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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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Rapid loading of  the TG-insensitive store. (A)  After depletion of the TGsensitive store, 20 mM Ca2+  was added twice for 8 s (solid  trace) or 10 s (dotted trace) in  two separate experiments. 5 s  after the second application  of Ca2+, 4 μM ionomycin was  added to assay the content of  the TG-insensitive store.  Each trace is the average of  40 cells. (B) The initial falling  phases of the two 10-s transients from (A) are aligned  on an expanded timescale.  Ionomycin releases stored  Ca2+ after the second transient (dotted trace). (C) Data  from two cells displaying either a high (dotted trace) or a  low (solid trace) response to  ionomycin. For each cell, the  initial falling phases of the  two transients are aligned as in B to enable detection of the Ca2+ released by ionomycin. (D) Ca2+ dependence of store loading in response to brief Ca2+ transients. Cells from four experiments were analyzed as described in C. The first pulse was subtracted from the  second one, and the peak of the resulting “ionomycin release transient” was taken as a measure of store loading. This is plotted as a  function of the peak [Ca2+]i during the second transient before ionomycin. The shaded bar shows the range of ionomycin response (40– 80 nM) observed in resting cells before store loading.
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Figure 4: Rapid loading of the TG-insensitive store. (A) After depletion of the TGsensitive store, 20 mM Ca2+ was added twice for 8 s (solid trace) or 10 s (dotted trace) in two separate experiments. 5 s after the second application of Ca2+, 4 μM ionomycin was added to assay the content of the TG-insensitive store. Each trace is the average of 40 cells. (B) The initial falling phases of the two 10-s transients from (A) are aligned on an expanded timescale. Ionomycin releases stored Ca2+ after the second transient (dotted trace). (C) Data from two cells displaying either a high (dotted trace) or a low (solid trace) response to ionomycin. For each cell, the initial falling phases of the two transients are aligned as in B to enable detection of the Ca2+ released by ionomycin. (D) Ca2+ dependence of store loading in response to brief Ca2+ transients. Cells from four experiments were analyzed as described in C. The first pulse was subtracted from the second one, and the peak of the resulting “ionomycin release transient” was taken as a measure of store loading. This is plotted as a function of the peak [Ca2+]i during the second transient before ionomycin. The shaded bar shows the range of ionomycin response (40– 80 nM) observed in resting cells before store loading.

Mentions: In order for the TG-insensitive store to contribute to Ca2+ signaling under a range of physiological conditions, it must be able to take up Ca2+ during transient as well as sustained periods of [Ca2+]i elevation. We therefore determined the [Ca2+]i dependence of store loading during short applications of Ca2+ (8–10 s). This short pulse of Ca2+ elicited a brief spike of [Ca2+]i that declined as Ca2+ was pumped across the plasma membrane (Fig. 4 A). After complete recovery, Ca2+ was applied again; immediately after the end of the pulse, ionomycin was applied to release any Ca2+ that had accumulated in the TG-insensitive store. By aligning the initial falling phases of the two Ca2+ spikes (Fig. 4 B), it is possible to detect Ca2+ released by ionomycin as a result of store loading. As shown by the average response of 40 cells (Fig. 4 B) and in two single cells (Fig. 4 C), [Ca2+]i elevation for 8–10 s causes significant loading of the TG-insensitive store. Results from 62 cells are summarized in Fig. 4 D as a plot of the Ca2+ released by ionomycin against the peak [Ca2+]i immediately before ionomycin (a rough measure of the Ca2+accessible to the store for loading). The amount of Ca2+ released by ionomycin after all Ca2+ spikes >300 nM exceeded the amount released in resting cells (40–80 nM; shaded line), and loading increased nonlinearly above ∼1.5 μM cytosolic Ca2+. The ability of the TG-insensitive store to sequester Ca2+ in response to small, brief Ca2+ spikes suggests that it may participate in modulating transient as well as sustained Ca2+ signals.


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

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

Rapid loading of  the TG-insensitive store. (A)  After depletion of the TGsensitive store, 20 mM Ca2+  was added twice for 8 s (solid  trace) or 10 s (dotted trace) in  two separate experiments. 5 s  after the second application  of Ca2+, 4 μM ionomycin was  added to assay the content of  the TG-insensitive store.  Each trace is the average of  40 cells. (B) The initial falling  phases of the two 10-s transients from (A) are aligned  on an expanded timescale.  Ionomycin releases stored  Ca2+ after the second transient (dotted trace). (C) Data  from two cells displaying either a high (dotted trace) or a  low (solid trace) response to  ionomycin. For each cell, the  initial falling phases of the  two transients are aligned as in B to enable detection of the Ca2+ released by ionomycin. (D) Ca2+ dependence of store loading in response to brief Ca2+ transients. Cells from four experiments were analyzed as described in C. The first pulse was subtracted from the  second one, and the peak of the resulting “ionomycin release transient” was taken as a measure of store loading. This is plotted as a  function of the peak [Ca2+]i during the second transient before ionomycin. The shaded bar shows the range of ionomycin response (40– 80 nM) observed in resting cells before store loading.
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Related In: Results  -  Collection

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Figure 4: Rapid loading of the TG-insensitive store. (A) After depletion of the TGsensitive store, 20 mM Ca2+ was added twice for 8 s (solid trace) or 10 s (dotted trace) in two separate experiments. 5 s after the second application of Ca2+, 4 μM ionomycin was added to assay the content of the TG-insensitive store. Each trace is the average of 40 cells. (B) The initial falling phases of the two 10-s transients from (A) are aligned on an expanded timescale. Ionomycin releases stored Ca2+ after the second transient (dotted trace). (C) Data from two cells displaying either a high (dotted trace) or a low (solid trace) response to ionomycin. For each cell, the initial falling phases of the two transients are aligned as in B to enable detection of the Ca2+ released by ionomycin. (D) Ca2+ dependence of store loading in response to brief Ca2+ transients. Cells from four experiments were analyzed as described in C. The first pulse was subtracted from the second one, and the peak of the resulting “ionomycin release transient” was taken as a measure of store loading. This is plotted as a function of the peak [Ca2+]i during the second transient before ionomycin. The shaded bar shows the range of ionomycin response (40– 80 nM) observed in resting cells before store loading.
Mentions: In order for the TG-insensitive store to contribute to Ca2+ signaling under a range of physiological conditions, it must be able to take up Ca2+ during transient as well as sustained periods of [Ca2+]i elevation. We therefore determined the [Ca2+]i dependence of store loading during short applications of Ca2+ (8–10 s). This short pulse of Ca2+ elicited a brief spike of [Ca2+]i that declined as Ca2+ was pumped across the plasma membrane (Fig. 4 A). After complete recovery, Ca2+ was applied again; immediately after the end of the pulse, ionomycin was applied to release any Ca2+ that had accumulated in the TG-insensitive store. By aligning the initial falling phases of the two Ca2+ spikes (Fig. 4 B), it is possible to detect Ca2+ released by ionomycin as a result of store loading. As shown by the average response of 40 cells (Fig. 4 B) and in two single cells (Fig. 4 C), [Ca2+]i elevation for 8–10 s causes significant loading of the TG-insensitive store. Results from 62 cells are summarized in Fig. 4 D as a plot of the Ca2+ released by ionomycin against the peak [Ca2+]i immediately before ionomycin (a rough measure of the Ca2+accessible to the store for loading). The amount of Ca2+ released by ionomycin after all Ca2+ spikes >300 nM exceeded the amount released in resting cells (40–80 nM; shaded line), and loading increased nonlinearly above ∼1.5 μM cytosolic Ca2+. The ability of the TG-insensitive store to sequester Ca2+ in response to small, brief Ca2+ spikes suggests that it may participate in modulating transient as well as sustained Ca2+ signals.

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