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  • Title: Calcium flux measurements in apoptosis.
    Author: McConkey DJ, Nutt LK.
    Journal: Methods Cell Biol; 2001; 66():229-46. PubMed ID: 11396005.
    Abstract:
    Early studies in apoptosis implicated an increase in cytosolic Ca2+ as a direct mediator of DNA fragmentation. However, efforts to delineate targets for this increase in Ca2+ have been slow in evolving. Several previous studies have implicated ER Ca2+ pool depletion in the initiation of apoptosis. Our own preliminary studies confirm that many (but not all) apoptotic stimuli empty the ER store via a mechanism that is blocked by BCL-2 expression. Furthermore, ER pool depletion is not affected by broad spectrum caspase inhibitors, indicating that it occurs via a caspase-independent mechanism. Finally, our data demonstrate that ER pool depletion occurs prior to release of cytochrome c from mitochondria. Given previous work demonstrating close coordination of ER and mitochondrial Ca2+ levels, we speculate that ER-dependent changes in mitochondrial Ca2+ serve as important signals for cytochrome c release. Alternative mechanisms include activation of caspase-12 and/or the JNK pathway, both of which can be directly stimulated by depletion of the ER Ca2+ pool. Although substantial improvements in intracellular Ca2+ imaging have emerged, compelling answers to many of the present questions related to the role of Ca2+ in apoptosis await future technical improvements. The development of organelle-specific, recombinant Ca2+ probes (targeted aequorins and cameleons) certainly should facilitate some of this work, although the target cell of interest must be amenable to molecular manipulation (transfection), which precludes straightforward analysis of primary cells. Pharmacological tools (i.e., thapsigargin and DBHQ) can provide conclusive data on ER pool status without requiring an overly sophisticated image analysis system. However, confocal microscopy allows for the effective analysis of Ca2+ pools as long as dye localization is homogeneous and properly controlled. However, current techniques should be considered semiquantitative at best and will remain so until specific organelle-targeted fluorescent dyes are developed and widely available.
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