 0.1–10 μM (Harris et al., 1983; Ikeda et al., 1984; Raeder et al., 1985). Therefore the concentrations of amiodarone that inhibit INCX are within the therapeutic range of the drug. Is this effect beneficial or harmful in the amiodarone therapy? The Na+/Ca2+ exchanger is a bi‐directional transporter and can operate both in the Ca2+ efflux and Ca2+ entry modes. Our results indicate that amiodarone inhibits the exchange current in both directions equally. When the cell depolarizes in the presence of an augmented intracellular Na+ concentration, the Na+/Ca2+ exchanger may reverse and induce Ca2+ entry. An intracellular Na+ accumulation during anoxia or ischaemia precedes Ca2+ overload most likely via Na+/Ca2+ exchange (Tani, 1990). In heart failure, Na+/Ca2+ exchanger mRNA and protein levels increase (Studer et al., 1994). The Na+/Ca2+ exchanger appears to contribute to the occurrence of delayed afterdepolarization and triggered activity (Lakatta, 1992). Under such pathological conditions, suppression of Na+/Ca2+ exchanger may be beneficial and prevent Ca2+ overload. Indeed, Aomine & Fukui (1993) demonstrated that ouabain‐induced increase in contraction was decreased by amiodarone. Therefore the inhibitory effect on the Na+/Ca2+ exchanger may contribute to the cardioprotective effect of the drug. More work will be necessary to elucidate the molecular mechanism of the blocking effect of amiodarone on the Na+/Ca2+ exchanger.
We thank Dr Isao Matsuoka for his helpful discussion, Dr Tomoyuki Ono and Ms Sanae Sato for their technical assistance. This work was supported by grants‐in‐aids from Ministry of Education, Science, Sports and Culture of Japan (09670098, 11670096, 11357020).
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