Apoptosis is an important mechanism of cell demise in multicellular organisms and Cl− transport has an important role in the progression of the apoptotic volume decrease (AVD). DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate) is one of the most commonly used Cl− transport inhibitors that eliminates or reduces different apoptotic hallmarks such as AVD, caspase-3 activity and DNA fragmentation. DIDS is also a protein crosslinker that alkylates either amino or thiol groups. Since caspases are thiol proteases, our aim was to study whether DIDS could directly inhibit the activity of these proteases. Here, we show that caspase activity induced by 4 h incubation with staurosporine was inhibited by DIDS in HeLa cells that were maintained in the absence of serum for 24 h. Interestingly, the caspase-inhibitory effect of DIDS is downstream to the inhibition of cytochrome c release, suggesting that DIDS might be also acting at the apoptosome. Moreover, DIDS was able to inhibit capase-3, -9, and -8 activities in cell lysates, implying that DIDS can react with and directly block caspases. Our data suggest that antiapoptotic activity of DIDS involves not only inhibition of the voltage-dependent anion channel (VDAC) at the mitochondria and Cl− channels at the plasma membrane, but also a third mechanism based on the direct inhibition of caspases.

Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na+ and loss of K+ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA+) and 4-aminopyridine-sensitive K+ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K+] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K+]i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K+ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K+]i associated with apoptosis.

In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca 2+ ] ER), changes in the cytoplasmic calcium concentration ([Ca 2+ ] 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 [Ca 2+ ] i and with Mag-fluo-4 to measure variations in the [Ca 2+ ] 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 [Ca 2+ ] i , but only K5 immediately reduced the [Ca 2+ ] ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca 2+ ] i and they decreased both the [Ca 2+ ] ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca 2+ ] 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 Ca 2+ channels, that are not dihydropyridine-sensitive, load the ER with Ca 2+ and this Ca 2+ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca 2+ ] ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.

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