A technique for quantifying intracellular free sodium ion using a microplate reader in combination with sodium-binding benzofuran isophthalate and probenecid in cultured neonatal rat cardiomyocytes.
A technique for quantifying intracellular free sodium ion using a microplate reader in combination with sodium-binding benzofuran isophthalate and probenecid in cultured neonatal rat cardiomyocytes.
复制标题
DOI：
10.1186/1756-0500-6-556
复制
发表时间：
2013-12-26
影响因子：
1.8
通讯作者：
Yoshimura M
中科院分区：
文献类型：
--
作者：
Katoh D;Hongo K;Ito K;Yoshino T;Kayama Y;Komukai K;Kawai M;Date T;Yoshimura M
细胞内钠（[Na+]i）动力学参与心脏疾病，包括缺血、心力衰竭和肥大。由于[Na+]i在调节心脏的电活动和收缩活动中起着至关重要的作用，因此对[Na+]i进行定量具有很大的意义。使用荧光显微镜和钠结合苯并呋喃三乙酸盐（SBFI）是测量[Na+]i的最常用方法。然而，与该技术相关的一个限制是，该测试不能同时评价几种类型或不同浓度的化合物对[Na+]i的影响。此外，关于化合物对培养细胞中[Na+]i的长期影响的报道很少，尽管已经广泛讨论了在几秒钟或几分钟内[Na+]i的快速变化。我们建立了一种新的技术，定量[Na+]i在培养的新生大鼠心肌细胞连接到96孔板使用酶标仪结合SBFI和丙磺舒。我们表明丙磺舒是不可缺少的准确测量，因为它可以防止染料从细胞泄漏。我们通过量化哇巴因的作用进一步证实了该系统的可靠性，哇巴因已知会短暂改变[Na+]i。为了说明新方法的实用性，我们还研究了醛固酮对培养的心肌细胞中[Na+]i的慢性影响。我们的技术可以快速测量[Na+]i的准确性和灵敏度与传统的显微镜为基础的方法。结果表明，这种基于96孔板的测量具有优点，特别是用于筛选调节[Na+]i的化合物的测试，并且有助于阐明心肌细胞中改变的[Na+]i处理的机制和后果。
Intracellular sodium ([Na+]i) kinetics are involved in cardiac diseases including ischemia, heart failure, and hypertrophy. Because [Na+]i plays a crucial role in modulating the electrical and contractile activity in the heart, quantifying [Na+]i is of great interest. Using fluorescent microscopy with sodium-binding benzofuran isophthalate (SBFI) is the most commonly used method for measuring [Na+]i. However, one limitation associated with this technique is that the test cannot simultaneously evaluate the effects of several types or various concentrations of compounds on [Na+]i. Moreover, there are few reports on the long-term effects of compounds on [Na+]i in cultured cells, although rapid changes in [Na+]i during a period of seconds or several minutes have been widely discussed. We established a novel technique for quantifying [Na+]i in cultured neonatal rat cardiomyocytes attached to a 96-well plate using a microplate reader in combination with SBFI and probenecid. We showed that probenecid is indispensable for the accurate measurement because it prevents dye leakage from the cells. We further confirmed the reliability of this system by quantifying the effects of ouabain, which is known to transiently alter [Na+]i. To illustrate the utility of the new method, we also examined the chronic effects of aldosterone on [Na+]i in cultured cardiomyocytes. Our technique can rapidly measure [Na+]i with accuracy and sensitivity comparable to the traditional microscopy based method. The results demonstrated that this 96-well plate based measurement has merits, especially for screening test of compounds regulating [Na+]i, and is useful to elucidate the mechanisms and consequences of altered [Na+]i handling in cardiomyocytes.