Department of Cell Biology, University of Salzburg, Salzburg, Austria.
Eur J Neurosci. 2009 Jul;30(2):196-206. doi: 10.1111/j.1460-9568.2009.06828.x. Epub 2009 Jul 15.
Under physiological conditions, astrocytes take up L-glutamate from the synaptic gap, metabolize it to L-glutamine and return it to neurons, where L-glutamine is metabolized to L-glutamate and stored in neurotransmitter vesicles. However, under pathological conditions, such as hepatic failure, L-glutamine and ammonium are elevated globally in the brain. The Trojan horse hypothesis of L-glutamine toxicity assumes that intramitochondrial hydrolysis of L-glutamine enhances ammonium locally and leads to mitochondrial dysfunction. In the present study, we show that exposure of murine primary microglia as well as of the microglial cell-line BV-2 to L-glutamine promotes chromatin condensation and formation of crescent-like structures in the nucleus. Furthermore, L-glutamine induced an increase in annexin-V labelling, cell shrinkage (apoptotic volume decrease), cell fragmentation and formation of apoptotic bodies. Inhibition of the phosphate-activated glutaminase with 6-diazo-5-oxo-L-norleucine suppressed chromatin condensation and annexin-V labelling in L-glutamine-exposed cells. In addition, inhibition of the glutamine synthetase with L-methionine sulfoximine suppressed chromatin condensation and annexin-V labelling in ammonium-exposed cells. L-glutamine and ammonium enhanced production of reactive oxygen species, as detected with CM-H(2)DCFDA. Apoptosis, induced by L-glutamine, was inhibited either by the radical scavenger alpha-tocopherol or by the nitric oxide synthase blocker N (G)-methyl-L-arginine. Cyclosporin A, a ligand of the permeability transition pore complex component cyclophilin D, prevented L-glutamine-triggered apoptosis. Furthermore, blockade of caspase-9 activity with Z-LEHD-FMK prevented L-glutamine-triggered apoptosis. Taken together, our results indicate that hydrolysis of l-glutamine and, accordingly, accumulation of ammonium in mitochondria induce the intrinsic pathway of apoptosis, characterized by mitochondrial dysfunction and activation of caspase-9, which activates caspase-3.
在生理条件下，星形胶质细胞从突触间隙摄取 L-谷氨酸，将其代谢为 L-谷氨酰胺并将其返回神经元，在神经元中，L-谷氨酰胺被代谢为 L-谷氨酸并储存在神经递质小泡中。然而，在病理条件下，如肝衰竭，脑内 L-谷氨酰胺和铵整体升高。L-谷氨酰胺毒性的特洛伊木马假说假设 L-谷氨酰胺的线粒体水解增强局部铵并导致线粒体功能障碍。在本研究中，我们表明，暴露于鼠原代小胶质细胞以及小胶质细胞系 BV-2 的 L-谷氨酰胺促进染色质凝聚，并在核中形成新月形结构。此外，L-谷氨酰胺诱导 annexin-V 标记增加、细胞收缩（凋亡体积减小）、细胞碎裂和凋亡小体形成。用 6-二氮-5-氧-L-正亮氨酸抑制磷酸激活的谷氨酰胺酶抑制 L-谷氨酰胺暴露细胞中的染色质凝聚和 annexin-V 标记。此外，用 L-蛋氨酸亚砜抑制谷氨酰胺合成酶抑制铵暴露细胞中的染色质凝聚和 annexin-V 标记。L-谷氨酰胺和铵增强活性氧的产生，如用 CM-H（2）DCFDA 检测到的。由 L-谷氨酰胺诱导的细胞凋亡被自由基清除剂α-生育酚或一氧化氮合酶抑制剂 N（G）-甲基-L-精氨酸抑制。亲环素 D 组成的通透性转换孔复合体成分的配体环孢菌素 A 可防止 L-谷氨酰胺引发的细胞凋亡。此外，用 Z-LEHD-FMK 阻断半胱天冬酶-9 活性可防止 L-谷氨酰胺引发的细胞凋亡。总之，我们的结果表明，L-谷氨酰胺的水解和相应的线粒体中铵的积累诱导了具有线粒体功能障碍和半胱天冬酶-9 激活特征的细胞凋亡的内在途径，半胱天冬酶-9 激活激活了半胱天冬酶-3。
