Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Abstract

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl- transport inhibitor DIDS and the K+ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl- flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl- fluxes rather than blocking cell shrinkage or K+ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

“…Our results with staurosporine also support the conclusions about volume shrinkage made by previous investigators who analyzed suspended cells by electron microscopy [4] and by Coulter analysis [31,34,46]. Adherent cells had been studied as well, with volume estimated from the overall cell shape or from the attachment area [33,43,53].…”

Intracellular K+ Concentration Decrease Is Not Obligatory for Apoptosis

“…Thus, in Xenopus oocytes caspase-3 activation could possibly be triggered by an altered ionic strength mediated by ions other than K ϩ . Other studies show that STS-induced caspase-3 activation and apoptosis are not prevented by various K ϩ channel blockers in rat cortical neurons (30), rat cerebellar granule cells (31), mouse neuroblastoma cells (32), salmonid cells, and hepatocytes (33). Even though the intracellular K ϩ concentration was not monitored in these studies, the reported caspase-3 activity despite the use of K ϩ channel blockers suggests that further cell types have a K ϩ -independent mechanism for inducing apoptosis.…”

DIDS Attenuates Staurosporine-induced Cardiomyocyte Apoptosis by PI3K/Akt Signaling Pathway: Activation of eNOS/NO and Inhibition of Bax Translocation

“…More importantly, staurosporine (STS)-induced apoptotic death of salmonid cells can be rescued by DIDS [6], and in U937, HeLa, NG108-15, and PC12 cells, DIDS has also been reported to reduce apoptosis induced by STS and tumor necrosis factor (TNF) [7]. Similarly, recent experimental results suggested that DIDS rescued cardiomyocytes from multiple other apoptotic stimuli, such as doxorubicin [8] and ischemia-reperfusion [9].…”