title: Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis.
abstract: In cyanide-induced apoptosis, an increase in cytosolic free Ca2+ and generation of reactive oxygen species are initiation stimuli for apoptotic cell death. Previous studies have shown that cyanide-stimulated translocation of Bax (Bcl-associated X protein) to mitochondria is linked with release of cytochrome c and subsequent activation of a caspase cascade [Shou, Li, Prabhakaran, Borowitz and Isom (2003) Toxicol. Sci. 75, 99-107]. In the present study, the relationship of the cyanide-induced increase in cytosolic free Ca2+ to activation of Bad ( Bcl-2/Bcl-X(L)- antagonist, causing cell death) was determined in cortical cells. Bad is a Ca2+-sensitive pro-apoptotic Bcl-2 protein, which on activation translocates from cytosol to mitochondria to initiate cytochrome c release. In cultured primary cortical cells, cyanide produced a concentration- and time-dependent translocation of Bad from cytosol to mitochondria. Translocation occurred early in the apoptotic response, since mitochondrial Bad was detected within 1 h of cyanide treatment. Mitochondrial levels of the protein continued to increase up to 12 h post-cyanide exposure. Concurrent with Bad translocation, a Ca2+-sensitive increase in cellular calcineurin activity was observed. Increased cytosolic Ca2+ and calcineurin activation stimulated Bad translocation since BAPTA [bis-(o-aminophenoxy)ethane-N, N, N', N'-tetra-acetic acid], an intracellular Ca2+ chelator, and cyclosporin A, a calcineurin inhibitor, significantly reduced translocation. BAPTA also blocked release of cytochrome c from mitochondria as well as apoptosis. Furthermore, treatment of cells with the calcineurin inhibitors cyclosporin A or FK506 blocked the apoptotic response, linking calcineurin activation and the subsequent translocation of Bad to cell death. These observations show that by inducing a rapid increase in cytosolic free Ca2+, cyanide can partially initiate the apoptotic cascade through a calcineurin-mediated translocation of Bad to mitochondria.
authors.affiliation: Neurotoxicology Laboratory, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shou
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Krishnan
authors.initials: K
authors.lastname: Prabhakaran
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Borowitz
authors.forename: Gary E
authors.initials: GE
authors.lastname: Isom
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000378:metabolism / Q000494:pharmacology; D003486:Cyanides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D051025:bcl-Associated Death Protein; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: C494821:Bad protein, rat; D065095:Calcineurin Inhibitors; D002352:Carrier Proteins; D002614:Chelating Agents; D003486:Cyanides; D016328:NF-kappa B; D051025:bcl-Associated Death Protein; D045304:Cytochromes c; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019703:Calcineurin; D002118:Calcium