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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 releases additional intracellular Ca2+ from antigen-stimulated mucosal mast cells. (A) RBL-2H3 cells in suspension were stimulated with antigen (1 μg/ml); extracellular Ca2+ was then chelated with 2 mM EGTA. Subsequent addition of 2 μM 4-Br A23187 (I) induced a transient Ca2+ response due to incomplete depletion of antigen-sensitive stores or release from a different pool. (B) Addition of thapsigargin (1 μM) after antigen almost completely abolished the subsequent response to 4-Br A23187 in the absence of extracellular Ca2+. (C) Cells were exposed to the mitochondrial inhibitors, antimycin A (0.1 μM) and oligomycin B (1.2 μM), for ∼40 min (in the presence of glucose) before stimulating with antigen and thapsigargin. After EGTA addition, a very small Ca2+ response was still observed in response to 4-Br A23187. Similar results were obtained in four separate experiments.
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Figure 4: 4-Br A23187 releases additional intracellular Ca2+ from antigen-stimulated mucosal mast cells. (A) RBL-2H3 cells in suspension were stimulated with antigen (1 μg/ml); extracellular Ca2+ was then chelated with 2 mM EGTA. Subsequent addition of 2 μM 4-Br A23187 (I) induced a transient Ca2+ response due to incomplete depletion of antigen-sensitive stores or release from a different pool. (B) Addition of thapsigargin (1 μM) after antigen almost completely abolished the subsequent response to 4-Br A23187 in the absence of extracellular Ca2+. (C) Cells were exposed to the mitochondrial inhibitors, antimycin A (0.1 μM) and oligomycin B (1.2 μM), for ∼40 min (in the presence of glucose) before stimulating with antigen and thapsigargin. After EGTA addition, a very small Ca2+ response was still observed in response to 4-Br A23187. Similar results were obtained in four separate experiments.

Mentions: The residual Ca2+ signal seen in both depolarized and SK&F 96365–treated RBL-2H3 cells could result from incomplete inhibition of ICRAC. This is of concern since subsequent addition of the ionophore 4-Br A23187 might further deplete stores and activate additional CRAC channels. This would then argue against our assumption that the increase in [Ca2+]i is due solely to ionophore-mediated Ca2+ entry across the plasma membrane. In order to test this hypothesis, [Ca2+]i measurements in RBL-2H3 cell populations were performed in the absence of extracellular Ca2+. Fig. 4 A confirms that, after antigen addition, chelation of extracellular Ca2+ with EGTA completely abolishes the sustained increase in Ca2+, which is due to Ca2+ influx. Subsequent addition of 4-Br A23187 elicited a transient increase in [Ca2+]i, which could be due to incomplete Ca2+ release from stores in response to antigen. Indeed, when the Ca2+–ATPase inhibitor thapsigargin was added to antigen-stimulated cells, addition of 4-Br A23187 in the presence of EGTA elicited a much smaller Ca2+ response (Fig. 4 B). Prior exposure of the cells to the mitochondrial inhibitors, antimycin and oligomycin, caused a slight further reduction in the Ca2+ response to 4-Br A23187 after antigen and thapsigargin (Fig. 4 C). This suggests that only a very small part of the Ca2+ response to 4-Br A23187 could be due to release of Ca2+ from mitochondria. Since mitochondria are known to accumulate Ca2+ when the cells are stimulated with antigen (Mohr and Fewtrell 1990) or thapsigargin (Ali et al. 1994), this contribution might be greater if the Ca2+ ionophore was added while intracellular [Ca2+]i was still high, and the mitochondria were loaded with calcium. However, under the conditions used in our experiments (Fig. 2Fig. 3Fig. 4Fig. 5), intracellular [Ca2+]i had returned close to resting levels before the ionophore was added. The mitochondria should therefore have released most of their calcium. The tiny residual response to 4-Br A23187 seen in Fig. 4 C presumably originates from a different population of stores (Pizzo et al. 1997; Huang and Putney 1998).


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 releases additional intracellular Ca2+ from antigen-stimulated mucosal mast cells. (A) RBL-2H3 cells in suspension were stimulated with antigen (1 μg/ml); extracellular Ca2+ was then chelated with 2 mM EGTA. Subsequent addition of 2 μM 4-Br A23187 (I) induced a transient Ca2+ response due to incomplete depletion of antigen-sensitive stores or release from a different pool. (B) Addition of thapsigargin (1 μM) after antigen almost completely abolished the subsequent response to 4-Br A23187 in the absence of extracellular Ca2+. (C) Cells were exposed to the mitochondrial inhibitors, antimycin A (0.1 μM) and oligomycin B (1.2 μM), for ∼40 min (in the presence of glucose) before stimulating with antigen and thapsigargin. After EGTA addition, a very small Ca2+ response was still observed in response to 4-Br A23187. Similar results were obtained in four separate experiments.
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Related In: Results  -  Collection

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Figure 4: 4-Br A23187 releases additional intracellular Ca2+ from antigen-stimulated mucosal mast cells. (A) RBL-2H3 cells in suspension were stimulated with antigen (1 μg/ml); extracellular Ca2+ was then chelated with 2 mM EGTA. Subsequent addition of 2 μM 4-Br A23187 (I) induced a transient Ca2+ response due to incomplete depletion of antigen-sensitive stores or release from a different pool. (B) Addition of thapsigargin (1 μM) after antigen almost completely abolished the subsequent response to 4-Br A23187 in the absence of extracellular Ca2+. (C) Cells were exposed to the mitochondrial inhibitors, antimycin A (0.1 μM) and oligomycin B (1.2 μM), for ∼40 min (in the presence of glucose) before stimulating with antigen and thapsigargin. After EGTA addition, a very small Ca2+ response was still observed in response to 4-Br A23187. Similar results were obtained in four separate experiments.
Mentions: The residual Ca2+ signal seen in both depolarized and SK&F 96365–treated RBL-2H3 cells could result from incomplete inhibition of ICRAC. This is of concern since subsequent addition of the ionophore 4-Br A23187 might further deplete stores and activate additional CRAC channels. This would then argue against our assumption that the increase in [Ca2+]i is due solely to ionophore-mediated Ca2+ entry across the plasma membrane. In order to test this hypothesis, [Ca2+]i measurements in RBL-2H3 cell populations were performed in the absence of extracellular Ca2+. Fig. 4 A confirms that, after antigen addition, chelation of extracellular Ca2+ with EGTA completely abolishes the sustained increase in Ca2+, which is due to Ca2+ influx. Subsequent addition of 4-Br A23187 elicited a transient increase in [Ca2+]i, which could be due to incomplete Ca2+ release from stores in response to antigen. Indeed, when the Ca2+–ATPase inhibitor thapsigargin was added to antigen-stimulated cells, addition of 4-Br A23187 in the presence of EGTA elicited a much smaller Ca2+ response (Fig. 4 B). Prior exposure of the cells to the mitochondrial inhibitors, antimycin and oligomycin, caused a slight further reduction in the Ca2+ response to 4-Br A23187 after antigen and thapsigargin (Fig. 4 C). This suggests that only a very small part of the Ca2+ response to 4-Br A23187 could be due to release of Ca2+ from mitochondria. Since mitochondria are known to accumulate Ca2+ when the cells are stimulated with antigen (Mohr and Fewtrell 1990) or thapsigargin (Ali et al. 1994), this contribution might be greater if the Ca2+ ionophore was added while intracellular [Ca2+]i was still high, and the mitochondria were loaded with calcium. However, under the conditions used in our experiments (Fig. 2Fig. 3Fig. 4Fig. 5), intracellular [Ca2+]i had returned close to resting levels before the ionophore was added. The mitochondria should therefore have released most of their calcium. The tiny residual response to 4-Br A23187 seen in Fig. 4 C presumably originates from a different population of stores (Pizzo et al. 1997; Huang and Putney 1998).

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