Abstract
Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.
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Abbreviations
- I k :
-
outward delayed rectifier K+ current
- TEA:
-
tetraethylammoniumchloride
- pHi:
-
intracellular pH
- NHE:
-
Na+/H+ exchanger
- Hoe 642:
-
4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate
- EIPA:
-
5-(N-ethyl-N-isopropyl)-amiloride
- TUNEL:
-
terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling
- IGF-1:
-
insulin-like growth factor
- cAMP:
-
cyclo-adenosine-monophosphate
- Bcl-XL:
-
B cell leukemia gene
- CHX:
-
cycloheximide
- DIV:
-
day in vitro
- NMDA:
-
N-methyl-D-aspartate
- HK:
-
culture cell medium containing 25 mM potassium
- LK:
-
culture medium containing 5 mM potassium
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Acknowledgements
This study was supported by a grant from the German Research Foundation to JBS (SFB 430, Teilprojekt B8).
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Schneider, D., Gerhardt, E., Bock, J. et al. Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis. Cell Death Differ 11, 760–770 (2004). https://doi.org/10.1038/sj.cdd.4401377
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DOI: https://doi.org/10.1038/sj.cdd.4401377
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