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Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Published on: May 1, 2018
Differential effect of calmodulin antagonists on MG132-induced mitochondrial dysfunction and cell death in PC12 cells
Chung Soo Lee1, Eun Sook Han, Young Su Han
1Department of Pharmacology, College of Medicine, Chung-Ang University, Dong-jak Gu, Seoul 156-756, South Korea. leecs@cau.ac.kr
Abstract:
Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.
Insights
Calmodulin antagonists like trifluoperazine protect PC12 cells from proteasome inhibitor MG132 toxicity. These compounds reduce cell death by inhibiting caspase activation and preserving mitochondrial function.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Proteasome dysfunction is implicated in neurodegenerative diseases.
- PC12 cells are a neuronal model for studying cellular stress responses.
Purpose of the Study:
- To investigate the protective effects of calmodulin antagonists against proteasome inhibitor-induced cell damage.
- To elucidate the mechanisms underlying MG132 toxicity and the role of calmodulin.
Main Methods:
- PC12 cells were treated with MG132, a proteasome inhibitor, and various calmodulin antagonists.
- Cell viability, mitochondrial dysfunction, caspase activation, reactive oxygen species (ROS), and intracellular calcium levels were assessed.
Main Results:
- Calmodulin antagonists (trifluoperazine, W-7) at 0.5-1 microM attenuated MG132-induced cell death and GSH depletion.
- These antagonists reduced nuclear damage, mitochondrial membrane potential loss, cytochrome c release, ROS formation, and elevated intracellular Ca(2+) levels.
- MG132 toxicity was mediated by caspase-8, -9, and -3 activation.
Conclusions:
- Calmodulin antagonists protect PC12 cells from proteasome inhibitor toxicity.
- Protection involves calmodulin inhibition, suppression of mitochondrial permeability changes, and reduced intracellular calcium.
- Targeting calmodulin may offer therapeutic strategies for neuroprotection.
