Wang G J, Chung H J, Schnuer J, Lea E, Robinson M B, Potthoff W K, Aizenman E, Rosenberg P A
Department of Neurology, Children's Hospital & Harvard Medical School, Boston MA 02115, USA.
Eur J Neurosci. 1998 Aug;10(8):2523-31. doi: 10.1046/j.1460-9568.1998.00256.x.
Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l-serine-O-sulphate, but not by l-alpha-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20-24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 +/- 9.8% in neuronal cultures (P < 0.001; n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05; n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 +/- 4.9 micrometer, compared to 1.2 +/- 0.3 micrometer in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 micrometer MK-801, 10 micrometer tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm) inhibited the 45calcium uptake stimulated by 30 micrometer N-methyl-d-aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced approximately 25% inhibition of 30 micrometer NMDA-induced current in cortical neurons. Dihydrokainate (1 mm) alone generated a small current (17% of the current produced by 30 micrometer NMDA) that was blocked by 30 micrometer 5,7-dichlorokynurenate and only weakly by 10 micrometer cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.
在培养的几乎纯的大鼠皮层神经元（星形胶质细胞少于0.2%）中，谷氨酸转运受到二氢卡因酸、L-丝氨酸-O-硫酸盐的强烈抑制，但不受L-α-氨基己二酸的抑制。该系统使得可以对谷氨酸转运对于保护神经元免受内源性突触释放谷氨酸毒性至关重要这一假说进行检验。支持这一假说的是，在神经元培养物中，暴露于1 mM二氢卡因酸20 - 24小时后，细胞存活率仅降至14.8±9.8%（P < 0.001；n = 3），尽管它对富含星形胶质细胞的培养物中的神经元存活率没有影响（P > 0.05；n = 3）。二氢卡因酸还显著导致皮层神经元培养物细胞外培养基中谷氨酸的积累（6.6±4.9微摩尔，而对照中为1.2±0.3微摩尔，n = 14，P < 0.01）。二氢卡因酸的神经毒性被10微摩尔MK - 801、10微摩尔河豚毒素以及一种降解细胞外谷氨酸的酶系统所阻断。后两者也消除了细胞外培养基中谷氨酸的积累。二氢卡因酸（1 mM）抑制了由30微摩尔N - 甲基 - D - 天冬氨酸（NMDA）刺激的45钙摄取，但对更高浓度则无抑制作用，这与二氢卡因酸在NMDA受体上的弱拮抗作用一致。全细胞记录显示，1 mM二氢卡因酸对皮层神经元中30微摩尔NMDA诱导的电流产生约25%的抑制。单独的二氢卡因酸（1 mM）产生小电流（为30微摩尔NMDA产生电流的17%），该电流被30微摩尔5,7 - 二氯犬尿氨酸阻断，仅被10微摩尔氰基 - 7 - 硝基喹喔啉 - 2,3 - 二酮（CNQX）微弱阻断。这些结果表明，二氢卡因酸在神经元培养物中的毒性是由于其阻断这些培养物中谷氨酸转运的能力，并且对二氢卡因酸敏感的神经元谷氨酸转运可能在保护神经元免受突触释放谷氨酸的毒性方面很重要。
