Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine.
Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine.
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发表时间：
1993-04
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通讯作者：
Roberto Imberti;A. Nieminen;Brian Herman;J. Lemasters
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作者：
Roberto Imberti;A. Nieminen;Brian Herman;J. Lemasters
在分离的线粒体中，叔丁基过氧化氢（t-BuOOH）和其他促氧化剂引起渗透性转变，其特征在于对小离子的渗透性增加、膜电位的溶胀和损失。环孢菌素A和三氟拉嗪抑制这种渗透性转变。在这里，我们研究了线粒体通透性转换的作用，在致命的细胞损伤t-BuOOH。通过胶原酶灌注分离禁食大鼠的肝细胞，并通过碘化丙啶荧光法评估细胞活力。t-BuOOH引起剂量和时间依赖性细胞杀伤。果糖是糖酵解ATP形成的底物，在较低浓度（≤ 100 μ M）时受到保护，但在较高浓度的t-BuOOH时不受保护。在果糖处理的细胞中，寡霉素（10微克/毫升）延迟100至300 μ M t-BuOOH后的细胞杀伤，而环孢菌素A（0.5 μ M）加三氟拉嗪（5 μ M）甚至更有效地减少致死性损伤。在肝细胞悬液中，100 μ M t-BuOOH引起线粒体去极化，通过释放罗丹明123测定。环孢菌素A加三氟拉嗪在果糖的存在下大大减少了罗丹明123的释放。同样，在单个培养的肝细胞通过激光扫描共聚焦显微镜观察，叔丁氧醇引起泄漏的罗丹明123从线粒体，一个事件之前的细胞死亡，并延迟果糖与环孢素A加三氟拉嗪。在1 mM时，t-BuOOH抑制糖酵解，果糖与寡霉素或环孢菌素A加三氟拉嗪的组合仅具有短暂的保护作用。总之，t-BuOOH毒性随着剂量的增加而逐渐增加。在低t-BuOOH（<或= 50 μ M）时，线粒体ATP合成能力受到抑制，但未解偶联。（250字处删节）
In isolated mitochondria, t-butylhydroperoxide (t-BuOOH) and other pro-oxidants cause a permeability transition characterized by increased permeability to small ions, swelling and loss of membrane potential. Cyclosporin A and trifluoperazine inhibit this permeability transition. Here, we investigated the role of the mitochondrial permeability transition in lethal cellular injury from t-BuOOH. Hepatocytes from fasted rats were isolated by collagenase perfusion, and cell viability was assessed by propidium iodide fluorescence. t-BuOOH caused dose- and time-dependent cell killing. Fructose, a substrate for glycolytic ATP formation, protected at lower (< or = 100 microM), but not at higher concentrations of t-BuOOH. In fructose-treated cells, oligomycin (10 micrograms/ml) delayed cell killing after 100 to 300 microM t-BuOOH, whereas cyclosporin A (0.5 microM) plus trifluoperazine (5 microM) even more potently reduced lethal injury. In hepatocyte suspensions, 100 microM t-BuOOH caused mitochondrial depolarization as determined by release of rhodamine 123. Cyclosporin A plus trifluoperazine in the presence of fructose substantially reduced release of rhodamine 123. Similarly, in single cultured hepatocytes viewed by laser scanning confocal microscopy, t-BuOOH caused leakage of rhodamine 123 from mitochondria, an event which preceded cell death and which was delayed by fructose in combination with cyclosporin A plus trifluoperazine. At 1 mM, t-BuOOH inhibited glycolysis, and fructose in combination with either oligomycin or cyclosporin A plus trifluoperazine had only a short-lived protective effect. In conclusion, t-BuOOH toxicity was progressive with increasing dosages. At low t-BuOOH (< or = 50 microM), mitochondrial ATP synthetic capacity was inhibited, but not uncoupled.(ABSTRACT TRUNCATED AT 250 WORDS)