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Ca(2+)-induced, phospholipase-independent injury during reoxygenation of anoxic mitochondria
T Inoue1, Y Yoshida, M Nishimura
1Department of Physiological Chemistry, Osaka University Medical School, Japan.
Abstract:
Reoxygenation of rat-liver mitochondria after anoxic incubation induced release of matrix proteins. As assessed by release of a matrix enzyme, it was proportional to the rate of H2O2 production. The release was not observed with low concentrations of extramitochondrial free Ca2+, indicating a Ca(2+)-dependent pathway. Phospholipase A2 was not involved in the reoxygenation injury, because non-esterified fatty acids did not increase on reoxygenation even when re-acylation was inhibited and because inhibitors of phospholipase A2 had little effect on enzyme release. Cyclosporin A, ATP, ADP and inhibitors of pyridine nucleotide oxidation had a protective effect, strongly suggesting involvement of so-called Ca(2+)-dependent permeability transition. Ca2+ was also released from reoxygenated mitochondria and inhibition of reuptake of released Ca2+ attenuated the enzyme release. Similar releases of aspartate aminotransferase and Ca2+ were observed with mitochondria in an oxygen radical-generating system, hypoxanthine and xanthine oxidase. In this system, lecithin-cardiolipin liposomes also released entrapped Ca2+ without disruption of the membrane. From these results, we conclude that during reoxygenation, Ca2+ release and subsequent reuptake induced permeability transition of mitochondria, resulting in reoxygenation injury.
Insights
Reoxygenation causes rat liver mitochondria to release matrix proteins, linked to hydrogen peroxide production. Calcium (Ca2+) release and reuptake trigger mitochondrial permeability transition, leading to cell injury.
Area of Science:
- Mitochondrial Biology
- Cellular Physiology
- Biochemistry
Background:
- Reoxygenation following anoxia can cause cellular damage.
- Mitochondria play a critical role in cellular energy metabolism and are susceptible to injury during reoxygenation.
- The precise mechanisms underlying mitochondrial dysfunction during reoxygenation are not fully understood.
Purpose of the Study:
- To investigate the mechanisms of mitochondrial matrix protein release during reoxygenation in rat liver mitochondria.
- To elucidate the role of calcium (Ca2+) and reactive oxygen species in reoxygenation-induced mitochondrial injury.
Main Methods:
- Isolated rat liver mitochondria were subjected to anoxic incubation followed by reoxygenation.
- Matrix protein release was assessed by measuring the activity of a matrix enzyme.
- Hydrogen peroxide (H2O2) production was monitored.
- The effects of varying extramitochondrial free Ca2+ concentrations, phospholipase A2 activity, and specific inhibitors (Cyclosporin A, ATP, ADP, pyridine nucleotide oxidation inhibitors) were evaluated.
- Ca2+ release and reuptake by mitochondria were measured.
- An oxygen radical-generating system (hypoxanthine/xanthine oxidase) was used as a comparative model.
Main Results:
- Reoxygenation induced the release of matrix proteins, proportional to H2O2 production.
- Ca2+ release and subsequent reuptake were critical for this process, indicating a Ca2+-dependent pathway.
- Phospholipase A2 was not implicated in the injury.
- Protective agents like Cyclosporin A and ATP suggested the involvement of the Ca2+-dependent permeability transition.
- Ca2+ release from mitochondria was observed during reoxygenation, and inhibiting its reuptake attenuated enzyme release.
- Similar Ca2+ and enzyme release occurred in an oxygen radical-generating system.
Conclusions:
- Reoxygenation triggers mitochondrial Ca2+ release and subsequent reuptake.
- This Ca2+ flux induces the mitochondrial permeability transition, leading to matrix protein release and reoxygenation injury.
- The findings highlight a critical role for mitochondrial Ca2+ handling in reoxygenation-induced cellular damage.