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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 activates glycolysis and reduces ATP production by OxPhos in dPC12 cells. a In the cells treated with 0.25 μM Baf and CMA for 30 min, cytosolic pH decreases. Live-cell imaging of the BCECF distribution within the cells reveals focal condensation of the probe. b Extracellular acidification rate (ECA), which is linked to the rate of glycolysis, increases in the cells treated with 0.25 μM Baf to the levels characteristic of valinomycin (Val, 0.25 μM) or FCCP (FCCP, 0.5 μM) uncoupling. CMA (0.25 μM) does not change the extracellular acidification. c, ddPC12 cells grown in galactose(+) conditions show a decrease in their ATP levels in a Baf concentration-dependent manner (c) and a time-dependent manner (d). In glucose(+) medium Baf does not affect cellular ATP levels. Inhibition of the F0F1 ATPase by oligomycin (10 μM) further reduces the ATP levels. DMSO was used as a negative control. Asterisks significant differences
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Fig3: Baf activates glycolysis and reduces ATP production by OxPhos in dPC12 cells. a In the cells treated with 0.25 μM Baf and CMA for 30 min, cytosolic pH decreases. Live-cell imaging of the BCECF distribution within the cells reveals focal condensation of the probe. b Extracellular acidification rate (ECA), which is linked to the rate of glycolysis, increases in the cells treated with 0.25 μM Baf to the levels characteristic of valinomycin (Val, 0.25 μM) or FCCP (FCCP, 0.5 μM) uncoupling. CMA (0.25 μM) does not change the extracellular acidification. c, ddPC12 cells grown in galactose(+) conditions show a decrease in their ATP levels in a Baf concentration-dependent manner (c) and a time-dependent manner (d). In glucose(+) medium Baf does not affect cellular ATP levels. Inhibition of the F0F1 ATPase by oligomycin (10 μM) further reduces the ATP levels. DMSO was used as a negative control. Asterisks significant differences

Mentions: To maintain their energy status, uncoupled cells normally increase ATP production through glycolysis, thus elevating lactate production and increasing the rates of cytosolic and extracellular acidification. Inhibition of the V-ATPase activity can also contribute to both intra- and extracellular acidification due to impaired proton transport. In dPC12 cells treated with Baf for >30 min distinct acidification of the cytosol was observed, detected with the pH probe BCECF (Fig. 3a), with the distribution of BCECF showing a punctate pattern. Cells treated with CMA revealed similar changes in the intensity and distribution of BCECF. We therefore concluded that cytosol acidification by Baf is mainly due to the inhibition of V-ATPase activity.Fig. 3


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 activates glycolysis and reduces ATP production by OxPhos in dPC12 cells. a In the cells treated with 0.25 μM Baf and CMA for 30 min, cytosolic pH decreases. Live-cell imaging of the BCECF distribution within the cells reveals focal condensation of the probe. b Extracellular acidification rate (ECA), which is linked to the rate of glycolysis, increases in the cells treated with 0.25 μM Baf to the levels characteristic of valinomycin (Val, 0.25 μM) or FCCP (FCCP, 0.5 μM) uncoupling. CMA (0.25 μM) does not change the extracellular acidification. c, ddPC12 cells grown in galactose(+) conditions show a decrease in their ATP levels in a Baf concentration-dependent manner (c) and a time-dependent manner (d). In glucose(+) medium Baf does not affect cellular ATP levels. Inhibition of the F0F1 ATPase by oligomycin (10 μM) further reduces the ATP levels. DMSO was used as a negative control. Asterisks significant differences
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Fig3: Baf activates glycolysis and reduces ATP production by OxPhos in dPC12 cells. a In the cells treated with 0.25 μM Baf and CMA for 30 min, cytosolic pH decreases. Live-cell imaging of the BCECF distribution within the cells reveals focal condensation of the probe. b Extracellular acidification rate (ECA), which is linked to the rate of glycolysis, increases in the cells treated with 0.25 μM Baf to the levels characteristic of valinomycin (Val, 0.25 μM) or FCCP (FCCP, 0.5 μM) uncoupling. CMA (0.25 μM) does not change the extracellular acidification. c, ddPC12 cells grown in galactose(+) conditions show a decrease in their ATP levels in a Baf concentration-dependent manner (c) and a time-dependent manner (d). In glucose(+) medium Baf does not affect cellular ATP levels. Inhibition of the F0F1 ATPase by oligomycin (10 μM) further reduces the ATP levels. DMSO was used as a negative control. Asterisks significant differences
Mentions: To maintain their energy status, uncoupled cells normally increase ATP production through glycolysis, thus elevating lactate production and increasing the rates of cytosolic and extracellular acidification. Inhibition of the V-ATPase activity can also contribute to both intra- and extracellular acidification due to impaired proton transport. In dPC12 cells treated with Baf for >30 min distinct acidification of the cytosol was observed, detected with the pH probe BCECF (Fig. 3a), with the distribution of BCECF showing a punctate pattern. Cells treated with CMA revealed similar changes in the intensity and distribution of BCECF. We therefore concluded that cytosol acidification by Baf is mainly due to the inhibition of V-ATPase activity.Fig. 3

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.

Show MeSH
Related in: MedlinePlus