Inhibitory Profile of SEA0400 [2-[4-[(2,5- Difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline] Assessed on the Cardiac Na -Ca Exchanger, NCX1.1

SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na -Ca exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na -Ca exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na -Ca exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na Ca exchange currents with high affinity (IC50 78 15 and 23 4 nM for peak and steady-state currents, respectively). Considerably less inhibitory potency (i.e., micromolar) was observed for inward currents. The inhibitory profile was reexamined after proteolytic treatment of excised patches with -chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC50 value of 1.2 0.6 M was estimated for outward currents, whereas inward currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 on outward exchange currents were evident at both high and low concentrations of regulatory Ca , although distinct features were noted. SEA0400 accelerated the inactivation rate of outward currents. Based on paired pulse experiments, SEA0400 altered the recovery of exchangers from the Na i-dependent inactive state, particularly at higher regulatory Ca i concentrations. Finally, the inhibitory potency of SEA0400 was strongly dependent on the intracellular Na concentration. Our data confirm that SEA0400 is the most potent inhibitor of the cardiac Na Ca exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity. In cardiac muscle, Na -Ca exchange is the primary mechanism for trans-sarcolemmal Ca removal, a process essential for muscle relaxation (Blaustein and Lederer, 1999; Hryshko, 2002). In general, the Na -Ca exchanger removes the same quantity of Ca that enters the myocyte through L-type Ca channels on a beat-to-beat basis (Bers, 2000). As a reversible ion counter-transporter, the exchanger can also serve as a Ca entry mechanism and can potentially contribute to Ca -induced Ca release from the sarcoplasmic reticulum (Leblanc and Hume, 1990; Bers, 2000). During ischemia-reperfusion, reverse mode Na -Ca exchange is established as an important contributor to cellular injury (Mochizuki and Jiang, 1998; Seki et al., 2002). The general sequelae associated with ischemia-reperfusion injury are as follows: intracellular acidosis occurs during anaerobic metabolism associated with ischemia; upon reperfusion, the Na -H exchanger (NHE) alleviates the acidosis with concomitant increases in intracellular Na ([Na ])I; consequent diminution of the transmembrane Na gradient decreases the driving force for forward mode Na -Ca exchange and This study was supported by operating grants from the Heart and Stroke Foundation of Manitoba and the Canadian Institutes of Health Research (CIHR) and by a Canada Research Chair Award (to L.V.H.). C.L. was supported by a CIHR Postdoctoral Fellowship Award and the Tailored Advanced Collaborative Training in Cardiovascular Science for Research Fellows (TACTICS) Partnership Program of the CIHR and the Heart and Stroke Foundation. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.070805. ABBREVIATIONS: NHE, Na -H exchanger; [Ca ]i, intracellular Ca 2 ; KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; MES, 4-morpholineethanesulfonic acid; TEA, tetraethylammonium; MOPS, 4-morpholinepropanesulfonic acid; , rate of current decay; FSS, extent of current inactivation. 0022-3565/04/3112-748–757$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 311, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 70805/1174295 JPET 311:748–757, 2004 Printed in U.S.A. 748 at A PE T Jornals on July 0, 2017 jpet.asjournals.org D ow nladed from favors the occurrence of reverse mode; and intracellular Ca ([Ca ]i) increases and, depending upon severity, may produce Ca i overload and/or cell death. Under these conditions, inhibition of reverse mode Na -Ca exchange represents a promising approach toward attenuating ischemiareperfusion injury (Nakamura et al., 1998; Ladilov et al., 1999; Mukai et al., 2000; Schafer et al., 2001; Shigekawa and Iwamoto, 2001; Inserte et al., 2002; Seki et al., 2002). The critical roles of Na -Ca exchange in both physiological and pathophysiological cardiac function make it an attractive target for pharmacological manipulation (Kusuoka et al., 1993; Winslow et al., 1999; Shigekawa and Iwamoto, 2001; Sipido et al., 2002; Pogwizd, 2003). Two agents have recently been described as potent Na -Ca exchange inhibitors. The first compound, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate (KB-R7943), was described in 1996 and inhibits the exchanger with micromolar potency in different assay systems (Iwamoto et al., 1996; Watano et al., 1996). KB-R7943 also offers protection against arrhythmogenesis and contractile dysfunction associated with ischemia-reperfusion injury (Ladilov et al., 1999; Mukai et al., 2000; Elias et al., 2001; Schafer et al., 2001; Seki et al., 2002). Mechanistically, most studies have demonstrated that KB-R7943 preferentially inhibits the reverse mode of Na Ca exchange (Iwamoto et al., 1996; Watano et al., 1996; Elias et al., 2001), although another study has reported that the extent of inhibition is independent of transport direction under bidirectional ionic conditions (Kimura et al., 1999). However, KB-R7943 also inhibits several unrelated transport systems including Na , Ca , and K channels, complicating the interpretation of its cardioprotective actions (Sobolevsky and Khodorov, 1999; Arakawa et al., 2000; Pintado et al., 2000). Exemplifying this point, KB-R7943 inhibits Ca transients in myotubes obtained from NCX1.1 knockout mice, where its effects are clearly distinct from any actions on the Na -Ca exchanger (Reuter et al., 2002). A second inhibitor of the Na -Ca exchanger has recently been described with greatly increased potency (i.e., nanomolar range) compared with KB-R7943 (Matsuda et al., 2001; Tanaka et al., 2002). This compound, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), inhibits Na -Ca exchange activity in both neuronal (Matsuda et al., 2001) and cardiac (Tanaka et al., 2002; Magee et al., 2003; Takahashi et al., 2003) preparations, and exhibits protective effects against cerebral (Matsuda et al., 2001), myocardial (Takahashi et al., 2003), and renal ischemia-reperfusion injury (Ogata et al., 2003). Several other transport systems were examined, and SEA0400 was largely selective for the Na -Ca exchanger (Matsuda et al., 2001; Tanaka et al., 2002). Notably, however, SEA0400 also inhibits Ca transients in heart tubes from NCX1 knockout mice, arguing against strict specificity (Reuter et al., 2002). Based on these characteristics, the authors suggested that other ion channels might be targeted by SEA0400. Our study was undertaken to gain insight into the inhibitory mechanism of SEA0400. NCX1.1 was expressed in Xenopus laevis oocytes, and exchange activity was measured using the giant excised patch-clamp technique. Distinct experimental conditions were employed to assess the transport mode selectivity of SEA0400 and to determine whether its inhibitory effects involved the intrinsic ionic regulatory mechanisms of the exchanger. Materials and Methods Preparation of X. laevis Oocytes and Synthesis of NCX1.1 cRNA. X. laevis oocytes were prepared and stored as previously described (Elias et al., 2001). Complementary DNA encoding NCX1.1, residing in pBluescript II SK( ) (Stratagene, La Jolla, CA), was linearized with HindIII (New England Biolabs, Beverly, MA), and cRNA was synthesized using T3 mMessage mMachine in vitro transcription kits (Ambion, Austin, TX) according to the manufacturer’s instructions. Following injection with 23 ng of cRNA encoding NCX1.1, oocytes were maintained at 18°C, and electrophysiological measurements were obtained from days 3 to 6 postinjection. Measurement of Na -Ca Exchange Activity. Na -Ca exchange current measurements were obtained using the giant excised patch-clamp technique (Hilgemann, 1989), as previously described (Elias et al., 2001). Borosilicate glass pipettes were pulled and polished to a final diameter of 20 to 30 m and coated with a Parafilm/ mineral oil mixture to enhance patch stability and reduce electrical noise. The vitellin layer was removed by dissection, and oocytes were placed in a solution containing: 100 mM KOH, 100 mM MES, 20 mM HEPES, 5 mM EGTA, and 5 to 10 mM MgCl2, pH 7.0 at room temperature (with MES). Gigaohm seals were formed by suction and membrane patches (inside-out configuration) were excised by progressive movements of the pipette tip. Rapid solution changes were accomplished using a computer-controlled, 20-channel solution switcher. Axon Instruments Inc. (Union City, CA) hardware (Axopatch 200A) and software (AxoTape) were used for data acquisition and analysis, and Origin software was used for curve-fitting (e.g., determination of IC50 values) and statistical analyses. A holding potential of 0 mV was employed for all current measurements. For outward Na -Ca exchange current measurements, pipette (i.e., extracellular) solutions contained: 100 mM N-methyl-D-glucamineMES, 30 mM HEPES, 30 mM TEA-OH, 16 mM sulfamic acid, 8.0 mM CaCO3, 6 mM KOH, 0.25 mM ouabain, 0.1 mM niflumic acid, and 0.1 mM flufenamic acid, pH 7.0 at room temperature (with MES). Outward currents were elicited by switching from Li to Na -based bath solutions containing: 100 mM [Na Li]-aspartate, 20 mM CsOH, 20 mM MOPS, 20 mM TEA-OH, 10 mM EGTA, 0 to 9.91 mM CaCO3, and 1.0 to 1.5 mM Mg(OH)2, pH 7.0 at 30°C (with MES or LiOH). Free Mg and Ca were adjusted to yield free concentrations of 1.0 and 0 to 30 M, respectively, using MAXC software (Bers et al., 1994). For inward Na -Ca exchange current measurements, pipettes contained: 100 mM Na-MES, 20 mM CsOH, 20 mM TEA-OH, 10 mM EGTA, 10 mM HEPES, 8 mM sulfamic acid, 4 mM Mg(OH)2, 0.25 mM ouabain, 0.1 mM niflumic acid, and 0.1 mM flufenamic acid, pH 7.0 at 30°C (with MES). Inward currents were activated by switching between Ca -free and -containing, Li -based bath solutions, described above. For combined inward-outward current measurements, pipettes contained: 100 mM Na-MES, 20 mM CsOH, 20 mM HEPES, 20 mM TEA-OH, 4 mM sulfamic acid, 2 mM CaCO3, 0.25 mM ouabain, 0.1 mM niflumic acid, and 0.1 mM flufenamic acid, pH 7.0 at 30°C (with MES). Outward and inward currents were activated using the same solutions as those for initiating pure outward and pure inward currents, described above. Under Results, only the Na and Ca concentrations of experimental solutions are described, for brevity. All experiments were conducted at 30 1°C. SEA0400 was a generous gift from Taisho Pharmaceutical Co., Ltd. (Tokyo, Japan) and was prepared as a 20 mM stock solution in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide never exceeded 0.1% and was typically much lower. SEA0400 was applied to the cytoplasmic surface of the patch for all experiments.