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.

Fig. 1.

Staurosporine induces hyperpolarization of rat hippocampal neurons. a, Cultured rat hippocampal neurons exposed to STS for the indicated period of time were loaded with TMRE (100 nm, 15 min), and fluorescence was quantified. The TMRE uptake increased significantly at all time points measured. Data are means ± SEM from n = 27–150 neurons fromn = 5–20 separate experiments per time point. Different from controls *p < 0.05.b, STS-induced increase in TMRE fluorescence was not inhibited by blocking outward potassium currents. Hippocampal neurons were treated with 300 nm STS for 6 hr. Subsequently, cultures were exposed to HBS or HBS + TEA + clofilium tosylate (Clo) for a further 15 min. After a 10 min loading period, cellular TMRE fluorescence was acquired. Data were not expressed as an SD and therefore were presented in median/quartile form and represent values from n = 82–185 cells. *,^#p < 0.05 compared with respective controls.

Fig. 2.

Inhibition of cytochrome c release in rat hippocampal neurons by dissipation of mitochondrial potassium and proton gradients. Cultured rat hippocampal neurons were exposed to vehicle (a), 10 nm Val (b), 10 μm FCCP (c), 300 nm STS (d), Val + STS (e), or FCCP + STS (f) for 6 hr. Distribution of cytochrome c is shown by immunofluorescence analysis. Only cultures exposed to 300 nm STS showed a diffuse cytochrome c staining pattern (d). Scale bar, 50 μm.

Fig. 3.

Effect of oligomycin (a) and STS (b–e) on cellular TMRE uptake in D283 medulloblastoma cells and simulation of TMRE fluorescence changes according to Ward et al. (2000). The dotted linesrepresent the time lapse of total cellular fluorescence in percentage of baseline after equilibration with 10 nm(a–c) or 100 nm (d, e) TMRE. After equilibration of the TMRE signal, cells were exposed to 5 μm oligomycin (a) or 3 μm STS (b–e). The onset of treatment is indicated by the arrows on the left. After the respective treatments, mitochondria were depolarized by the addition of 2 μm FCCP plus 5 μm oligomycin (arrows on the right). Simulations based on the Nernst equation were calculated with immediate changes in ΔΨ_M (solid lines in a,b, and d; left arrow,hyperpolarization from −150 to −190 mV) or ΔΨ_P(solid lines in c and e;left arrow, hyperpolarization from −60 to −90 mV). In each case, this was followed by a simulation of mitochondrial depolarization after addition of FCCP plus oligomycin (right arrows, depolarization from −190 to 0 mV). Note that the time lapse of the oligomycin and STS treatments have the same shape. Simulations based on changes in ΔΨ_P are less convincing, and the peak after FCCP plus oligomycin in cells loaded with 100 nm TMRE in d does not occur ine (solid lines). Traces are means from all cells within the microscope field in a typical experiment (n = 18–26 cells). The experiments were performed in duplicate (10 nm TMRE experiments) and triplicate (100 nm TMRE experiment) with similar results.

Fig. 4.

Cytochrome c release induced by staurosporine and valinomycin in human medulloblastoma D283 cells. Cultures were incubated for the indicated periods of time with staurosporine (STS) (a), valinomycin (Val) (b), or the vehicle. Cells were fractionated into a cytosolic and a mitochondria-containing nuclear–heavy membrane fraction (Pellet). Immunoblot analysis was performed using an anti-cytochrome c antibody. Blots were incubated with an anti-VDAC antibody to exclude contaminations of the cytosolic fraction with mitochondria. An anti-α-tubulin antibody was used as loading control. The experiment was repeated three times with similar results. c, Immunoblot analysis of whole-cell lysates using an anti-cytochrome c and an anti-α-tubulin antibody after exposure to STS, Val, or vehicle for the indicated periods of time. The experiment was repeated twice with similar results.

Fig. 5.

Overexpression of Bcl-xL inhibits staurosporine-induced mitochondrial hyperpolarization and cytochrome c release. a–d, D283 cells were exposed to vehicle or STS. After 30 min exposure to STS, cytochrome c immunofluorescence remained mitochondrial (d), whereas CMXRos uptake increased significantly (b). Scale bar, 10 μm. Experiments were performed three times with comparable results.e–l, D283 medulloblastoma cells were stably transfected with pSFFV-Neo-Bcl-xL (D283/Bcl-xL) or empty plasmid pSFFV-Neo (D283/Neo). Overexpres- sion of Bcl-xL was confirmed by immunoblotting using an anti-Bcl-x antibody (f, inset).e, Quantification of CMXRos fluorescence in D283/Neo cultures confirmed an early increase in CMXRos uptake after the exposure to STS. In contrast, the increase of CMXRos uptake was inhibited in D283/Bcl-xL cells. Data are means ± SEM fromn = 8 cultures; *p < 0.05 with respect to control. Experiment was performed in triplicate and yielded comparable results. f, Quantification of TMRE uptake in D283/Neo cultures confirmed the early increase of fluorescence obtained with CMXRos. The increase of TMRE uptake was inhibited in D283/Bcl-xL cells. Data are means ± SEM from n = 8 cultures; *p < 0.05 with respect to control. Experiment was repeated twice with comparable results.g–j, Cytochrome c distribution was visualized by immunofluorescence analysis after 3 hr exposure to STS or vehicle. Note that the STS-induced cytochrome c release was significantly reduced in D283/Bcl-xL cells (j) compared with D283/SFFV cells (h). Scale bar, 10 μm (g). k, Control cells and Bcl-xL-overexpressing cells were treated with vehicle (Con) or STS for 4 and 6 hr. Caspase-3-like protease activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Activities are presented as increase in AMC fluorescence (in arbitrary fluorescence units) over 60 min per microgram of protein. Data are means ± SEM from n = 8 cultures. The experiment was repeated three times with similar results. Different from controls, *p < 0.05.l, Quantification of cell death after exposure to STS. D283 cells were incubated with STS or vehicle for up to 24 hr. Cells were stained simultaneously with 1 μm calcein AM and 2 μm ethidium homodimer (EthD-1). Live (green) and dead (red) cells were counted. n = 4 cultures (430–3000 cells) per time point. Different from controls, *p < 0.05.

Fig. 6.

Overexpression of Bcl-xL does not inhibit valinomycin-induced mitochondrial depolarization and cytochrome c release. a–d, Representative images of vehicle-treated (a, c) and Val-treated (b, d) D283 medulloblastoma cells fixed after 15 min of exposure. a,b, Changes of mitochondrial membrane potential were determined by CMXRos uptake. Note that the mitochondrial staining pattern was lost after treatment with Val (b).c, d, Distribution of cytochrome c was determined by immunofluorescence analysis. Cytochrome c immunofluorescence remained intact after exposure to Val (d). Scale bar, 10 μm. e, Quantification of cell death after exposure to Val. D283/Neo and D283/Bcl-xL cells were exposed to vehicle or Val for 12 and 24 hr and stained simultaneously with 1 μm calcein AM and 2 μm ethidium homodimer. The percentage of dead cells was determined. n = 4 cultures (600–3000 cells) per time point. Different from controls, *p < 0.05.n.s., Not statistically significant.f–m, Cells were treated with Val or vehicle for 6 hr. Cytochrome c distribution was visualized by immunofluorescence analysis. Val-induced cytochrome c release was not inhibited in Bcl-xL-overexpressing cells (l, arrowheads) compared with control cells (h). Mitochondria appeared swollen in control and Bcl-xL-overexpressing cells (h, l; arrows). Overexpression of Bcl-xL could also not inhibit loss of mitochondrial membrane potential (i, m). Scale bar (in f): f–m, 10 μm.n–p, High magnification of mitochondria and mitochondria-rich regions in vehicle- and Val-treated cultures (6 hr) stained with the cytochrome c antibody. Overexpression of Bcl-xL does not inhibit large-scale mitochondrial swelling induced by Val. Mitochondria of vehicle-treated D283/Neo and D283/Bcl-xL cells were indistinguishable, therefore only mitochondria of a D283/Bcl-xL cell are shown. Images were deconvoluted using No Neighbor Deblurring software, which applies the algorithm of Monck et al. (1992) to reduce image background haze attributable to light originating from unsharp areas of the specimen. Scale bar (in n): n–p; 5 μm. N, Nucleus. All experiments were repeated twice with comparable results. q–r, Images of mitochondria in vehicle- and Val-treated (6 hr) rat primary astrocytes stained with the anti-cytochrome c antibody. Images were deconvoluted using the above-mentioned software. Scale bar, 5 μm (q).

Fig. 7.

Valinomycin does not activate neural apoptosis.a, D283 medulloblastoma cells were treated with vehicle (Con), Val, or STS for up to 24 hr. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Activities are presented as increase in AMC fluorescence (in arbitrary fluorescence units) over 60 min per microgram of protein. Data are means ± SEM from n = 8 cultures, and experiments were repeated three times with similar results. Different from controls, *p < 0.05. b, Immunoblot probed with an antibody recognizing procaspase-3 and active caspase-3. Cultures were exposed to vehicle (Con), Val, and STS for 6 hr. Experiment was repeated twice with similar results.c–e, Hoechst staining of D283 cells treated with vehicle (c) or Val (d) for 20 hr. Exposure to Val induced no chromatin condensation in contrast to cells treated with STS for 6 hr (e). Scale bar, 50 μm.

Fig. 8.

Valinomycin inhibits staurosporine-induced hyperpolarization and cytochrome c release. a–d, Cultures were exposed for 30 min to vehicle (Con) (a), STS (b), Val (c), or to a combination of STS and Val (d), loaded with 2 μm R123 for 30 min and washed with HBS. STS induced an increased uptake of R123 into mitochondria (b), whereas treatment with Val decreased R123 uptake into mitochondria (c). Combined exposure to STS and Val reduced the STS-induced increase in R123 uptake (d). Scale bar, 25 μm. Experiments were repeated four times with comparable results. e, Cultures were incubated for 4 hr with vehicle, Val, STS, or a combination of Val and STS. Cells were fractionated into a cytosolic and a mitochondria-containing nuclear–heavy membrane fraction (Pellet). Immunoblot analysis of cytosolic fractions was performed using an anti-cytochrome c antibody. Blots were incubated with an anti-VDAC antibody to exclude contaminations of the cytosolic fractions with mitochondria and with an anti-α-tubulin antibody to confirm equal loading of each sample. Control pellet of vehicle-treated cells is shown. The experiment was performed in triplicate and yielded comparable results.

Fig. 9.

Valinomycin induces mitochondrial swelling and inhibits staurosporine induced cytochrome c release in MCF-7/Casp-3 cells stably transfected with cytochrome c–EGFP. Cultures were treated with vehicle (Con; a,e), STS (b, f), Val (c,g), and STS and Val in combination (d, h). Digital images were acquired before (a–d) and 6 hr after (e–h) treatment. Exposure to STS induced a significant cytochrome c release resulting in a diffuse staining of cytoplasm and nucleus (f). Val alone induced cytochrome c release (arrowheads), as well as mitochondrial swelling (arrows) (g). Exposure to Val inhibited STS-induced cytochrome c release (h). Scale bar, 10 μm. Experiments were repeated six times with similar results.

Fig. 10.

Valinomycin reduces staurosporine-induced increase in caspase-3 like protease activity and apoptotic nuclear morphology. a, Apoptotic cell death was induced in D283 cells by an exposure to STS for 6 hr. Val or vehicle was added as indicated. Caspase-3 like protease activity was measured by cleavage of the Ac-DEVD-AMC substrate. Activities are represented as increase in AMC fluorescence over 60 min per microgram of protein. Val significantly reduced STS-induced Ac-DEVD-AMC cleavage. Data are means from n = 8 cultures; experiment was repeated twice with comparable results, *p < 0.05 different from controls.b, Immunoblot analysis of cultures treated with STS, Val, STS + Val or vehicle for 6 hr using an anti-caspase-3 antibody. Addition of Val to cultures treated with STS reduced cleavage of pro-caspase-3 compared with cultures treated with STS alone. Experiment was repeated twice with similar results. c, D283 cells were exposed to STS for 4 hr. Morphology of nuclei was visualized by Hoechst 33258 staining. Percentage of nuclei with fragmented chromatin was determined. n = 4 cultures (700–800 nuclei). Different from controls, *p < 0.05.d, Cell death was induced by exposure to STS for 6 hr. Vehicle or FCCP were added as indicated. Caspase-3 like activity was measured by cleavage of the Ac-DEVD-AMC substrate. Data are means from n = 8 cultures; experiments were done in duplicate and yielded similar results, *p < 0.05 different from controls. e, Determination of cellular ATP content in D283 cells exposed to vehicle, STS, Val, or STS + Val for 3 and 6 hr. Luciferase activity of cultures treated with vehicle was set to 100% activity. Treatment with STS, Val, and STS + Val resulted in a decrease in luciferase activity. Data are means fromn = 4–6 cultures per treatment.