Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death.
Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death.
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发表时间：
1989-07
影响因子：
3.6
通讯作者：
H. Manev;M. Favaron;A. Guidotti;E. Costa
中科院分区：
文献类型：
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作者：
H. Manev;M. Favaron;A. Guidotti;E. Costa
在 7-8 天大的大鼠小脑颗粒细胞原代培养物中研究了谷氨酸引发的迟发性神经毒性的机制。在不含 Mg2+ 的培养基中用 50 µM 谷氨酸处理培养物 15 分钟，然后去除兴奋毒素，导致神经元死亡，这种死亡在谷氨酸处理终止后 2-3 小时开始出现。在接下来的几个小时内，死亡神经元的数量逐渐增加，24小时后发现80-85%的神经元死亡。 N-甲基-D-天冬氨酸敏感谷氨酸受体拮抗剂（苯环己哌啶）或1.2 mM MgCl2，但不是N-甲基-D-天冬氨酸不敏感谷氨酸受体拮抗剂（6-氰基-7-硝基喹喔啉-2,3-二酮），消除了红藻氨酸的神经毒性作用。谷氨酸诱导的神经元死亡的发生在很大程度上取决于 Ca2+。在谷氨酸暴露终止后和神经元死亡早期迹象（谷氨酸后时期）出现之前立即去除细胞外Ca2+（用1mM乙二醇-双-（β-氨基乙基醚）-N,N,N',N'-四乙酸）可显着减少神经元变性。谷氨酸的神经毒性浓度导致后谷氨酸时期 45Ca2+ 摄取持续增加。 45Ca2+摄取的延迟增加以及延迟的神经毒性不受苯环己哌啶、二苯并环庚烯亚胺谷氨酸后处理的影响； DL-2-氨基-5-磷酸戊酸，或 MgCl2 或电压依赖性 Ca2+ 通道阻滞剂（尼群地平、维拉帕米、地尔硫卓）。神经毒性浓度的谷氨酸还诱导 [3H]佛波醇-12,13-二丁酸结合延迟持续增加，反映了谷氨酸后时期蛋白激酶 C (PKC) 从细胞质到细胞膜的易位增加。用神经节苷脂 GT1b（三唾液酸神经节四糖基神经酰胺）预处理神经元，然后从培养介质中去除游离的 GT1b，可防止 PKC 易位、谷氨酸后时期 45Ca2+ 摄取的持续增加以及延迟的神经元死亡。我们认为，谷氨酸受体刺激引发的 PKC 持续激活和易位可能是导致神经元 Ca2+ 流入持续增加的触发事件。这种流入对电压依赖性 Ca2+ 通道阻滞剂和谷氨酸受体拮抗剂不敏感。看来 Ca2+ 流入的延迟增加可能在导致神经元死亡中很重要。
The mechanism of delayed neurotoxicity, triggered by glutamate, was studied in 7-8-day-old primary cultures of rat cerebellar granule cells. Treatment of cultures for 15 min with 50 microM glutamate in Mg2+ -free medium, followed by removal of the excitoxin, resulted in neuronal death, which started to appear 2-3 hr after the termination of glutamate treatment. The number of dead neurons increased gradually in the next few hours and 80-85% of neurons were found dead 24 hr later. Antagonists of N-methyl-D-aspartate-sensitive glutamate receptors (phencyclidine) or 1.2 mM MgCl2, but not the antagonist of N-methyl-D-asparatate-insensitive glutamate receptors (6-cyano-7-nitroquinoxaline-2,3-dione), abolished the neurotoxic effect of kainate. Development of glutamate-induced neuronal death depends strongly on Ca2+. Removal of extracellular Ca2+ (with 1mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) immediately after the termination of glutamate exposure and before the appearance of the early signs of neuronal death (post-glutamate period) dramatically reduced neuronal degeneration. Neurotoxic concentrations of glutamate induced sustained increase of 45Ca2+ uptake in the post-glutamate period. The delayed increase of 45Ca2+ uptake, as well as the delayed neurotoxicity, were not affected by post-glutamate treatment with phencyclidine, dibenzocyclohepteneimine; DL-2-amino-5-phosphonovalerate, or MgCl2 or with voltage-dependent Ca2+ channel blockers (nitrendipine, verapamil, diltiazem). Neurotoxic concentrations of glutamate also induced a delayed sustained increase of [3H]phorbol-12,13-dibutyrate binding, reflecting an increased translocation of protein kinase C (PKC) from cytosol to the cell membrane during the post-glutamate period. Pretreatment of neurons with the ganglioside GT1b (trisialosylgangliotetraglycosylceramide), followed by removal of free GT1b from the incubation medium, prevented PKC translocation, the sustained increase of 45Ca2+ uptake in the post-glutamate period, and the delayed neuronal death. We suggest that the sustained activation and translocation of PKC primed by glutamate receptor stimulation may be the triggering event causing the protracted increase of neuronal Ca2+ influx. This influx is insensitive to voltage-dependent Ca2+ channel blockers and glutamate receptor antagonists. It appears that this delayed increase of Ca2+ influx may be important in causing neuronal death.