Cyanide induces apoptosis through cytochrome c activated caspase cascade in primary cultured cortical neurons. The underlying mechanism for cytochrome c release from mitochondria after cyanide treatment is still unclear. In this study, the roles of endogenous Bcl-2 proteins in cyanide-induced apoptosis were investigated. After cyanide (100-500 lM) treatment for 24 h, two pro-apoptotic Bcl-2 proteins, Bcl-X S and Bax were up-regulated as shown by western blot and RT-PCR analysis. The expression levels of two antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-X L , remained unchanged after cyanide treatment, whereas the mRNA levels of Bcl-X S and Bax began to increase within 2 h and their protein levels increased 6 h after treatment. NF-jB, a redox-sensitive transcription factor activated after cyanide treatment, is responsible for the up-regulation of Bcl-X S and Bax. SN50, which is a synthetic peptide that blocks translocation of NF-jB from cytosol to nucleus, inhibited the up-regulation of Bcl-X S and Bax. Similar results were obtained using a specific jB decoy DNA. NMDA receptor activation and reactive oxygen species (ROS) generation are upstream events of NF-jB activation, as blockade of these two events by MK801, L-NAME or PBN inhibited cyanide-induced up-regulation of Bcl-X S and Bax. Up-regulation of pro-apoptotic Bcl-X S and Bax contributed to cyanide-induced cytochrome c release, because SN50 and a specific Bax antisense oligodeoxynucleotide significantly reduced release of cytochrome c from mitochondria as shown by western blot analysis. It was concluded that NF-jB-mediated up-regulation of Bcl-X S and Bax is involved in regulating cytochrome c release in cyanideinduced apoptosis.

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In cyanide-induced apoptosis, an increase in cytosolic free Ca 2+ and generation of reactive oxygen species are initiation stimuli for apoptotic cell death. Previous studies have shown that cyanidestimulated 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 Ca 2+ to activation of Bad (Bcl-2/Bcl-X L -antagonist, causing cell death) was determined in cortical cells. Bad is a Ca 2+ -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 Ca 2+ -sensitive increase in cellular calcineurin activity was observed. Increased cytosolic Ca 2+ and calcineurin activation stimulated Bad translocation since BAPTA [bis-(o-aminophenoxy)ethane-N,N,N ,N -tetra-acetic acid], an intracellular Ca 2+ 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 Ca 2+ , cyanide can partially initiate the apoptotic cascade through a calcineurin-mediated translocation of Bad to mitochondria.

Execution of cyanide-induced apoptosis is mediated by release of cytochrome c from mitochondria. To determine how cyanide initiates cytochrome c release, Bax translocation was investigated in primary cultures of cortical neurons. Under nonapoptotic (control) conditions, Bax resided predominantly in the cytoplasm. After 300-microM cyanide treatment for 1 h, Bax translocated to the mitochondria, as shown by immunocytochemical staining and subcellular fractionation; Western blot analysis confirmed "cytosol-to-mitochondria" translocation of Bax. Temporal analysis showed that Bax translocation preceded cytochrome c release from the mitochondria, which was initiated 3 h after cyanide treatment. In double-immunofluorescence labeling for both Bax and cytochrome c, it was observed that cytochrome c was released only in cells showing Bax in mitochondria. The role of p38 mitogen-activated protein (MAP) kinase in Bax translocation was studied. The p38 MAP kinase was activated 30 min after cyanide, and its phosphorylation level of activity began to decrease 3 h later. SB203580, a p38 MAP kinase inhibitor, blocked translocation of Bax to mitochondria, whereas SB202474, a control peptide, had no effect on translocation. Inhibition of p38 MAP kinase by SB203580 blocked all downstream effects of Bax translocation, including cytochrome c release, caspase activation, and internucleosomal DNA fragmentation. These results demonstrated that Bax translocation is critical for cyanide-induced cytochrome c release and that p38 MAP kinase regulates Bax translocation from cytosol to mitochondria.

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