Naunyn-Schmiedeberg's Arch Pharmacol (2016) 389:1205–1214 DOI 10.1007/s00210-016-1282-y

ORIGINAL ARTICLE

Inhibitory effect of YM-244769, a novel Na+/Ca2+ exchanger inhibitor on Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes Kanna Yamashita 1 & Yasuhide Watanabe 1 & Satomi Kita 2 & Takahiro Iwamoto 2 & Junko Kimura 3

Received: 11 May 2016 / Accepted: 21 July 2016 / Published online: 1 August 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Recently, YM-244769 (N-(3-aminobenzyl)6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na+/Ca2+ exchange (NCX) inhibitor by using various cells transfected with NCX using the 45Ca2+ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (INCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional INCX in a concentration-dependent manner. The IC50 values of YM-244769 for the bidirectional outward and inward INCX were both about 0.1 μM. YM-244769 suppressed the unidirectional outward INCX (Ca2+ entry mode) with an IC50 value of 0.05 μM. The effect on the unidirectional inward INCX (Ca2+ exit mode) was less potent, with 10 μM of YM244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na+ concentration, YM-244769 suppressed INCX more potently than it did at 0 mM [Na+]i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca2+ entry mode of NCX more potently than the Ca2+ exit mode, and inhibition by YM244769 is [Na+]i-dependent and trypsin-insensitive. These

* Yasuhide Watanabe [email protected]

1

Division of Pharmacological Science, Department of Health Science, Hamamatsu University School of Medicine, Hamamatsu, Japan

2

Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan

3

Department of Pharmacology, School of Medicine, Fukushima Medical University, Fukushima, Japan

characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400. Keyword Na+/Ca2+ exchange current (INCX) . YM-244769 . Benzyloxyphenyl derivative . Cardiac myocytes . Membrane current . Patch-clamp method

Introduction The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either the Ca2+ exit or Ca2+ entry mode, depending on the ion gradients of both Na+ and Ca2+ across the plasma membrane and membrane potential. It plays an important role in regulating intracellular Ca2+ homeostasis in various tissues. The NCX stoichiometry is 3Na+/1Ca2+. In the cardiac action potential (AP), at potentials negative to the NCX reversal potential (ENCX), NCX generates an inward INCX. At potentials positive to ENCX, NCX generates an outward INCX. In the heart, NCX is abundantly expressed and contributes to about a 20 to 30 % reduction of intracellular Ca2+ concentration ([Ca2+]i) by expelling Ca2+ from the cytoplasm during normal diastole in large mammals such as guinea pig, rabbit, dog, and human ventricle, but only about 7 % or less in mice or rat myocardium. The Ca2+ pump of sarcoplasmic reticulum (SR) removes most of the remaining intracellular Ca2+ (Blaustein and Lederer 1999; Amran et al. 2003). Under cardiac pathological conditions, including ischemia/ reperfusion injury, arrhythmia, early and delayed afterdepolarization, hypertrophy, and heart failure, NCX leads to intracellular Ca2+ overload (Blaustein and Lederer 1999). While the Ca2+ entry mode of NCX contributes to Ca2+

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overload under certain pathophysiological conditions (notably hypertrophy and heart failure), early and delayed afterdepolarizations (DADs) can be a consequence of the Ca2+ exit mode of NCX activation and, in fact, are a mechanism to reduce Ca2+ overload. Thus, NCX inhibition may be useful to avoid Ca2+ overload by blocking the Ca2+ entry mode of NCX and may also reduce arrhythmogenic consequences of Ca2+ overload such as DADs. Under such pathological conditions, selective NCX inhibitors would be useful, although inhibiting the Ca2+ exit mode of NCX would be expected to increase Ca2+ overload. There have been four modestly selective NCX inhibitors developed by different pharmaceutical companies: KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl-]ethyl]isothiourea methanesulfonate), SEA0400(2-[4-[(2,5-difuorophenyl) methoxy]phenoxy]-5-ethoxyaniline), SN-6 (2-[4-(4nitrobenzyloxy) benzyl] thiazolidine-4-carboxylic acid ethyl ester) and YM-244769 (N-(3-aminobenzyl)-6-{4-[(3fluorobenzyl)oxy]phenoxy} nicotinamide) (Fig. 1). In 1996, KB-R7943 was developed as a prototype NCX inhibitor. However, it was later found that KB-R7943 was not specific to NCX but could affect various proteins. Only when most ion channels and transporters are inhibited, KB-R7943 can be used as a specific NCX inhibitor with an IC50 about 3 μM in the heart (Watano et al. 1996). Next, SN-6 and SEA0400 were developed as more specific NCX inhibitors. SN-6 also blocks NCX with the IC50 of about 2 μM in the heart (Niu et al. 2007). SEA0400 blocks NCX with IC50 of 30 to 40 nM (Tanaka et al. 2002). However, SEA0400 at 10 μM inhibited ICa in canine ventricular cells (Birinyi et al. 2005). In 2006, Iwamoto and Kita reported YM-244769 as a novel more specific NCX inhibitor. It was reported that KB-R7943, SN-6, and SEA0400 inhibited preferentially the unidirectional outward INCX (Ca2+ influx mode) rather than the unidirectional INCX (Ca2+ efflux mode) in isolated guinea pig cardiac ventricular cells and the cloned cardiac NCX1.1 expressed in Xenopus laevis (Kimura et al. 1999; Lee et al. 2004; Niu et al. 2007). Iwamoto et al. (2007) reported that the four benzyloxyphenyl derivative NCX inhibitors have [Na+]i-dependent blocking profile. However, the electrophysiological properties of YM-244769 in the heart have not yet been reported. NCX1 is expressed ubiquitously but is particularly abundant in the heart. NCX2 and NCX3 are expressed in different regions of the brain and NCX3 in the skeletal muscle. These benzyloxyphenyl derivative NCX inhibitors have different selectivities on the three NCX subtypes, i.e., NCX1, NCX2, and NCX3 (Iwamoto and Kita 2006; Iwamoto et al. 2007). KB-R7943, SN-6 and SEA0400 have been shown to have cardioprotective effects against digitalis-induced arrhythmia and ischemia-reperfusion injury (Iwamoto et al. 2007). Recently, YM-244769 was used as a selective NCX inhibitor in animal experiments (Gotoh et al. 2015; Jackson and Robinson 2015).

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In the present study, we examined the inhibitory effects of YM-244769 on the NCX1 current (INCX) in single guinea pig cardiac ventricular myocytes using the whole-cell clamp technique.

Methods All experiments were performed in compliance with the regulations of the Animal Research Committee of the Hamamatsu University School of Medicine and the Guide for the Care and use of Laboratory Animals (NIH publication). Cell isolation Guinea pigs weighing 250 to 400 g were anesthetized by intraperitoneal injection of pentobarbital. The chest was opened under artificial ventilation using a respirator for small animals (SN-480-7, Shinano, Tokyo, Japan), 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 (Type XIII, Sigma Chemical Co, St. Louis, MO). After digestion for about 15 to 20 min, the enzymes were washed out by perfusing a high K + , low Cl − solution (modified KB solution; Kimura et al. 1999). The ventricular tissue was cut into the modified KB solution and vigorously shaken to isolate single ventricular cells. The suspension of the isolated cells was stored in a refrigerator (4 °C) for later use. Patch-clamp recording Whole-cell membrane currents (bidirectional INCX and unidirectional INCX) were measured by the whole-cell patch-clamp technique as reported previously (Watanabe and Kimura 2000; Tanaka et al. 2002; Niu et al. 2007). Single cardiac ventricular cells were placed in a recording chamber (200 μl volume) attached to an inverted microscope (Nikon, Tokyo, Japan) and were superfused with the Tyrode solution at a rate of 5 ml min−1. The temperature of the external solution was maintained at 36 ± 0.5 °C. The Tyrode solution contained the following (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 modified KB solution contained the following (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 forged from 1.5-mm diameter glass capillaries with a microelectrode puller (pp-83, Narishige, Tokyo, Japan). The pipette resistance was approximately 2 to 3 MΩ

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Fig. 1 Chemical structures of benzyloxyphenyl derivative Na+/ Ca2+ exchange inhibitors (KBR7943, SN-6, SEA0400 and YM244769)

O

NH2

N N H

YM-244769

R Benzyloxyphenyl derivatives

when filled with the pipette solution which contained the following (in mM): NaCl 20, BAPTA 20, CaCl2 10 (free Ca2+ concentration 226 nM), CsCl 120, MgCl2 3, aspartic acid 50, MgATP 5, and HEPES 10 (pH 7.2 with CsOH). The extracellular solution contained the following (in mM): NaCl 140, CaCl2 2, MgCl2 1, ouabain 0.02, nifedipine 0.01, ryanodine 0.01, and 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. Membrane currents were recorded using pCLAMP8 software (Axon Instruments, Foster City, CA).

Bidirectional INCX recording Bidirectional INCX was induced by 140 mM Na+ and 1 mM Ca2+ in the extracellular solution and 20 mM Na+ and 226 nM free Ca2+ in the intracellular solution. The ramp pulse was initially depolarized from the holding potential of −60 to +30 mV, then hyperpolarized to −150 mV, and then depolarized back to −60 mV at a rate of 680 mV/s. The descending limb (from +30 to −150 mV) was plotted as the current-voltage (I-V) relationship without capacitance compensation. A ramp pulse was given every 10 s. The “bidirectional” extracellular solution contained the following (in mM): NaCl 140, CaCl2 1, MgCl2 1, nifedipine 0.01, ouabain 0.02, ryanodine 0.01, and HEPES 5 (pH 7.2). The Ca2+ current (ICa), K+ currents, Na+-K+ pump current, and sarcoplasmic reticulum Ca2+ release channels were blocked by nifedipine, Cs+, ouabain, and ryanodine, respectively, in the extracellular or the intracellular solution. In experiments with varying intracellular Na+ concentration, we used CsCl as a substitute for NaCl. The I-V relationships for the effect of YM-244769 at different [Na+]i and for the effect of trypsin on YM-244769 inhibition were obtained by ramp pulses as follows (Watanabe

and Kimura 2000; Watanabe et al. 2001). The holding potential was set at −60 mV. The membrane was initially depolarized from −60 to 60 mV, then hyperpolarized from 60 to −110 mV and depolarized back to −60 mV at a rate of 640 mV s−1. The descending limb (from 60 to −110 mV) was plotted in the I-V relationship without capacitance compensation.

Unidirectional INCX recording For recording the outward unidirectional INCX, the ramp pulse was initially depolarized from −50 to 30 mV, and then hyperpolarized to −50 mV at a rate of 680 mV/s. The depolarizing phase was employed for taking the I-V relation. For recording the inward unidirectional INCX, the ramp pulse was initially depolarized from −50 to 30 mV, then hyperpolarized to −100 mV, and then depolarized back to −50 mV, at a rate of 680 mV/s. The hyperpolarizing phase was employed for the I-V relation. The ramp pulse was given every 5 to 10 s. The unidirectional outward INCX or the Ca2+ entry mode of NCX was induced by transiently changing [Ca2+]o from 0 to 2 mM in the presence of 20 mM [Na+]i and 140 mM [Na+]o with 96 nM free [Ca2+]i (20 mM BAPTA and 6 mM CaCl2) to minimize the Ca2+ exit mode of NCX. Other components of the pipette solution were (in mM) CsOH 120, MgCl2 3, aspartic acid 50, MgATP 5, and HEPES 20 (pH 7.2 with aspartic acid). The unidirectional inward INCX or the Ca2+ exit mode of NCX was induced by changing [Na+]o from 0 (substituted by 140 mM [Li+]o) to 140 mM in the presence of 226 nM free [Ca2+]i (20 mM BAPTA and 10 mM CaCl2) and 1 mM [Ca2+]o without [Na+]i. Other components of the pipette solution were (in mM) CsCl 30, CsOH 100, MgCl2 3, aspartic acid 50, MgATP 5, and HEPES 20 (pH 7.2 with aspartic acid).

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Drugs Ouabain, ryanodine, and nifedipine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). KB-R7943 was a gift from Kanebo Co. Ltd. (Osaka, Japan). YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) was developed by Iwamoto and Kita (coauthors) and synthesized by Astellas Pharma Inc. (Tsukuba, Japan). YM-244769, nifedipine, and KB-R7943 were initially dissolved in dimethylsulfoxide (DMSO) as stock solutions and added to the extracellular solution. The final concentration of DMSO was ≦0.1 %, which did not affect INCX. Trypsin (2.5 μg/ml) (Sigma Chemical Co, St. Louis, MO, USA) was directly dissolved in the pipette solution. All the chemicals used were of the highest grade available. Data analysis All the values are presented as mean ± standard error (number of experiments). Student’s t test, ANOVA, and Mann-Whitney test 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 using DeltaGraph Professional (Polaroid Computing, Tokyo, Japan). Percent inhibition of the INCX at various concentrations of YM-244769 was fitted by the following logistic equation: n o Percent inhibition ¼ 100  1= 1 þ ðIC50 =½DÞnH Here, [D] is the concentration of YM-244769, IC50 is the half-maximum concentration for the inhibition of the drug, and nH is an empirical parameter describing the steepness of the fit and is equivalent to the Hill coefficient.

Results Effects of YM-244769 on the bi-directional and unidirectional INCX Bidirectional I NCX was induced by 1 mM Ca 2+ and 140 mM Na+ in the external solution and 20 mM Na+ and 226 nM free Ca2+ in the pipette solution. Under these ionic conditions, the reversal potential of the exchange current with a 3Na+/1Ca2+ stoichiometry was calculated to be −68 mV. After establishing the whole-cell clamp configuration, the external solution was switched from Tyrode solution to the control external solution, while monitoring the increase in the current until it reached a steady state. Figure 2a shows a concatenated current in response to ramp pulses given every 10 s. When the current became stable, the control external solution was

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switched to one containing YM-244769. YM-244769 at 3 μM suppressed both outward and inward components of bidirectional I NCX . After the effect of YM-244769 reached a steady state, a high concentration of 100 μM KB-R7943, which is a relatively selective NCX blocker, was applied to completely block INCX. Figure 2b illustrates the current-voltage (I-V) relationships obtained in control (a) and after exposure to 3 μM YM-244769 (b). The I-V curves recorded in the presence of 3 μM YM244769 intersected with the control I-V curve at around −60 mV. The I-V curve obtained in the presence of 100 μM KB-R7943 (c) also intersected with the control I-V curve at around −60 mV (Fig. 2b), suggesting that the YM-244769-sensitive current was INCX. Figure 2c illustrates the net I-V curves of INCX obtained by subtracting the I-V curves with 100 μM KB-R7943 (c) from those before (a) and after (b) the application of 3 μM YM244769. The current magnitude was measured at 20 mV for the outward INCX component and at −140 mV for the inward INCX component, and the percent inhibition was calculated assuming that 100 μM KB-R7943 completely inhibited each direction of INCX. In this cell, 3 μM YM244769 inhibited the bidirectional outward and inward INCX by about 93 and 100 %, respectively. Similar results were obtained in two other cells. YM-244769 at 3 μM inhibited the bidirectional outward and inward INCX by 92.6 ± 1.0 and 100 ± 0 %, respectively. In the presence of 20 mM Na+ and 96 nM free Ca2+ in the pipette solution, unidirectional outward INCX was induced by changing [Ca2+]o from 0 to 2 mM for 1 to 3 min (Fig. 3a, top). Ramp pulses were given every 5 or 10 s. Then, in the same cell, 5 to 6 min, after returning to the 0 [Ca2+]o external solution, 1 μM YM-244769 was added to the external solution. Concatenated currents and I-V curves of control and in the presence of 1 μM YM-244769 are illustrated in Fig. 3b, top. In this cell, 1 μM YM-244769 inhibited the unidirectional outward INCX by about 90 % at +20 mV (Fig. 3a, b, bottom). Similar results were obtained in three other cells. YM-244769 at 1 μM inhibited the unidirectional outward INCX by 87.8 ± 1.5 %. With the pipette solution containing 0 mM Na+ and 226 nM free Ca2+, the unidirectional inward INCX was induced by changing the external solution from 0 mM [Na+]o replaced by 140 mM [Li+]o to 140 mM [Na+]o for 1 to 2 min. Then, after returning to the 0 mM Na+ solution, 1 μM YM-244769 was added to the external solution for 5 to 6 min and [Na+]o was reintroduced in the external solution in the presence of YM-244769. Control concatenated currents and I-V curves of the control (Fig. 4a) and in the presence of 1 μM YM-244769 (Fig. 4b) are illustrated. In this cell, 1 μM YM-244769 inhibited the unidirectional inward INCX by only about 40 %. Similar results were obtained in 20 other cells.

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a

YM-244769 3 µM KB-R7943 100 µM

0.4 nA 0

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0

nA 0.3

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mV

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-60

-120

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Fig. 2 Effect of YM-244769 on the bidirectional INCX. a Concatenated current trace. Pulse interval was 10 s. Bars above indicate where 3 nM YM-244769 and 100 μM KB-R7943 were superfused. b Current-voltage relationship curves (I-V curves) obtained from corresponding labels in a.

Control (a), in the presence of 3 μM YM-244769 (b), and 100 μM KBR7943 (c). c Differences of I-V curves between a and b; a–b, and between b and c; b–c shown in b. YM-244769-sensitive current reversed at similar voltages as the KB-R7943-sensitive current, indicating that both are INCX

YM-244769 at 10 μM inhibited the unidirectional inward INCX by 50.8 ± 5.3 % (n = 16). Concentration-inhibition curves were obtained between 0.01 and 3 μM YM-244769 for the bidirectional and unidirectional outward or inward INCX (Fig. 5). YM-244769 suppressed the bidirectional and unidirectional outward INCX in a concentration-dependent manner. The bidirectional outward and inward INCX were measured at 20 and −140 mV, respectively. The IC50 values of YM-244769 against the bidirectional outward and inward INCX were 0.12 and 0.1 μM, respectively, with Hill coefficients of 0.7 and 0.8, respectively. The unidirectional outward INCX was measured at +20 mV. The

IC50 value for the unidirectional outward INCX was 0.05 μM with a Hill coefficient of 0.9. In contrast, even 1 and 5 μM YM-244769 inhibited the unidirectional inward INCX only by about 40 to 55 %. Thus, YM-244769 is a strongly directiondependent inhibitor, and the unidirectional outward INCX was the most potently suppressed.

a

Effect of YM-244769 at different [Na+]i SEA0400 inhibited INCX in a [Na+]i-dependent manner in Xenopus laevis oocytes expressing cloned cardiac NCX (Lee et al. 2004). [Na+]i-dependent inhibition was also

b YM244769 1 µM

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Fig. 3 Effect of YM-244769 on the unidirectional outward INCX. a Unidirectional outward INCX was induced by raising external Ca2+ from 0 to 2 mM in control (top). After returning to 0 mM Ca2+ in the same cell, 1 μM YM-244769 was applied and external Ca2+ was raised to 2 mM. b

-50 mV

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I-V curves of (a) and (b) from the control chart recording in the absence of 1 μM YM-244769 (top). a, b I-V curves of (c) and (d) from the chart recording in the presence of 1 μM YM-244769 (bottom)

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a

Fig. 4 Effect of YM-244769 on the unidirectional inward INCX. a, b Unidirectional inward INCX was induced by raising external Na+ from 0 mM (140 mM Li+) to 140 mM Na+ in the control and in the absence of 1 μM YM-244769 (top). a, b I-V curves obtained from control chart recording at 0 mM Na+ (a) and 140 mM Na+ (b) (bottom). c I-V curves from the chart recording in the presence of 1 μM YM-244769; (c) at 0 mM Na+ and at 140 mM Na+ (d)

YM244769 1 M

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observed with KB-R7943 and SN-6 in guinea pig isolated ventricular myocytes (Niu et al. 2007). We examined this property with YM-244769. Figure 6a illustrates the net IV curves of INCX obtained by subtracting the I-V curves with 100 μM KB-R7943 from those before and after 1 μM YM-244769 application at 10, 20, and 30 mM [Na+]i in the pipette solution. Figure 6b summarizes the effect of 1 μM YM-244769 on the bidirectional outward Bi-outward INCX Bi-inward INCX Uni-outward INCX Uni-inward INCX

50

0

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Fig. 5 Concentration-response curves of YM-244769 on I NCX . Comparison of concentration-response curves of YM-244769 for bidirectional and unidirectional outward and inward INCX. IC50 values of YM-244769 were 0.1, 0.12, and 0.05 μM for the bidirectional outward and inward I NCX and the unidirectional outward I NCX , respectively (n = 3 to 6). In contrast, 1 and 10 μM YM-244769 inhibited the unidirectional inward INCX by only 45 and 50 % (n = 21, 16). The magnitudes of the bidirectional outward and inward NCX currents were measured at −140 and +20 mV, respectively. The unidirectional outward INCX was measured at +50 mV. The magnitude of the unidirectional inward INCX was measured at −100 mV. The ratio of inhibition by YM-244769 on the unidirectional outward and inward INCX is expressed as a percentage of the control current. The values are expressed as means ± s.e. mean of 3 to 21 experiments

20

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0

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INCX at 10, 20m and 30 mM [Na+]i. The bidirectional outward INCX was measured at +20 mV. Although the rate of inhibition was similar between 20 and 30 mM [Na+]i, it was significantly smaller at 10 mM [Na+]i. The inhibitory potency of 1 μM YM-244769 is as follows: 30 mM [Na+]i > 20 mM [Na+]i > 10 mM [Na+]i. The inhibitory effect of YM-244769 on the bidirectional outward INCX are [Na+]idependent, similarly to other benzyloxyphenyl derivatives such as KB-R7943 and SN-6 (Niu et al. 2007). We also examined the [Na+]i-dependent inhibition of YM244769 on the unidirectional inward INCX. Figure 6c summarizes the inhibition of YM-244769 on the unidirectional inward INCX between 0 and 5 mM [Na+]i.. The inhibition of YM-244769 at 1 μM and 10 μM on the unidirectional inward INCX indicated a more potent tendency at 5 mM [Na+]i than at 0 mM [Na+]i. However, these results indicate that there was no significant difference in the inhibitory effect of YM24769 on INCX between 0 and 5 mM [Na+]i.

Effect of trypsin on YM-244769 inhibition of INCX We examined whether YM-244769 inhibition was sensitive to trypsin. Trypsin (2.5 μg/ml) was introduced to the cell via the pipette solution. 2,3-butanedione monoxime (BDM) was used as an indicator of trypsin effectiveness, because 10 mM BDM is known to inhibit INCX by about 90 %, but not in the presence of trypsin in the pipette solution (Watanabe et al. 2001). Confirming with BDM that trypsin was effectively perfused in the cell, 3 μM YM-244769 was applied in the bath solution (Fig. 7a). YM-244769 inhibited INCX even in the presence of trypsin (Fig. 7a). Finally, KB-R7943, a trypsin-insensitive NCX inhibitor (Watanabe et al. 2006), was applied to completely inhibit INCX. Figure 7b indicates I-V curves

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a 20 mM [Na]i

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% Inhibition of bi-directional INCX

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Fig. 6 [Na ]i-dependent inhibitory effect of YM-244769 on INCX. a Difference I-V curves of 1 μM YM-244769 on bidirectional outward INCX at 10, 20, and 30 mM [Na+]i. Each I-V curve was after subtraction of the current recorded in the presence of 100 μM KB-R7943. b Comparison of inhibitory effect of 1 μM YM-244769 on bidirectional outward INCX at 10, 20, and 30 mM [Na+]i. The magnitude of the outward

INCX was measured at +20 mV. c Effect of 1 or 10 μM YM-244769 on the unidirectional inward INCX at 0 or 5 mM [Na+]i. The inward INCX was measured at −100 mV. The values are expressed as means ± s.e. mean of 3 to 21 experiments. The numbers of cells are indicated in parenthesis. **significant difference (p < 0.01)

of the control (a), in the presence of BDM (b), with added YM-244769 (c) and with added KB-R7943 (d). YM244769 at 3 μM inhibited the INCX by about 90 % in this cell (Fig. 7b). As summarized in Fig. 7c, 3 μM YM244769 blocked INCX by 91.2 ± 1.7 % (n = 4) after trypsin treatment and by 92.6 ± 1.0 % (n = 3) without trypsin treatment. This suggests that YM-244769 is a trypsininsensitive NCX inhibitor.

selective NCX1 inhibitors. Therefore, we examined about the electrophysiology properties of NCX1 of a new selective NCX inhibitor, YM-244769, reported by Iwamoto and Kita (2006). In the present study, using isolated guinea pig cardiac ventricular cells, we found that YM-244769 inhibited INCX in a concentration- and [Na+]i-dependent manner. The IC50 value of YM-244769 against the unidirectional outward INCX was 50 nM. The IC50 values against the bidirectional outward and inward INCX were similar and approximately 100 nM with a Hill coefficient of about 1. YM-244769 is a novel synthesized high-potential benzyloxyphenyl NCX inhibitor and preferentially inhibits NCX3 rather than NCX1 or NCX2 (Iwamoto 2004; Iwamoto and Kita 2006). By measuring 45Ca2+ flux using CCL39 fibroblasts stably expressing NCX isoforms or mutants, Iwamoto and Kita (2006) reported that the IC50 of YM-244769 was 18 nM against NCX3, which was higher than the 68 nM against NCX1 and similar to the IC50 of SEA0400 at 56 nM against NCX 1. The four benzyloxyphenyl NCX inhibitors, KB-R7943, SEA0400, SN-6, and YM-244769, have different selectivities for the three NCX isoforms (Iwamoto et al. 2007). KB-R7943 inhibits NCX1 with IC50 4.3 μM, NCX2 with IC50 4.7 μM, and NCX3 with IC50 1.5 μM (Iwamoto

+

Discussions NCX1 leads to intracellular Ca2+ overload under pathological conditions, including ischemia/reperfusion injury, arrhythmia, triggered activity, hypertrophy, and heart failure. Under such pathological conditions, selective NCX1 inhibitors would be useful by preventing intracellular Ca2+ overload in heart (Watanabe et al. 2006). In small animal models, the specific NCX inhibitors generally have benefits by reducing excessive calcium overload owing to various cardiac pathological events such as ischemia/reperfusion injury, arrhythmia, triggered activity, hypertrophy, and heart failure. And then, in larger animal models with established cardiac pathologies, it is needed to evaluate the therapeutic efficacy and safety of the

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Naunyn-Schmiedeberg's Arch Pharmacol (2016) 389:1205–1214

a

Trypsin

pip

BDM

10 mM

YM-244769 3 µM KBR-7943 100 µM

pA 100 0

(a)

1 min

200 -100 mV 100

(b) (a) (c)-60 (d)

100

% Inhibition of INCX

c

b pA

(c)

(b)

(d)

(3)

(4)

Trypsin (-)

Trypsin (+)

50

0 YM-244769 3 µM

Fig. 7 Effects of trypsin on the YM-244769 inhibition of INCX. a Chart recording of the current with the pipette solution containing trypsin (2.5 μg ml−1). The horizontal bars above the current indicate where the drugs indicated were applied. b I-V curves obtained at the corresponding labels in a. a is control, b in the presence of BDM, c in the presence of

YM-244769, and d in the presence of KB-R7943. c Summarized data. The effect of YM-244769 was not affected by trypsin. The values are expressed as means ± s.e.mean of 3 to 4 experiments. The numbers of cells are indicated in parenthesis

et al. 2001; Iwamoto 2004; Iwamoto and Kita 2006). The IC50 values of SEA0400 are 56 nM against NCX1 and 980 nM against NCX2, without inhibition of NCX3 (Iwamoto 2004). The IC50 values of SN-6 are 2.9 μM against NCX1, 16 μM against NCX2, and 8.6 μM against NCX3 (Iwamoto et al. 2004a). Therefore, YM-244769 and SEA0400 inhibit NCX1 with similar potency, which is more potent than KB-R7943 or SN-6. These inhibitors did not inhibit the extracellular Na+-dependent Ca2+ exit via NCX1 (Iwamoto 2004; Iwamoto and Kita 2006; Iwamoto et al. 2007). In this study, to confirm the [Na+]i,-dependent inhibition of YM-244769 on bidirectional I NCX , we used supraphysiological intracellular concentration such as 10, 20, and 30 mM Na+ as reported previously (Niu et al. 2007). YM-244769 inhibited bidirectional I NCX in a [Na+]i,-dependent manner. The inhibitory effects of YM244769 on unidirectional inward I NCX were similar at 0 mM [Na+]i. Our results indicate that the inhibition of I NCX by YM-244769 was more potent as [Na + ] i was higher for the bidirectional NCX1 current. Hilgemann et al. (1992) reported that the steady state of inward NCX current is inhibited by cytoplasmic sodium ([Na+]idependent inactivation). Iwamoto et al. (2004b) suggested that the site of [Na+]i-sensitive inactivation of the exchanger molecule is at the large cytoplasmic loop of the plasma membrane. SN-6, KB-R7943, and SEA0400 also inhibited Ca2+ uptake via the NCX in a [Na+]i-dependent

manner (Lee et al. 2005; Niu et al. 2007; Ozdemir et al. 2008). Recently, two groups published ten transmembrane segment (TMS) topology models of mammalian NCX1; the major difference between them is in the orientation of the three C-terminal TMs, but not in the large intracellular loop between TMS 5 and 6 (Ren and Philipson 2013; Szerencsei et al. 2013). NCX1 in canine cardiac myocytes consists of 938 amino acids with a large intracellular loop between TMS 5 and 6, which contains essential segments for the regulation of NCX1 activity. It has been suggested that the exchanger inhibitory peptide (XIP) region in the large intracellular loop connecting TMS 5 and 6 of NCX1 is involved in the intracellular Na+-dependent inactivation (I1 inactivation) (Iwamoto et al. 2004b). YM-244769 may stabilize the I1 inactive state or accelerate the rate of I1 inactivation in a similar manner to that suggested for KBR7943, SEA0400, and SN-6 (Iwamoto 2004; Lee et al. 2005; Iwamoto and Kita 2006; Iwamoto et al. 2007; Niu et al. 2007). However, this agent at 1 or 10 μM did not significantly inhibit the unidirectional INCX in a [Na+]i,dependent manner but had a inhibiting trend. The Hill coefficient of unity among the four benzyloxyphenyl inhibitors including YM-244769 may suggest that a single-molecule binding is enough to express their inhibitory effect (Kimura et al. 1999; Tanaka et al. 2002; Niu et al. 2007). Mutational analyses have indicated that the XIP region (I1 inactivation site-mapped Val-227 and Tyr-228), XIP

Naunyn-Schmiedeberg's Arch Pharmacol (2016) 389:1205–1214

neighboring regions (mapped Phe-213) in TMS 5, and the α-2 repeat mapped Gly-833 at the re-entrant loop of TMS 7 and 8 may commonly contribute to interact as the inhibitory domain with YM-244769, SN-6, KB-R7943, and SEA0400 (Iwamoto 2004; Iwamoto et al. 2007). Interestingly, the ion transport pathway of NCX is suggested to consist of membrane loops with α-1 and α-2 regions that face each other and TMS at both sides of two membrane loops (Iwamoto et al. 2000; Qiu et al. 2001). NCX α-2 region includes the I1 inactivation site in the cytoplasmic long loop between TMS 7 and 8 (Iwamoto et al. 2007). Therefore, we conjecture that YM-244769 may inhibit INCX by simultaneously blocking both the I1 inactivation site and α-2 region from the cytoplasm side or extracellular side. Recently, Birinyi et al. (2005) reported that, in canine ventricular cardiomyocytes, there was a significant difference in the KB-R7943-induced suppression between bidirectional outward and inward I NCX , but not for the SEA0400-induced suppression. Ozdemir et al. (2008) also reported that the inhibition effect of SEA0400 on the Ca2+ entry mode of the bidirectional INCX was larger than on the Ca2+ exit mode in pig or mouse ventricular myocytes. In the present study, however, we did not find any significant difference in the inhibition of YM244769 between bidirectional outward and inward INCX, as previously described (Watano et al. 1996; Kimura et al. 1999; Watanabe and Kimura 2000; Watanabe et al. 2001; Tanaka et al. 2002; Niu et al. 2007). Although mammalian hearts express only a single isoform NCX1, alternatively spliced isoforms are found in various tissues and species. The NCX1 gene contains six exons, A, B, C, D, E, and F, which make up the alternatively spliced regions (Schulze et al. 2002). The alternative splicing might result in differences in drug sensitivity. NCX inhibitors can be classified into two groups: one is trypsin-sensitive and another trypsin-insensitive. The trypsinsensitive NCX inhibitors include BDM and antiarrhythmic agents, such as amiodarone and bepridil. The trypsininsensitive NCX inhibitors are aprindine, dronedarone, azimilide, cibenzoline, KB-R7943, SEA0400, and SN-6 (Watanabe et al. 2006). YM-244769 is trypsin-insensitive, like the other three benzyloxyphenyl inhibitors. NCX inhibitors may be useful in preventing Ca2+ overload during ischemia-reperfusion injury and arrhythmias associated with heart failure. In this study, we showed that YM-244769, a novel and selective NCX blocker, inhibits the cardiac INCX in a similar manner to SEA0400. YM-244769 inhibits not only NCX1 but also NCX3, which is an NCX subtype expressed in neurons and skeletal muscles. This drug may be a powerful new tool for studying the function of NCX in the physiology and pathology of various organs.

1213 Acknowledgements This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (17590223, 21590288) to J.K. and a grant-in-aid from the Smoking Research Foundation (KI18003) to J.K. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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