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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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NAADP does not contribute to the effects of lysosomes on carbachol-evoked Ca2+ release in HEK cells.(A) HEK cells loaded with dextran-conjugates of Oregon Green (OG, pH-sensitive probe) and Texas Red (TR, inert marker) were stimulated with CCh (1 mM). Results (means ± S.E. from 27 ROI on a single coverslip, representative of at least 3 independent experiments) show that CCh causes the pH of the lysosome lumen to increase. Addition of HBS did not affect OG or TR fluorescence [30]. (B) Similar experiments with and without NED-19 (10 µM, 1 h) show that it has no significant effect on the peak increase in lysosomal pH evoked by CCh. Results (means ± S.E. from 7 experiments) show the peak change in OG fluorescence (ΔRFU, relative fluorescence units). (C) [Ca2+]i was recorded from HEK cells stimulated with CCh (1 mM) alone or with NED-19 (10 µM, 1 h). Results show means ± S.E. from 3 wells in one experiment, typical of 3 experiments. (D) Summary results show the lack of effect of NED-19 on the peak Ca2+ signals evoked by CCh. Results are means ± S.E. from 3 experiments.
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pone-0111275-g002: NAADP does not contribute to the effects of lysosomes on carbachol-evoked Ca2+ release in HEK cells.(A) HEK cells loaded with dextran-conjugates of Oregon Green (OG, pH-sensitive probe) and Texas Red (TR, inert marker) were stimulated with CCh (1 mM). Results (means ± S.E. from 27 ROI on a single coverslip, representative of at least 3 independent experiments) show that CCh causes the pH of the lysosome lumen to increase. Addition of HBS did not affect OG or TR fluorescence [30]. (B) Similar experiments with and without NED-19 (10 µM, 1 h) show that it has no significant effect on the peak increase in lysosomal pH evoked by CCh. Results (means ± S.E. from 7 experiments) show the peak change in OG fluorescence (ΔRFU, relative fluorescence units). (C) [Ca2+]i was recorded from HEK cells stimulated with CCh (1 mM) alone or with NED-19 (10 µM, 1 h). Results show means ± S.E. from 3 wells in one experiment, typical of 3 experiments. (D) Summary results show the lack of effect of NED-19 on the peak Ca2+ signals evoked by CCh. Results are means ± S.E. from 3 experiments.

Mentions: In sea urchin eggs, IP3-evoked Ca2+ release triggers a rapid increase in the luminal pH of lysosomes [28]. We observed a similar response in CCh-stimulated HEK cells [30] (Figure 2A) and attributed it to an exchange of lysosomal H+ for cytosolic Ca2+[30]. Morgan et al., however, suggest a different interpretation for their results. They argue that Ca2+ release from sea urchin lysosomes increases lysosomal pH, and that IP3-evoked Ca2+ release elicits the same response by locally stimulating formation of NAADP and perhaps also by a direct effect of cytosolic Ca2+ on NAADP-evoked Ca2+ release [28]. It is unlikely that such interactions contribute to the effects of lysosomes on IP3-evoked Ca2+ signals in HEK cells. Firstly, active lysosomes attenuate IP3-evoked Ca2+ signals in HEK cells (Figure 1), while they are proposed to amplify them in sea urchin eggs [28]. Secondly, NED-19, an antagonist of NAADP [36], had no effect on the alkalinization of lysosomal pH during stimulation of HEK cells with CCh (Figure 2B). Furthermore, NED-19 did not affect the time course of the Ca2+ signals evoked by a maximally effective concentration of CCh (Figure 2C) or the peak response to any concentration of CCh (Figure 2D).


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

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

NAADP does not contribute to the effects of lysosomes on carbachol-evoked Ca2+ release in HEK cells.(A) HEK cells loaded with dextran-conjugates of Oregon Green (OG, pH-sensitive probe) and Texas Red (TR, inert marker) were stimulated with CCh (1 mM). Results (means ± S.E. from 27 ROI on a single coverslip, representative of at least 3 independent experiments) show that CCh causes the pH of the lysosome lumen to increase. Addition of HBS did not affect OG or TR fluorescence [30]. (B) Similar experiments with and without NED-19 (10 µM, 1 h) show that it has no significant effect on the peak increase in lysosomal pH evoked by CCh. Results (means ± S.E. from 7 experiments) show the peak change in OG fluorescence (ΔRFU, relative fluorescence units). (C) [Ca2+]i was recorded from HEK cells stimulated with CCh (1 mM) alone or with NED-19 (10 µM, 1 h). Results show means ± S.E. from 3 wells in one experiment, typical of 3 experiments. (D) Summary results show the lack of effect of NED-19 on the peak Ca2+ signals evoked by CCh. Results are means ± S.E. from 3 experiments.
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pone-0111275-g002: NAADP does not contribute to the effects of lysosomes on carbachol-evoked Ca2+ release in HEK cells.(A) HEK cells loaded with dextran-conjugates of Oregon Green (OG, pH-sensitive probe) and Texas Red (TR, inert marker) were stimulated with CCh (1 mM). Results (means ± S.E. from 27 ROI on a single coverslip, representative of at least 3 independent experiments) show that CCh causes the pH of the lysosome lumen to increase. Addition of HBS did not affect OG or TR fluorescence [30]. (B) Similar experiments with and without NED-19 (10 µM, 1 h) show that it has no significant effect on the peak increase in lysosomal pH evoked by CCh. Results (means ± S.E. from 7 experiments) show the peak change in OG fluorescence (ΔRFU, relative fluorescence units). (C) [Ca2+]i was recorded from HEK cells stimulated with CCh (1 mM) alone or with NED-19 (10 µM, 1 h). Results show means ± S.E. from 3 wells in one experiment, typical of 3 experiments. (D) Summary results show the lack of effect of NED-19 on the peak Ca2+ signals evoked by CCh. Results are means ± S.E. from 3 experiments.
Mentions: In sea urchin eggs, IP3-evoked Ca2+ release triggers a rapid increase in the luminal pH of lysosomes [28]. We observed a similar response in CCh-stimulated HEK cells [30] (Figure 2A) and attributed it to an exchange of lysosomal H+ for cytosolic Ca2+[30]. Morgan et al., however, suggest a different interpretation for their results. They argue that Ca2+ release from sea urchin lysosomes increases lysosomal pH, and that IP3-evoked Ca2+ release elicits the same response by locally stimulating formation of NAADP and perhaps also by a direct effect of cytosolic Ca2+ on NAADP-evoked Ca2+ release [28]. It is unlikely that such interactions contribute to the effects of lysosomes on IP3-evoked Ca2+ signals in HEK cells. Firstly, active lysosomes attenuate IP3-evoked Ca2+ signals in HEK cells (Figure 1), while they are proposed to amplify them in sea urchin eggs [28]. Secondly, NED-19, an antagonist of NAADP [36], had no effect on the alkalinization of lysosomal pH during stimulation of HEK cells with CCh (Figure 2B). Furthermore, NED-19 did not affect the time course of the Ca2+ signals evoked by a maximally effective concentration of CCh (Figure 2C) or the peak response to any concentration of CCh (Figure 2D).

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