Limits...
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.

Show MeSH

Related in: MedlinePlus

AR42J cells also require sharp increases in cytosolic calcium for induction of apoptosis. AR42J cells were loaded with 25 nm TMRM-AM and 10 μm Fluo-4-AM (A and B). Fluorescence was measured over time before and after application of 30 μm menadione in cells pretreated with 10 μm thapsigargin (A, n = 6) or 200 nm thapsigargin followed by 10 μm thapsigargin (B, n = 6). Changes in TMRM fluorescence after menadione treatment were compared in C (mean ± S.E., *, p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2742844&req=5

Figure 3: AR42J cells also require sharp increases in cytosolic calcium for induction of apoptosis. AR42J cells were loaded with 25 nm TMRM-AM and 10 μm Fluo-4-AM (A and B). Fluorescence was measured over time before and after application of 30 μm menadione in cells pretreated with 10 μm thapsigargin (A, n = 6) or 200 nm thapsigargin followed by 10 μm thapsigargin (B, n = 6). Changes in TMRM fluorescence after menadione treatment were compared in C (mean ± S.E., *, p < 0.05).

Mentions: To confirm the Rhod-2 results shown above we transfected cells with mitochondria-specific Ca2+ sensitive pericam (41). The results obtained with the mitochondrial pericam were essentially similar to those with Rhod-2. However, transfection with pericam is highly inefficient in freshly isolated acinar cells and the number of positive cells extremely low. To bypass these problems, we have used the AR42J cell line. First, experiments depicted in Fig. 1 were repeated using AR42J cells loaded with Fluo4 and TMRM (Fig. 3 and supplemental Fig. S1A). Effects of the low and high doses of thapsigargin on cytosolic Ca2+ and TMRM fluorescence reported above for the freshly isolated acinar cells were also observed in AR42J cells. In particular, as shown in Fig. 3, A and C addition of 10 μm thapsigargin induced a sharp and large increase in cytosolic Ca2+ concentration and after menadione application, a substantial mitochondrial membrane depolarization (Δfluorescence = −0.212 ± 0.033, n = 6) (Fig. 3, A and C). The other protocol (200 nm followed by 10 μm of thapsigargin) caused a small and slow cytosolic Ca2+ increase and no depolarization as opposed to fast Ca2+ release data (n = 6, Fig. 3, B and C).


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)

AR42J cells also require sharp increases in cytosolic calcium for induction of apoptosis. AR42J cells were loaded with 25 nm TMRM-AM and 10 μm Fluo-4-AM (A and B). Fluorescence was measured over time before and after application of 30 μm menadione in cells pretreated with 10 μm thapsigargin (A, n = 6) or 200 nm thapsigargin followed by 10 μm thapsigargin (B, n = 6). Changes in TMRM fluorescence after menadione treatment were compared in C (mean ± S.E., *, p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: AR42J cells also require sharp increases in cytosolic calcium for induction of apoptosis. AR42J cells were loaded with 25 nm TMRM-AM and 10 μm Fluo-4-AM (A and B). Fluorescence was measured over time before and after application of 30 μm menadione in cells pretreated with 10 μm thapsigargin (A, n = 6) or 200 nm thapsigargin followed by 10 μm thapsigargin (B, n = 6). Changes in TMRM fluorescence after menadione treatment were compared in C (mean ± S.E., *, p < 0.05).
Mentions: To confirm the Rhod-2 results shown above we transfected cells with mitochondria-specific Ca2+ sensitive pericam (41). The results obtained with the mitochondrial pericam were essentially similar to those with Rhod-2. However, transfection with pericam is highly inefficient in freshly isolated acinar cells and the number of positive cells extremely low. To bypass these problems, we have used the AR42J cell line. First, experiments depicted in Fig. 1 were repeated using AR42J cells loaded with Fluo4 and TMRM (Fig. 3 and supplemental Fig. S1A). Effects of the low and high doses of thapsigargin on cytosolic Ca2+ and TMRM fluorescence reported above for the freshly isolated acinar cells were also observed in AR42J cells. In particular, as shown in Fig. 3, A and C addition of 10 μm thapsigargin induced a sharp and large increase in cytosolic Ca2+ concentration and after menadione application, a substantial mitochondrial membrane depolarization (Δfluorescence = −0.212 ± 0.033, n = 6) (Fig. 3, A and C). The other protocol (200 nm followed by 10 μm of thapsigargin) caused a small and slow cytosolic Ca2+ increase and no depolarization as opposed to fast Ca2+ release data (n = 6, Fig. 3, B and C).

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