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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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Na+ depletion attenuates the Ca2+ plateau but enhances loading of the TG-insensitive store. (A) In the continuous  absence of Na+, 2 mM Ca2+ was readded to TG-treated cells. The  plateau was attenuated by Na+ removal, yet ionomycin released a  large amount of Ca2+ from the store. (B) Comparison of the average responses of cells in the presence (dotted trace; 17/191 cells)  or absence (solid trace; 131/313 cells) of Na+, in which the mean  [Ca2+]i was selected to be <500 nM during the last half of the  Ca2+ readdition period. Despite similar [Ca2+]i profiles during  the loading period, cells bathed in Na+-free solution retain significantly more Ca2+ in the TG-insensitive store. (C) 2 mM Ca2+  was readded to TG-treated cells in Na+-free Ringer's solution as  depicted in A. Subsequent addition of Na+ in Ca2+-free Ringer's  released Ca2+ from the TG-insensitive store.
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Figure 8: Na+ depletion attenuates the Ca2+ plateau but enhances loading of the TG-insensitive store. (A) In the continuous absence of Na+, 2 mM Ca2+ was readded to TG-treated cells. The plateau was attenuated by Na+ removal, yet ionomycin released a large amount of Ca2+ from the store. (B) Comparison of the average responses of cells in the presence (dotted trace; 17/191 cells) or absence (solid trace; 131/313 cells) of Na+, in which the mean [Ca2+]i was selected to be <500 nM during the last half of the Ca2+ readdition period. Despite similar [Ca2+]i profiles during the loading period, cells bathed in Na+-free solution retain significantly more Ca2+ in the TG-insensitive store. (C) 2 mM Ca2+ was readded to TG-treated cells in Na+-free Ringer's solution as depicted in A. Subsequent addition of Na+ in Ca2+-free Ringer's released Ca2+ from the TG-insensitive store.

Mentions: A large proportion of Ca2+ export from mitochondria is carried out by a Na+/Ca2+ exchanger in the mitochondrial inner membrane (Crompton et al., 1976; Carafoli, 1987; Gunter and Pfeiffer, 1990; Cox and Matlib, 1993; Gunter et al., 1994). As a further test of whether the TG-insensitive store is mitochondrial, we asked whether the depletion of intracellular Na+, a condition that inhibits Na+/ Ca2+ exchange in mitochondria (Crompton et al., 1976; Gunter and Pfeiffer, 1990; Thayer and Miller, 1990; Gunter et al., 1994), enhances the accumulation of Ca2+ in the TG-insensitive store. The removal of external Na+ to deplete Na+i diminished the Ca2+ plateau evoked by Ca2+ readdition (Fig. 8 A). Similar results were obtained when Na+ was removed 600 s (Fig. 8 A) or 30 s before the readdition of Ca2+. Na+ depletion also enhanced store loading; this is most clearly evident from a comparison of ionomycin-induced release in cells bathed in 0 Na+ (Fig. 8 B, solid trace) or in normal Na+ selected for a comparably low plateau [Ca2+]i (dotted trace). Readdition of Na+ evoked the export of intracellular Ca2+ after a delay of ∼30 s (Fig. 8 C), consistent with the reactivation of mitochondrial Na+/ Ca2+ exchange. Na+ readdition did not elicit a [Ca2+]i rise in cells not previously exposed to Ca2+, i.e., cells in which the TG-insensitive store was empty (data not shown). These results support the idea that mitochondria constitute the TG-insensitive store, and they suggest that Ca2+ export from mitochondria is essential for maintenance of the high Ca2+ plateau.


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

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

Na+ depletion attenuates the Ca2+ plateau but enhances loading of the TG-insensitive store. (A) In the continuous  absence of Na+, 2 mM Ca2+ was readded to TG-treated cells. The  plateau was attenuated by Na+ removal, yet ionomycin released a  large amount of Ca2+ from the store. (B) Comparison of the average responses of cells in the presence (dotted trace; 17/191 cells)  or absence (solid trace; 131/313 cells) of Na+, in which the mean  [Ca2+]i was selected to be <500 nM during the last half of the  Ca2+ readdition period. Despite similar [Ca2+]i profiles during  the loading period, cells bathed in Na+-free solution retain significantly more Ca2+ in the TG-insensitive store. (C) 2 mM Ca2+  was readded to TG-treated cells in Na+-free Ringer's solution as  depicted in A. Subsequent addition of Na+ in Ca2+-free Ringer's  released Ca2+ from the TG-insensitive store.
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Related In: Results  -  Collection

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Figure 8: Na+ depletion attenuates the Ca2+ plateau but enhances loading of the TG-insensitive store. (A) In the continuous absence of Na+, 2 mM Ca2+ was readded to TG-treated cells. The plateau was attenuated by Na+ removal, yet ionomycin released a large amount of Ca2+ from the store. (B) Comparison of the average responses of cells in the presence (dotted trace; 17/191 cells) or absence (solid trace; 131/313 cells) of Na+, in which the mean [Ca2+]i was selected to be <500 nM during the last half of the Ca2+ readdition period. Despite similar [Ca2+]i profiles during the loading period, cells bathed in Na+-free solution retain significantly more Ca2+ in the TG-insensitive store. (C) 2 mM Ca2+ was readded to TG-treated cells in Na+-free Ringer's solution as depicted in A. Subsequent addition of Na+ in Ca2+-free Ringer's released Ca2+ from the TG-insensitive store.
Mentions: A large proportion of Ca2+ export from mitochondria is carried out by a Na+/Ca2+ exchanger in the mitochondrial inner membrane (Crompton et al., 1976; Carafoli, 1987; Gunter and Pfeiffer, 1990; Cox and Matlib, 1993; Gunter et al., 1994). As a further test of whether the TG-insensitive store is mitochondrial, we asked whether the depletion of intracellular Na+, a condition that inhibits Na+/ Ca2+ exchange in mitochondria (Crompton et al., 1976; Gunter and Pfeiffer, 1990; Thayer and Miller, 1990; Gunter et al., 1994), enhances the accumulation of Ca2+ in the TG-insensitive store. The removal of external Na+ to deplete Na+i diminished the Ca2+ plateau evoked by Ca2+ readdition (Fig. 8 A). Similar results were obtained when Na+ was removed 600 s (Fig. 8 A) or 30 s before the readdition of Ca2+. Na+ depletion also enhanced store loading; this is most clearly evident from a comparison of ionomycin-induced release in cells bathed in 0 Na+ (Fig. 8 B, solid trace) or in normal Na+ selected for a comparably low plateau [Ca2+]i (dotted trace). Readdition of Na+ evoked the export of intracellular Ca2+ after a delay of ∼30 s (Fig. 8 C), consistent with the reactivation of mitochondrial Na+/ Ca2+ exchange. Na+ readdition did not elicit a [Ca2+]i rise in cells not previously exposed to Ca2+, i.e., cells in which the TG-insensitive store was empty (data not shown). These results support the idea that mitochondria constitute the TG-insensitive store, and they suggest that Ca2+ export from mitochondria is essential for maintenance of the high Ca2+ plateau.

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