In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca²⁺]_ER), changes in the cytoplasmic calcium concentration ([Ca²⁺]_i), alteration of the mitochondrial membrane potential, and the ER stress in the activation of caspase-3 in neonatal cerebellar granule cells (CGN). The cells were loaded with Fura-2 to detect changes in the [Ca²⁺]_i and with Mag-fluo-4 to measure variations in the [Ca²⁺]_ER or with TMRE to follow modifications in the mitochondrial membrane potential in response to five different inducers of CGN cell death. These inducers were staurosporine, thapsigargin, tunicamycin, nifedipine and plasma membrane repolarization by switching culture medium from 25 mM KCl (K25) to 5 mM KCl (K5). Additionally, different markers of ER stress were determined and all these parameters were correlated with the activation of caspase-3. The different inducers of cell death in CGN resulted in three different levels of activation of caspase-3. The highest caspase-3 activity occurred in response to K5. At the same time, staurosporine, nifedipine, and tunicamycin elicited an intermediate activation of caspase-3. Importantly, thapsigargin did not activate caspase-3 at any time. Both K5 and nifedipine rapidly decreased the [Ca²⁺]_i, but only K5 immediately reduced the [Ca²⁺]_ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca²⁺]_i and they decreased both the [Ca²⁺]_ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca²⁺]_i, but it took 10 min to observe any decrease in the mitochondrial membrane potential. Three cell death inducers -K5, staurosporine, and thapsigargin- elicited ER stress, but they took 30 min to have any effect. Thapsigargin, as expected, displayed the highest efficacy activating PERK. Moreover, a specific PERK inhibitor did not have any impact on cell death triggered by these cell death inducers. Our data suggest that voltage-gated Ca²⁺ channels, that are not dihydropyridine-sensitive, load the ER with Ca²⁺ and this Ca²⁺ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca²⁺]_ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.

在这项研究中，我们评估了减少内质网钙浓度（[Ca ²⁺ ] _ER），改变细胞质钙浓度（[Ca ²⁺ ] _i），改变线粒体膜电位和ER的作用。新生小脑颗粒细胞（CGN）中caspase-3激活过程中的应激反应。细胞中装有Fura-2以检测[Ca ²⁺ ] _i的变化，并装有Mag-fluo-4以测量[Ca ²⁺ ] _ER的变化或使用TMRE来响应五种不同的CGN细胞死亡诱导物，从而改变线粒体膜电位。通过将培养基从25 mM KCl（K25）切换到5 mM KCl（K5），这些诱导剂为星形孢菌素，毒胡萝卜素，衣霉素，硝苯地平和质膜复极化。另外，确定了ER应激的不同标志物，并且所有这些参数都与caspase-3的激活相关。CGN中细胞死亡的不同诱导物导致caspase-3激活的三种不同水平。最高的caspase-3活性是对K5的反应。同时，星形孢菌素，硝苯地平和衣霉素引起caspase-3的中间活化。重要的是，thapsigargin在任何时候都不会激活caspase-3。K5和硝苯地平均迅速降低[Ca²⁺ ] _i，但只有K5立即降低[Ca ²⁺ ] _ER和线粒体膜电位。星形孢菌素和衣霉素增加了[Ca ²⁺ ] _i，并且降低了[Ca ²⁺ ] _ER和线粒体膜电位，但速率远低于K5。Thapsigargin大大提高了[Ca ²⁺ ] _i，但花了10分钟才能观察到线粒体膜电位的任何降低。三种细胞死亡诱导剂-K5，星形孢菌素和毒胡萝卜素可引起内质网应激，但它们需要30分钟才能发挥作用。如预期的那样，thapsigargin显示出最高的激活PERK的功效。此外，特定的PERK抑制剂对这些细胞死亡诱导剂触发的细胞死亡没有任何影响。我们的数据表明，对二氢吡啶不敏感的电压门控Ca ²⁺通道会给ER加载Ca ^2+，并且此Ca ²⁺通量在保持线粒体膜电位极化方面起关键作用。[Ca ²⁺ ] _ER迅速下降 导致线粒体膜快速去极化和caspase-3的强活化，而无需在CGN中干预ER应激。