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 cytochrome c release from mitochondria and activation of the executioner caspase-3. Measurements of cellular tetramethylrhodamine ethyl ester fluorescence and subsequent simulation of fluorescence changes based on Nernst calculations of fluorescence in the extracellular, cytoplasmic, and mitochondrial compartments revealed that the release of cytochrome c was preceded by mitochondrial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-xL, but not pharmacological blockade of outward potassium currents, inhibited staurosporine-induced hyperpolarization and apoptosis. Dissipation of mitochondrial potassium and proton gradients by valinomycin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine-induced hyperpolarization, cytochrome c release, and caspase activation. This effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se also caused release of cytochrome c from mitochondria. In contrast to staurosporine, however, valinomycin-induced cytochrome c release and cell death were not associated with caspase-3 activation and insensitive to Bcl-xL overexpression. Our data suggest two distinct mechanisms for mitochondrial cytochrome c release: (1) active cytochrome c release associated with early mitochondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.
Poppe
M
M
Interdisciplinary Center for Clinical Research, Research Group "Apoptosis and Cell Death", Westphalian Wilhelms-University, D-48149 Münster, Germany.
Reimertz
C
C
Düssmann
H
H
Krohn
A J
AJ
Luetjens
C M
CM
Böckelmann
D
D
Nieminen
A L
AL
Kögel
D
D
Prehn
J H
JH
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D007476:Ionophores / Q000494:pharmacology; D008527:Medulloblastoma / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D011522:Protons; D051381:Rats; D011916:Rats, Inbred F344; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014634:Valinomycin / Q000494:pharmacology; D051020:bcl-X Protein
C494813:Bcl2l1 protein, rat; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; D019253:Proto-Oncogene Proteins c-bcl-2; D011522:Protons; D051020:bcl-X Protein; D014634:Valinomycin; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine; D011188:Potassium