Fukuda A, Deshpande S B, Shimano Y, Nishino H
Department of Physiology, Nagoya City University Medical School, Nagoya, Japan.
Neuroscience. 1998 Nov;87(2):497-507. doi: 10.1016/s0306-4522(98)00139-0.
The differential effects of 3-nitropropionic acid on cultured neurons and astrocytes (of cortical and striatal origin) were examined by studying intracellular Ca2+ changes using imaging techniques with fura-2. The neurons and astrocytes whose intracellular Ca2+ concentration was recorded were identified later by immunocytochemical staining for microtubule-associated protein 2 and glial fibrillary acidic protein, respectively. 3-Nitropropionic acid (1.7 mM) irreversibly increased intracellular Ca2+ in astrocytes (27%) and, to a significantly smaller extent, in neurons (10%). The latency to onset of the intracellular Ca2+ increase was longer in neurons (45 min) than in astrocytes (29 min). Thus, a differential susceptibility of astrocytes and neurons was observed. The 3-nitropropionic acid-induced astrocytic and neuronal Ca2+ accumulations were both due to influx of Ca2+, as the increases were absent in Ca2+-free medium. An inhibitor of the Na+-Ca2+ exchanger (2',4'-dichlorobenzamil), greatly reduced the intracellular Ca2+ increase in astrocytes, but not in neurons. This indicates that the intracellular Ca2+ increase in astrocytes is primarily mediated by a reverse operation of the Na+-Ca2+ exchange system, whereas in neurons it is mediated by a different mechanism. In addition, we noted that astrocytic cell death occurred in 9% of cells at 60 min or more after the start of a 40 min perfusion with 3-nitropropionic acid, while only 4% of neurons died. In astrocytes, cell death was preceded by blebbing of the cell membrane, and by a sustained increase in intracellular Ca2+ followed by an abrupt further elevation occurring just before cellular collapse. The present results indicate that astrocytes are more vulnerable than neurons to 3-nitropropionic acid-induced cellular Ca2+ overload and toxicity, and hence support the hypothesis that, in part, 3-nitropropionic acid-induced neurotoxicity could be secondary to astrocytic cell death caused by Ca2+ overload.
通过使用fura-2成像技术研究细胞内Ca2+变化，检测了3-硝基丙酸对培养的（皮质和纹状体来源的）神经元和星形胶质细胞的不同作用。随后，分别通过对微管相关蛋白2和胶质纤维酸性蛋白进行免疫细胞化学染色，鉴定出记录其细胞内Ca2+浓度的神经元和星形胶质细胞。3-硝基丙酸（1.7 mM）使星形胶质细胞内Ca2+不可逆地增加（27%），而在神经元中增加的程度明显较小（10%）。神经元内Ca2+增加开始的潜伏期（45分钟）比星形胶质细胞（29分钟）长。因此，观察到星形胶质细胞和神经元的敏感性存在差异。3-硝基丙酸诱导的星形胶质细胞和神经元Ca2+积累均归因于Ca2+的内流，因为在无Ca2+培养基中没有增加。Na+-Ca2+交换体抑制剂（2',4'-二氯苯甲酰胺）极大地减少了星形胶质细胞内Ca2+的增加，但对神经元没有影响。这表明星形胶质细胞内Ca2+的增加主要由Na+-Ca2+交换系统的反向运作介导，而在神经元中则由不同机制介导。此外，我们注意到在用3-硝基丙酸进行40分钟灌注开始后60分钟或更长时间，9%的星形胶质细胞发生细胞死亡，而只有4% 的神经元死亡。在星形胶质细胞中，细胞死亡之前细胞膜出现泡状突起，细胞内Ca2+持续增加，随后在细胞崩溃前突然进一步升高。目前的结果表明，星形胶质细胞比神经元更容易受到3-硝基丙酸诱导的细胞Ca2+过载和毒性的影响，因此支持这样的假设，即3-硝基丙酸诱导的神经毒性部分可能继发于Ca2+过载导致的星形胶质细胞死亡。
