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(13) DYSFUNCTION OF MITOCHONDRIA AND CYTOCHROME C RELEASE DURING APOPTOSIS R.A. Eliseev and T.E. Gunter
Mitochondria have been shown to play a significant role in apoptosis. Release of the mitochondrial intermembrane protein, cytochrome c, can lead to activation of late caspases. During apoptosis, cytochrome c is released from the intermembrane space along with other intermembrane proteins including apoptosis inducing factor and some caspases. The goal of our study was to elucidate the mechanism of mitochondrial dysfunction and cytochrome c release in apoptosis. The hypotheses on the mechanism currently being discussed in the literature, suggest either the MPT-mediated swelling of the matrix space followed by mechanical rupture of the outer membrane or a specific transporter. We induced apoptosis in HL-60 cells with etoposide. At different times after the induction of apoptosis, we measured mitochondrial membrane potential, cellular ATP, release of mitochondrial proteins and mannitol accessible volume. We detected a progressive decrease in mitochondrial membrane potential in apoptotic cells coincident with the decrease in cellular ATP and with the appearance of cytochrome c and another intermembrane protein-adenylate kinase in the cytosol. Depolarization of mitochondria and the decrease in ATP production indicated mitochondrial dysfunction. The release of adenylate kinase along with cytochrome c demonstrated that there was non-specific permeabilization of the outer mitochondrial membrane, most likely because of tearing due to the swelling. Also the mannitol accessible volume increased and the soluble matrix enzyme, malate dehydrogenase, was released, marking the opening of the inner mitochondrial membrane. We also carried out electron microscopy of apoptotic cells which revealed mitochondrial swelling followed by fragmentation. All this might indicate the onset of the MPT during apoptosis. To further check if this was the mechanism responsible for cytochrome c release we added etoposide in the presence of specific MPT inhibitor cyclosporin a (CsA). CsA failed to reverse apoptosis, cytochrome c and AK release, mitochondrial swelling, depolarization and ATP fall. However, it prevented the malate dehydrogenase release and mannitol accessible volume increase – the signs of permeabilization of the inner mitochondrial membrane. We therefore conclude that although the MPT seems to be activated during apoptosis, this is not the primary mechanism of mitochondrial swelling leading to cytochrome c release. Other possible mechanisms of mitochondrial swelling are being currently investigated. This work was supported by AHA-NYS affiliate 960166, by NIH P30 ES 01247, and by support from the University of Rochester Medical School.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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