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Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening.

Baumgartner HK, Gerasimenko JV, Thorne C, Ferdek P, Pozzan T, Tepikin AV, Petersen OH, Sutton R, Watson AJ, Gerasimenko OV - J. Biol. Chem. (2009)

Bottom Line: However, if mitochondria were prevented from loading with Ca2+ with 10 mum RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores.These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam.We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, School of Biomedical Sciences, Liverpool University, Liverpool L69 3BX, United Kingdom.

ABSTRACT
We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 mum RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

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Menadione can induce release of calcium from mitochondria of AR42J cells (pericam measurements). AR42J cells were transfected with fluorescent mitochondrial ratiometric calcium pericam. Fluorescence was measured over time before and after menadione in cells pretreated for 10 min with 10 μm thapsigargin (A) or for 10 min with 200 nm and subsequently with 10 μm thapsigargin (B). The ratio of pericam fluorescence is shown in Aa and B, while original traces of pericam fluorescence (488 nm and 430 nm excitation) are shown in Ab. The change in fluorescence after menadione treatment under each condition (A and B) was compared (C) (mean ± S.E., n = 8–11 per group, *, p < 0.05).
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Figure 4: Menadione can induce release of calcium from mitochondria of AR42J cells (pericam measurements). AR42J cells were transfected with fluorescent mitochondrial ratiometric calcium pericam. Fluorescence was measured over time before and after menadione in cells pretreated for 10 min with 10 μm thapsigargin (A) or for 10 min with 200 nm and subsequently with 10 μm thapsigargin (B). The ratio of pericam fluorescence is shown in Aa and B, while original traces of pericam fluorescence (488 nm and 430 nm excitation) are shown in Ab. The change in fluorescence after menadione treatment under each condition (A and B) was compared (C) (mean ± S.E., n = 8–11 per group, *, p < 0.05).

Mentions: As shown in Fig. 4A, a high dose of thapsigargin showed a large, sharp and transient influx of Ca2+ into the mitochondria. When cells were then treated with menadione, a substantial decrease in mitochondrial Ca2+ was observed (Fig. 4A). Vice versa, when cells were first exposed to low dose of thapsigargin (before high dose), much smaller and slower Ca2+ influx was observed, and the Ca2+ release in response to menadione was practically abolished (Fig. 4B). On average, the change in ratio of pericam fluorescence shows a more significant decrease in fluorescence of cells treated with menadione in the presence of a high dose of thapsigargin (Δratio fluorescence −0.081 ± 0.021, n = 8) as compared with the cells challenged with low dose followed by high dose of thapsigargin (Δratio fluorescence −0.005 ± 0.006, n = 12, p < 0.0002, Fig. 4C). These experiments were also performed in pancreatic acinar cells expressing pericam using a nucleofection technique with similar results (supplemental Fig. S1, B and C). The change in ratio of pericam fluorescence after menadione shows a noticeable decrease in the presence of a high dose of thapsigargin (Δratio fluorescence −0.081 ± 0.021, n = 8) as compared with the absence of change after low dose followed by high dose of thapsigargin (Δratio fluorescence −0.005 ± 0.006, n = 12, p < 0.0002).


Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening.

Baumgartner HK, Gerasimenko JV, Thorne C, Ferdek P, Pozzan T, Tepikin AV, Petersen OH, Sutton R, Watson AJ, Gerasimenko OV - J. Biol. Chem. (2009)

Menadione can induce release of calcium from mitochondria of AR42J cells (pericam measurements). AR42J cells were transfected with fluorescent mitochondrial ratiometric calcium pericam. Fluorescence was measured over time before and after menadione in cells pretreated for 10 min with 10 μm thapsigargin (A) or for 10 min with 200 nm and subsequently with 10 μm thapsigargin (B). The ratio of pericam fluorescence is shown in Aa and B, while original traces of pericam fluorescence (488 nm and 430 nm excitation) are shown in Ab. The change in fluorescence after menadione treatment under each condition (A and B) was compared (C) (mean ± S.E., n = 8–11 per group, *, p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2742844&req=5

Figure 4: Menadione can induce release of calcium from mitochondria of AR42J cells (pericam measurements). AR42J cells were transfected with fluorescent mitochondrial ratiometric calcium pericam. Fluorescence was measured over time before and after menadione in cells pretreated for 10 min with 10 μm thapsigargin (A) or for 10 min with 200 nm and subsequently with 10 μm thapsigargin (B). The ratio of pericam fluorescence is shown in Aa and B, while original traces of pericam fluorescence (488 nm and 430 nm excitation) are shown in Ab. The change in fluorescence after menadione treatment under each condition (A and B) was compared (C) (mean ± S.E., n = 8–11 per group, *, p < 0.05).
Mentions: As shown in Fig. 4A, a high dose of thapsigargin showed a large, sharp and transient influx of Ca2+ into the mitochondria. When cells were then treated with menadione, a substantial decrease in mitochondrial Ca2+ was observed (Fig. 4A). Vice versa, when cells were first exposed to low dose of thapsigargin (before high dose), much smaller and slower Ca2+ influx was observed, and the Ca2+ release in response to menadione was practically abolished (Fig. 4B). On average, the change in ratio of pericam fluorescence shows a more significant decrease in fluorescence of cells treated with menadione in the presence of a high dose of thapsigargin (Δratio fluorescence −0.081 ± 0.021, n = 8) as compared with the cells challenged with low dose followed by high dose of thapsigargin (Δratio fluorescence −0.005 ± 0.006, n = 12, p < 0.0002, Fig. 4C). These experiments were also performed in pancreatic acinar cells expressing pericam using a nucleofection technique with similar results (supplemental Fig. S1, B and C). The change in ratio of pericam fluorescence after menadione shows a noticeable decrease in the presence of a high dose of thapsigargin (Δratio fluorescence −0.081 ± 0.021, n = 8) as compared with the absence of change after low dose followed by high dose of thapsigargin (Δratio fluorescence −0.005 ± 0.006, n = 12, p < 0.0002).

Bottom Line: However, if mitochondria were prevented from loading with Ca2+ with 10 mum RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores.These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam.We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, School of Biomedical Sciences, Liverpool University, Liverpool L69 3BX, United Kingdom.

ABSTRACT
We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 mum RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

Show MeSH
Related in: MedlinePlus