Zeevalk G D, Bernard L P, Nicklas W J
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Department of Neurology, Piscataway 08854, USA.
Neuroscience. 2000;96(2):309-16. doi: 10.1016/s0306-4522(99)00567-9.
Past studies have shown that inhibiting energy metabolism with malonate in mesencephalic cultures damages neurons by mechanisms involving N-methyl-D-aspartate receptors and free radicals. Overstimulation of N-methyl-D-aspartate receptors is known to produce free radicals. This study was, therefore, carried out to determine if N-methyl-D-aspartate receptor activation triggered by energy impairment was a significant contributor to the oxidative stress generated during energy inhibition. Exposure of mesencephalic cultures to malonate for the minimal time required to produce toxicity, i.e. 6h, resulted in an increase in the efflux of both oxidized and reduced glutathione, and a decrease in tissue levels of reduced glutathione. In contrast, exposure to 1mM glutamate for 1h caused an increased efflux of reduced glutathione, but no changes in intra- or extracellular oxidized glutathione or intracellular reduced glutathione. Blocking N-methyl-D-aspartate receptors with MK-801 (0.5 microM) during malonate exposure did not modify malonate-induced alterations in glutathione status or free radical generation as monitored by dihydrochlorofluorescein diacetate and dihydrorhodamine 123 fluorescence. In contrast, the increase in dihydrorhodamine fluorescence caused by glutamate was completely blocked by MK-801. Reduction of tissue glutathione with a 24h pretreatment with 10 microM buthionine sulfoxamine, as shown previously, greatly potentiated malonate-induced toxicity to dopamine and GABA neurons, but had no potentiating effect on toxicity due to glutamate. The findings indicate that although oxidative stress mediates damage due either to energy deprivation or excitotoxicity, N-methyl-D-aspartate receptor over-stimulation does not contribute significantly to the oxidative stress that is incurred during malonate exposure.
以往的研究表明，在中脑培养物中用丙二酸抑制能量代谢会通过涉及N-甲基-D-天冬氨酸受体和自由基的机制损害神经元。已知N-甲基-D-天冬氨酸受体的过度刺激会产生自由基。因此，本研究旨在确定能量损伤引发的N-甲基-D-天冬氨酸受体激活是否是能量抑制期间产生的氧化应激的重要促成因素。将中脑培养物暴露于丙二酸产生毒性所需的最短时间，即6小时，导致氧化型和还原型谷胱甘肽的外流增加，以及组织中还原型谷胱甘肽水平降低。相比之下，暴露于1mM谷氨酸1小时会导致还原型谷胱甘肽外流增加，但细胞内或细胞外氧化型谷胱甘肽或细胞内还原型谷胱甘肽没有变化。在丙二酸暴露期间用MK-801（0.5 microM）阻断N-甲基-D-天冬氨酸受体，并未改变丙二酸诱导的谷胱甘肽状态改变或自由基生成，这通过二氢氯荧光素二乙酸酯和二氢罗丹明123荧光监测。相反，谷氨酸引起的二氢罗丹明荧光增加被MK-801完全阻断。如先前所示，用10 microM丁硫氨酸亚砜胺进行24小时预处理降低组织谷胱甘肽，极大地增强了丙二酸对多巴胺能和GABA能神经元的毒性，但对谷氨酸引起的毒性没有增强作用。这些发现表明，尽管氧化应激介导了能量剥夺或兴奋性毒性所致的损伤，但N-甲基-D-天冬氨酸受体的过度刺激对丙二酸暴露期间产生的氧化应激没有显著贡献。
