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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 increases susceptibility of dPC12 to excitatory stimulation. a Pretreatment with 0.25 μM Baf dramatically increases the respiratory response to ΔΨp depolarization by high K+. The response is not reduced by the SERCA inhibitor thapsigargin. The dashed line shows the level of deoxygenation induced by K+ in the Baf(−) cells treated with thapsigargin. b In Ca2+-free medium Baf(+) cells do not respond to high K+ and rapidly loose their respiratory activity. c Pretreatment with Baf (25 μM, 30 min) in glucose(+) medium reduces elevation of ATP in response to ΔΨp depolarization by high K+, whereas in galactose(+) medium K+ stimulation strongly decreases cellular ATP. The effect of CMA (0.25 μM) on the ATP response to K+ is less pronounced. Valinomycin (Val, 0.25 μM) in galactose(+) conditions reduces the ATP by >90%, irrespective of K+ treatment. d Pretreatment of the cells with 25 μM Baf for 30 min amplifies the dose-dependent response to glutamate (5–20 mM), while CMA does not affect the response to glutamate. e Relative decrease in cellular ATP (measured 20 h after glutamate treatment) reveals a negative effect of Baf on the viability of dPC12 cells, exposed to glutamate. DMSO was used as a negative control. Asterisks significant differences
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Fig7: Baf increases susceptibility of dPC12 to excitatory stimulation. a Pretreatment with 0.25 μM Baf dramatically increases the respiratory response to ΔΨp depolarization by high K+. The response is not reduced by the SERCA inhibitor thapsigargin. The dashed line shows the level of deoxygenation induced by K+ in the Baf(−) cells treated with thapsigargin. b In Ca2+-free medium Baf(+) cells do not respond to high K+ and rapidly loose their respiratory activity. c Pretreatment with Baf (25 μM, 30 min) in glucose(+) medium reduces elevation of ATP in response to ΔΨp depolarization by high K+, whereas in galactose(+) medium K+ stimulation strongly decreases cellular ATP. The effect of CMA (0.25 μM) on the ATP response to K+ is less pronounced. Valinomycin (Val, 0.25 μM) in galactose(+) conditions reduces the ATP by >90%, irrespective of K+ treatment. d Pretreatment of the cells with 25 μM Baf for 30 min amplifies the dose-dependent response to glutamate (5–20 mM), while CMA does not affect the response to glutamate. e Relative decrease in cellular ATP (measured 20 h after glutamate treatment) reveals a negative effect of Baf on the viability of dPC12 cells, exposed to glutamate. DMSO was used as a negative control. Asterisks significant differences

Mentions: Depolarization of the ΔΨp by high K+ triggers a rapid transient decrease in the O2 level and substantial elevation of ATP level in dPC12 cells, which reaches a maximum in 2–5 min after K+ addition [28]. Preincubation with Baf significantly increased the respiratory response to K+ (Fig. 7a, b). This increase was further augmented by thapsigargin. Under extracellular Ca2+-free conditions the response of Baf-treated cells to K+ was abolished (Fig. 7b). While measuring the changes in ATP levels induced by high K+, we found that in Baf-treated cells grown on glucose the characteristic increase in ATP was partially inhibited (Fig. 7c), whereas under galactose(+) conditions the addition of K+ caused a significant drop in cellular ATP. Pretreatment with CMA did not change the respiratory response and slightly reduced the ATP response to K+. For comparison, valinomycin (0.25 μM) did not affect the ATP response to K+ in glucose(+) medium, while under galactose(+) conditions it dramatically reduced ATP irrespective of K+ addition.Fig. 7


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 increases susceptibility of dPC12 to excitatory stimulation. a Pretreatment with 0.25 μM Baf dramatically increases the respiratory response to ΔΨp depolarization by high K+. The response is not reduced by the SERCA inhibitor thapsigargin. The dashed line shows the level of deoxygenation induced by K+ in the Baf(−) cells treated with thapsigargin. b In Ca2+-free medium Baf(+) cells do not respond to high K+ and rapidly loose their respiratory activity. c Pretreatment with Baf (25 μM, 30 min) in glucose(+) medium reduces elevation of ATP in response to ΔΨp depolarization by high K+, whereas in galactose(+) medium K+ stimulation strongly decreases cellular ATP. The effect of CMA (0.25 μM) on the ATP response to K+ is less pronounced. Valinomycin (Val, 0.25 μM) in galactose(+) conditions reduces the ATP by >90%, irrespective of K+ treatment. d Pretreatment of the cells with 25 μM Baf for 30 min amplifies the dose-dependent response to glutamate (5–20 mM), while CMA does not affect the response to glutamate. e Relative decrease in cellular ATP (measured 20 h after glutamate treatment) reveals a negative effect of Baf on the viability of dPC12 cells, exposed to glutamate. DMSO was used as a negative control. Asterisks significant differences
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Fig7: Baf increases susceptibility of dPC12 to excitatory stimulation. a Pretreatment with 0.25 μM Baf dramatically increases the respiratory response to ΔΨp depolarization by high K+. The response is not reduced by the SERCA inhibitor thapsigargin. The dashed line shows the level of deoxygenation induced by K+ in the Baf(−) cells treated with thapsigargin. b In Ca2+-free medium Baf(+) cells do not respond to high K+ and rapidly loose their respiratory activity. c Pretreatment with Baf (25 μM, 30 min) in glucose(+) medium reduces elevation of ATP in response to ΔΨp depolarization by high K+, whereas in galactose(+) medium K+ stimulation strongly decreases cellular ATP. The effect of CMA (0.25 μM) on the ATP response to K+ is less pronounced. Valinomycin (Val, 0.25 μM) in galactose(+) conditions reduces the ATP by >90%, irrespective of K+ treatment. d Pretreatment of the cells with 25 μM Baf for 30 min amplifies the dose-dependent response to glutamate (5–20 mM), while CMA does not affect the response to glutamate. e Relative decrease in cellular ATP (measured 20 h after glutamate treatment) reveals a negative effect of Baf on the viability of dPC12 cells, exposed to glutamate. DMSO was used as a negative control. Asterisks significant differences
Mentions: Depolarization of the ΔΨp by high K+ triggers a rapid transient decrease in the O2 level and substantial elevation of ATP level in dPC12 cells, which reaches a maximum in 2–5 min after K+ addition [28]. Preincubation with Baf significantly increased the respiratory response to K+ (Fig. 7a, b). This increase was further augmented by thapsigargin. Under extracellular Ca2+-free conditions the response of Baf-treated cells to K+ was abolished (Fig. 7b). While measuring the changes in ATP levels induced by high K+, we found that in Baf-treated cells grown on glucose the characteristic increase in ATP was partially inhibited (Fig. 7c), whereas under galactose(+) conditions the addition of K+ caused a significant drop in cellular ATP. Pretreatment with CMA did not change the respiratory response and slightly reduced the ATP response to K+. For comparison, valinomycin (0.25 μM) did not affect the ATP response to K+ in glucose(+) medium, while under galactose(+) conditions it dramatically reduced ATP irrespective of K+ addition.Fig. 7

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