Olafsdottir K, Pascoe G A, Reed D J
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Arch Biochem Biophys. 1988 May 15;263(1):226-35. doi: 10.1016/0003-9861(88)90631-5.
In this study the Ca2+ ionophore, A23187, was used to determine the effects of disrupted Ca2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca2+ and A23187 to induce accumulation or loss of cellular Ca2+. These treatments resulted in loss of mitochondrial and cytosolic glutathione (GSH), loss of protein-thiols, and cell injury. This injury was dependent on the concentrations of ionophore and extracellular Ca2+. A correlation was found between cell injury and the loss of mitochondrial GSH, while the loss of cytosolic glutathione preceded both these events. The time course of protein-thiol loss appeared secondary to the loss of non-protein thiols. In the absence of extracellular Ca2+, the antioxidants alpha-tocopherol and diphenyl-p-phenylenediamine both totally prevented A23187-induced cell injury and loss of mitochondrial GSH, and thus protected the cells from the effects of mobilization of intracellular Ca2+. In the presence of extracellular Ca2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca2+. Leupeptin, an inhibitor of Ca2+-activated proteases, and dibucaine, a phospholipase inhibitor, did not affect cytotoxicity. Our results indicate that the level of mitochondrial GSH may be important for cell survival during ionophore-induced perturbation of cellular Ca2+ homeostasis.
在本研究中，使用钙离子载体A23187来确定Ca2+稳态破坏对细胞巯基的影响。将分离的大鼠肝细胞与不同浓度的细胞外Ca2+和A23187一起孵育，以诱导细胞Ca2+的积累或丧失。这些处理导致线粒体和胞质谷胱甘肽（GSH）丧失、蛋白质巯基丧失以及细胞损伤。这种损伤取决于离子载体和细胞外Ca2+的浓度。发现细胞损伤与线粒体GSH丧失之间存在相关性，而胞质谷胱甘肽的丧失先于这两个事件。蛋白质巯基丧失的时间进程似乎继发于非蛋白质巯基的丧失。在没有细胞外Ca2+的情况下，抗氧化剂α-生育酚和二苯基对苯二胺都完全防止了A23187诱导的细胞损伤和线粒体GSH丧失，从而保护细胞免受细胞内Ca2+动员的影响。在存在细胞外Ca2+的情况下，抗氧化剂处理仅部分防止了细胞损伤以及线粒体GSH的丧失。线粒体Ca2+通道阻滞剂钌红在没有细胞外Ca2+的情况下保护肝细胞免受A23187诱导的损伤。亮抑酶肽（一种Ca2+激活蛋白酶的抑制剂）和丁卡因（一种磷脂酶抑制剂）不影响细胞毒性。我们的结果表明，在离子载体诱导的细胞Ca2+稳态扰动期间，线粒体GSH水平可能对细胞存活很重要。
