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Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

López Sanjurjo CI, Tovey SC, Taylor CW - PLoS ONE (2014)

Bottom Line: The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1.Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release.We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

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Lysosomes rapidly recycle the Ca2+ sequestered after IP3-evoked Ca2+ release.(A, B) HEK cells were repetitively stimulated with CCh (1 mM, 30 s) alone or with bafilomycin A1 (1 µM, 1 h) in nominally Ca2+-free HBS without (A) or with Gd3+ (1 mM) (B). Results show means ± S.E. for ≥ 45 cells from a single experiment, typical of at least 3 similar experiments. The inset to panel A shows how a high concentration of Gd3+ (1 mM) effectively insulates the cell from exchanging Ca2+ with the extracellular environment by blocking Ca2+ entry and extrusion [37]. Under these conditions, repetitive responses to CCh are entirely dependent on recycling of intracellular Ca2+ (dashed lines). (C) Summary results show effects of Gd3+ on the peak increase in [Ca2+]i evoked by each challenge with CCh in the absence of bafilomycin A1. (D) Predicted effects of bafilomycin A1 on the Ca2+ signals evoked by repetitive CCh challenges of Gd3+-insulated cells. The predicted results represent an idealized situation in which Gd3+ entirely insulates the cell from Ca2+ exchanges with the extracellular environment (in practise the insulation is incomplete), and then shows the results predicted for situations where lysosomes either accumulate (upper panel) or entirely recycle (lower panel) the sequestered Ca2+ (see Figure S1A). (E) Peak increases in [Ca2+]i evoked by the first CCh challenge under the conditions shown. *p <0.05, paired Students's t-test. (F) Effects of bafilomycin A1 on the peak increases in [Ca2+]i evoked by successive CCh challenges in nominally Ca2+-free HBS containing 1 mM Gd3+. Results are normalized to the first CCh challenge for each condition (the raw data and the results obtained in the absence of Gd3+ are shown in Figure S1B and S1C). Results (C, E and F) are means ± S.E. from at least 4 independent experiments.
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pone-0111275-g005: Lysosomes rapidly recycle the Ca2+ sequestered after IP3-evoked Ca2+ release.(A, B) HEK cells were repetitively stimulated with CCh (1 mM, 30 s) alone or with bafilomycin A1 (1 µM, 1 h) in nominally Ca2+-free HBS without (A) or with Gd3+ (1 mM) (B). Results show means ± S.E. for ≥ 45 cells from a single experiment, typical of at least 3 similar experiments. The inset to panel A shows how a high concentration of Gd3+ (1 mM) effectively insulates the cell from exchanging Ca2+ with the extracellular environment by blocking Ca2+ entry and extrusion [37]. Under these conditions, repetitive responses to CCh are entirely dependent on recycling of intracellular Ca2+ (dashed lines). (C) Summary results show effects of Gd3+ on the peak increase in [Ca2+]i evoked by each challenge with CCh in the absence of bafilomycin A1. (D) Predicted effects of bafilomycin A1 on the Ca2+ signals evoked by repetitive CCh challenges of Gd3+-insulated cells. The predicted results represent an idealized situation in which Gd3+ entirely insulates the cell from Ca2+ exchanges with the extracellular environment (in practise the insulation is incomplete), and then shows the results predicted for situations where lysosomes either accumulate (upper panel) or entirely recycle (lower panel) the sequestered Ca2+ (see Figure S1A). (E) Peak increases in [Ca2+]i evoked by the first CCh challenge under the conditions shown. *p <0.05, paired Students's t-test. (F) Effects of bafilomycin A1 on the peak increases in [Ca2+]i evoked by successive CCh challenges in nominally Ca2+-free HBS containing 1 mM Gd3+. Results are normalized to the first CCh challenge for each condition (the raw data and the results obtained in the absence of Gd3+ are shown in Figure S1B and S1C). Results (C, E and F) are means ± S.E. from at least 4 independent experiments.

Mentions: To address this issue, HEK cells were stimulated with CCh under conditions (1 mM GdCl3 in the extracellular medium) that inhibit both Ca2+ extrusion across the plasma membrane and Ca2+ entry [37] (Figure 5A inset). Comparison of the black traces in Figures 5A and 5B, where HEK cells in nominally Ca2+-free HBS were repeatedly stimulated with brief pulses of a maximally effective concentration of CCh (1 mM), demonstrates that the approach is effective, albeit without fully preventing loss of Ca2+ from stimulated cells. The incomplete inhibition of Ca2+ loss by Gd3+ contrasts with a previous analysis of HEK cells where CCh-evoked Ca2+ oscillations persisted for many minutes with undiminished amplitude in Ca2+-free medium supplemented with 1 mM Gd3+[37]. The different results probably result from the much higher concentration of CCh used in our experiments (1 mM) relative to that used to evoke Ca2+ oscillations (1–5 µM) [37]. In Ca2+-free HBS, cells responded robustly to the first CCh challenge, but not to subsequent challenges (Figure 5A). In the same HBS supplemented with Gd3+, even the fourth challenge with CCh evoked a detectable increase in [Ca2+]i (Figures 5B and 5C). These results confirm that a substantial fraction of the Ca2+ released from intracellular stores by IP3 is normally extruded from the cell. That Ca2+ would normally be replenished by SOCE, but in the absence of extracellular Ca2+ the stores are unable to refill. A high concentration of Gd3+, by inhibiting Ca2+ exchanges across the plasma membrane (both influx and efflux), allows Ca2+ to be recycled within the cell and thereby allows the ER to respond to repeated CCh challenges (Figures 5B and 5C).


Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

López Sanjurjo CI, Tovey SC, Taylor CW - PLoS ONE (2014)

Lysosomes rapidly recycle the Ca2+ sequestered after IP3-evoked Ca2+ release.(A, B) HEK cells were repetitively stimulated with CCh (1 mM, 30 s) alone or with bafilomycin A1 (1 µM, 1 h) in nominally Ca2+-free HBS without (A) or with Gd3+ (1 mM) (B). Results show means ± S.E. for ≥ 45 cells from a single experiment, typical of at least 3 similar experiments. The inset to panel A shows how a high concentration of Gd3+ (1 mM) effectively insulates the cell from exchanging Ca2+ with the extracellular environment by blocking Ca2+ entry and extrusion [37]. Under these conditions, repetitive responses to CCh are entirely dependent on recycling of intracellular Ca2+ (dashed lines). (C) Summary results show effects of Gd3+ on the peak increase in [Ca2+]i evoked by each challenge with CCh in the absence of bafilomycin A1. (D) Predicted effects of bafilomycin A1 on the Ca2+ signals evoked by repetitive CCh challenges of Gd3+-insulated cells. The predicted results represent an idealized situation in which Gd3+ entirely insulates the cell from Ca2+ exchanges with the extracellular environment (in practise the insulation is incomplete), and then shows the results predicted for situations where lysosomes either accumulate (upper panel) or entirely recycle (lower panel) the sequestered Ca2+ (see Figure S1A). (E) Peak increases in [Ca2+]i evoked by the first CCh challenge under the conditions shown. *p <0.05, paired Students's t-test. (F) Effects of bafilomycin A1 on the peak increases in [Ca2+]i evoked by successive CCh challenges in nominally Ca2+-free HBS containing 1 mM Gd3+. Results are normalized to the first CCh challenge for each condition (the raw data and the results obtained in the absence of Gd3+ are shown in Figure S1B and S1C). Results (C, E and F) are means ± S.E. from at least 4 independent experiments.
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Related In: Results  -  Collection

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pone-0111275-g005: Lysosomes rapidly recycle the Ca2+ sequestered after IP3-evoked Ca2+ release.(A, B) HEK cells were repetitively stimulated with CCh (1 mM, 30 s) alone or with bafilomycin A1 (1 µM, 1 h) in nominally Ca2+-free HBS without (A) or with Gd3+ (1 mM) (B). Results show means ± S.E. for ≥ 45 cells from a single experiment, typical of at least 3 similar experiments. The inset to panel A shows how a high concentration of Gd3+ (1 mM) effectively insulates the cell from exchanging Ca2+ with the extracellular environment by blocking Ca2+ entry and extrusion [37]. Under these conditions, repetitive responses to CCh are entirely dependent on recycling of intracellular Ca2+ (dashed lines). (C) Summary results show effects of Gd3+ on the peak increase in [Ca2+]i evoked by each challenge with CCh in the absence of bafilomycin A1. (D) Predicted effects of bafilomycin A1 on the Ca2+ signals evoked by repetitive CCh challenges of Gd3+-insulated cells. The predicted results represent an idealized situation in which Gd3+ entirely insulates the cell from Ca2+ exchanges with the extracellular environment (in practise the insulation is incomplete), and then shows the results predicted for situations where lysosomes either accumulate (upper panel) or entirely recycle (lower panel) the sequestered Ca2+ (see Figure S1A). (E) Peak increases in [Ca2+]i evoked by the first CCh challenge under the conditions shown. *p <0.05, paired Students's t-test. (F) Effects of bafilomycin A1 on the peak increases in [Ca2+]i evoked by successive CCh challenges in nominally Ca2+-free HBS containing 1 mM Gd3+. Results are normalized to the first CCh challenge for each condition (the raw data and the results obtained in the absence of Gd3+ are shown in Figure S1B and S1C). Results (C, E and F) are means ± S.E. from at least 4 independent experiments.
Mentions: To address this issue, HEK cells were stimulated with CCh under conditions (1 mM GdCl3 in the extracellular medium) that inhibit both Ca2+ extrusion across the plasma membrane and Ca2+ entry [37] (Figure 5A inset). Comparison of the black traces in Figures 5A and 5B, where HEK cells in nominally Ca2+-free HBS were repeatedly stimulated with brief pulses of a maximally effective concentration of CCh (1 mM), demonstrates that the approach is effective, albeit without fully preventing loss of Ca2+ from stimulated cells. The incomplete inhibition of Ca2+ loss by Gd3+ contrasts with a previous analysis of HEK cells where CCh-evoked Ca2+ oscillations persisted for many minutes with undiminished amplitude in Ca2+-free medium supplemented with 1 mM Gd3+[37]. The different results probably result from the much higher concentration of CCh used in our experiments (1 mM) relative to that used to evoke Ca2+ oscillations (1–5 µM) [37]. In Ca2+-free HBS, cells responded robustly to the first CCh challenge, but not to subsequent challenges (Figure 5A). In the same HBS supplemented with Gd3+, even the fourth challenge with CCh evoked a detectable increase in [Ca2+]i (Figures 5B and 5C). These results confirm that a substantial fraction of the Ca2+ released from intracellular stores by IP3 is normally extruded from the cell. That Ca2+ would normally be replenished by SOCE, but in the absence of extracellular Ca2+ the stores are unable to refill. A high concentration of Gd3+, by inhibiting Ca2+ exchanges across the plasma membrane (both influx and efflux), allows Ca2+ to be recycled within the cell and thereby allows the ER to respond to repeated CCh challenges (Figures 5B and 5C).

Bottom Line: The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1.Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release.We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

Show MeSH
Related in: MedlinePlus