Jayakumar A R, Panickar K S, Norenberg M D
Department of Pathology, University of Miami School of Medicine, Miami, Florida, USA Veterans Affairs Medical Center, Miami, Florida 33101, USA.
J Neurochem. 2002 Dec;83(5):1226-34. doi: 10.1046/j.1471-4159.2002.01261.x.
The effect of peripheral benzodiazepine receptor (PBR) ligands on free radical production was investigated in primary cultures of rat brain astrocytes and neurons as well as in BV-2 microglial cell lines using the fluorescent dye dichlorofluorescein-diacetate. Free radical production was measured at 2, 30, 60 and 120 min of treatment with the PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and protoporphyrin IX (PpIX) (all at 10 nm). In astrocytes, all ligands showed a significant increase in free radical production at 2 min. The increase was short-lived with PK11195, whereas with Ro5-4864 it persisted for at least 2 h. PpIX caused an increase at 2 and 30 min, but not at 2 h. Similar results were observed in microglial cells. In neurons, PK11195 and PpIX showed an increase in free radical production only at 2 min; Ro5-4864 had no effect. The central-type benzodiazepine receptor ligand, clonazepam, was ineffective in eliciting free radical production in all cell types. As the PBR may be a component of the mitochondrial permeability transition (MPT) pore, and free radical production may occur following induction of the MPT, we further investigated whether cyclosporin A (CsA), an inhibitor of the MPT, could prevent free radical formation by PBR ligands. CsA (1 micro m) completely blocked free radical production following treatment with PK11195 and Ro5-4864 in all cell types. CsA was also effective in blocking free radical production in astrocytes following PpIX treatment, but it failed to do so in neurons and microglia. Our results indicate that exposure of neural cells to PBR ligands generates free radicals, and that the MPT may be involved in this process.
使用荧光染料二氯荧光素二乙酸酯，在大鼠脑星形胶质细胞和神经元的原代培养物以及BV - 2小胶质细胞系中研究了外周苯二氮䓬受体（PBR）配体对自由基产生的影响。在用PBR配体1 -（2 - 氯苯基 - N - 甲基丙基）- 3 - 异喹啉甲酰胺（PK11195）、7 - 氯 - 5 -（4 - 氯苯基）- 1,3 - 二氢 - 1 - 甲基 - 2H - 1,4 - 苯并二氮杂䓬 - 2 - 酮（Ro5 - 4864）和原卟啉IX（PpIX）（均为10 nM）处理2、30、60和120分钟时测量自由基产生情况。在星形胶质细胞中，所有配体在2分钟时均显示自由基产生显著增加。PK11195引起的增加是短暂的，而Ro5 - 4864引起的增加持续至少2小时。PpIX在2分钟和30分钟时引起增加，但在2小时时未引起增加。在小胶质细胞中观察到类似结果。在神经元中，PK11195和PpIX仅在2分钟时显示自由基产生增加；Ro5 - 4864没有影响。中枢型苯二氮䓬受体配体氯硝西泮在所有细胞类型中均不能有效引发自由基产生。由于PBR可能是线粒体通透性转换（MPT）孔的一个组成部分，并且自由基产生可能在MPT诱导后发生，我们进一步研究了MPT抑制剂环孢素A（CsA）是否可以阻止PBR配体形成自由基。CsA（1 μM）在所有细胞类型中完全阻断了用PK11195和Ro5 - 4864处理后的自由基产生。CsA在用PpIX处理后的星形胶质细胞中也有效阻断自由基产生，但在神经元和小胶质细胞中未能做到。我们的结果表明，神经细胞暴露于PBR配体可产生自由基，并且MPT可能参与此过程。
