Molecular indentification of the pathway for amino acids relased from swollen cell

神经胶质细胞释放谷氨酸在脑缺血状态下具有病理生理作用。谷氨酸释放的途径尚不清楚。我们研究了低渗肿胀胶质瘤细胞系（C6）谷氨酸释放的特性。首先，利用谷氨酸脱氢酶和NAD显色底物建立了谷氨酸的高效测定方法。用这种方法测定了低渗溶液中C6细胞释放的谷氨酸。细胞外谷氨酸浓度由等渗条件下的~ 3uM增加到低渗条件下的~ 10um。在16 ~ 36℃范围内，谷氨酸的释放对温度的敏感性较低，说明谷氨酸可能是通过通道途径释放的，而不是通过转运体或胞外作用释放的。由于体积敏感的Cl通道是谷氨酸释放途径的候选通道，我们测试了几种试剂，包括根皮素和花生四烯酸，据报道可以阻断体积敏感的Cl通道，发现这些试剂不能抑制谷氨酸从C6细胞释放。Gd是肿胀细胞释放ATP的阻滞剂，也不能阻断谷氨酸的释放。综上所述，我们得出结论，谷氨酸在低渗条件下通过通道途径从C6细胞释放，这与Cl通道或ATP的渗透途径不同。

Glutamic acid released from glial cells has pathophysiological roles in brain under ischemic conditions. The pathways for the glutamic acid release are still unknown. We investigate the characteristics of the glutamic acid release from the hyposmotically swollen glioma cell line (C6). First we established the efficient method for the measurement of glutamic acid using glutamic acid dehydorgenease and the chromogenic substrate for NAD. With this method, we measured the glutamic acid released from C6 cells in the hypotonic solutions. The extracellular concentration of glutamic acid increased from 〜3uM under the isotonic condition to 〜10 uM under the hypotonic condition. The glutamic acid release was less sensitive to the temperature between 16℃ and 36℃, suggesting that glutamic acid might be released via the channel pathway, not via transporters nor exocytosis. Since volume-sensitive Cl channels are candidates for the pathway for glutamic acid release, we tested several reagents, including phloretin and arachidonic acid, reported to block volume-sensitive Cl channels and found that those failed to inhibit glutamic acid release from C6 cells. Gd, the blocker for ATP release from swollen cells, also failed to block glutamic acid release. Taken together, we conclude that glutamic acid is released from C6 cells under hypotonic condition via the channel pathway, which is distinct from Cl channels or the permeation pathway for ATP.