Churn S B, Sombati S, Taft W C, DeLorenzo R J
Department of Neurology, Medical College of Virginia, Richmond 23298.
Stroke. 1993 Feb;24(2):271-7; discussion 277-8. doi: 10.1161/01.str.24.2.271.
Glutamate-induced excitotoxicity has been implicated as a causative factor for selective neuronal loss in ischemia and hypoxia. Toxic exposure of neurons to glutamate results in an extended neuronal depolarization that precedes delayed neuronal death. Because both delayed neuronal death and extended neuronal depolarization are dependent on calcium, we examined the effect of glutamate exposure on extended neuronal depolarization and calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity.
Three-week-old cortical cell cultures from embryonic rats were exposed to 500 microM glutamate and 10 microM glycine or to control medium for 10 minutes. Cells were examined for neuronal toxicity, electrophysiology, and biochemical alterations. In one set of experiments, whole-cell current clamp recording was performed throughout the experiment. In a parallel experiment, cortical cultures were allowed to recover from glutamate exposure for 1 hour, at which time the cells were homogenized and CaM kinase II activity was assayed using standard techniques.
Excitotoxic exposure to glutamate resulted in extended neuronal depolarization, which remained after removal of the glutamate. Glutamate exposure also resulted in delayed neuronal death, which was preceded by significant inhibition of CaM kinase II activity. The excitotoxic inhibition of CaM kinase II correlated with neuronal loss, was N-methyl-D-aspartate receptor-mediated, and was not due to autophosphorylation of the enzyme.
Glutamate-induced delayed neuronal toxicity correlates with extended neuronal depolarization and inhibition of CaM kinase II activity. Because inhibition of CaM kinase II activity significantly preceded the histological loss of neurons, the data suggest that modulation of CaM kinase II activity may be involved in the cascade of events resulting in loss of calcium homeostasis and delayed neuronal death.
谷氨酸诱导的兴奋性毒性被认为是缺血和缺氧时选择性神经元丢失的一个致病因素。神经元暴露于谷氨酸毒性环境会导致神经元去极化延长，随后出现延迟性神经元死亡。由于延迟性神经元死亡和延长的神经元去极化均依赖于钙，我们研究了谷氨酸暴露对延长的神经元去极化及钙/钙调蛋白依赖性蛋白激酶II（CaM激酶II）活性的影响。
将来自胚胎大鼠的3周龄皮质细胞培养物暴露于500微摩尔/升谷氨酸和10微摩尔/升甘氨酸中或对照培养基中10分钟。检测细胞的神经元毒性、电生理学和生化改变。在一组实验中，整个实验过程中进行全细胞电流钳记录。在平行实验中，使皮质培养物从谷氨酸暴露中恢复1小时，此时将细胞匀浆，并用标准技术测定CaM激酶II活性。
谷氨酸兴奋性毒性暴露导致神经元去极化延长，去除谷氨酸后仍持续存在。谷氨酸暴露还导致延迟性神经元死亡，其之前CaM激酶II活性受到显著抑制。CaM激酶II的兴奋性毒性抑制与神经元丢失相关，由N-甲基-D-天冬氨酸受体介导，并非由于该酶的自身磷酸化。
谷氨酸诱导的延迟性神经元毒性与延长的神经元去极化及CaM激酶II活性抑制相关。由于CaM激酶II活性抑制明显先于神经元的组织学丢失，数据表明CaM激酶II活性的调节可能参与了导致钙稳态丧失和延迟性神经元死亡的一系列事件。
