Real-time detection of L-glutamate released from C6 glioma cells using a modified enzyme-luminescence method

摘要

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 (approximate to 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 mu M). 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.