(1) TWO PATHWAYS OF Ca2+-INDUCED CYTOCHROME C RELEASE FROM MITOCHONDRIA

A. Andreyev and G. Fiskum
Dept. of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD 21201

At pathologically elevated levels of intracellular Ca2+, mitochondrial Ca2+ uptake can lead to release of the apoptotic factor cytochrome c (Cyt C) into the cytosol. In a physiologically relevant KCl-based medium maintained at 37 degrees and containing ATP and Mg2+, accumulation of 300 nmol Ca2+/mg prot. by isolated rat brain mitochondria triggers, within seconds, a maximal release of Cyt C into the medium (~40% total), as assayed by Western blots. This process is not associated with high-amplitude swelling as measured by light scattering and by electron microscopy. Following completion of Ca2+ uptake, the membrane potential is restored. Under these conditions an inhibitor of the permeability transition (PT), cyclosporin A (CsA) does not affect Ca2+ fluxes, membrane potential changes or Cyt C release. In contrast, under the same conditions rat liver mitochondria undergo classical PT resulting in high amplitude swelling, depolarization and complete loss of Cyt C. CsA completely prevents swelling and depolarization but only partially prevents release of Cyt C. In the absence of the PT inhibitors ATP and Mg2+ both types of mitochondria undergo permeabilization at different extent (liver > brain) that results in swelling and Cyt C release. These results indicate that Ca2+ induces mitochondrial Cyt C release by both PT-independent and PT-dependent mechanisms and that the activity of these pathways is tissue specific. We propose that the PT-independent pathway of Cyt C release is more consistent with the pattern of energy metabolism typical of delayed apoptotic cell death. (Supported by NS34152)