|
|
(2) Ca2+ UPTAKE AND RELEASE OF CYTOCHROME C IN NEURAL CELL MITOCHONDRIA A.Y. Andreyev, S.E. Wiley and
A.N. Murphy
It is well established that diverse apoptotic stimuli converge at the level of mitochondria to induce release of cytochrome c (CytC) into the cytosol, thus triggering activation of caspases, culminating in apoptosis. Ca2+-dependent mitochondrial permeability transition (MPT) resulting in swelling and outer membrane rupture is implicated as a mechanism of CytC release. Here we report that under physiologically realistic conditions (KCl-based medium, pH 7.0, NADH-linked respiratory substrates and mM levels of ATP and Mg) Ca2+ accumulation in mitochondria of neural cells induces respiratory inhibition and Cyt c release independent of the MPT. By using digitonin-permeabilized cells at high densities (20-30 million SY5Y cells per ml) we were able to study Ca2+ accumulation in the presence of endogenous cytosolic component. We found that cytosolic factors substantially modify mechanisms of Ca2+ uptake and release as well as maximal Ca2+ accumulation capacity. Trying to address selectivity of CytC release we designed SY5Y cell line expressing adenylate kinase isoform 2 with small (hemagglutinin) tag. This chimera localizes properly to mitochondria and is used for immunodetection of non-selective release of proteins from intermembrane space. In the presence of ATP and Mg2+, Ca2+ induces CytC release independently of the classical MPT, as indicated from the lack of swelling, lack of DY collapse and retention of accumulated Ca2+. Cyclosporin A (CsA) has no effect on Ca2+ uptake and Ca2+-induced CytC release, whereas inhibitors of adenine nucleotide translocator carboxyatractylate and bongkrekate enhance Cyt c release. Bcl-2 protect against Cyt c release but this protection is reversed by carboxyatractylate. CytC release is selective, as indicated by the lack of release of adenylate kinase. The pore-forming molecule alamethicin mimics MPT and causes complete loss of both proteins as a result of mitochondrial swelling. In the presence of ATP and Mg2+, MPT and swelling-dependent Cyt c release is accompanied by adenylate kinase release. These results indicate that the mechanism of Ca2+-induced CytC release from neural cell mitochondria can occur by the MPT, but can also involve a selective permeabilization of the outer membrane. We hypothesize that proteins present at the contact sites of inner and outer mitochondrial membranes (which are known to include the adenine nucleotide translocase and Bcl-2) serve as machinery for MPT-independent CytC translocation across the outer membrane. Supported in part by NIH 34154 to ANM.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
||