The regulatory mechanism of amino acid release form swollen cells by aquaporins

我们研究了代谢抑制引起的肿胀细胞释放氨基酸的机制。在氨基酸中，由于谷氨酸在脑缺氧中的重要性，我们将重点放在谷氨酸上。首先，我们开发了检测谷氨酸的方法。我们使用四唑盐（WST-8，DOJIN）和谷氨酸脱氢酶来形成甲瓒。通过这种组合，我们可以使用酶标仪测量谷氨酸盐的浓度。我们将NaN 3应用于胶质瘤细胞系（C6），并测量细胞外溶液中的谷氨酸。细胞外谷氨酸从初始条件下的3μM增加到30 min后的10μM。氯通道阻断剂不能抑制谷氨酸的升高。我们还测试了HgC 12，它可以阻断水通道蛋白的水通路。通过HgC 12的应用，我们可以观察到即使没有代谢抑制剂，谷氨酸的升高。这可能是由于HgC_（12）对细胞膜的损伤所致。在本项目中，我们还发现了C6细胞在施加胞外葡萄糖的作用下释放谷氨酸。这一发现可能有助于高血糖引起的组织损伤机制的研究。

We investigate the mechanism of amino acid release from swollen cells induced by the metabolic inhibition. Among the amino acids, we focused on the glutamate because of the importance in the brain anoxia. First we developed the method of detecting glutamate. We used the tetrazolium salts (WST-8, DOJIN) and glutamate dehydrogenase to form the formazan. With this combination, we could measure the concentration of glutamate using the plate reader. We applied the NaN3 to the glioma cell line (C6) and measured the glutamate in the extracellular solution. Extracellular gutamate increased from 3μM in the initial condition to 10μM after 30min. The rise of glutamate could not be suppressed by chloride channel blockers. We also tested HgC12 which could block aquaporin water pathway. By the application of HgC12, we could observed the rise of glutamate even without the metabolic inhibitor. This effect might be explained by the damage of cell membrane by HgC12. During this project, we also found the glutamate released form C6 cells by the application of extracellular glucose. This finding might contribute the investigation of mechanism of tissue damage caused by the high plasma glucose.