Newcomb R, Sun X, Taylor L, Curthoys N, Giffard R G
Neurex Corporation, Menlo Park, California 94025, USA.
J Biol Chem. 1997 Apr 25;272(17):11276-82. doi: 10.1074/jbc.272.17.11276.
Elevated extracellular concentrations of the excitatory transmitter glutamate are an important cause of neuronal death in a variety of disorders of the nervous system. The concentrations and rates of clearance and production of extracellular glutamate were measured in the medium of primary cultures from mouse neocortex containing neurons, astrocytes, or both cell types. Measurements were performed in the presence and absence of 2 mM glutamine with or without neuronal injury caused by 5-h exposure to hypoxia or 500 microM N-methyl-D-aspartate or a freeze-thaw cycle. High rates of glutamate generation (0.5-0.8 microM/min in the 0.4-ml culture well) occurred if neurons were both damaged and exposed to glutamine. Intact neurons or glia exposed to glutamine generated only small amounts of glutamate (0.03 microM/min). Glutamate generation by damaged neurons was dependent on the presence of glutamine, activated by phosphate, and inhibited by 6-diazo-5-oxo-L-norleucine and p-chloromercuriphenylsulfonic acid (pCMPS), strongly implicating the mitochondrial glutaminase. Following 5-h exposure to 500 microM N-methyl-D-aspartate, the glutaminase was localized to fragments of damaged neurons and was accessible to inhibition by the membrane-impermeant pCMPS. The glutaminase activity from damaged neurons is sufficient to account for the neurotoxic concentrations of glutamate in hypoxic mixed neuronal-glial cultures exposed to 2 mM glutamine. Finally, pCMPS is neuroprotective and also prevents the increased rate of generation of glutamate observed in neuronal cultures after prolonged exposure to glutamine. The cumulative data indicate the following: 1) excitotoxic neuronal death activates the hydrolysis of extracellular glutamine by the mitochondrial glutaminase, and 2) the glutaminase in damaged neurons is sufficient to cause neuronal death in in vitro models of neuronal injury.
在多种神经系统疾病中，兴奋性神经递质谷氨酸细胞外浓度升高是神经元死亡的重要原因。在含有神经元、星形胶质细胞或两种细胞类型的小鼠新皮质原代培养物的培养基中，测量细胞外谷氨酸的浓度、清除率和生成率。在存在和不存在2 mM谷氨酰胺的情况下进行测量，同时伴有或不伴有因5小时暴露于缺氧、500 μM N-甲基-D-天冬氨酸或冻融循环引起的神经元损伤。如果神经元既受损又暴露于谷氨酰胺，则会出现高谷氨酸生成率（在0.4 ml培养孔中为0.5 - 0.8 μM/分钟）。完整的神经元或暴露于谷氨酰胺的胶质细胞仅产生少量谷氨酸（0.03 μM/分钟）。受损神经元产生谷氨酸依赖于谷氨酰胺的存在，受磷酸盐激活，并被6-重氮-5-氧代-L-正亮氨酸和对氯汞苯磺酸（pCMPS）抑制，这强烈表明与线粒体谷氨酰胺酶有关。在暴露于500 μM N-甲基-D-天冬氨酸5小时后，谷氨酰胺酶定位于受损神经元的片段，并且可被膜不透性的pCMPS抑制。受损神经元的谷氨酰胺酶活性足以解释在暴露于2 mM谷氨酰胺的缺氧混合神经元-胶质细胞培养物中谷氨酸的神经毒性浓度。最后，pCMPS具有神经保护作用，还可防止在长时间暴露于谷氨酰胺后神经元培养物中观察到的谷氨酸生成率增加。累积数据表明：1）兴奋性毒性神经元死亡通过线粒体谷氨酰胺酶激活细胞外谷氨酰胺的水解，2）受损神经元中的谷氨酰胺酶足以在神经元损伤的体外模型中导致神经元死亡。
