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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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Mitochondrial inhibitors have little effect on secretion. White bars show spontaneous and antigen-stimulated β-hexosaminidase secretion, the inhibitory effect of lanthanum and the secretion observed when the Ca2+ ionophore 4-Br A23187 was also added to adherent RBL-2H3 mucosal mast cells. Black bars show data obtained from cells that had been exposed to the mitochondrial inhibitors antimycin A (0.1 μM) and oligomycin B (1.2 μM) for 20–30 min (in the presence of glucose) before stimulation. Cells were stimulated with antigen (1 μg/ml), with or without lanthanum (10 μM), and 4-Br A23187 (2 μM) for 10–12 min. Each column shows the mean ± SEM of triplicate determinations from four separate experiments. Stimulated secretion was abolished when cells were incubated with mitochondrial inhibitors in the absence of glucose (not shown), thus confirming that the inhibitors were effective.
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Figure 6: Mitochondrial inhibitors have little effect on secretion. White bars show spontaneous and antigen-stimulated β-hexosaminidase secretion, the inhibitory effect of lanthanum and the secretion observed when the Ca2+ ionophore 4-Br A23187 was also added to adherent RBL-2H3 mucosal mast cells. Black bars show data obtained from cells that had been exposed to the mitochondrial inhibitors antimycin A (0.1 μM) and oligomycin B (1.2 μM) for 20–30 min (in the presence of glucose) before stimulation. Cells were stimulated with antigen (1 μg/ml), with or without lanthanum (10 μM), and 4-Br A23187 (2 μM) for 10–12 min. Each column shows the mean ± SEM of triplicate determinations from four separate experiments. Stimulated secretion was abolished when cells were incubated with mitochondrial inhibitors in the absence of glucose (not shown), thus confirming that the inhibitors were effective.

Mentions: The possibility that mitochondrial Ca2+ sequestration and/or release could affect secretion (Montero et al. 2000) in response to 4-Br A23187 was also explored. Fig. 6 shows the secretory responses of control cells and of cells that had been exposed to antimycin and oligomycin in the continued presence of glucose. Under the latter conditions, ATP levels are maintained by glycolysis, but mitochondrial function is inhibited and mitochondria are no longer able to sequester Ca2+ or synthesize ATP (Mohr and Fewtrell 1990). As expected, inhibition of antigen-induced Ca2+ influx with lanthanum completely abolished β-hexosaminidase secretion from control cells, unless the Ca2+ ionophore 4-Br A23187 was used to carry Ca2+ into the cells (Fig. 6, white bars). Inhibiting mitochondrial function (Fig. 6, black bars) only slightly reduced secretion in response to antigen, which is consistent with the somewhat lower Ca2+ response seen under these conditions (Fig. 4 C). Again, blocking CRAC channels with lanthanum completely inhibited antigen-induced secretion. Furthermore, the Ca2+ ionophore was still able to restore the secretory response in cells whose mitochondria were inhibited and therefore unable to sequester Ca2+ (Fig. 6, black bars). This clearly confirms that secretion in response to 4-Br A23187 is not due to release of Ca2+ from mitochondria.


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

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

Mitochondrial inhibitors have little effect on secretion. White bars show spontaneous and antigen-stimulated β-hexosaminidase secretion, the inhibitory effect of lanthanum and the secretion observed when the Ca2+ ionophore 4-Br A23187 was also added to adherent RBL-2H3 mucosal mast cells. Black bars show data obtained from cells that had been exposed to the mitochondrial inhibitors antimycin A (0.1 μM) and oligomycin B (1.2 μM) for 20–30 min (in the presence of glucose) before stimulation. Cells were stimulated with antigen (1 μg/ml), with or without lanthanum (10 μM), and 4-Br A23187 (2 μM) for 10–12 min. Each column shows the mean ± SEM of triplicate determinations from four separate experiments. Stimulated secretion was abolished when cells were incubated with mitochondrial inhibitors in the absence of glucose (not shown), thus confirming that the inhibitors were effective.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169467&req=5

Figure 6: Mitochondrial inhibitors have little effect on secretion. White bars show spontaneous and antigen-stimulated β-hexosaminidase secretion, the inhibitory effect of lanthanum and the secretion observed when the Ca2+ ionophore 4-Br A23187 was also added to adherent RBL-2H3 mucosal mast cells. Black bars show data obtained from cells that had been exposed to the mitochondrial inhibitors antimycin A (0.1 μM) and oligomycin B (1.2 μM) for 20–30 min (in the presence of glucose) before stimulation. Cells were stimulated with antigen (1 μg/ml), with or without lanthanum (10 μM), and 4-Br A23187 (2 μM) for 10–12 min. Each column shows the mean ± SEM of triplicate determinations from four separate experiments. Stimulated secretion was abolished when cells were incubated with mitochondrial inhibitors in the absence of glucose (not shown), thus confirming that the inhibitors were effective.
Mentions: The possibility that mitochondrial Ca2+ sequestration and/or release could affect secretion (Montero et al. 2000) in response to 4-Br A23187 was also explored. Fig. 6 shows the secretory responses of control cells and of cells that had been exposed to antimycin and oligomycin in the continued presence of glucose. Under the latter conditions, ATP levels are maintained by glycolysis, but mitochondrial function is inhibited and mitochondria are no longer able to sequester Ca2+ or synthesize ATP (Mohr and Fewtrell 1990). As expected, inhibition of antigen-induced Ca2+ influx with lanthanum completely abolished β-hexosaminidase secretion from control cells, unless the Ca2+ ionophore 4-Br A23187 was used to carry Ca2+ into the cells (Fig. 6, white bars). Inhibiting mitochondrial function (Fig. 6, black bars) only slightly reduced secretion in response to antigen, which is consistent with the somewhat lower Ca2+ response seen under these conditions (Fig. 4 C). Again, blocking CRAC channels with lanthanum completely inhibited antigen-induced secretion. Furthermore, the Ca2+ ionophore was still able to restore the secretory response in cells whose mitochondria were inhibited and therefore unable to sequester Ca2+ (Fig. 6, black bars). This clearly confirms that secretion in response to 4-Br A23187 is not due to release of Ca2+ from mitochondria.

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