Calcium-induced Cytochrome c release from ratbrain mitochondria is altered by digitonin

doi:10.1016/S0304-3940(02)00948-5

Neuroscience Letters

Volume 332, Issue 2, 31 October 2002, Pages 91-94

https://doi.org/10.1016/S0304-3940(02)00948-5 Get rights and content

Abstract

To determine if calcium could release Cytochrome c (Cyt c) from brain mitochondria without activating the permeability transition (mPT), brain mitochondria were prepared in two different ways. Digitonin was used to lyse synaptosomes and release synaptosomal mitochondria or a Percoll gradient was used to separate non-synaptosomal mitochondria from the synaptosomes. In gradient-purified mitochondria, low levels of added digitonin produced swelling and Cyt c release. Digitonin augmented Ca²⁺-induced Cyt c release that was insensitive to the mPT inhibitors, cyclosporin A CsA and ADP. Similarly, in mitochondria prepared with digitonin, these inhibitors also failed to prevent Ca²⁺-induced Cyt c release. Thus the mPT-independent, Ca²⁺-induced Cyt c release pathway was attributable to alteration of the permeability properties of the outer mitochondrial membrane by digitonin.

Section snippets

Acknowledgements

We would like to thank Dr Calvin Roff of R&D Systems for his support. This work was funded by the HDSA, NINDS 39414 to J.M.D., AHA 9804691X and 0130497Z to N.B., and NIH 1R21-AI45090 to R.J.

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To access mitochondria within synaptosomes digitonin may be used (Sims and Blass, 1986). However it has been demonstrated that low levels of digitonin produces swelling and cytochrome c release in brain mitochondria, and that brain mitochondria isolated using digitonin have altered permeability properties of the outer mitochondrial membrane (Brustovetsky et al., 2002). For these reasons digitonin was not included in present experimental set-up.
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Characteristics of the succinate-supported H₂O₂ formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H₂O₂ generation measured in mitochondria prepared with digitonin (600.6 ± 26.8 pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H₂O₂ formation was significantly smaller in Percoll-purified mitochondria (252.6 ± 17.3 pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H₂O₂ formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185 ± 3.2 mV) and addition of BSA (0.025%) to the assay medium increased H₂O₂ generation by only 50%. In Percoll-purified mitochondria ΔΨm was more depolarized (171 ± 2 mV) and BSA caused hyperpolarization by 10.7 ± 1.9 mV. H₂O₂ formation, which was largely independent of ΔΨm, was stimulated by 400%, became highly dependent on ΔΨm and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10 mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on ΔΨm.
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Calcium-induced Cytochrome c release from ratbrain mitochondria is altered by digitonin

Abstract

Section snippets

Acknowledgements

Anal. Biochem.

Biochem. Biophy. Acta

Methods Enzymol.

Biochem. Med. Metab. Biol.

FEBS Lett.

Calcium induced release of mitochondrial Cytochrome c by different mechanisms selective for brain versus liver

Cell Death Diff.

Calcium-induced Cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane

J. Neurochem.

Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites

Biochem. J.

BAX-induced Cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions

J. Cell Biol.

Bax directly induces release of cytochrome c from isolated mitochondria

Proc. Natl. Acad. Sci. USA

BAX-induced Cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg²⁺ ions