Drug assessment based on detection of L-glutamate released from C6 glioma cells using an enzyme-luminescence method.

Monitoring of excitation activity of nerve cells is very useful for not only brain research but also assessment of the effects of various chemicals, including drugs and toxins. We previously reported a novel enzyme-luminescence method for real-time monitoring of l-glutamate release from C6 glioma cells with high levels of sensitivity ( approximately 10 nM) and temporal resolution (<1 s) using a luminescence plate reader. In the present study, we tested the applicability of this novel system for assessment of effects of drugs in vitro. Several drugs (e.g., veratridine and 4-aminopyridine) were administered to C6 glioma cells for inducing glutamate release. Moreover, antagonists of voltage-dependent Ca (2+) channels (e.g., nifedipine, flunarizine, and NiCl 2) and Na (+) channels (e.g., carbamazepine and lidocaine) were applied separately for evaluating the effects of these chemicals on glutamate release from the cells. The combined effect of carbamazepine and lidocaine was also investigated by using our method, and the combined effect was found to be more potent than that of single drug administration. These results indicated that the glutamate release from C6 cells was modulated by these drugs in a way similar to that found by using several conventional analytical techniques. We therefore conclude that the developed monitoring system for real-time detection of dynamic l-glutamate release from cells could be very useful for application to assessment of drugs acting on the nervous system.

Real-time detection of dopamine released from a nerve model cell by an enzyme-catalyzed luminescence method and its application to drug assessment.

A real-time observation of neurotransmitter release from a nerve cell is a useful method for not only neuroscience research, but also assessing of the influence of chemicals, including drugs, on the human nervous system. In this study, a more simple and sensitive method for real-time monitoring of dopamine release from a nerve model cell was developed. Highly sensitive detection of dopamine was performed by using tyramine oxidase for dopamine oxidation, which was followed by a luminol luminescence reaction. This enzyme-catalyzed luminescence method was applied to observe dopamine release from the PC12 cell as a nerve model cell upon stimulation with acetylcholine and an acetylcholine receptor agonist. The results demonstrated that the real-time monitoring of the activation of the PC12 cell was easily performed by this method. This method possessed many advantages, such as high sensitivity, rapid measurement and no pretreatment for cells. It might be applied to drug screening and the assessment of harmful influences of food additives and pesticides on the nerves.