Limke Tobi L, Otero-Montañez James K L, Atchison William D
Department of Pharmacology and Toxicology, Institute for Environmental Toxicology, and Neuroscience Program, Michigan State University, East Lansing, Michigan, USA.
J Pharmacol Exp Ther. 2003 Mar;304(3):949-58. doi: 10.1124/jpet.102.042457.
Acute exposure to methylmercury (MeHg) causes severe disruption of intracellular Ca(2+) (Ca(2+)) regulation, which apparently contributes to neuronal death. Activation of the mitochondrial permeability transition pore (MTP) evidently contributes to this effect. We examined in more detail the contribution of mitochondrial Ca(2+) (Ca(2+)) to elevations of Ca(2+) caused by acute exposure to a low concentration of MeHg in primary cultures of rat cerebellar granule neurons. In particular, we sought to determine whether interactions occurred between Ca(2+)(i) pools in response to MeHg. Prior depletion of Ca(2+)(m) using carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin significantly decreased the amplitude of Ca(2+) release from intracellular stores, and delayed the onset of whole-cell Ca(2+) elevations, caused by 0.5 microM MeHg. CCCP alone hastened the MeHg-induced release of Ca(2+) within the cell, whereas oligomycin alone delayed the MeHg-induced influx of extracellular Ca(2+). In granule cells loaded with rhod-2 acetoxymethylester to measure changes in Ca(2+), MeHg exposure caused a biphasic increase in fluorescence. The initial increase in fluorescence occurred in the absence of extracellular Ca(2+) and was abolished by mitochondrial depolarization. The secondary increase was associated with spreading of the dye from punctate staining to whole-cell distribution, and was delayed significantly by the MTP inhibitor cyclosporin A and the smooth endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin. We conclude that MeHg causes release of Ca(2+) from the mitochondria through opening of the MTP, which contributes the bulk of the elevated Ca(2+) observed during MeHg neurotoxicity. Additionally, the Ca(2+) that enters the mitochondria seems to originate in the smooth endoplasmic reticulum, providing a mechanism for the observed mitochondrial Ca(2+) overload.
急性暴露于甲基汞（MeHg）会导致细胞内钙离子（Ca(2+)）调节严重紊乱，这显然是神经元死亡的原因之一。线粒体通透性转换孔（MTP）的激活显然促成了这种效应。我们更详细地研究了线粒体钙离子（Ca(2+)）对大鼠小脑颗粒神经元原代培养物中急性暴露于低浓度MeHg所引起的Ca(2+)升高的作用。特别是，我们试图确定响应MeHg时Ca(2+)(i)池之间是否发生相互作用。预先使用羰基氰化物间氯苯腙（CCCP）和寡霉素耗尽Ca(2+)(m)，显著降低了细胞内储存库释放的Ca(2+)幅度，并延迟了由0.5微摩尔MeHg引起的全细胞Ca(2+)升高的起始时间。单独使用CCCP会加速MeHg诱导的细胞内Ca(2+)释放，而单独使用寡霉素则会延迟MeHg诱导的细胞外Ca(2+)内流。在用罗丹明-2乙酰甲酯加载以测量Ca(2+)变化的颗粒细胞中，暴露于MeHg会导致荧光双相增加。荧光的初始增加发生在细胞外无Ca(2+)的情况下，并通过线粒体去极化而消除。第二次增加与染料从点状染色扩散到全细胞分布有关，并被MTP抑制剂环孢菌素A和平滑内质网Ca(2+)ATP酶抑制剂毒胡萝卜素显著延迟。我们得出结论，MeHg通过打开MTP导致线粒体释放Ca(2+)，这是MeHg神经毒性期间观察到的Ca(2+)升高的主要原因。此外，进入线粒体的Ca(2+)似乎起源于平滑内质网，为观察到的线粒体Ca(2+)过载提供了一种机制。
