Inhibitory profile of SEA0400 [2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline] assessed on the cardiac Na+-Ca2+ exchanger, NCX1.1
Inhibitory profile of SEA0400 [2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline] assessed on the cardiac Na+-Ca2+ exchanger, NCX1.1
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DOI：
10.1124/jpet.104.070805
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发表时间：
2004-11-01
影响因子：
3.5
通讯作者：
Hryshko, LV
中科院分区：
文献类型：
--
作者：
Lee, C;Visen, NS;Hryshko, LV
SEA0400（2-[4-[(2,5-二氟苯基）甲氧基]苯氧基]-5-乙氧基苯胺）最近被描述为心脏、神经元和肾脏制剂中 Na+-Ca2+ 交换的有效和选择性抑制剂。研究了 SEA0400 对非洲爪蟾卵母细胞中表达的克隆心脏 Na+-Ca2+ 交换器 NCX1.1 的抑制作用，以深入了解其抑制机制。使用巨型切除贴片技术测量Na+-Ca2+交换电流，并使用评估内向和外向电流的条件。 SEA0400 以高亲和力抑制向外的 Na+-Ca2+ 交换电流（峰值电流和稳态电流的 IC50 分别为 78 +/- 15 和 23 +/- 4 nM）。观察到内向电流的抑制效力（即微摩尔）相当低。用α-胰凝乳蛋白酶对切除的斑块进行蛋白水解处理后，重新检查抑制谱，这是一种消除离子调节机制的过程。经过这种处理后，估计外向电流的 IC50 值为 1.2 +/- 0.6 muM，而内向电流对 SEA0400 几乎变得不敏感。尽管注意到了不同的特征，但 SEA0400 对外向交换电流的抑制作用在高浓度和低浓度的调节性 Ca2+ 下都很明显。 SEA0400加速了外向电流的灭活速度。基于配对脉冲实验，SEA0400 改变了交换器从 Na-i(+) 依赖性非活性状态的恢复，特别是在较高的调节 Ca-i(2+) 浓度下。最后，SEA0400 的抑制效力强烈依赖于细胞内 Na+ 浓度。我们的数据证实，SEA0400 是迄今为止描述的心脏 Na+-Ca2+ 交换器最有效的抑制剂，并为其明显的转运模式选择性提供了合理的解释。
SEA0400 (2-[4-[(2,5-difluorophenyl) methoxy] phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na+-Ca2+ exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na+-Ca2+ exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na+-Ca2+ exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na+-Ca2+ 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 alpha-chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC50 value of 1.2 +/- 0.6 muM 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 Ca2+, 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(2+) 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+-Ca2+ exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity.