The role of the mitochondrial permeability transition pore in cell death and calcium(2+) signaling

This dissertation examines (I) the role of cycolphilin D in the regulation of the mitochondrial permeability transition pore during cell death, (II) the role of cyclophilin D in mitochondrial Ca 2+ signaling, (III) the regulation of the mitochondrial permeability transition pore by peripheral benzodiazepine receptor ligands.; Using live cell imaging and two photon microscopy, we report that overexpression of cyclophilin D desensitizes HEK293 cells and rat glioma C6 cells to apoptotic stimuli. By site directed mutagenesis of cyclophilin D that compromises peptidyl-prolyl cis-trans isomerase activity, we demonstrate that the mechanism involved in this protective effect requires peptidyl-prolyl cis-trans isomerase activity. Finally, using glutathione-S-transferase pulled down assays, we demonstrate that cyclophilin D binding to the adenine nucleotide translocator, which is considered to be the core component of the mitochondrial permeability transition pore, is not affected by the loss of peptidyl-prolyl cis-trans isomerase activity. Collectively, our data suggest that cyclophilin D should be viewed as a cell survival-signaling molecule and indicate a protective role of cyclophilin D against apoptosis that is mediated by target (s) other than the adenine nucleotide translocator.; We examine how cyclophilin D overexpression changes both mitochondrial transmembrane potential and cytosolic Ca2+ release. Our results show that overexpression of cyclophilin D can hyperpolarize the mitochondrial potential under resting conditions. The amplitude and half decay time (t _1/2) of IP₃ induced cytosolic Ca2+ waves is significantly decreased, consistent with an increased mitochondrial Ca 2+ uptake.; To further understand the functional role of peripheral benzodiazepine receptors on the mitochondrial permeability transition pore, we examined how PK11195 and Ro5-4864 affect the timing of mitochondrial potential collapse under conditions of oxidative stress. We found that both PK11195 and Ro5-4864 sensitize HEK293 cells to oxidative stress-induced potential collapse. In addition ATP induced strong mitochondrial Ca2+ uptake under oxidative stress. PK11195 significantly prolonged the rise time of the mitochondrial Ca2+. Finally under oxidative stress conditions, ATP-induced Ca2+ release sensitized HEK293 cells to PK11195 and Ro5-4864 treatment. (Abstract shortened by UMI.)...