Dissipation of potassium and proton gradients inhibits mitochondrial hyperpolarization and cytochrome c release during neural apoptosis

Exposure of rat hippocampal neurons or human D283 medulloblastoma cells to the apoptosis-inducing kinase inhibitor staurosporine induced rapid cytochr ome c release from mitochondria and activation of the executioner caspase-3 . Measurements of cellular tetramethylrhodamine ethyl ester fluorescence an d subsequent simulation of fluorescence changes based on Nernst calculation s of fluorescence in the extracellular, cytoplasmic, and mitochondrial comp artments revealed that the release of cytochrome c was preceded by mitochon drial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-x L, but not pharmacological blockade of outward potassium currents, inhibite d staurosporine-induced hyperpolarization and apoptosis. Dissipation of mit ochondrial potassium and proton gradients by valinomycin or carbonyl cyanid e p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine- induced hyperpolarization, cytochrome c release, and caspase activation. Th is effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se al so caused release of cytochrome c from mitochondria. In contrast to stauros porine, however, valinomycin-induced cytochrome c release and cell death we re not associated with caspase-3 activation and insensitive to Bcl-xL overe xpression. Our data suggest two distinct mechanisms for mitochondrial cytoc hrome c release: (1) active cytochrome c release associated with early mito chondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.