The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition

Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpresion of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA- preventable loss of the mitochondrial membrane potential (ΔΨ(m)). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP- ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of ΔΨ(m) and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore

The overexpression of Bax kills cells by a mechanism that depends on induction of the mitochondrial permeability transition (MPT) (Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol Chem. 273, 7770-7775). In the present study, purified, recombinant Bax opened the mitochondrial permeability transition pore (PTP). Depending on its concentration, Bax had two distinct effects. Al a concentration of 125 nM, Bax caused the release of the intermembranous proteins cytochrome c and adenylate kinase and the release from the matrix of sequestered calcein, effects prevented by the inhibitor of the PTP cyclosporin A (CSA), AL this concentration of Bax, there was no detectable mitochondrial swelling or depolarization. These effects of low Bax concentrations are interpreted as the consequence of transient, non-synchronous activation of the PTP followed by a prompt recovery of mitochondrial integrity, By contrast, Bax concentrations between 250 nM and 1 mu M caused a sustained opening of the PTP with consequent persistent mitochondrial swelling and deenergization (the MPT). CSA prevented the RIFT induced by fax, Increasing concentrations of calcium caused a greater proportion of the mitochondrial to undergo the MPT in the presence of Bax. Importantly, two known mediators of apoptosis, ceramide and GD3 ganglioside, potentiated the induction by Bax of the MPT, The data imply that Bax mediates the opening of the mitochondrial PTP with the resultant release of cytochrome c from the intermembranous space