Huang J, Philbert M A
Department of Pharmacology and Toxicology, Rutgers College of Pharmacy, Piscataway, NJ 08854, USA.
Brain Res. 1996 Mar 4;711(1-2):184-92. doi: 10.1016/0006-8993(95)01376-8.
Glutathione (GSH) and glutathione-related enzyme systems in astrocytes play an important role in cellular defense against oxidative stress in the nervous system. The present study was designed to characterize the cellular responses of cultured astrocytes to chemically-induced perturbations of mitochondrial and cytosolic GSH homeostasis. Treatment of astrocytes in culture with ethacrynic acid (EA), a mitochondrion-penetrating thiol reagent, induced rapid and extensive depletion of both cytosolic and mitochondrial pools of GSH. Concomitant with the effects of EA on cellular GSH were significant and concentration-dependent increases in intracellular generation of reactive oxygen species (ROS) as indicated by the oxidation of preloaded 2',7'-dichlorofluorescein diacetate. Significant elevation of intracellular ROS occurred by 15 min following exposure to 100 microM EA and reached peak levels by 30 min which were approximately 7-fold higher than corresponding control levels. Ethacrynic acid-induced GSH depletion and intracellular ROS elevation was followed by marked decreases in glutathione reductase (GR) activity in mitochondria, and to a lesser extent, in cytosolic fractions of cultured astrocytes. This inhibitory effect was time- and concentration-dependent, and other GSH-related enzymes, glutathione peroxidase and glutathione S-transferase, were not or only slightly affected. Kinetic studies showed that EA markedly diminished V(max) values of both mitochondrial and cytosolic GR without affecting K(m), suggesting noncompetitive inhibition of this thiol-dependent enzyme. Another thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase was also markedly inhibited by EA in a time-dependent fashion. Subsequent decline of mitochondrial transmembrane potential (rhodamine 123 uptake) and cellular ATP production following EA treatment occurred prior to the onset of loss of cell viability as indicated by lactate dehydrogenase leakage. These results suggest that the loss of mitochondrial GSH may render the astrocytes unable to combat the pathological sequelae of endogenous oxidative stress, leading to perturbations of thiol-dependent enzyme activities, mitochondrial function and energy metabolism.
星形胶质细胞中的谷胱甘肽（GSH）和谷胱甘肽相关酶系统在神经系统细胞抵御氧化应激中发挥重要作用。本研究旨在表征培养的星形胶质细胞对化学诱导的线粒体和胞质GSH稳态扰动的细胞反应。用线粒体穿透性硫醇试剂依他尼酸（EA）处理培养的星形胶质细胞，可诱导胞质和线粒体池中的GSH迅速且大量消耗。与EA对细胞GSH的影响相伴的是，如预加载的2',7'-二氯荧光素二乙酸酯的氧化所示，细胞内活性氧（ROS）生成显著且呈浓度依赖性增加。暴露于100μM EA后15分钟，细胞内ROS显著升高，并在30分钟达到峰值水平，约比相应对照水平高7倍。依他尼酸诱导的GSH消耗和细胞内ROS升高之后，培养的星形胶质细胞线粒体中的谷胱甘肽还原酶（GR）活性显著降低，胞质部分的降低程度较小。这种抑制作用具有时间和浓度依赖性，其他GSH相关酶谷胱甘肽过氧化物酶和谷胱甘肽S-转移酶未受影响或仅受到轻微影响。动力学研究表明，EA显著降低了线粒体和胞质GR的V(max)值，而不影响K(m)，表明对这种硫醇依赖性酶的非竞争性抑制。另一种硫醇依赖性酶甘油醛-3-磷酸脱氢酶也被EA以时间依赖性方式显著抑制。EA处理后线粒体跨膜电位（罗丹明123摄取）和细胞ATP产生的随后下降发生在细胞活力丧失（如乳酸脱氢酶泄漏所示）开始之前。这些结果表明，线粒体GSH的丧失可能使星形胶质细胞无法对抗内源性氧化应激的病理后果，导致硫醇依赖性酶活性、线粒体功能和能量代谢的扰动。
