Chatterjee S, Noack H, Possel H, Keilhoff G, Wolf G
Institute for Medical Neurobiology, Otto-von-Guericke Universität, Magdeburg, Germany.
Glia. 1999 Aug;27(2):152-61.
Because glutathione (GSH) levels in glia play an important role in cellular defense against oxidative and nitrosative stress, the present study was designed to study GSH levels in the primary glial cell cultures. Here we used fluorescence microscopy and spectroscopy with monochlorobimane for measurement of intracellular glutathione content. Monochlorobimane showed high specificity for GSH with very little binding to protein sulphydryls as ascertained from the low fluorescence intensity of the protein fraction of the cells as well as from the low fluorescence of the GSH-depleted cells. The formation of the monochlorobimane-glutathione conjugate was observed to be enzymatically catalyzed as seen from its higher rate of formation in the presence of cell homogenate. A monochlorobimane concentration of 60 microM was used for conjugation of cellular GSH; at higher mBCl concentrations there was no appreciable increase in fluorescence. Therefore, cultures were treated with 60 microM mBCl for an incubation time of 20 min (beyond this time, export of the bimane-glutathione adduct was significantly large) and examined by fluorescence microscopy. This adduct could be fixed with a mixture of paraformaldehyde and glutaraldehyde, and excellent fixation was observed with 4% paraformaldehyde and 0.2% glutaraldehyde. Analysis of the fluorescence images revealed differences in fluorescence intensity between astro- and microglial cells, which were identified by glial fibrilliary acidic protein and OX42 staining, respectively. Microglial cells isolated from primary glial cultures were found to have higher GSH content than astrocytes. Biochemical determination of GSH levels in microglia isolated from primary glial cultures corroborated this fact. From our findings it seems that owing to the greater intracellular concentration of reactive oxygen and nitrogen species to which microglia are subjected, especially under conditions of inflammation, this cell type is fortified with higher GSH levels as a means to combat oxidative and nitrosative stress.
由于神经胶质细胞中的谷胱甘肽（GSH）水平在细胞抵御氧化应激和亚硝化应激中发挥重要作用，本研究旨在研究原代神经胶质细胞培养物中的GSH水平。在此，我们使用荧光显微镜和单氯双硫腙光谱法测量细胞内谷胱甘肽含量。从细胞蛋白质部分的低荧光强度以及GSH耗尽细胞的低荧光可以确定，单氯双硫腙对GSH具有高度特异性，与蛋白质巯基的结合极少。从其在细胞匀浆存在下较高的形成速率可以看出，单氯双硫腙 - 谷胱甘肽共轭物的形成是由酶催化的。使用60微摩尔的单氯双硫腙浓度来共轭细胞内的GSH；在更高的mBCl浓度下，荧光没有明显增加。因此，培养物用60微摩尔的mBCl处理20分钟（超过这个时间，双硫腙 - 谷胱甘肽加合物的输出显著增加），并通过荧光显微镜检查。这种加合物可以用多聚甲醛和戊二醛的混合物固定，用4%多聚甲醛和0.2%戊二醛观察到了良好的固定效果。荧光图像分析显示星形胶质细胞和小胶质细胞之间的荧光强度存在差异，分别通过胶质纤维酸性蛋白和OX42染色进行鉴定。从原代神经胶质细胞培养物中分离出的小胶质细胞被发现比星形胶质细胞具有更高的GSH含量。对从原代神经胶质细胞培养物中分离出的小胶质细胞中GSH水平的生化测定证实了这一事实。从我们的研究结果来看，似乎由于小胶质细胞所遭受的细胞内活性氧和氮物种浓度更高，特别是在炎症条件下，这种细胞类型通过更高的GSH水平来强化自身，作为对抗氧化应激和亚硝化应激的一种手段。
