The glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures
The glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures
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DOI：
10.1111/j.1460-9568.1996.tb01328.x
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发表时间：
1996-09-01
影响因子：
3.4
通讯作者：
Rosenberg, PA
中科院分区：
文献类型：
--
作者：
Blitzblau, R;Gupta, S;Rosenberg, PA
由于充分证明谷氨酸摄取在保护神经元免受谷氨酸毒性方面的重要性，我们有兴趣测试l-反式吡咯烷-2,4-二羧酸盐（PDC）对大鼠皮层培养的影响。该化合物是谷氨酸转运体的底物，是一种有效的谷氨酸转运抑制剂，不与谷氨酸受体显著相互作用。暴露30分钟，并评估20-24小时后的神经元存活率，PDC在常规的富含星形胶质细胞的皮层培养物中具有神经毒性，这些培养物的EC（50）为320 +/- 157 μ M。在星形胶质细胞缺乏的培养物中，PDC的EC（50）为50 +/- 5 μ M。在星形胶质细胞富集和星形胶质细胞缺乏的培养中，PDC的神经毒性均被NMDA拮抗剂MK-801阻断，而非NMDA受体拮抗剂6-氰-7-硝基喹啉-2,3-二酮（CNQX）无法阻断。我们使用几种方法测试了PDC的神经毒性可能是由于释放兴奋性氨基酸的可能性。在用谷氨酸的非代谢类似物[H-3]- d -天冬氨酸预加载细胞后，我们首先证明了PDC引起了[H-3]-o-天冬氨酸的大量外排。这种作用依赖于胞外钠。与谷氨酸神经毒性相反，PDC神经毒性通过去除细胞外钠而被抑制。在1 mM PDC存在的情况下，钠引起神经毒性，EC（50）为18 +/- 7.6 mM。河河鱼毒素对PDC的神经毒性和PDC诱发的[H-3]- d -天冬氨酸释放均无影响。在没有神经元存在的星形胶质细胞培养中，pdcs诱导的[H-3]- d -天冬氨酸的释放是明显的。PDC还能诱发内源性谷氨酸的释放。最后，通过与谷氨酸-丙酮酸转氨酶和丙酮酸共孵育来降解细胞外谷氨酸，阻断PDC的神经毒性。这些结果证明了PDC的神经毒性，并提示这种毒性的机制是谷氨酸在细胞外空间的谷氨酸转运体依赖性积累。
Because of the well-documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20-24 h, PDC was neurotoxic in conventional astrocyte-rich cortical cultures, with an EC(50) in these cultures of 320 +/- 157 mu M. In astrocyte-poor cultures, an EC(50) for PDC of 50 +/- 5 mu M was determined. The neurotoxicity of PDC in both astrocyte-rich and astrocyte-poor cultures was blocked by the NMDA antagonist MK-801, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre-loading cells with the non-metabolizable analogue of glutamate, [H-3]-D-aspartate, first we demonstrated that PDC caused significant efflux of [H-3]-o-aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC(50) of 18 +/- 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC-evoked [H-3]-D-aspartate release. PDC-evoked release of [H-3]-D-aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate-pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter-dependent accumulation of glutamate in the extracellular space.