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

肿胀细胞释放氨基酸途径的分子鉴定

基本信息

批准号：
12670051
负责人：
HAZAMA Akihiro
金额：
$ 2.11万
依托单位：
Okazaki National Research Institutes,Center for Integrative Bioscience
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670051/
关键词：
glutamic acid cell swelling hyototonic ischemia C6 glia channek chioride ATP

项目摘要

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.

神经胶质细胞释放的谷氨酸在脑缺血条件下具有病理生理作用。谷氨酸释放的途径仍然未知。我们研究了低渗肿胀胶质瘤细胞系（C6）中谷氨酸释放的特性。首先建立了以谷氨酸脱氢酶和NAD为显色底物测定谷氨酸的方法。用此方法测定了低渗溶液中C6细胞释放的谷氨酸。细胞外谷氨酸浓度从等渗条件下的1.3uM增加到低渗条件下的1.10uM。在16 ~ 36℃温度范围内，谷氨酸的释放对温度的敏感性较低，表明谷氨酸的释放可能是通过通道途径，而不是通过转运蛋白或胞吐作用。由于体积敏感性氯离子通道是谷氨酸释放途径的候选者，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性氯离子通道，并发现这些试剂未能抑制C6细胞的谷氨酸释放。Gd，从肿胀细胞释放ATP的阻断剂，也未能阻断谷氨酸的释放。综上所述，我们得出结论，谷氨酸从C6细胞在低渗条件下通过通道途径，这是不同的Cl通道或ATP的渗透途径。