Okutsu Saeko, Hatakeyama Hiroyasu, Kanzaki Makoto, Tsubokawa Hiroshi, Nagatomi Ryoichi
Department of Medicine and Science in Sports and Exercise, Tohoku University Graduate School of Medicine, Sendai, Japan.
Tohoku J Exp Med. 2008 Jun;215(2):181-7. doi: 10.1620/tjem.215.181.
Excess glutamate and Ca(2+) influx into neurons exacerbate brain damage such as ischemia. Astrocytes at the site of damage proliferate and attenuate the glutamate- and Ca(2+)-induced neuronal damage by removing excess glutamate and Ca(2+) through the N-methyl-D-aspartate (NMDA) glutamate receptor and the L-type Ca(2+) channel, respectively. Fibroblasts are commonly mobilized to the site of damage, probably supporting the restoration process. Notably, fibroblasts express the L-type voltage-sensitive Ca(2+) channel, but not central nervous system-specific NMDA glutamate receptor. We examined if electric pulse stimulation (EPS) was capable of inducing NMDA receptor on fibroblasts by way of Ca(2+) channel activation, so that they could potentially have a neuroprotective role. To activate L-type Ca(2+) channel, we delivered electric pulse to cultured NIH3T3 mouse fibroblasts. EPS of 20 V with a pulse duration of 2 msec at a frequency of 1 Hz for more than 1 h up to 24 h successfully introduced Ca(2+) into NIH3T3 fibroblasts as detected by Fluo-4AM calcium imaging, which was totally inhibited by a L-type Ca(2+) channel inhibitor, verapamil. Remarkable expression of NMDA receptor mRNA in the fibroblasts after 24-h EPS was demonstrated by RT-PCR. Verapamil treatment during EPS totally abrogated the EPS-induced NMDA receptor mRNA expression. To the best of our knowledge, this is the first report showing that electric pulse is able to induce sustained Ca(2+) influx via L-type Ca(2+) channel in a non-excitatory fibroblast, which leads to the expression CNS-specific NMDA receptor mRNA. Neuroprotective role of NMDA receptor induced in fibroblasts needs to be further examined.
过量的谷氨酸和钙离子流入神经元会加剧诸如局部缺血等脑损伤。损伤部位的星形胶质细胞会增殖，并通过分别经由N-甲基-D-天冬氨酸（NMDA）谷氨酸受体和L型钙离子通道清除过量的谷氨酸和钙离子，来减轻谷氨酸和钙离子诱导的神经元损伤。成纤维细胞通常会被募集到损伤部位，可能对修复过程起到支持作用。值得注意的是，成纤维细胞表达L型电压敏感性钙离子通道，但不表达中枢神经系统特异性的NMDA谷氨酸受体。我们研究了电脉冲刺激（EPS）是否能够通过激活钙离子通道在成纤维细胞上诱导NMDA受体的产生，从而使其可能具有神经保护作用。为了激活L型钙离子通道，我们对培养的NIH3T3小鼠成纤维细胞施加电脉冲。通过Fluo-4AM钙成像检测发现，以1 Hz的频率施加20 V、脉冲持续时间为2毫秒的EPS超过1小时直至24小时，成功地将钙离子导入NIH3T3成纤维细胞，而L型钙离子通道抑制剂维拉帕米完全抑制了这种导入。RT-PCR证实了24小时EPS处理后成纤维细胞中NMDA受体mRNA的显著表达。EPS期间维拉帕米处理完全消除了EPS诱导的NMDA受体mRNA表达。据我们所知，这是首次报道电脉冲能够通过L型钙离子通道在非兴奋性成纤维细胞中诱导持续的钙离子内流，进而导致中枢神经系统特异性NMDA受体mRNA的表达。成纤维细胞中诱导产生的NMDA受体的神经保护作用有待进一步研究。
