Department of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine, 9 Leontovicha Street, Kiev 01601, Ukraine.
Neurochem Int. 2010 Feb;56(3):466-78. doi: 10.1016/j.neuint.2009.12.006. Epub 2009 Dec 16.
The low level of ambient glutamate is important for the brain's spontaneous activity and proper synaptic transmission. Cholesterol deficiency has been implicated in the pathogenesis of several neurodegenerative disorders. It was examined whether membrane cholesterol modulated the extracellular glutamate level in nerve terminals and the processes responsible for its maintenance. The ambient L-[(14)C]glutamate level, being an equilibrium between Na(+)-dependent uptake and tonic release, was increased from 0.193+/-0.013 nmol/mg protein to 0.282+/-0.013 (extracellular endogenous glutamate-from 6.9+/-2.0 to 16.6+/-2.0, respectively) in rat brain synaptosomes treated with a cholesterol acceptor methyl-beta-cyclodextrin (MbetaCD). This alteration was not due to the change in the activity of glutamine synthetase that was shown with the specific blocker L-methionine sulfoximine. In the presence of DL-threo-beta-benzyloxyaspartate, which significantly reduced the contribution of glutamate transporters, net tonic release of L-[(14)C]glutamate was decreased by 38% and release in low-Na(+) medium was attenuated by 41% after cholesterol extraction. Also, cholesterol-deficient synaptosomes showed a reduced content of cytosolic L-[(14)C]glutamate and a lower initial velocity of L-[(14)C]glutamate uptake. We suggested that cholesterol deficiency altered the intra-to-extracellular glutamate ratio by the reduction of the cytosolic level of the neurotransmitter and the augmentation of the ambient glutamate level, thereby favoring a decrease in tonic glutamate release. Thus, increased extracellular glutamate in cholesterol-deficient nerve terminals was not a result of the changes in tonic release and/or glutamine synthetase activity, but was set by lack function of glutamate transporters.
细胞外谷氨酸水平较低对于大脑的自发性活动和正常的突触传递很重要。胆固醇缺乏与几种神经退行性疾病的发病机制有关。本研究旨在探讨神经末梢的细胞外谷氨酸水平及其维持过程是否受膜胆固醇调节。环境中的 L-[(14)C]谷氨酸水平是钠离子依赖性摄取和基础释放之间的平衡，用胆固醇接受体甲基-β-环糊精（MbetaCD）处理大鼠脑突触体后，其水平从 0.193+/-0.013 nmol/mg 蛋白增加到 0.282+/-0.013（细胞外内源性谷氨酸分别从 6.9+/-2.0 增加到 16.6+/-2.0）。这种变化不是由于谷氨酸合成酶活性的变化引起的，因为用特定的抑制剂 L-甲硫氨酸亚砜亚胺已经证明了这一点。在 DL-threo-β-苯甲氧基天冬氨酸存在下，谷氨酸转运体的作用明显减少，胆固醇提取后，净基础释放的 L-[(14)C]谷氨酸减少了 38%，低钠介质中的释放减少了 41%。此外，胆固醇缺乏的突触体显示细胞溶质 L-[(14)C]谷氨酸含量减少，L-[(14)C]谷氨酸摄取的初始速度降低。我们认为，胆固醇缺乏通过减少神经递质的细胞溶质水平和增加环境谷氨酸水平来改变细胞内-细胞外谷氨酸的比值，从而有利于基础谷氨酸释放的减少。因此，胆固醇缺乏的神经末梢中细胞外谷氨酸的增加不是由于基础释放和/或谷氨酰胺合成酶活性的变化引起的，而是由于谷氨酸转运体功能缺失所致。
