Dissipation of Potassium and Proton Gradients Inhibits Mitochondrial Hyperpolarization and Cytochrome c Release during Neural Apoptosis

Abstract

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.

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…TMRM has been widely used to monitor changes in ⌬⌿ m in models of injury (36,37,45,46,59). However, TMRM is also sensitive to alterations in ⌬⌿ p ; therefore care has to be taken in the interpretation of TMRM fluorescent signals.…”

Protein Kinase C-α and ERK1/2 Mediate Mitochondrial Dysfunction, Decreases in Active Na+ Transport, and Cisplatin-induced Apoptosis in Renal Cells

“…Recent studies suggest that the initiation of apoptotic events is not always associated with MPTP but, in some cases, with mitochondrial membrane hyperpolarization and mitochondrial shrinkage (46,47). Apoptosis caused by the withdrawal of growth factors or interlukin-3 from cultured cells is associated with cytosolic acidification and hyperpolarization of the inner mitochondrial membrane, which are dependent on ATP synthase activity (48,49).…”

“…Apoptosis caused by the withdrawal of growth factors or interlukin-3 from cultured cells is associated with cytosolic acidification and hyperpolarization of the inner mitochondrial membrane, which are dependent on ATP synthase activity (48,49). Furthermore, staurosporine-induced apoptosis in neuronal cells is associated with hyperpolarization of the mitochondrial inner membrane, which precedes the release of cytochrome c (46,50). Dissipation of mitochondrial K ϩ and H ϩ gradients inhibits staurosporine-induced cytochrome c release and attenuates apoptosis (46).…”

Suppression of Endothelial Cell Apoptosis by High Density Lipoproteins (HDL) and HDL-associated Lysosphingolipids

“…Subcellular fractionation was performed as described (18). Briefly, cells were homogenized using a glass Dounce (10 strokes) and were centrifuged at 15,000 ϫ g for 15 min at 4°C.…”

“…No such decrease was observed in endothelial cells preincubated with 1.0 g/L HDL at this time. To investigate the specificity of cytochrome c release during apoptosis we examined the effect of GF deprivation on VDAC, a membrane channel expressed in the mitochondrium as described previously (18). As shown in Fig.…”