Jiang S, Chow S C, Nicotera P, Orrenius S
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Exp Cell Res. 1994 May;212(1):84-92. doi: 10.1006/excr.1994.1121.
The endoplasmic reticular Ca(2+)-ATPase inhibitor, thapsigargin, was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition, confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus, we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis.
内质网Ca(2+)-ATP酶抑制剂毒胡萝卜素被用于研究胞质游离钙浓度（[Ca2+]i）升高作为激活胸腺细胞凋亡信号的作用。用毒胡萝卜素处理大鼠胸腺细胞导致[Ca2+]i早期持续升高，随后出现广泛的DNA片段化。琼脂糖凝胶电泳显示，DNA片段的模式是典型的核酸内切酶介导的核小体间切割。此外，共聚焦显微镜研究显示在毒胡萝卜素处理的胸腺细胞中形成了凋亡细胞核。诱导胸腺细胞DNA片段化和[Ca2+]i升高所需的毒胡萝卜素浓度相同，毒胡萝卜素诱导的DNA片段化和凋亡细胞核形成的动力学也相同。激活凋亡所需的毒胡萝卜素最低浓度为1 nM。在名义上无Ca(2+)的培养基中孵育的细胞或用细胞内Ca2+螯合剂双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸/乙酰氧甲酯预处理的细胞中，毒胡萝卜素诱导的[Ca2+]i升高和胸腺细胞凋亡受到抑制。在加入毒胡萝卜素后的不同时间点用5 mM EGTA去除细胞外游离Ca2+，结果显示[Ca2+]i持续升高触发胸腺细胞凋亡具有约2小时的时间依赖性。因此，我们得出结论，毒胡萝卜素诱导的[Ca2+]i升高所提供的信号足以激活胸腺细胞凋亡。
