Durkin J P, Tremblay R, Buchan A, Blosser J, Chakravarthy B, Mealing G, Morley P, Song D
Cellular Neurobiology Group, National Research Council of Canada, Ottawa, Ontario, Canada.
J Neurochem. 1996 Mar;66(3):951-62. doi: 10.1046/j.1471-4159.1996.66030951.x.
Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15-min exposure of day 14-18 cortical neurons to 100 microM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30-50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC-selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral Ischemia in vivo.
多项证据表明，蛋白激酶C（PKC）活性的快速丧失可能在脑缺血后神经元的延迟死亡中起重要作用。然而，在原代神经元培养中，据报道，通过免疫印迹和传统活性方法测量，细胞毒性水平的谷氨酸不会导致PKC丧失。这一明显的矛盾尚未得到充分解决。在本研究中，使用一种仅测量在分离膜中具有活性的PKC的测定方法，确定了细胞毒性水平的谷氨酸、N-甲基-D-天冬氨酸（NMDA）和α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）对皮质神经元膜PKC活性的影响，该方法可用于区分活性酶与处于非活性状态的膜相关酶。将第14 - 18天的皮质神经元暴露于100微摩尔的谷氨酸、AMPA或NMDA 15分钟，导致膜PKC活性迅速且持续丧失，到4小时时降至对照培养物中活性的30 - 50%。然而，尽管酶活性丧失，但在此期间这些膜中存在的酶量保持不变。在短暂谷氨酸处理后的完整神经元中，PKC选择性底物MARCKS蛋白的磷酸化减少，这证实了PKC活性的失活。相比之下，反式-（1S，3R）-1-氨基-1，3-环戊烷二羧酸对代谢型谷氨酸受体的激活没有神经毒性，并且在神经元中诱导了PKC活性的强烈且持久的激活。NMDA和AMPA导致的PKC失活依赖于细胞外钙离子（Ca2+），但对钠离子（Na+）的依赖性较小，尽管这些药物诱导的细胞死亡依赖于这两种离子。PKC活性的丧失可能是通过特定途径的钙离子内流导致的，因为钙离子载体离子霉素引起的细胞内游离钙离子（[Ca2+]）大量增加并未导致PKC失活。结果表明，在体外被谷氨酸、NMDA和AMPA杀死的神经元中PKC活性模式与在体内受脑缺血损伤的神经元中观察到的一致。
