title: Calcium and its role in the nuclear translocation and activation of cytosolic phospholipase A2 in cells rendered sensitive to TNF-induced apoptosis by cycloheximide.
abstract: In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A(2) (cPLA(2)). 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 Ca(2+) 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 cPLA(2) 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 [(3)H]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 cPLA(2) activation by diverse apoptotic stimuli.
authors.affiliation: Department of Microbiology, North Carolina State University, Raleigh, NC 27606, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Draper
authors.forename: Virginia G
authors.initials: VG
authors.lastname: Harris
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Culver
authors.forename: Scott M
authors.initials: SM
authors.lastname: Laster
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000276:immunology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology*; D016718:Arachidonic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D003513:Cycloheximide / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D006868:Hydrolysis / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D010718:Phosphatidylserines / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D014316:Tritium; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D010718:Phosphatidylserines; D014409:Tumor Necrosis Factor-alpha; D014316:Tritium; D016718:Arachidonic Acid; D003513:Cycloheximide; D014700:Verapamil; D010741:Phospholipases A; D054467:Phospholipases A2; D020169:Caspases; D002118:Calcium