Limits...
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.

Show MeSH

Related in: MedlinePlus

Lysosomes do not accumulate Ca2+ entering cells via store-operated Ca2+ entry evoked by carbachol.(A) Ca2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP3Rs from which some Ca2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca2+-free HBS alone (B) or with bafilomycin A1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca2+ signal attributable to Ca2+ entry (ΔΔ[Ca2+]i) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca2+]i evoked by CCh in normal or Ca2+-free HBS, with and without bafilomycin A1-treatment. Results (percentages of the responses to CCh alone in Ca2+-free HBS) are means ± S.E. from 4 experiments. *p <0.05, paired Students's t-test using the raw data. (E) Similar analysis (means ± S.E., n  =  4) shows ΔΔ[Ca2+]i recorded 2 min after CCh addition. (F) Cells were stimulated with CCh (1 mM, 15 min) in nominally Ca2+-free HBS with or without bafilomycin A1 (1 µM, 1 h) before restoration of the indicated concentrations of extracellular Ca2+. Results (means ± S.E., n  =  4) show the sustained increase in [Ca2+]i.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206489&req=5

pone-0111275-g004: Lysosomes do not accumulate Ca2+ entering cells via store-operated Ca2+ entry evoked by carbachol.(A) Ca2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP3Rs from which some Ca2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca2+-free HBS alone (B) or with bafilomycin A1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca2+ signal attributable to Ca2+ entry (ΔΔ[Ca2+]i) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca2+]i evoked by CCh in normal or Ca2+-free HBS, with and without bafilomycin A1-treatment. Results (percentages of the responses to CCh alone in Ca2+-free HBS) are means ± S.E. from 4 experiments. *p <0.05, paired Students's t-test using the raw data. (E) Similar analysis (means ± S.E., n  =  4) shows ΔΔ[Ca2+]i recorded 2 min after CCh addition. (F) Cells were stimulated with CCh (1 mM, 15 min) in nominally Ca2+-free HBS with or without bafilomycin A1 (1 µM, 1 h) before restoration of the indicated concentrations of extracellular Ca2+. Results (means ± S.E., n  =  4) show the sustained increase in [Ca2+]i.

Mentions: Our previous analysis established that lysosomes selectively accumulate Ca2+ released from the ER, but not Ca2+ entering cells via SOCE evoked by thapsigargin [30]. It is not known whether lysosomes affect SOCE evoked by CCh. The question is important because Ca2+ entering the cell via SOCE can locally regulate specific intracellular events [43], [44], but it is unclear whether it can also pass through the ER and so re-enter the cytosol via IP3Rs [32], [33]. The latter route is impossible when the SR/ER Ca2+-ATPase (SERCA) is inhibited by thapsigargin (Figure 4A). We therefore considered the possibility that CCh-evoked SOCE might be modulated by lysosomal Ca2+ uptake systems if a significant fraction of the Ca2+ entering by SOCE passed through the ER via SERCA and IP3Rs (Figure 4A). Evidence that CCh-evoked Ca2+ entry in HEK-PR1 cells is mediated by SOCE (Figure 3) [37] allows this issue to be addressed


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

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

Lysosomes do not accumulate Ca2+ entering cells via store-operated Ca2+ entry evoked by carbachol.(A) Ca2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP3Rs from which some Ca2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca2+-free HBS alone (B) or with bafilomycin A1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca2+ signal attributable to Ca2+ entry (ΔΔ[Ca2+]i) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca2+]i evoked by CCh in normal or Ca2+-free HBS, with and without bafilomycin A1-treatment. Results (percentages of the responses to CCh alone in Ca2+-free HBS) are means ± S.E. from 4 experiments. *p <0.05, paired Students's t-test using the raw data. (E) Similar analysis (means ± S.E., n  =  4) shows ΔΔ[Ca2+]i recorded 2 min after CCh addition. (F) Cells were stimulated with CCh (1 mM, 15 min) in nominally Ca2+-free HBS with or without bafilomycin A1 (1 µM, 1 h) before restoration of the indicated concentrations of extracellular Ca2+. Results (means ± S.E., n  =  4) show the sustained increase in [Ca2+]i.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111275-g004: Lysosomes do not accumulate Ca2+ entering cells via store-operated Ca2+ entry evoked by carbachol.(A) Ca2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP3Rs from which some Ca2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca2+-free HBS alone (B) or with bafilomycin A1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca2+ signal attributable to Ca2+ entry (ΔΔ[Ca2+]i) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca2+]i evoked by CCh in normal or Ca2+-free HBS, with and without bafilomycin A1-treatment. Results (percentages of the responses to CCh alone in Ca2+-free HBS) are means ± S.E. from 4 experiments. *p <0.05, paired Students's t-test using the raw data. (E) Similar analysis (means ± S.E., n  =  4) shows ΔΔ[Ca2+]i recorded 2 min after CCh addition. (F) Cells were stimulated with CCh (1 mM, 15 min) in nominally Ca2+-free HBS with or without bafilomycin A1 (1 µM, 1 h) before restoration of the indicated concentrations of extracellular Ca2+. Results (means ± S.E., n  =  4) show the sustained increase in [Ca2+]i.
Mentions: Our previous analysis established that lysosomes selectively accumulate Ca2+ released from the ER, but not Ca2+ entering cells via SOCE evoked by thapsigargin [30]. It is not known whether lysosomes affect SOCE evoked by CCh. The question is important because Ca2+ entering the cell via SOCE can locally regulate specific intracellular events [43], [44], but it is unclear whether it can also pass through the ER and so re-enter the cytosol via IP3Rs [32], [33]. The latter route is impossible when the SR/ER Ca2+-ATPase (SERCA) is inhibited by thapsigargin (Figure 4A). We therefore considered the possibility that CCh-evoked SOCE might be modulated by lysosomal Ca2+ uptake systems if a significant fraction of the Ca2+ entering by SOCE passed through the ER via SERCA and IP3Rs (Figure 4A). Evidence that CCh-evoked Ca2+ entry in HEK-PR1 cells is mediated by SOCE (Figure 3) [37] allows this issue to be addressed

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