| The goal of this study was to elucidate the mechanism(s) of cytochrome c release from mitochondria during apoptosis. We found that during etoposide-induced apoptosis in HL-60 cells, cytochrome c release, which generally occurred about 2 hours after addition of etoposide, was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition (MPT) was also observed; however, it was not the primary cause of cytochrome c release. While the MPT inhibitor cyclosporin A had little effect on cytochrome c release, caspase 3 activation, and DNA laddering, it did prevent permeabilization of the inner membrane.; Uptake of potassium and mitochondrial swelling during apoptosis were blocked by bcl-2 overexpression, resulting in a very large reduction in cytochrome c release and a major delay in the development of the characteristics of apoptosis. Bcl-2 was found to both inhibit the increased potassium uptake during apoptosis and to upregulate mitochondrial potassium efflux through the potassium/proton exchanger, thereby preventing potassium accumulation, swelling, and cytochrome c release. Direct activation of the potassium uniporter with diazoxide was also found to be sufficient to release cytochrome c and to trigger apoptosis. This could be inhibited, not only by the inhibitor of the potassium uniporter, 5-hydroxydecanoate, but also by bcl-2. Residual cytochrome c release in the absence of swelling in bcl-2 overexpressing cells could be due to Bax translocation into mitochondria which was particularly observed during the period 2 to 4 hours after etoposide addition.; This study suggests several novel aspects of apoptotic signaling: (1) Potassium related swelling of mitochondria during apoptosis; (2) Regulation of both potassium uptake and exchange by bcl-2; (3) The co-existence within one system of multiple mechanisms of cytochrome c release: potassium- and MPT-driven mitochondrial swelling and also swelling-independent release, possibly through Bax-mediated permeabilization of the outer mitochondrial membrane. This latter observation reflects multiple possible pathways leading to the same endpoint—the full expression of apoptosis. |
