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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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Frequency of exocytotic events at different [Ca2+]i. Histogram analysis of the Ca2+ dependence of serotonin secretion in response to activation of ICRAC by antigen (black bars) and when Ca2+ influx occurred via the ionophore 4-Br A23187 (white bars). The data are from 15 RBL-2H3 mucosal mast cells, including the ones shown in Fig. 2, Fig. 3, and Fig. 5. Analyses for each cell were conducted from the time of the first secretory event in response to antigen to the point at which the inhibitor was added (for antigen) and from the time between the first and last secretory events after the addition of 4-Br A23187 (for Ca2+ ionophore). The number of secretory events between these times for all of the cells were expressed as a function of the aggregate length of time the cells spent at the indicated Ca2+ concentrations during each of these time windows.
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Figure 7: Frequency of exocytotic events at different [Ca2+]i. Histogram analysis of the Ca2+ dependence of serotonin secretion in response to activation of ICRAC by antigen (black bars) and when Ca2+ influx occurred via the ionophore 4-Br A23187 (white bars). The data are from 15 RBL-2H3 mucosal mast cells, including the ones shown in Fig. 2, Fig. 3, and Fig. 5. Analyses for each cell were conducted from the time of the first secretory event in response to antigen to the point at which the inhibitor was added (for antigen) and from the time between the first and last secretory events after the addition of 4-Br A23187 (for Ca2+ ionophore). The number of secretory events between these times for all of the cells were expressed as a function of the aggregate length of time the cells spent at the indicated Ca2+ concentrations during each of these time windows.

Mentions: The Ca2+ dependence of secretion in response to activation of ICRAC by antigen and when Ca2+ influx occurs via the ionophore 4-Br A23187 is shown in Fig. 7. It is clear that the mean [Ca2+]i levels at which secretory events are detected are quite similar for the two pathways of Ca2+ influx. When CRAC channels were activated, the frequency of secretory events peaked between 0.6 and 0.8 μM Ca2+. When influx occurred via the Ca2+ ionophore 4-Br A23187, the frequency of secretion was somewhat greater at all Ca2+ concentrations, and it peaked between 0.4 and 0.6 μM Ca2+. We have previously shown that secretion is not seen in the troughs between Ca2+ oscillations or in cells where Ca2+ oscillates from baseline levels (Kim et al. 1997). It is therefore possible that the enhanced secretion seen with the Ca2+ ionophore occurs because intracellular Ca2+ levels remain elevated and no longer oscillate, as they usually do in response to antigen. Alternatively, we found that the average delay between the initial increase in intracellular Ca2+ and the onset of exocytosis in response to antigen was decreased from 34 to 17 s when protein kinase C was preactivated with phorbol myristate acetate (Kim et al. 1997). If, as these results suggest, protein kinase C is not fully activated during the early part of the Ca2+ response to antigen, this might explain why the frequency of secretion is somewhat higher when the Ca2+ ionophore is subsequently added.


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

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

Frequency of exocytotic events at different [Ca2+]i. Histogram analysis of the Ca2+ dependence of serotonin secretion in response to activation of ICRAC by antigen (black bars) and when Ca2+ influx occurred via the ionophore 4-Br A23187 (white bars). The data are from 15 RBL-2H3 mucosal mast cells, including the ones shown in Fig. 2, Fig. 3, and Fig. 5. Analyses for each cell were conducted from the time of the first secretory event in response to antigen to the point at which the inhibitor was added (for antigen) and from the time between the first and last secretory events after the addition of 4-Br A23187 (for Ca2+ ionophore). The number of secretory events between these times for all of the cells were expressed as a function of the aggregate length of time the cells spent at the indicated Ca2+ concentrations during each of these time windows.
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Related In: Results  -  Collection

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

Figure 7: Frequency of exocytotic events at different [Ca2+]i. Histogram analysis of the Ca2+ dependence of serotonin secretion in response to activation of ICRAC by antigen (black bars) and when Ca2+ influx occurred via the ionophore 4-Br A23187 (white bars). The data are from 15 RBL-2H3 mucosal mast cells, including the ones shown in Fig. 2, Fig. 3, and Fig. 5. Analyses for each cell were conducted from the time of the first secretory event in response to antigen to the point at which the inhibitor was added (for antigen) and from the time between the first and last secretory events after the addition of 4-Br A23187 (for Ca2+ ionophore). The number of secretory events between these times for all of the cells were expressed as a function of the aggregate length of time the cells spent at the indicated Ca2+ concentrations during each of these time windows.
Mentions: The Ca2+ dependence of secretion in response to activation of ICRAC by antigen and when Ca2+ influx occurs via the ionophore 4-Br A23187 is shown in Fig. 7. It is clear that the mean [Ca2+]i levels at which secretory events are detected are quite similar for the two pathways of Ca2+ influx. When CRAC channels were activated, the frequency of secretory events peaked between 0.6 and 0.8 μM Ca2+. When influx occurred via the Ca2+ ionophore 4-Br A23187, the frequency of secretion was somewhat greater at all Ca2+ concentrations, and it peaked between 0.4 and 0.6 μM Ca2+. We have previously shown that secretion is not seen in the troughs between Ca2+ oscillations or in cells where Ca2+ oscillates from baseline levels (Kim et al. 1997). It is therefore possible that the enhanced secretion seen with the Ca2+ ionophore occurs because intracellular Ca2+ levels remain elevated and no longer oscillate, as they usually do in response to antigen. Alternatively, we found that the average delay between the initial increase in intracellular Ca2+ and the onset of exocytosis in response to antigen was decreased from 34 to 17 s when protein kinase C was preactivated with phorbol myristate acetate (Kim et al. 1997). If, as these results suggest, protein kinase C is not fully activated during the early part of the Ca2+ response to antigen, this might explain why the frequency of secretion is somewhat higher when the Ca2+ ionophore is subsequently added.

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