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Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria.

Zhdanov AV, Dmitriev RI, Papkovsky DB - Cell. Mol. Life Sci. (2010)

Bottom Line: This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm.Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm.Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Republic of Ireland. a.zhdanov@ucc.ie

ABSTRACT
Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca(2+) and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 ((d)PC12), (d)SH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm. The response to Baf is regulated by cytosolic Ca(2+) fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.

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The effect of Baf on the respiration of dPC12 cells is attributed to mitochondrial uncoupling. a As a result of sustained activation of respiration by Baf (0.25 μM), cellular O2 levels steadily decrease for 60–80 min and then remain elevated for hours. b Dose-dependent decrease in cellular O2 measured 45 min after Baf application reveals that the respiratory response reaches a maximum at 0.5–0.8 μM Baf. c Cell deoxygenation induced by Baf increases when glucose in the medium is replaced with galactose. In contrast, 0.25 μM CMA has no effect. d Complete dissipation of the acidic compartments after 30 min treatment with 0.25 μM Baf is confirmed by LysoTracker Red staining. e The intensity of the ΔΨm-sensitive probe TMRM decreases by 30–50% when the cells are incubated with 0.25 μM Baf for 30 min. f In the cells treated with Baf or CMA for 4 h under starving conditions the level of LC3 II degradation decreases due to inhibition of autophagy. DMSO was used as a negative control. In d and e representative live cell confocal images are shown. Bar 20 μm; asterisks significant differences
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Fig1: The effect of Baf on the respiration of dPC12 cells is attributed to mitochondrial uncoupling. a As a result of sustained activation of respiration by Baf (0.25 μM), cellular O2 levels steadily decrease for 60–80 min and then remain elevated for hours. b Dose-dependent decrease in cellular O2 measured 45 min after Baf application reveals that the respiratory response reaches a maximum at 0.5–0.8 μM Baf. c Cell deoxygenation induced by Baf increases when glucose in the medium is replaced with galactose. In contrast, 0.25 μM CMA has no effect. d Complete dissipation of the acidic compartments after 30 min treatment with 0.25 μM Baf is confirmed by LysoTracker Red staining. e The intensity of the ΔΨm-sensitive probe TMRM decreases by 30–50% when the cells are incubated with 0.25 μM Baf for 30 min. f In the cells treated with Baf or CMA for 4 h under starving conditions the level of LC3 II degradation decreases due to inhibition of autophagy. DMSO was used as a negative control. In d and e representative live cell confocal images are shown. Bar 20 μm; asterisks significant differences

Mentions: Although uncoupling of isolated mitochondria by Baf has been described [23], its effects on cell respiration have not been investigated. Using the intracellular O2 sensing technique [29], we found that exposure of dPC12 cells to 0.25 μM Baf gradually decreased the oxygenation of the cell monolayer from 140–160 μM down to 80±5 μM O2 (Fig. 1a) due to increased respiration. Maximal respiration was achieved in 50–100 min, and then it gradually decreased but still remained elevated after >5 h. The effect was dose-dependent, becoming significant at 50 nM and reaching a maximum at 0.5–0.8 μM Baf after 45 min of treatment (Fig. 1b). In the presence of mitochondrial complex III inhibitor antimycin A the effect was abolished (not shown). If Baf was subsequently removed from the medium, respiration returned to the basal level.Fig. 1


Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria.

Zhdanov AV, Dmitriev RI, Papkovsky DB - Cell. Mol. Life Sci. (2010)

The effect of Baf on the respiration of dPC12 cells is attributed to mitochondrial uncoupling. a As a result of sustained activation of respiration by Baf (0.25 μM), cellular O2 levels steadily decrease for 60–80 min and then remain elevated for hours. b Dose-dependent decrease in cellular O2 measured 45 min after Baf application reveals that the respiratory response reaches a maximum at 0.5–0.8 μM Baf. c Cell deoxygenation induced by Baf increases when glucose in the medium is replaced with galactose. In contrast, 0.25 μM CMA has no effect. d Complete dissipation of the acidic compartments after 30 min treatment with 0.25 μM Baf is confirmed by LysoTracker Red staining. e The intensity of the ΔΨm-sensitive probe TMRM decreases by 30–50% when the cells are incubated with 0.25 μM Baf for 30 min. f In the cells treated with Baf or CMA for 4 h under starving conditions the level of LC3 II degradation decreases due to inhibition of autophagy. DMSO was used as a negative control. In d and e representative live cell confocal images are shown. Bar 20 μm; asterisks significant differences
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Related In: Results  -  Collection

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Fig1: The effect of Baf on the respiration of dPC12 cells is attributed to mitochondrial uncoupling. a As a result of sustained activation of respiration by Baf (0.25 μM), cellular O2 levels steadily decrease for 60–80 min and then remain elevated for hours. b Dose-dependent decrease in cellular O2 measured 45 min after Baf application reveals that the respiratory response reaches a maximum at 0.5–0.8 μM Baf. c Cell deoxygenation induced by Baf increases when glucose in the medium is replaced with galactose. In contrast, 0.25 μM CMA has no effect. d Complete dissipation of the acidic compartments after 30 min treatment with 0.25 μM Baf is confirmed by LysoTracker Red staining. e The intensity of the ΔΨm-sensitive probe TMRM decreases by 30–50% when the cells are incubated with 0.25 μM Baf for 30 min. f In the cells treated with Baf or CMA for 4 h under starving conditions the level of LC3 II degradation decreases due to inhibition of autophagy. DMSO was used as a negative control. In d and e representative live cell confocal images are shown. Bar 20 μm; asterisks significant differences
Mentions: Although uncoupling of isolated mitochondria by Baf has been described [23], its effects on cell respiration have not been investigated. Using the intracellular O2 sensing technique [29], we found that exposure of dPC12 cells to 0.25 μM Baf gradually decreased the oxygenation of the cell monolayer from 140–160 μM down to 80±5 μM O2 (Fig. 1a) due to increased respiration. Maximal respiration was achieved in 50–100 min, and then it gradually decreased but still remained elevated after >5 h. The effect was dose-dependent, becoming significant at 50 nM and reaching a maximum at 0.5–0.8 μM Baf after 45 min of treatment (Fig. 1b). In the presence of mitochondrial complex III inhibitor antimycin A the effect was abolished (not shown). If Baf was subsequently removed from the medium, respiration returned to the basal level.Fig. 1

Bottom Line: This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm.Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm.Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Republic of Ireland. a.zhdanov@ucc.ie

ABSTRACT
Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca(2+) and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 ((d)PC12), (d)SH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm. The response to Baf is regulated by cytosolic Ca(2+) fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.

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