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Effects of Ca2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following
J C Papadimitriou1, P C Phelps, M L Shin
1Department of Pathology, University of Maryland School of Medicine, Baltimore.
Insights
Complement component 5b-9 (C5b-9) channels cause rapid cell death by increasing cytosolic calcium ([Ca2+]i) and mitochondrial potential loss. Preventing calcium influx delays death, suggesting metabolic depletion is the cause.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Lytic C5b-9 channels on tumor cells trigger adenine nucleotide depletion and cell death.
- Extracellular calcium chelation delays cell death but enhances nucleotide leakage.
Purpose of the Study:
- To investigate the temporal relationship between cytosolic calcium ([Ca2+]i), mitochondrial membrane potential (Δψm), and cell death during C5b-9 attack.
- To elucidate the role of calcium influx in C5b-9-mediated cell death.
Main Methods:
- Digital imaging fluorescence microscopy (DIFM) was used to monitor individual cells.
- Measurements included ionized cytosolic calcium ([Ca2+]i) and mitochondrial membrane potential (Δψm).
- Cells were exposed to C5b-9 attack with and without extracellular calcium chelation.
Main Results:
- A rapid, >20-fold increase in [Ca2+]i was observed immediately following C5b-9 attack.
- This was followed by dissipation of Δψm and acute cell death.
- Extracellular calcium chelation significantly delayed these events, while nominally calcium-free medium did not.
- Propidium iodide labeling was only observed in dying cells, not viable cells with C5b-9 channels.
Conclusions:
- Calcium influx through C5b-9 channels is responsible for the rapid rise in [Ca2+]i and subsequent Δψm dissipation, leading to acute cell death.
- When [Ca2+]i increase is prevented, cell death is likely due to metabolic depletion.
Abstract:
We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+]e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+]i), mitochondrial membrane potential (delta psi m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+]i, rapidly followed by dissipation of delta psi m and subsequent acute cell death. These events were markedly delayed by chelation of Ca2+e, but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+]i, as well as for the rapid loss of delta psi m, followed by acute cell death. When this [Ca2+]i increase is prevented, the cell death is probably related to metabolic depletion.