Role of mitochondrial Na+ concentration, measured by CoroNa Red, in the protection of metabolically inhibited MDCK cells
Role of mitochondrial Na+ concentration, measured by CoroNa Red, in the protection of metabolically inhibited MDCK cells
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DOI：
10.1681/asn.2005010075
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发表时间：
2005-12-01
影响因子：
13.6
通讯作者：
Smets, I
中科院分区：
文献类型：
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作者：
Baron, S;Caplanusi, A;Smets, I
在缺血或缺氧组织中，胞浆钙水平升高可诱导致死过程。除了内质网外，线粒体在清除细胞内过多的Ca ~（2+）中起着关键作用。在先前的研究中，有人提出，在肾上皮细胞中代谢抑制（MI）约18分钟后，细胞溶质Ca 2+的清除通过线粒体Na+/Ca 2+交换器（NCX）的反向作用发生。为了进一步研究潜在的机制，在代谢抑制的MDCK细胞中监测线粒体Na+浓度（[Na+]，）的变化。钠敏感荧光探针CoroNa Red用于监测[Na+]（m）。在MI的前15分钟，观察到[Na+]（m）增加两倍，达到113 +/- 7 mM，而胞质Na+浓度（[Na+]（c））升高三倍，达到65 +/- 6 mM的水平。在接下来的MI的45分钟，[Na+]（m）下降到91 +/- 7 mM，而[Na+]（c）进一步增加到91 +/-4 mM.[Na+]（m）的显著增加可能足以维持线粒体通过NCX摄取Ca 2+的驱动力。此外，当在MI期间应用线粒体NCX的特异性抑制剂CGP-37157时，[Na+]（m）的第二相下降被完全消除。所获得的结果支持的假设，线粒体NCX逆转后约15分钟的MI。此外，由于MI后细胞内稳态可以恢复，线粒体可能通过逆转线粒体NCX保护MDCK细胞免受MI期间的损伤。这项研究是第一个报告[Na+]（m）的测量在nonpermeabilized活细胞。
In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+],) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+](m). In the first 15 min of MI, a twofold increase of [Na+](m) was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+](c)) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+](m) dropped to 91 +/- 7 mM, whereas [Na+](c) further increased to 91 +/- 4 mM. The striking rise in [Na+](m) is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+](m) was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+](m) measurements in nonpermeabilized living cells.