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Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells.

Mahmoud SF, Fewtrell C - J. Cell Biol. (2001)

Bottom Line: Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion.Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion.Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
We have previously shown that store-associated microdomains of high Ca(2+) are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca(2+) microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca(2+) influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca(2+) release-activated Ca(2+) (CRAC) current (I(CRAC)) through CRAC channels. In the second, a Ca(2+) ionophore was used to transport Ca(2+) randomly across the plasma membrane. Since store depletion by Ca(2+) ionophore will also activate I(CRAC), different means of inhibiting I(CRAC) before ionophore addition were used. Ca(2+) responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.

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4-Br A23187 also triggers exocytosis when ICRAC is inhibited by depolarizing the cells in high K+. The increase in [Ca2+]i and exocytosis induced by 1 μg/ml antigen (Ag) were inhibited in individual RBL-2H3 cells upon exposure to high potassium saline (140 mM). Addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I) was effective in triggering additional secretory events at [Ca2+]i levels similar to those induced by antigen. Again, no secretory events were observed when [Ca2+]i reached very high levels in response to 4-Br A23187. Individual cell responses showed that depolarization in high K+ did not entirely inhibit the Ca2+ response, whereas exocytotic events were completely abolished. Addition of 0.05% saponin (S) lysed the cells and released the remaining serotonin. The 4 cells shown are representative of 12 cells in which secretion was seen. 10 cells responded to both antigen and ionophore, one responded to antigen but not ionophore, whereas one cell failed to secrete in response to antigen but did so when ionophore was added.
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Figure 3: 4-Br A23187 also triggers exocytosis when ICRAC is inhibited by depolarizing the cells in high K+. The increase in [Ca2+]i and exocytosis induced by 1 μg/ml antigen (Ag) were inhibited in individual RBL-2H3 cells upon exposure to high potassium saline (140 mM). Addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I) was effective in triggering additional secretory events at [Ca2+]i levels similar to those induced by antigen. Again, no secretory events were observed when [Ca2+]i reached very high levels in response to 4-Br A23187. Individual cell responses showed that depolarization in high K+ did not entirely inhibit the Ca2+ response, whereas exocytotic events were completely abolished. Addition of 0.05% saponin (S) lysed the cells and released the remaining serotonin. The 4 cells shown are representative of 12 cells in which secretion was seen. 10 cells responded to both antigen and ionophore, one responded to antigen but not ionophore, whereas one cell failed to secrete in response to antigen but did so when ionophore was added.

Mentions: A residual Ca2+ signal was usually observed after ICRAC inhibition by SK&F 96365 (Fig. 2). Although this might simply be due to an elevation in the resting level of Ca2+, as described earlier (Fig. 1 B), it could also result from incomplete inhibition of ICRAC. Other means of inhibiting Ca2+ influx via CRAC channels were therefore explored. Depolarizing the cells in high potassium is known to inhibit Ca2+ influx (Mohr and Fewtrell 1987), and we confirmed that both the increase in [Ca2+]i and secretory events induced by antigen are inhibited in single RBL-2H3 cells upon depolarization in a high potassium (140 mM) saline solution (Fig. 3). Subsequent addition of 4-Br A23187 increased [Ca2+]i and was effective in triggering additional secretory events. Individual RBL-2H3 cell responses show that depolarization in high K+ does not completely inhibit the Ca2+ response at the physiological Ca2+ concentration used (1.8 mM), whereas exocytotic events were completely abolished (Fig. 3).


Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells.

Mahmoud SF, Fewtrell C - J. Cell Biol. (2001)

4-Br A23187 also triggers exocytosis when ICRAC is inhibited by depolarizing the cells in high K+. The increase in [Ca2+]i and exocytosis induced by 1 μg/ml antigen (Ag) were inhibited in individual RBL-2H3 cells upon exposure to high potassium saline (140 mM). Addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I) was effective in triggering additional secretory events at [Ca2+]i levels similar to those induced by antigen. Again, no secretory events were observed when [Ca2+]i reached very high levels in response to 4-Br A23187. Individual cell responses showed that depolarization in high K+ did not entirely inhibit the Ca2+ response, whereas exocytotic events were completely abolished. Addition of 0.05% saponin (S) lysed the cells and released the remaining serotonin. The 4 cells shown are representative of 12 cells in which secretion was seen. 10 cells responded to both antigen and ionophore, one responded to antigen but not ionophore, whereas one cell failed to secrete in response to antigen but did so when ionophore was added.
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Related In: Results  -  Collection

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Figure 3: 4-Br A23187 also triggers exocytosis when ICRAC is inhibited by depolarizing the cells in high K+. The increase in [Ca2+]i and exocytosis induced by 1 μg/ml antigen (Ag) were inhibited in individual RBL-2H3 cells upon exposure to high potassium saline (140 mM). Addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I) was effective in triggering additional secretory events at [Ca2+]i levels similar to those induced by antigen. Again, no secretory events were observed when [Ca2+]i reached very high levels in response to 4-Br A23187. Individual cell responses showed that depolarization in high K+ did not entirely inhibit the Ca2+ response, whereas exocytotic events were completely abolished. Addition of 0.05% saponin (S) lysed the cells and released the remaining serotonin. The 4 cells shown are representative of 12 cells in which secretion was seen. 10 cells responded to both antigen and ionophore, one responded to antigen but not ionophore, whereas one cell failed to secrete in response to antigen but did so when ionophore was added.
Mentions: A residual Ca2+ signal was usually observed after ICRAC inhibition by SK&F 96365 (Fig. 2). Although this might simply be due to an elevation in the resting level of Ca2+, as described earlier (Fig. 1 B), it could also result from incomplete inhibition of ICRAC. Other means of inhibiting Ca2+ influx via CRAC channels were therefore explored. Depolarizing the cells in high potassium is known to inhibit Ca2+ influx (Mohr and Fewtrell 1987), and we confirmed that both the increase in [Ca2+]i and secretory events induced by antigen are inhibited in single RBL-2H3 cells upon depolarization in a high potassium (140 mM) saline solution (Fig. 3). Subsequent addition of 4-Br A23187 increased [Ca2+]i and was effective in triggering additional secretory events. Individual RBL-2H3 cell responses show that depolarization in high K+ does not completely inhibit the Ca2+ response at the physiological Ca2+ concentration used (1.8 mM), whereas exocytotic events were completely abolished (Fig. 3).

Bottom Line: Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion.Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion.Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
We have previously shown that store-associated microdomains of high Ca(2+) are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca(2+) microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca(2+) influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca(2+) release-activated Ca(2+) (CRAC) current (I(CRAC)) through CRAC channels. In the second, a Ca(2+) ionophore was used to transport Ca(2+) randomly across the plasma membrane. Since store depletion by Ca(2+) ionophore will also activate I(CRAC), different means of inhibiting I(CRAC) before ionophore addition were used. Ca(2+) responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.

Show MeSH
Related in: MedlinePlus