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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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Baf reduces ΔpHm and ΔΨm in dPC12 cells. a Inhibition of the F0F1 ATPase with oligomycin (10 μM) decreases Baf-specific cell deoxygenation by 10–20%. b Oligomycin treatment significantly elevates the fluorescence of the mtAlpHi probe in control cells, indicating a sustained matrix alkalinization. In Baf(+) cells the increase in ΔpH is lower and not sustainable. c TMRM profiles show a gradual decrease in probe fluorescence in Baf(+) cells upon addition of oligomycin. d Uncoupling with 0.5 μM FCCP causes a drop in TMRM and mtAlpHi fluorescence in Baf(+) cells, demonstrating a decrease in the ΔΨm and ΔpH, respectively (F0/F represents the ratio of fluorescence before and 3 min after FCCP addition). The decrease in mtAlpHi signal does not depend on Baf treatment; in contrast, the relative drop in TMRM signal in Baf(+) cells is significantly smaller than in the control. In all experiments except that shown in a, cells were preincubated with 0.25 μM Baf for 45 min prior to the addition of oligomycin or FCCP. DMSO was used as a negative control. Bars 20 μm; asterisks significant differences
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Fig4: Baf reduces ΔpHm and ΔΨm in dPC12 cells. a Inhibition of the F0F1 ATPase with oligomycin (10 μM) decreases Baf-specific cell deoxygenation by 10–20%. b Oligomycin treatment significantly elevates the fluorescence of the mtAlpHi probe in control cells, indicating a sustained matrix alkalinization. In Baf(+) cells the increase in ΔpH is lower and not sustainable. c TMRM profiles show a gradual decrease in probe fluorescence in Baf(+) cells upon addition of oligomycin. d Uncoupling with 0.5 μM FCCP causes a drop in TMRM and mtAlpHi fluorescence in Baf(+) cells, demonstrating a decrease in the ΔΨm and ΔpH, respectively (F0/F represents the ratio of fluorescence before and 3 min after FCCP addition). The decrease in mtAlpHi signal does not depend on Baf treatment; in contrast, the relative drop in TMRM signal in Baf(+) cells is significantly smaller than in the control. In all experiments except that shown in a, cells were preincubated with 0.25 μM Baf for 45 min prior to the addition of oligomycin or FCCP. DMSO was used as a negative control. Bars 20 μm; asterisks significant differences

Mentions: To confirm this, we analysed the changes in respiration, ΔpHm and ΔΨm, induced in Baf(+) dPC12 cells by oligomycin. Normally, inhibition of ATP synthase (the main consumer of the Η+ gradient across the mitochondrial membrane) reduces O2 consumption and leads to a rapid and sustained increase in ΔpHm [45]. We observed a 10–20% decrease in the respiratory response to Baf in the cells pretreated with oligomycin (Fig. 4a). Using the mitochondrial pH probe mtAlpHi [33], we found that oligomycin transiently elevated mitochondrial pH in Baf(+) cells (Fig. 4b). Being significantly smaller than in the control, this increase suggests that complexes I–IV of the electron transport chain (ETC) generate notable H+ gradients it the mitochondria uncoupled by Baf. This result also shows that in the presence of Baf, ATP synthase still works in direct mode utilising H+ gradients for ATP production. On the other hand, after oligomycin addition, the intensity of TMRM in Baf(+) cells gradually decreased (Fig. 4c), indicating that the complex V also participates in the maintenance of ΔΨm acting as an ATPase.Fig. 4


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)

Baf reduces ΔpHm and ΔΨm in dPC12 cells. a Inhibition of the F0F1 ATPase with oligomycin (10 μM) decreases Baf-specific cell deoxygenation by 10–20%. b Oligomycin treatment significantly elevates the fluorescence of the mtAlpHi probe in control cells, indicating a sustained matrix alkalinization. In Baf(+) cells the increase in ΔpH is lower and not sustainable. c TMRM profiles show a gradual decrease in probe fluorescence in Baf(+) cells upon addition of oligomycin. d Uncoupling with 0.5 μM FCCP causes a drop in TMRM and mtAlpHi fluorescence in Baf(+) cells, demonstrating a decrease in the ΔΨm and ΔpH, respectively (F0/F represents the ratio of fluorescence before and 3 min after FCCP addition). The decrease in mtAlpHi signal does not depend on Baf treatment; in contrast, the relative drop in TMRM signal in Baf(+) cells is significantly smaller than in the control. In all experiments except that shown in a, cells were preincubated with 0.25 μM Baf for 45 min prior to the addition of oligomycin or FCCP. DMSO was used as a negative control. Bars 20 μm; asterisks significant differences
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Fig4: Baf reduces ΔpHm and ΔΨm in dPC12 cells. a Inhibition of the F0F1 ATPase with oligomycin (10 μM) decreases Baf-specific cell deoxygenation by 10–20%. b Oligomycin treatment significantly elevates the fluorescence of the mtAlpHi probe in control cells, indicating a sustained matrix alkalinization. In Baf(+) cells the increase in ΔpH is lower and not sustainable. c TMRM profiles show a gradual decrease in probe fluorescence in Baf(+) cells upon addition of oligomycin. d Uncoupling with 0.5 μM FCCP causes a drop in TMRM and mtAlpHi fluorescence in Baf(+) cells, demonstrating a decrease in the ΔΨm and ΔpH, respectively (F0/F represents the ratio of fluorescence before and 3 min after FCCP addition). The decrease in mtAlpHi signal does not depend on Baf treatment; in contrast, the relative drop in TMRM signal in Baf(+) cells is significantly smaller than in the control. In all experiments except that shown in a, cells were preincubated with 0.25 μM Baf for 45 min prior to the addition of oligomycin or FCCP. DMSO was used as a negative control. Bars 20 μm; asterisks significant differences
Mentions: To confirm this, we analysed the changes in respiration, ΔpHm and ΔΨm, induced in Baf(+) dPC12 cells by oligomycin. Normally, inhibition of ATP synthase (the main consumer of the Η+ gradient across the mitochondrial membrane) reduces O2 consumption and leads to a rapid and sustained increase in ΔpHm [45]. We observed a 10–20% decrease in the respiratory response to Baf in the cells pretreated with oligomycin (Fig. 4a). Using the mitochondrial pH probe mtAlpHi [33], we found that oligomycin transiently elevated mitochondrial pH in Baf(+) cells (Fig. 4b). Being significantly smaller than in the control, this increase suggests that complexes I–IV of the electron transport chain (ETC) generate notable H+ gradients it the mitochondria uncoupled by Baf. This result also shows that in the presence of Baf, ATP synthase still works in direct mode utilising H+ gradients for ATP production. On the other hand, after oligomycin addition, the intensity of TMRM in Baf(+) cells gradually decreased (Fig. 4c), indicating that the complex V also participates in the maintenance of ΔΨm acting as an ATPase.Fig. 4

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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