Bai G, Rama Rao K V, Murthy C R, Panickar K S, Jayakumar A R, Norenberg M D
Department of Pathology, Veterans Affairs Medical Center, University of Miami School of Medicine, Miami, FL 33101, USA.
J Neurosci Res. 2001 Dec 1;66(5):981-91. doi: 10.1002/jnr.10056.
Ammonia is a toxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and the astrocyte appears to be the principal target of ammonia toxicity. The specific neurochemical mechanisms underlying HE, however, remain elusive. One of the suggested mechanisms for ammonia toxicity is impaired cellular bioenergetics. Because there is evidence that the mitochondrial permeability transition (MPT) is associated with mitochondrial dysfunction, we determined whether the MPT might be involved in the bioenergetic alterations related to ammonia toxicity. Accordingly, we examined the mitochondrial membrane potential (Deltapsi(m)) in cultured astrocytes and neurons using laser-scanning confocal microscopy after loading the cells with the voltage-sensitive dye JC-1. We found that ammonia induced a dissipation of the Deltapsi(m) in a time- and concentration-dependent manner. These findings were supported by flow cytometry using the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Cyclosporin A, a specific inhibitor of the MPT, completely blocked the ammonia-induced dissipation of the Deltapsi(m). We also found an increase in the mitochondrial permeability to 2-deoxyglucose in astrocytes that had been exposed to 5 mM NH(4)Cl, further supporting the concept that ammonia induces the MPT in these cells. Pretreatment with methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the ammonia-induced collapse of Deltapsi(m), suggesting a role of glutamine in this process. Over a 24-hr period, ammonia had no effect on the Deltapsi(m) in cultured neurons. Collectively, our data indicate that ammonia induces the MPT in cultured astrocytes, which may be a factor in the mitochondrial dysfunction associated with HE and other hyperammonemic states.
氨是一种与肝性脑病（HE）发病机制密切相关的毒素，星形胶质细胞似乎是氨毒性的主要靶点。然而，HE潜在的具体神经化学机制仍不清楚。氨毒性的一种推测机制是细胞生物能量代谢受损。由于有证据表明线粒体通透性转换（MPT）与线粒体功能障碍有关，我们研究了MPT是否可能参与与氨毒性相关的生物能量代谢改变。因此，在用电压敏感染料JC-1加载细胞后，我们使用激光扫描共聚焦显微镜检查了培养的星形胶质细胞和神经元中的线粒体膜电位（ΔΨm）。我们发现氨以时间和浓度依赖性方式诱导ΔΨm的消散。使用电压敏感染料四甲基罗丹明乙酯（TMRE）的流式细胞术支持了这些发现。MPT的特异性抑制剂环孢素A完全阻断了氨诱导的ΔΨm消散。我们还发现，暴露于5 mM NH4Cl的星形胶质细胞中线粒体对2-脱氧葡萄糖的通透性增加，进一步支持了氨在这些细胞中诱导MPT的观点。用谷氨酰胺合成酶抑制剂蛋氨酸亚砜亚胺预处理可阻断氨诱导的ΔΨm崩溃，提示谷氨酰胺在此过程中起作用。在24小时内，氨对培养的神经元中的ΔΨm没有影响。总体而言，我们的数据表明氨在培养的星形胶质细胞中诱导MPT，这可能是与HE和其他高氨血症状态相关的线粒体功能障碍的一个因素。
