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-XL-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.
Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis
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Yan SHOU, Li LI, Krishnan PRABHAKARAN, Joseph L. BOROWITZ, Gary E. ISOM; Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis. Biochem J 1 May 2004; 379 (3): 805–813. doi: https://doi.org/10.1042/bj20031107
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