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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 accumulate Ca2+ released from intracellular stores by IP3 alone or IP3 with cAMP.(A) CCh stimulates M3 muscarinic receptors leading to activation of PLC and IP3-evoked Ca2+ release from the ER. PTH, via type 1 PTH receptors, stimulates adenylyl cyclase. Cyclic AMP sensitizes IP3Rs to IP3 and thereby potentiates the Ca2+ release evoked by CCh. We suggest that cAMP is delivered to IP3Rs at high concentrations within signalling junctions [34] and that the IP3Rs that respond to CCh alone are activated by locally delivered IP3[31]. This local signalling allows CCh alone and CCh in combination with PTH to release Ca2+ from different stores [31]. (B) Bafilomycin A1 (Baf A1) inhibits the V-ATPase that mediates H+ accumulation by lysosomes, and thereby prevents lysosomal Ca2+ uptake. The latter may be mediated by H+-Ca2+ exchange. (C) Populations of HEK-PR1 cells were stimulated with CCh (1 mM) and then PTH (1 µM) with or without prior treatment with bafilomycin A1 (1 µM, 1 h). BAPTA (10 mM) was added as shown to chelate extracellular Ca2+. Results are means ± S.E. from 3 wells from one experiment, typical of 3 similar experiments. (D) Summary results show effects of bafilomycin A1 on the amplitudes of the peak Ca2+ signals evoked by addition of CCh, or PTH after CCh. Results (as percentages of the responses without bafilomycin A1) are means ± S.E. from 3 independent experiments. (E) Experiments similar to those in C, show the effects of bafilomycin A1 on the concentration-dependent effects of PTH on CCh-evoked Ca2+ signals. Results are means ± S.E. from 3 independent experiments. (F) The results suggest that lysosomes (LY) accumulate Ca2+ released via IP3Rs activated by IP3 alone or IP3 with cAMP.
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pone-0111275-g001: Lysosomes accumulate Ca2+ released from intracellular stores by IP3 alone or IP3 with cAMP.(A) CCh stimulates M3 muscarinic receptors leading to activation of PLC and IP3-evoked Ca2+ release from the ER. PTH, via type 1 PTH receptors, stimulates adenylyl cyclase. Cyclic AMP sensitizes IP3Rs to IP3 and thereby potentiates the Ca2+ release evoked by CCh. We suggest that cAMP is delivered to IP3Rs at high concentrations within signalling junctions [34] and that the IP3Rs that respond to CCh alone are activated by locally delivered IP3[31]. This local signalling allows CCh alone and CCh in combination with PTH to release Ca2+ from different stores [31]. (B) Bafilomycin A1 (Baf A1) inhibits the V-ATPase that mediates H+ accumulation by lysosomes, and thereby prevents lysosomal Ca2+ uptake. The latter may be mediated by H+-Ca2+ exchange. (C) Populations of HEK-PR1 cells were stimulated with CCh (1 mM) and then PTH (1 µM) with or without prior treatment with bafilomycin A1 (1 µM, 1 h). BAPTA (10 mM) was added as shown to chelate extracellular Ca2+. Results are means ± S.E. from 3 wells from one experiment, typical of 3 similar experiments. (D) Summary results show effects of bafilomycin A1 on the amplitudes of the peak Ca2+ signals evoked by addition of CCh, or PTH after CCh. Results (as percentages of the responses without bafilomycin A1) are means ± S.E. from 3 independent experiments. (E) Experiments similar to those in C, show the effects of bafilomycin A1 on the concentration-dependent effects of PTH on CCh-evoked Ca2+ signals. Results are means ± S.E. from 3 independent experiments. (F) The results suggest that lysosomes (LY) accumulate Ca2+ released via IP3Rs activated by IP3 alone or IP3 with cAMP.

Mentions: The mechanisms responsible for Ca2+ uptake into lysosomes are not known, although they require the pH gradient established across lysosomal membranes by the V-ATPase that pumps H+ into the lumen of lysosomes [14]. We [30] and others [28] recently provided evidence that lysosomes can also shape the Ca2+ signals evoked by IP3-evoked Ca2+ release from the ER. In our analysis, we demonstrated that dynamic lysosomes are associated with ER and that they selectively accumulate Ca2+ released by IP3Rs. But lysosomes do not sequester Ca2+ entering the cell via SOCE activated pharmacologically by inhibition of the SR/ER Ca2+-ATPase (SERCA) or by buffering of ER luminal Ca2+[30]. Collectively, these observations suggest that lysosomes, like mitochondria [11], dynamically and intimately associate with ER. These associations contribute to both shaping IP3-evoked Ca2+ signals and to providing lysosomes with Ca2+ that might regulate their behaviour [30]. Here, we address three further questions relating to the interaction between lysosomes and IP3-evoked Ca2+ signals. First, we have argued that receptors, like the endogenous M3 muscarinic receptors of human embryonic kidney (HEK) cells, locally deliver IP3 to IP3Rs within signalling junctions, whereas different ‘extra-junctional’ IP3Rs release Ca2+ from distinct Ca2+ stores in response to lower concentrations of IP3 when their sensitivity is increased by cAMP [31] (Figure 1A). Do lysosomes sequester Ca2+ released from each of these IP3-sensitive Ca2+ stores? Second, does the SOCE evoked by physiological stimuli (rather than thapsigargin) direct Ca2+ to lysosomes? The answer to this question is important because it addresses whether a significant fraction of the Ca2+ entering cells via SOCE then passes through the ER and IP3Rs before re-entering the cytosol [32], [33]. Finally, and most importantly, are lysosomes ‘dead-end’ compartments for Ca2+, or is the Ca2+ they accumulate rapidly recycled to sustain refilling of ER Ca2+ stores?


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

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

Lysosomes accumulate Ca2+ released from intracellular stores by IP3 alone or IP3 with cAMP.(A) CCh stimulates M3 muscarinic receptors leading to activation of PLC and IP3-evoked Ca2+ release from the ER. PTH, via type 1 PTH receptors, stimulates adenylyl cyclase. Cyclic AMP sensitizes IP3Rs to IP3 and thereby potentiates the Ca2+ release evoked by CCh. We suggest that cAMP is delivered to IP3Rs at high concentrations within signalling junctions [34] and that the IP3Rs that respond to CCh alone are activated by locally delivered IP3[31]. This local signalling allows CCh alone and CCh in combination with PTH to release Ca2+ from different stores [31]. (B) Bafilomycin A1 (Baf A1) inhibits the V-ATPase that mediates H+ accumulation by lysosomes, and thereby prevents lysosomal Ca2+ uptake. The latter may be mediated by H+-Ca2+ exchange. (C) Populations of HEK-PR1 cells were stimulated with CCh (1 mM) and then PTH (1 µM) with or without prior treatment with bafilomycin A1 (1 µM, 1 h). BAPTA (10 mM) was added as shown to chelate extracellular Ca2+. Results are means ± S.E. from 3 wells from one experiment, typical of 3 similar experiments. (D) Summary results show effects of bafilomycin A1 on the amplitudes of the peak Ca2+ signals evoked by addition of CCh, or PTH after CCh. Results (as percentages of the responses without bafilomycin A1) are means ± S.E. from 3 independent experiments. (E) Experiments similar to those in C, show the effects of bafilomycin A1 on the concentration-dependent effects of PTH on CCh-evoked Ca2+ signals. Results are means ± S.E. from 3 independent experiments. (F) The results suggest that lysosomes (LY) accumulate Ca2+ released via IP3Rs activated by IP3 alone or IP3 with cAMP.
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pone-0111275-g001: Lysosomes accumulate Ca2+ released from intracellular stores by IP3 alone or IP3 with cAMP.(A) CCh stimulates M3 muscarinic receptors leading to activation of PLC and IP3-evoked Ca2+ release from the ER. PTH, via type 1 PTH receptors, stimulates adenylyl cyclase. Cyclic AMP sensitizes IP3Rs to IP3 and thereby potentiates the Ca2+ release evoked by CCh. We suggest that cAMP is delivered to IP3Rs at high concentrations within signalling junctions [34] and that the IP3Rs that respond to CCh alone are activated by locally delivered IP3[31]. This local signalling allows CCh alone and CCh in combination with PTH to release Ca2+ from different stores [31]. (B) Bafilomycin A1 (Baf A1) inhibits the V-ATPase that mediates H+ accumulation by lysosomes, and thereby prevents lysosomal Ca2+ uptake. The latter may be mediated by H+-Ca2+ exchange. (C) Populations of HEK-PR1 cells were stimulated with CCh (1 mM) and then PTH (1 µM) with or without prior treatment with bafilomycin A1 (1 µM, 1 h). BAPTA (10 mM) was added as shown to chelate extracellular Ca2+. Results are means ± S.E. from 3 wells from one experiment, typical of 3 similar experiments. (D) Summary results show effects of bafilomycin A1 on the amplitudes of the peak Ca2+ signals evoked by addition of CCh, or PTH after CCh. Results (as percentages of the responses without bafilomycin A1) are means ± S.E. from 3 independent experiments. (E) Experiments similar to those in C, show the effects of bafilomycin A1 on the concentration-dependent effects of PTH on CCh-evoked Ca2+ signals. Results are means ± S.E. from 3 independent experiments. (F) The results suggest that lysosomes (LY) accumulate Ca2+ released via IP3Rs activated by IP3 alone or IP3 with cAMP.
Mentions: The mechanisms responsible for Ca2+ uptake into lysosomes are not known, although they require the pH gradient established across lysosomal membranes by the V-ATPase that pumps H+ into the lumen of lysosomes [14]. We [30] and others [28] recently provided evidence that lysosomes can also shape the Ca2+ signals evoked by IP3-evoked Ca2+ release from the ER. In our analysis, we demonstrated that dynamic lysosomes are associated with ER and that they selectively accumulate Ca2+ released by IP3Rs. But lysosomes do not sequester Ca2+ entering the cell via SOCE activated pharmacologically by inhibition of the SR/ER Ca2+-ATPase (SERCA) or by buffering of ER luminal Ca2+[30]. Collectively, these observations suggest that lysosomes, like mitochondria [11], dynamically and intimately associate with ER. These associations contribute to both shaping IP3-evoked Ca2+ signals and to providing lysosomes with Ca2+ that might regulate their behaviour [30]. Here, we address three further questions relating to the interaction between lysosomes and IP3-evoked Ca2+ signals. First, we have argued that receptors, like the endogenous M3 muscarinic receptors of human embryonic kidney (HEK) cells, locally deliver IP3 to IP3Rs within signalling junctions, whereas different ‘extra-junctional’ IP3Rs release Ca2+ from distinct Ca2+ stores in response to lower concentrations of IP3 when their sensitivity is increased by cAMP [31] (Figure 1A). Do lysosomes sequester Ca2+ released from each of these IP3-sensitive Ca2+ stores? Second, does the SOCE evoked by physiological stimuli (rather than thapsigargin) direct Ca2+ to lysosomes? The answer to this question is important because it addresses whether a significant fraction of the Ca2+ entering cells via SOCE then passes through the ER and IP3Rs before re-entering the cytosol [32], [33]. Finally, and most importantly, are lysosomes ‘dead-end’ compartments for Ca2+, or is the Ca2+ they accumulate rapidly recycled to sustain refilling of ER Ca2+ stores?

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