Abstract
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.
Highlights
The endoplasmic reticulum (ER) is one of the most important intracellular Ca2+ stores
We have evaluated the effect of different inducers of apoptosis on Ca2+ release from the ER in cultured neonatal cerebellar granule neurons (CGN) and we have looked into the correlation between this calcium release and caspase-3 activation, mitochondrial depolarization, and ER stress
Based on the present results, we propose that the survival of CGN requires a sustained Ca2+ transfer from the ER to the mitochondria to keep this organelle polarized and the inhibition of this Ca2+ transfer leads to a rapid mitochondrial partial depolarization that in turn leads to apoptotic death in this neuronal survival model
Summary
The endoplasmic reticulum (ER) is one of the most important intracellular Ca2+ stores. It is well documented that a sustained reduction in the luminal [Ca2+] of the ER can result in a condition known as ER stress that eventually can lead to cell death (Boyce and Yuan, 2006; Szegedi et al, 2006; Sun et al, 2013). This condition may result in the activation of the ER stress receptor pathways. One possibility is that the cytoplasmic Ca2+ reduction induced by potassium deprivation could lead to a decrease of the luminal ER [Ca2+], which in turn could cause an ER stress in this model
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