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Calcium and Its Role in the Nuclear Translocation and Activation of Cytosolic Phospholipase A₂ in Cells Rendered Sensitive to TNF-Induced Apoptosis by Cycloheximide ¹

David W. Draper^*, Virginia G. Harris, Carolyn A. Culver^* and Scott M. Laster^2,*

^* Department of Microbiology, North Carolina State University, Raleigh, NC 27606; and Science Applications International, Alexandria, VA 22314

In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A₂ (cPLA₂). As our model, we used a murine fibroblast cell line (C3HA) that was induced to undergo apoptosis by a combination of TNF and cycloheximide. Using fura 2 Ca²⁺ imaging, we found strong evidence for an intracellular calcium response after 1 h of treatment, which correlated with the onset of phosphatidylserine externalization, but preceded effector procaspase processing by several hours. The response was strongest in the perinuclear region, where mean levels rose 83% (144 ± 14 nM in untreated cells vs 264 ± 39 nM in treated), while cells displaying morphological evidence of apoptosis had the highest levels of calcium (250–1000 nM). Verapamil blocked this response, indicating an extracellular source for the calcium. Fluorescence microscopy revealed a pattern of nuclear translocation of cPLA₂ during apoptosis, which was also blocked by verapamil, indicating an important role for calcium in this process. In addition, we found that verapamil prevented the release of [³H]arachidonic acid from C3HA cells induced to undergo apoptosis by the chemotherapeutic agents vinblastine, melphalan, and cis-platinum. Together, these data suggest that calcium is important for cPLA₂ activation by diverse apoptotic stimuli.

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