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Mitochondrial regulation of store-operated calcium signaling in T lymphocytes.

Hoth M, Fanger CM, Lewis RS - J. Cell Biol. (1997)

Bottom Line: Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate.Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state.These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426, USA. mhoth@leland.stanford.edu

ABSTRACT
Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or "capacitative" Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG-insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

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CCCP attenuates the Ca2+ plateau and inhibits loading  of the TG-insensitive store. (A) 2 mM Ca2+ was readded to TGtreated cells in the presence (dotted trace) or absence (solid trace)  of 1 μM CCCP. After termination of the store loading period,  ionomycin was added to assay store content. (B) Average response of CCCP-treated cells selected for a final [Ca2+]i plateau  >1 μM from the experiment shown in A (43/264 cells). CCCP  prevents store loading even if [Ca2+]i is high. (C) 1 μM CCCP  was applied at different times during the plateau response in 2  mM Ca2+. Each trace represents the average of 250–300 cells.
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Figure 6: CCCP attenuates the Ca2+ plateau and inhibits loading of the TG-insensitive store. (A) 2 mM Ca2+ was readded to TGtreated cells in the presence (dotted trace) or absence (solid trace) of 1 μM CCCP. After termination of the store loading period, ionomycin was added to assay store content. (B) Average response of CCCP-treated cells selected for a final [Ca2+]i plateau >1 μM from the experiment shown in A (43/264 cells). CCCP prevents store loading even if [Ca2+]i is high. (C) 1 μM CCCP was applied at different times during the plateau response in 2 mM Ca2+. Each trace represents the average of 250–300 cells.

Mentions: To test further whether the store represents mitochondria, we examined the ability of several inhibitors of mitochondrial Ca2+ transport to affect the content of the ionomycinreleasable pool. As described below, these inhibitors did interfere with store loading and therefore enabled us to test whether the store also plays an active role in modulating capacitative Ca2+ entry. In a first series of experiments, CCCP (1 μM) was applied shortly before the readdition of Ca2+ to TG-treated cells. CCCP had two prominent effects: it greatly attenuated the size of the Ca2+ plateau produced by Ca2+ readdition, and it effectively prevented loading of the TG-insensitive store, as indicated by the small amount of Ca2+ released by ionomycin (Fig. 6 A and Table I). The inhibition of loading could result from the fact that CCCP reduced the [Ca2+]i plateau to a level near the threshold for Ca2+ uptake (see Fig. 2 B). However, the ionomycin response was similarly inhibited in the minority of cells that displayed a [Ca2+]i plateau >1 μM (Fig. 6 B), demonstrating that CCCP directly blocks uptake by the store. Similar results were obtained with a related protonophore, FCCP (2 μM). Application of CCCP had little or no effect on [Ca2+]i in resting Jurkat cells (data not shown). However, CCCP added during the high Ca2+ plateau evoked an immediate transient rise in the average [Ca2+]i (Fig. 6 C). The magnitude and the duration of the rise in [Ca2+]i induced by CCCP increased with the duration of store loading, demonstrating that even at these supramicromolar levels of [Ca2+]i, uptake by the store continues for at least 15 min.


Mitochondrial regulation of store-operated calcium signaling in T lymphocytes.

Hoth M, Fanger CM, Lewis RS - J. Cell Biol. (1997)

CCCP attenuates the Ca2+ plateau and inhibits loading  of the TG-insensitive store. (A) 2 mM Ca2+ was readded to TGtreated cells in the presence (dotted trace) or absence (solid trace)  of 1 μM CCCP. After termination of the store loading period,  ionomycin was added to assay store content. (B) Average response of CCCP-treated cells selected for a final [Ca2+]i plateau  >1 μM from the experiment shown in A (43/264 cells). CCCP  prevents store loading even if [Ca2+]i is high. (C) 1 μM CCCP  was applied at different times during the plateau response in 2  mM Ca2+. Each trace represents the average of 250–300 cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139882&req=5

Figure 6: CCCP attenuates the Ca2+ plateau and inhibits loading of the TG-insensitive store. (A) 2 mM Ca2+ was readded to TGtreated cells in the presence (dotted trace) or absence (solid trace) of 1 μM CCCP. After termination of the store loading period, ionomycin was added to assay store content. (B) Average response of CCCP-treated cells selected for a final [Ca2+]i plateau >1 μM from the experiment shown in A (43/264 cells). CCCP prevents store loading even if [Ca2+]i is high. (C) 1 μM CCCP was applied at different times during the plateau response in 2 mM Ca2+. Each trace represents the average of 250–300 cells.
Mentions: To test further whether the store represents mitochondria, we examined the ability of several inhibitors of mitochondrial Ca2+ transport to affect the content of the ionomycinreleasable pool. As described below, these inhibitors did interfere with store loading and therefore enabled us to test whether the store also plays an active role in modulating capacitative Ca2+ entry. In a first series of experiments, CCCP (1 μM) was applied shortly before the readdition of Ca2+ to TG-treated cells. CCCP had two prominent effects: it greatly attenuated the size of the Ca2+ plateau produced by Ca2+ readdition, and it effectively prevented loading of the TG-insensitive store, as indicated by the small amount of Ca2+ released by ionomycin (Fig. 6 A and Table I). The inhibition of loading could result from the fact that CCCP reduced the [Ca2+]i plateau to a level near the threshold for Ca2+ uptake (see Fig. 2 B). However, the ionomycin response was similarly inhibited in the minority of cells that displayed a [Ca2+]i plateau >1 μM (Fig. 6 B), demonstrating that CCCP directly blocks uptake by the store. Similar results were obtained with a related protonophore, FCCP (2 μM). Application of CCCP had little or no effect on [Ca2+]i in resting Jurkat cells (data not shown). However, CCCP added during the high Ca2+ plateau evoked an immediate transient rise in the average [Ca2+]i (Fig. 6 C). The magnitude and the duration of the rise in [Ca2+]i induced by CCCP increased with the duration of store loading, demonstrating that even at these supramicromolar levels of [Ca2+]i, uptake by the store continues for at least 15 min.

Bottom Line: Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate.Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state.These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426, USA. mhoth@leland.stanford.edu

ABSTRACT
Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or "capacitative" Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG-insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

Show MeSH
Related in: MedlinePlus