Zakir Hossain S M, Shinohara Hiroaki, Wang Feifei, Kitano Hiromi
Department of Advanced Nano and Biosciences, Graduate School of Innovative Life Science, University of Toyama, 3190 Gofuku, Toyama 930-8555. Japan.
Anal Bioanal Chem. 2007 Nov;389(6):1961-6. doi: 10.1007/s00216-007-1569-4. Epub 2007 Sep 12.
There is an increasing interest in new strategies to detect neurotransmitters released from nerve cells in real time for brain science, drug assessment, and so on. Previously we reported real-time monitoring of dopamine release from nerve model cells by enzyme-catalyzed luminescence measurement with tyramine oxidase and peroxidase. In the present study, the system was modified with glutamate oxidase instead of tyramine oxidase to detect L-glutamate sensitively ( approximately 10 nM) and rapidly with high temporal resolution (<1 s). We applied this modified method successfully to perform real-time monitoring of L-glutamate release from brain model cell (C6 glioma cell) using a luminescence plate reader upon stimulation with high concentration of KCl (>10 mM) or 5-hydroxytryptamine (>1 microM). The measurement solution was not toxic and therefore the L-glutamate release from the cell was measured by the second stimulation after exchanging the measurement solution. We conclude that the developed monitoring system is suitable for real-time detection of dynamic L-glutamate release from nerve cells in vitro and will be suitable for application in assessment of drugs acting on the nervous system.
对于实时检测神经细胞释放的神经递质的新策略，在脑科学、药物评估等领域的关注度日益增加。此前我们报道了通过酪氨酸氧化酶和过氧化物酶的酶催化发光测量对神经模型细胞中多巴胺释放进行实时监测。在本研究中，该系统用谷氨酸氧化酶替代酪氨酸氧化酶进行了改进，以灵敏地（约10 nM）且快速地（<1 s的高时间分辨率）检测L-谷氨酸。我们成功应用这种改进方法，使用发光酶标仪在高浓度KCl（>10 mM）或5-羟色胺（>1 microM）刺激下对脑模型细胞（C6胶质瘤细胞）中L-谷氨酸的释放进行实时监测。测量溶液无毒，因此在更换测量溶液后通过第二次刺激来测量细胞中L-谷氨酸的释放。我们得出结论，所开发的监测系统适用于体外神经细胞动态L-谷氨酸释放的实时检测，并且将适用于作用于神经系统的药物评估。
