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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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The Ca2+ ionophore 4-Br A23187 is effective in increasing [Ca2+]i and eliciting secretion when ICRAC is inhibited by SK&F 96365. Antigen (Ag) (1 μg/ml) stimulation of individual RBL-2H3 cells elicited an increase in intracellular Ca2+ (upper trace) as well as a burst of amperometric events (lower trace) corresponding to exocytosis of secretory granules. Four typical single-cell responses are shown. When Ca2+ influx was inhibited by adding 50 μM SK&F 96365, intracellular Ca2+ dropped, and secretion was immediately halted. Subsequent addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I, respectively) caused an increase in intracellular Ca2+ to levels similar to those seen in response to antigen and a resumption of secretion. When [Ca2+]i rose to higher levels, a burst of secretory events could often be seen on the rising phase (B and D). However, once the [Ca2+]i reached maximum levels (>1,200 nM), secretion was inhibited. Lysis of the cell with 0.05% saponin (S) released the remaining intracellular serotonin (B and C). The 4 cells shown are representative of 11 cells in which secretion was seen; all 11 cells secreted in response to both antigen and ionophore.
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Figure 2: The Ca2+ ionophore 4-Br A23187 is effective in increasing [Ca2+]i and eliciting secretion when ICRAC is inhibited by SK&F 96365. Antigen (Ag) (1 μg/ml) stimulation of individual RBL-2H3 cells elicited an increase in intracellular Ca2+ (upper trace) as well as a burst of amperometric events (lower trace) corresponding to exocytosis of secretory granules. Four typical single-cell responses are shown. When Ca2+ influx was inhibited by adding 50 μM SK&F 96365, intracellular Ca2+ dropped, and secretion was immediately halted. Subsequent addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I, respectively) caused an increase in intracellular Ca2+ to levels similar to those seen in response to antigen and a resumption of secretion. When [Ca2+]i rose to higher levels, a burst of secretory events could often be seen on the rising phase (B and D). However, once the [Ca2+]i reached maximum levels (>1,200 nM), secretion was inhibited. Lysis of the cell with 0.05% saponin (S) released the remaining intracellular serotonin (B and C). The 4 cells shown are representative of 11 cells in which secretion was seen; all 11 cells secreted in response to both antigen and ionophore.

Mentions: Antigen stimulation of individual RBL-2H3 cells elicited an increase in intracellular Ca2+ as well as a burst of secretory events (Fig. 2). Four typical cell responses are presented. As we have previously shown (Kim et al. 1997), secretion occurred when [Ca2+]i was elevated and, when [Ca2+]i oscillated, secretory events were clustered around the peaks of oscillations. When Ca2+ influx was inhibited by adding 50 μM SK&F 96365, intracellular [Ca2+]i dropped and secretion was immediately halted. When the ionophore 4-Br A23187 was subsequently added, an increase in intracellular Ca2+ and resumption of secretory events were observed. A concentration of 1–2 μM 4-Br A23187 was needed in order to achieve a significant increase in the mean [Ca2+]i after inhibition of ICRAC. These concentrations were not cytotoxic to the cells, as assessed by the retention of intracellular indo-1. In different responsive cells, the average [Ca2+]i that was effective in triggering secretion in response to 4-Br A23187 was in the range of 300–600 nM. This is equal to or less than the effective [Ca2+]i observed in response to antigen at the beginning of the experiment, which served as an internal control. However, when [Ca2+]i reached very high levels (>1,200 nM) during exposure to 4-Br A23187, secretion was never observed. Therefore, secretion is inhibited at extremely high concentrations of Ca2+, which do not usually occur in response to physiological stimuli. One notable feature, however, is that when [Ca2+]i rises rapidly to maximum levels, a burst of secretory events can often be seen on the rising phase (Fig. 2B and Fig. D). This supports the idea that, although an increase in [Ca2+]i is needed for secretion, once [Ca2+]i reaches unphysiologically high levels, the cells stop secreting. Under these conditions, the cells remain viable since there is no loss of intracellular indo-1. At the end of each experiment, saponin lysis released the remaining intracellular serotonin as well as the fluorescent dye indo-1.


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

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

The Ca2+ ionophore 4-Br A23187 is effective in increasing [Ca2+]i and eliciting secretion when ICRAC is inhibited by SK&F 96365. Antigen (Ag) (1 μg/ml) stimulation of individual RBL-2H3 cells elicited an increase in intracellular Ca2+ (upper trace) as well as a burst of amperometric events (lower trace) corresponding to exocytosis of secretory granules. Four typical single-cell responses are shown. When Ca2+ influx was inhibited by adding 50 μM SK&F 96365, intracellular Ca2+ dropped, and secretion was immediately halted. Subsequent addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I, respectively) caused an increase in intracellular Ca2+ to levels similar to those seen in response to antigen and a resumption of secretion. When [Ca2+]i rose to higher levels, a burst of secretory events could often be seen on the rising phase (B and D). However, once the [Ca2+]i reached maximum levels (>1,200 nM), secretion was inhibited. Lysis of the cell with 0.05% saponin (S) released the remaining intracellular serotonin (B and C). The 4 cells shown are representative of 11 cells in which secretion was seen; all 11 cells secreted in response to both antigen and ionophore.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169467&req=5

Figure 2: The Ca2+ ionophore 4-Br A23187 is effective in increasing [Ca2+]i and eliciting secretion when ICRAC is inhibited by SK&F 96365. Antigen (Ag) (1 μg/ml) stimulation of individual RBL-2H3 cells elicited an increase in intracellular Ca2+ (upper trace) as well as a burst of amperometric events (lower trace) corresponding to exocytosis of secretory granules. Four typical single-cell responses are shown. When Ca2+ influx was inhibited by adding 50 μM SK&F 96365, intracellular Ca2+ dropped, and secretion was immediately halted. Subsequent addition of 1 or 2 μM 4-Br A23187 (1 I or 2 I, respectively) caused an increase in intracellular Ca2+ to levels similar to those seen in response to antigen and a resumption of secretion. When [Ca2+]i rose to higher levels, a burst of secretory events could often be seen on the rising phase (B and D). However, once the [Ca2+]i reached maximum levels (>1,200 nM), secretion was inhibited. Lysis of the cell with 0.05% saponin (S) released the remaining intracellular serotonin (B and C). The 4 cells shown are representative of 11 cells in which secretion was seen; all 11 cells secreted in response to both antigen and ionophore.
Mentions: Antigen stimulation of individual RBL-2H3 cells elicited an increase in intracellular Ca2+ as well as a burst of secretory events (Fig. 2). Four typical cell responses are presented. As we have previously shown (Kim et al. 1997), secretion occurred when [Ca2+]i was elevated and, when [Ca2+]i oscillated, secretory events were clustered around the peaks of oscillations. When Ca2+ influx was inhibited by adding 50 μM SK&F 96365, intracellular [Ca2+]i dropped and secretion was immediately halted. When the ionophore 4-Br A23187 was subsequently added, an increase in intracellular Ca2+ and resumption of secretory events were observed. A concentration of 1–2 μM 4-Br A23187 was needed in order to achieve a significant increase in the mean [Ca2+]i after inhibition of ICRAC. These concentrations were not cytotoxic to the cells, as assessed by the retention of intracellular indo-1. In different responsive cells, the average [Ca2+]i that was effective in triggering secretion in response to 4-Br A23187 was in the range of 300–600 nM. This is equal to or less than the effective [Ca2+]i observed in response to antigen at the beginning of the experiment, which served as an internal control. However, when [Ca2+]i reached very high levels (>1,200 nM) during exposure to 4-Br A23187, secretion was never observed. Therefore, secretion is inhibited at extremely high concentrations of Ca2+, which do not usually occur in response to physiological stimuli. One notable feature, however, is that when [Ca2+]i rises rapidly to maximum levels, a burst of secretory events can often be seen on the rising phase (Fig. 2B and Fig. D). This supports the idea that, although an increase in [Ca2+]i is needed for secretion, once [Ca2+]i reaches unphysiologically high levels, the cells stop secreting. Under these conditions, the cells remain viable since there is no loss of intracellular indo-1. At the end of each experiment, saponin lysis released the remaining intracellular serotonin as well as the fluorescent dye indo-1.

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