Devinney Michael J, Reynolds Ian J, Dineley Kirk E
Department of Pharmacology, University of Pittsburgh School of Medicine, W1351 BST, Pittsburgh, PA 15261, USA.
Cell Calcium. 2005 Mar;37(3):225-32. doi: 10.1016/j.ceca.2004.10.003.
Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+]i and [Zn2+]i in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+]i is high relative to [Zn2+]i, we found that fura-2FF responds mostly to [Ca2+]i but is relatively unaffected by low [Zn2+]i. Moreover, fura-2FF ratio changes caused by high [Ca2+]i or high [Zn2+]i could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+]i and [Ca2+]j, respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells.
细胞内游离锌（[Zn2+]i）水平升高可能在涉及钙调节异常和氧化应激的损伤模型（如谷氨酸兴奋性毒性）中导致细胞死亡。然而，在活细胞中同时监测这两种离子是困难的。在此，我们提出一种利用荧光显微镜以及离子敏感指示剂fura-2FF和FluoZin-3来监测原代皮质神经元中[Ca2+]i和[Zn2+]i的新方法。我们发现新型单波长染料FluoZin-3对少量锌负荷反应强烈，对高浓度Ca2+或Mg2+不敏感，且相对不受低pH值或氧化剂的影响。比率型指示剂fura-2FF对Ca2+和Zn2+均敏感。然而，在类似于兴奋性毒性谷氨酸暴露的条件下，即[Ca2+]i相对于[Zn2+]i较高时，我们发现fura-2FF主要对[Ca2+]i作出反应，而相对不受低[Zn2+]i的影响。此外，由高[Ca2+]i或高[Zn2+]i引起的fura-2FF比率变化可以区分，因为每种离子产生不同的光谱响应。最后，双染料实验表明，在强烈谷氨酸暴露期间，FluoZin-3和fura-2FF在同一神经元中分别对[Zn2+]i和[Ca2+]i有强烈反应。这些研究提供了一种在细胞中同时检测钙和锌的新方法。
