Gleva G F, Goodglick L A, Kane A B
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912.
Am J Pathol. 1990 Jul;137(1):43-57.
Sequestration of calcium by mitochondria is an important mechanism to maintain normal intracellular calcium homeostasis. Anoxic or toxic damage to these organelles has been postulated to disrupt intracellular calcium compartmentalization, leading to cell death. The authors examined the potential relationship between mitochondrial dysfunction, altered calcium homeostasis, and irreversible injury in a model system of silica-induced toxicity to P388D1 cells. Exposure to toxic silica particles, but not to nontoxic latex heads, disrupted mitochondrial membrane potential, increased membrane-associated calcium, elevated free cytosolic calcium, and killed 50% to 60% of the cell population after 6 to 8 hours. To test whether disruption of the mitochondrial membrane potential was sufficient to cause irreversible injury, P388D1 cells were exposed to either the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or to the mitochondrial inhibitor, antimycin A. Over 90% of the treated cells showed depolarization of the mitochondrial membrane as indicated by the fluorescent probe rhodamine 123. Carbonyl cyanide p-trifluoromethoxyphenylbydrazone also caused an elevation in free cytosolic calcium as monitored by fura-2. However, even after 6 hours of exposure to these proton ionophores or mitochondrial inhibitors, P388D1 cells did not show increased chlorotetracycline (CTC)-induced fluorescence or loss of viability. P388D1 cells exposed to silica have been shown previously to lose 80% of their adenosine triphosphate (ATP) content. The effect of reduced ATP levels on intracellular calcium homeostasis and viability was assessed by exposing P338D1 cells to FCCP in the presence of sodium azide and 2-deoxyglucose, which reduced ATP content by more than 90%. Under these conditions, none of the cells were killed, and only 5.5% showed increased CTC-induced fluorescence after 6 hours. These data indicate that disruption of the mitochondrial membrane potential, even in combination with reduced ATP content, is not sufficient to kill P388D1 cells.
线粒体对钙的隔离是维持细胞内正常钙稳态的重要机制。据推测，这些细胞器的缺氧或毒性损伤会破坏细胞内钙的区室化，导致细胞死亡。作者在二氧化硅诱导的P388D1细胞毒性模型系统中研究了线粒体功能障碍、钙稳态改变与不可逆损伤之间的潜在关系。暴露于有毒的二氧化硅颗粒而非无毒的乳胶头，会破坏线粒体膜电位，增加膜相关钙，升高游离胞质钙，并在6至8小时后杀死50%至60%的细胞群体。为了测试线粒体膜电位的破坏是否足以导致不可逆损伤，将P388D1细胞暴露于质子离子载体羰基氰化物对三氟甲氧基苯腙（FCCP）或线粒体抑制剂抗霉素A。如荧光探针罗丹明123所示，超过90%的处理细胞显示线粒体膜去极化。羰基氰化物对三氟甲氧基苯腙还导致游离胞质钙升高，这通过fura-2监测。然而，即使在暴露于这些质子离子载体或线粒体抑制剂6小时后，P388D1细胞也未显示氯四环素（CTC）诱导的荧光增加或活力丧失。先前已表明，暴露于二氧化硅的P388D1细胞会损失80%的三磷酸腺苷（ATP）含量。通过在叠氮化钠和2-脱氧葡萄糖存在下将P338D1细胞暴露于FCCP来评估ATP水平降低对细胞内钙稳态和活力的影响，这使ATP含量降低了90%以上。在这些条件下，没有细胞被杀死，6小时后只有5.5%的细胞显示CTC诱导的荧光增加。这些数据表明，线粒体膜电位的破坏，即使与ATP含量降低相结合，也不足以杀死P388D1细胞。
