To live or to die? This crucial question eloquently reflects the dual role of Ca2+ in living organisms--survival factor or ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondriagenerated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of "free" cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.

During ischemic brain injury, glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca2+ overload and neuronal cell death. Here we show that glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells. During and shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. In these cells, mitochondrial membrane potential collapsed, nuclei swelled, and intracellular debris were scattered in the incubation medium. Neurons surviving the early necrotic phase recovered mitochondrial potential and energy levels. Later, they underwent apoptosis, as shown by the formation of apoptotic nuclei and by chromatin degradation into high and low molecular weight fragments. These results suggest that mitochondrial function is a critical factor that determines the mode of neuronal death in excitotoxicity.

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In addition to the established role of the mitochondria in energy metabolism, regulation of cell death has emerged as a second major function of these organelles. This seems to be intimately linked to their generation of reactive oxygen species (ROS), which have been implicated in mtDNA mutations, aging, and cell death. Mitochondrial regulation of apoptosis occurs by mechanisms, which have been conserved through evolution. Thus, many lethal agents target the mitochondria and cause release of cytochrome c and other pro-apoptotic proteins into the cytoplasm. Cytochrome c release is initiated by the dissociation of the hemoprotein from its binding to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and increases the level of soluble cytochrome c in the intermembrane space. Subsequent release of the hemoprotein occurs by pore formation mediated by pro-apoptotic Bcl-2 family proteins, or by Ca(2+) and ROS-triggered mitochondrial permeability transition, although the latter pathway might be more closely associated with necrosis. Taken together, these findings have placed the mitochondria in the focus of current cell death research.

Cytochrome c is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome c release requires a two-step process. Because cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome c involves a breaching of the electrostatic and͞or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome c is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome c with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome c release is significantly depressed when the electrostatic interaction between cytochrome c and cardiolipin remains intact. Our results indicate that the release of cytochrome c involves a distinct two-step process that is undermined when either step is compromised.

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The induction of apoptosis in Jurkat T-lymphocytes with 50 μΜ hydrogen peroxide was associated with caspase activation. Caspase activity was first detected 3 h after treatment, and the morphological features of apoptosis were apparent by 6 h. At higher concentrations of hydrogen peroxide there was no detectable caspase activity, and the cells died by necrosis. Cells treated with hydrogen peroxide were impaired in their ability to undergo Fas-mediated apoptosis. This appeared to be the result of direct inhibition of the cysteine-dependent caspases. The cells were able to recover and undergo apoptosis at later times. Therefore, hydrogen peroxide has two distinct effects. It initially inhibits the caspases and delays apoptosis. Then, depending on the degree of the initial oxidative stress, the caspases are activated and the cells die by apoptosis, or they remain inactive and necrosis occurs. We discuss the physiological implications of cells having to maintain a reducing environment during apoptosis to allow the caspases to function.