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

Authors


Address correspondence and reprint requests to Janet M. Dubinsky, Department of Neuroscience, University of Minnesota, 6–145 Jackson Hall, 321 Church St. SE, Minneapolis, MN, 55455, USA. E-mail: dubin001@tc.umn.edu

Abstract

The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 µmol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 µm glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.

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