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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 generation of ROS and apoptosis of dPC12 cells. a FACS analysis of ROS-sensitive carboxy-H2DCFDA probe fluorescence reveal an increase in ROS levels in the cells treated with Baf and CMA (both 0.25 μM) for 2 h. b Quantitative analysis of data presented in a as compared to the effect of classical ROS inducer tert-butyl hydroperoxide (TBHP, 250 μM). c Caspase-3 activity was increased in the cells treated with both V-ATPase inhibitors for 4 h; however, the effect of Baf was significantly stronger. Asterisks significant differences
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Fig2: The effect of Baf on generation of ROS and apoptosis of dPC12 cells. a FACS analysis of ROS-sensitive carboxy-H2DCFDA probe fluorescence reveal an increase in ROS levels in the cells treated with Baf and CMA (both 0.25 μM) for 2 h. b Quantitative analysis of data presented in a as compared to the effect of classical ROS inducer tert-butyl hydroperoxide (TBHP, 250 μM). c Caspase-3 activity was increased in the cells treated with both V-ATPase inhibitors for 4 h; however, the effect of Baf was significantly stronger. Asterisks significant differences

Mentions: Dissipation of lysosomes by Baf and CMA was coupled with significant inhibition of autophagic flux (seen as an increase in cellular LC3A/B II levels, Fig. 1f), and an increase in ROS production (Fig. 2a, b; Supplementary Fig. S2). Both inhibition of autophagy and elevation of ROS are known to activate apoptosis. We found that caspase-3 activity (marker of apoptosis) was increased by 150–200% and about 100% after 4 h of treatment with Baf and CMA, respectively (Fig. 2c). Under the same conditions, only minor changes in Smac/DIABLO location were observed (Supplementary Fig. S3).Fig. 2


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 generation of ROS and apoptosis of dPC12 cells. a FACS analysis of ROS-sensitive carboxy-H2DCFDA probe fluorescence reveal an increase in ROS levels in the cells treated with Baf and CMA (both 0.25 μM) for 2 h. b Quantitative analysis of data presented in a as compared to the effect of classical ROS inducer tert-butyl hydroperoxide (TBHP, 250 μM). c Caspase-3 activity was increased in the cells treated with both V-ATPase inhibitors for 4 h; however, the effect of Baf was significantly stronger. Asterisks significant differences
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037485&req=5

Fig2: The effect of Baf on generation of ROS and apoptosis of dPC12 cells. a FACS analysis of ROS-sensitive carboxy-H2DCFDA probe fluorescence reveal an increase in ROS levels in the cells treated with Baf and CMA (both 0.25 μM) for 2 h. b Quantitative analysis of data presented in a as compared to the effect of classical ROS inducer tert-butyl hydroperoxide (TBHP, 250 μM). c Caspase-3 activity was increased in the cells treated with both V-ATPase inhibitors for 4 h; however, the effect of Baf was significantly stronger. Asterisks significant differences
Mentions: Dissipation of lysosomes by Baf and CMA was coupled with significant inhibition of autophagic flux (seen as an increase in cellular LC3A/B II levels, Fig. 1f), and an increase in ROS production (Fig. 2a, b; Supplementary Fig. S2). Both inhibition of autophagy and elevation of ROS are known to activate apoptosis. We found that caspase-3 activity (marker of apoptosis) was increased by 150–200% and about 100% after 4 h of treatment with Baf and CMA, respectively (Fig. 2c). Under the same conditions, only minor changes in Smac/DIABLO location were observed (Supplementary Fig. S3).Fig. 2

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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