(5) COMPARISON OF Ca2+- AND PROTEIN-INDUCED CYTOCHROME C RELEASE FROM BRAIN MITOCHONDRIA IN VITRO AND IN SITU

N. Brustovetsky¹, T. Brustovetsky¹, R. Jemmerson² and J.M. Dubinsky^1
1Department of Neuroscience
²Department of Microbiology, University of Minnesota, Minneapolis, MN 55455

A solid phase ELISA was used to quantify Cytochrome c (Cyt c) release from Percoll-purified isolated brain mitochondria (Mtc) and neuronal mitochondria in situ.  Basal Cyt c release from untreated Mtc represented about 7% of maximum alamethecin-induced Cyt c release and was not inhibited by EGTA, cyclosporin A (CsA) or Koenig’s polyanion (KP), a VDAC inhibitor.  300 uM Ca2+ induced 2.5-3 fold increase of Cyt c release from brain Mtc incubated at 37ºC for 15 minutes in 75 but not 125 mM KCl.  In both cases similar depolarization and swelling, features of the mitochondrial permeability transition (mPT), were observed using a TPP+ electrode and light scattering measurements.  Transmission electron microscopy (TEM) revealed that initially isolated Mtc were in a very condensed state. Ca2+-induced swelling caused rupture of the outer membrane only in 75 mM KCl.  Ca2+-induced release of Cyt c in 75 mM KCl was almost completely inhibited by 1 uM Ru360, an inhibitor of the Ca2+ uniporter, or by a combination of mPT inhibitors, 1 uM oligomycin, 100 uM ADP and 1 uM CsA.  CsA alone was ineffective as well as KP or recombinant Bcl-xL.  In unpurified Mtc isolated with 0.01% digitonin (1) or purified Mtc in the presence of 0.001% digitonin, Ca2+ induced Cyt c efflux in 125 mM KCl without swelling in the presence of oligomycin, ADP and CsA.  Treatment of cultured cortical neurons by 500 uM glutamate for 5 minutes induced an increase of cytosolic Ca2+ and mitochondrial depolarization.  These changes were accompanied by 6-fold increase of Cyt c in the cytosol.  The same glutamate treatment of cultured astrocytes did not cause Cyt c translocation into cytosol.  The Cyt c release in neurons in situ was inhibited by 20 uM MK801, a glutamate receptor antagonist, or by 5 uM CsA but not by 5 uM FK506.  Incubation of neurons with 1 uM staurosporine for 6 hours caused Cyt c release into cytosol comparable to the release observed after glutamate.  Cytosolic extracts from HL-60 cells at an early stage of staurosporine-induced apoptosis, but not from untreated cells, evoked Cyt c release from isolated detergent-free brain Mtc without depolarization and swelling equivalent to that evoked by Ca2+.  TEM confirmed the lack of mitochondrial swelling.  The cytosol-induced Cyt c release was inhibited by 20 ug/ml of Koenig’s polyanion, indicating possible involvement of VDAC in apoptotic Cyt c translocation and by 10 ug/ml of Bcl-xL.  The combination of oligomycin, ADP and CsA failed to prevent cytosol-induced release.  In all experiments, Mtc retained a minimum of 60% of the alamethecin-releasable Cyt c.  Thus, brain Mtc possess two distinctly different mechanisms of Cyt c release: (i) Ca2+ induction of the mPT causing mitochondrial swelling and rupture of the outer membrane, and Cyt c release and (ii) apoptotic protein induction of Cyt c release associated with VDAC, independent of the mPT. 

Supported by the HDSA and the AHA.

1)  Rosenthal et al. (1987) JCBFM 7:752

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