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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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Carbachol evokes store-operated Ca2+ entry in HEK-PR1 cells.(A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca2+. For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca2+-free HBS before restoration of extracellular Ca2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca2+ ([Ca2+]e) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.
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pone-0111275-g003: Carbachol evokes store-operated Ca2+ entry in HEK-PR1 cells.(A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca2+. For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca2+-free HBS before restoration of extracellular Ca2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca2+ ([Ca2+]e) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.

Mentions: CCh evokes both IP3-mediated release of Ca2+ from intracellular stores (Figure 1A and 1C) and Ca2+ entry across the plasma membrane (Figure 3A). In most cells, including HEK cells (Figures 3B and 3C) [30], [37], depletion of intracellular Ca2+ stores activates SOCE [38]. But receptors that activate PLC can also stimulate additional Ca2+ entry pathways, including those that are regulated by arachidonic acid [39], [40]. Whether such Ca2+ entry pathways contribute to CCh-evoked Ca2+ entry in HEK cells is controversial [37], [41], [42]. In HEK-PR1 cells, CCh affected neither the time course of the Ca2+ signals evoked by restoration of extracellular Ca2+ to thapsigargin-treated cells, nor the amplitude of these signals when the extracellular Ca2+ concentration was varied (Figures 3B and 3C). These results suggest that the Ca2+ entry evoked by CCh in HEK-PR1 cells is mediated by SOCE.


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

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

Carbachol evokes store-operated Ca2+ entry in HEK-PR1 cells.(A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca2+. For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca2+-free HBS before restoration of extracellular Ca2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca2+ ([Ca2+]e) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206489&req=5

pone-0111275-g003: Carbachol evokes store-operated Ca2+ entry in HEK-PR1 cells.(A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca2+. For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca2+-free HBS before restoration of extracellular Ca2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca2+ ([Ca2+]e) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.
Mentions: CCh evokes both IP3-mediated release of Ca2+ from intracellular stores (Figure 1A and 1C) and Ca2+ entry across the plasma membrane (Figure 3A). In most cells, including HEK cells (Figures 3B and 3C) [30], [37], depletion of intracellular Ca2+ stores activates SOCE [38]. But receptors that activate PLC can also stimulate additional Ca2+ entry pathways, including those that are regulated by arachidonic acid [39], [40]. Whether such Ca2+ entry pathways contribute to CCh-evoked Ca2+ entry in HEK cells is controversial [37], [41], [42]. In HEK-PR1 cells, CCh affected neither the time course of the Ca2+ signals evoked by restoration of extracellular Ca2+ to thapsigargin-treated cells, nor the amplitude of these signals when the extracellular Ca2+ concentration was varied (Figures 3B and 3C). These results suggest that the Ca2+ entry evoked by CCh in HEK-PR1 cells is mediated by SOCE.

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