Crystal and microparticle effects on MDCK cell superoxide production: Oxalate-specific mitochondrial membrane potential changes
Crystal and microparticle effects on MDCK cell superoxide production: Oxalate-specific mitochondrial membrane potential changes
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DOI：
10.1016/j.freeradbiomed.2005.02.020
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发表时间：
2005-06-15
影响因子：
7.4
通讯作者：
Hothersall, JS
中科院分区：
文献类型：
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作者：
Meimaridou, E;Jacobson, J;Hothersall, JS
我们以前已经表明，草酸钙（COM）晶体引发超氧化物（O-2（.-））线粒体的反应。我们现在已经调查：（i）其他微粒是否可以引起相同的反应，（ii）是否涉及晶体的加工，以及（iii）草酸盐在线粒体功能的什么水平上起作用。O-2（.-）通过光泽精（10 μ M）化学发光在毛地黄皂苷透化的MDCK细胞中测量。[C-14]-COM解离在有或没有EDTA和使用替代螯合剂的情况下进行了检查。而线粒体O-2（.-）在COM处理的细胞中，与对照组相比，O-2增加了三到四倍，其他颗粒（尿酸，酵母聚糖和乳胶珠）也没有增加O-2（.-）或效果差得多（羟基磷灰石+50%，p < 0.01），均为28 μ g/cm（2）。游离草酸盐（750 μ M），从COM与EDTA（1 mM）释放的水平，增加O-2（.-）（+50%，p < 0.01）。省略EDTA会消除该信号，EGTA可以完全恢复该信号，抗坏血酸可以部分恢复该信号，但去铁胺或柠檬酸盐不能恢复该信号。磷酸盐缺失导致O-2（.-），涉及磷酸盐依赖的线粒体二羧酸转运。COM引起线粒体膜电位（Δ psi（m））的时间相关性增加，使用TMRM荧光和共聚焦显微镜测量。COM的应用程序Fura 2加载细胞诱导快速，大幅度的胞质Ca 2+瞬变，这是抑制毒胡萝卜素，表明COM诱导释放的Ca 2+从内部存储。因此，COM诱导的线粒体O-2（.-）需要释放游离草酸盐并有助于协同反应。COM的细胞内解离和线粒体二羧酸转运蛋白在O-2（.-）生产，这可能是由Δ psi（m）调节。(c)2005年爱思唯尔公司All rights reserved.
We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O-2(.-)) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O-2(.-) measured in digitonin-permeabilized MDCK cells by lucigenin (10 mu M) chemiluminescence. [C-14]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O-2(.-) in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O-2(.-) or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 mu g/cm(2). Free oxalate (750 mu M), at the level released from COM with EDTA (1 mM), increased O-2(.-) (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O-2(.-), implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Delta psi(m)) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca2+ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca2+ from internal stores. Thus, COM-induced mitochondrial O-2(.-) requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O-2(.-) production, which is probably regulated by Delta psi(m). (c) 2005 Elsevier Inc. All rights reserved.