Pfeiffer Annika, Jaeckel Martin, Lewerenz Jan, Noack Rebecca, Pouya Alireza, Schacht Teresa, Hoffmann Christina, Winter Jennifer, Schweiger Susann, Schäfer Michael K E, Methner Axel
Department of Neurology, University Medical Center and Focus Program Translational Neuroscience (FTN) of the Johannes Gutenberg-University Mainz, Mainz, Germany.
Br J Pharmacol. 2014 Apr;171(8):2147-58. doi: 10.1111/bph.12549.
The hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc-. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction.
We compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line.
Glutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery.
These results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.
海马细胞系HT22是研究内源性氧化应激后果的优秀模型。细胞外谷氨酸通过阻断谷氨酸/胱氨酸反向转运体系统xc-消耗细胞内谷胱甘肽。谷胱甘肽耗竭诱导了明确的细胞死亡程序，其特征是活性氧增加和线粒体功能障碍。
我们比较了基于对谷氨酸的抗性而选择的HT22细胞与谷氨酸敏感的亲本细胞系的线粒体形态、线粒体复合物丰度和线粒体呼吸。
抗谷氨酸的线粒体碎片化程度较低，并表现出看似矛盾的特征：线粒体钙和超氧化物增加，而高分辨率呼吸测定表明线粒体呼吸减少。这被解释为氧化应激下ATP合酶的反向活性，导致ATP水解以维持甚至升高线粒体膜电位，表明这些细胞忍受无效的能量代谢以保护其膜电位。抗谷氨酸的细胞也对ATP合酶抑制剂寡霉素有抗性，但对己糖激酶抑制剂脱氧葡萄糖敏感。用半乳糖替代葡萄糖使抗性细胞对寡霉素的敏感性提高了1000倍。这些结果，连同胞质己糖激酶1和2的强烈增加、乳酸产生减少以及葡萄糖-6-磷酸脱氢酶活性增加，表明抗谷氨酸的HT22细胞将大部分可用葡萄糖导向磷酸戊糖途径以增加谷胱甘肽的恢复。
这些结果表明线粒体和代谢适应在细胞对氧化应激的抗性中起重要作用。
