Mitochondria have received considerable attention recently with regard to their involvement in apoptosis. Interest has centered around the opening of mitochondrial permeability transition pores (PTP) and the release of proapoptotic factors. It has been shown that cytochrome c (cyto c) released from mitochondria into the cytosol is sufficient to initiate caspase activation. One of the major regulators of the PTP is mitochondrial matrix [Ca2+] ([Ca2+]m). Importantly, large increases of [Ca+]m are associated with cytosolic [Ca2+] spikes and oscillations evoked by IP3-linked agonists but, in health, these [Ca2+]m increases are used to control mitochondrial metabolism via activation of C2+-sensitive dehydrogenases rather than to open PTP. Our principal hypothesis is that in cells exposed to apoptotic stimuli such as TNF-alpha, ceramide, mitochondrial [Ca2+] signals evoked by IP3-linked cytosolic [Ca2+] oscillations trigger mitochondrial permeability transition and, in turn, cytochrome c release. We suggest that PTP closes after the decay of the Ca2+ spikes allowing mitochondrial metabolism to recover. This organization may provide an efficient mechanism to establish caspase activation and to allow mitochondrial metabolism to meet ATP requirements of apoptotic cell death. In order to determine the effects of IP3-linked cytosolic Ca2+ signals on the mitochondria and to dissect the underlying mechanism, we propose to utilize a hepatoma-derived cell line (HepG2 cells) as an experimental model and to measure dynamics of mitochondrial Ca2+ movements simultaneously with mitochondrial membrane potential responses or subcellular distribution of green fluorescent protein (GFP) fusion proteins (eg. cyto c-GFP) using fluorescence imaging approaches, including some novel techniques. Our preliminary studies show that [Ca2+] signals evoked by addition of large Ca2+ pulses or by IP3-mediated cytosolic [Ca2+] spikes trigger opening of PTP and in turn, cyto c release in ceramide treated cells. After the decay of Ca2+ spikes resealing of PTP and recovery of electron transport chain activity occur, whereas activation of caspases and in turn nuclear DNA fragmentation are triggered by cyto c released to the cytosol. Understanding the role calcium signaling plays in the process of apoptosis and dissecting the underlying mechanism at the molecular level not only affords insights into disease pathogenesis but may also open new avenues for developing diagnostic, prognostic and therapeutic tools.
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