Draper David W, Harris Virginia G, Culver Carolyn A, Laster Scott M
Department of Microbiology, North Carolina State University, Raleigh, NC 27606, USA.
J Immunol. 2004 Feb 15;172(4):2416-23. doi: 10.4049/jimmunol.172.4.2416.
In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A(2) (cPLA(2)). As our model, we used a murine fibroblast cell line (C3HA) that was induced to undergo apoptosis by a combination of TNF and cycloheximide. Using fura 2 Ca(2+) imaging, we found strong evidence for an intracellular calcium response after 1 h of treatment, which correlated with the onset of phosphatidylserine externalization, but preceded effector procaspase processing by several hours. The response was strongest in the perinuclear region, where mean levels rose 83% (144 +/- 14 nM in untreated cells vs 264 +/- 39 nM in treated), while cells displaying morphological evidence of apoptosis had the highest levels of calcium (250-1000 nM). Verapamil blocked this response, indicating an extracellular source for the calcium. Fluorescence microscopy revealed a pattern of nuclear translocation of cPLA(2) during apoptosis, which was also blocked by verapamil, indicating an important role for calcium in this process. In addition, we found that verapamil prevented the release of [(3)H]arachidonic acid from C3HA cells induced to undergo apoptosis by the chemotherapeutic agents vinblastine, melphalan, and cis-platinum. Together, these data suggest that calcium is important for cPLA(2) activation by diverse apoptotic stimuli.
在这些实验中，我们研究了钙作为第二信使在胞质磷脂酶A2（cPLA2）凋亡激活过程中的作用。作为我们的模型，我们使用了一种小鼠成纤维细胞系（C3HA），该细胞系通过肿瘤坏死因子（TNF）和放线菌酮的联合作用被诱导发生凋亡。使用fura 2钙成像技术，我们发现处理1小时后有强有力的证据表明存在细胞内钙反应，这与磷脂酰丝氨酸外翻的开始相关，但比效应半胱天冬酶的加工提前数小时。该反应在核周区域最强，此处平均水平升高了83%（未处理细胞中为144±14 nM，处理后为264±39 nM），而显示凋亡形态学证据的细胞具有最高的钙水平（250 - 1000 nM）。维拉帕米阻断了这种反应，表明钙的细胞外来源。荧光显微镜显示凋亡过程中cPLA2的核转位模式，这也被维拉帕米阻断，表明钙在这个过程中起重要作用。此外，我们发现维拉帕米可阻止由化疗药物长春碱、美法仑和顺铂诱导凋亡的C3HA细胞释放[3H]花生四烯酸。总之，这些数据表明钙对于多种凋亡刺激激活cPLA2很重要。
