FcepsilonRI cross-linking-induced actin assembly mediates calcium signalling in RBL-2H3 mast cells

Abstract

1. To determine the role of actin assembly in the Ca(2+) signalling of mast cells activated by cross-linking of FcepsilonRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization. 2. In the RBL-2H3 cells, F-actin content was increased by sensitization with anti-dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca(2+) ([Ca(2+)](i)); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments. 3. In the IgE-sensitized RBL-2H3 cells, DNP-human serum albumin (DNP-HSA) augmented actin assembly. DNP-HSA also increased the production of IP(3), [Ca(2+)](i) and degranulation. Cytochalasin D enhanced all of these DNP-HSA-induced effects. 4. In a Ca(2+)-free solution, DNP-HSA induced a transient increase in [Ca(2+)](i), and this increase was accelerated by cytochalasin D. After cessation of the DNP-HSA-induced Ca(2+) release, the re-addition of Ca(2+) induced a sustained increase in [Ca(2+)](i) through capacitative Ca(2+) entry (CCE), and this increase was enhanced by cytochalasin D. 5 The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C. 6. In contrast, neither the Ca(2+) release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D. 7. These results suggest that actin de-polymerization stimulates the FcepsilonRI-mediated signalling to augment the release of Ca(2+) from the endoplasmic reticulum in RBL-2H3 cells.

Figure 1

Effect of cytochalasin D on DNP-HSA-induced β-hexosaminidase degranulation. RBL-2H3 cells were preincubated without (Control) or with jasplakinolide (3 μM) for 45 min (+JP) in the presence (CytD+) or absence (CytD−) of cytochalasin D (300 nM) for the last 15 min followed by the addition of DNP-HSA (10 ng ml⁻¹) for 30 min to induce degranulation. The percentages of released β-hexosaminidase were calculated against the total amount of β-hexosaminidase. Results are expressed as means±s.e.mean of 10–14 experiments. ^**Significantly different with P<0.01. N.S. Not significantly different.

Figure 2

Effect of cytochalasin D on DNP-HSA-induced F-actin polymerization. (a) RBL-2H3 cells were treated without (IgE-untreated) or with IgE (0.05 μg ml⁻¹; IgE-sensitized) overnight. The F-actin content is shown as a percentage of the F-actin content in IgE-untreated cells. (b) IgE-sensitized RBL-2H3 cells were preincubated with (CytD+) or without cytochalasin D (300 nM) for 15 min (CytD−), followed by incubation without (Resting) or with DNP-HSA (10 ng ml⁻¹) for 5 min (+DNP-HSA). The F-actin content is shown as a percentage of the F-actin content at the resting state. Results are expressed as means±s.e.mean of 8–20 experiments. ^**Significantly different with P<0.01. N.S. Not significantly different.

Figure 3

Effect of actin disassembly on [Ca²⁺]_i in IgE-sensitized and untreated cells. Cytochalasin D (CytD) (300 nM) (a) or mycalolide B (MLB) (100 nM) (b) was added to the RBL-2H3 cells without a sensitization by anti-DNP IgE. The IgE-sensitized cells were incubated without (c) or with cytochalasin D for 15 min (d). Ca²⁺ was removed from the medium for 7 min, and re-added for 10 min. (e) The IgE-sensitized cells were incubated with mycalolide B for 15 min. (f) The IgE-sensitized cells were preincubated with jasplakinolide (JP) (3 μM) for 30 min and subsequently incubated with cytochalasin D for 15 min. The IgE-sensitized cells were preincubated with U73122 (g) or U73343 (h) for 10 min and subsequently incubated with cytochalasin D for 15 min. Typical recordings are shown (n=37–99).

Figure 4

Effect of actin disassembly on the DNP-HSA-induced increase in [Ca²⁺]_i. (a) Typical recording of 10 ng ml⁻¹ DNP-HSA-induced increase in [Ca²⁺]_i in the fura-PE3-loaded RBL-2H3 cells without (Control) or with cytochalasin D (CytD+). Cells were preincubated with cytochalasin D (300 nM) for 15 min. (b) Quantified results of [Ca²⁺]_i induced by DNP-HSA (10 ng ml⁻¹). The RBL-2H3 cells were preincubated without (Control) or with jasplakinolide (3 μM) for 45 min (+JP) in the presence (CytD+) or absence (CytD−) of cytochalasin D (300 nM) for the last 15 min. (c) AUC for 10 min after the DNP-HSA stimulation. Results are expressed as means±s.e.mean of 34–49 cells. ^**Significantly different with P<0.01. N.S. Not significantly different.

Figure 5

Effect of cytochalasin D on DNP-HSA-induced IP₃ production. Quantified results of IP₃ production induced by DNP-HSA. The RBL-2H3 cells were preincubated without (Control) or with 300 nM cytochalasin D (CytD+) for 15 min. IP₃ production was measured at ., 30 s, 1, 5 and 10 min after the DNP-HSA (10 ng ml⁻¹) stimulation. Results are expressed as means±s.e.mean of six experiments. Significantly different with ^*P<0.05, ^**P<0.01.

Figure 6

Effect of cytochalasin D on DNP-HSA-induced Ca²⁺ release. (a) Typical recordings of 10 ng ml⁻¹ DNP-HSA-induced Ca²⁺ release in the absence of extracellular Ca²⁺ and in the presence of 0.5 mM EGTA. DNP-HSA was applied 2 min after RBL-2H3 cells were exposed to Ca²⁺-free solution. (b) Typical recordings of DNP-HSA-induced Ca²⁺ release in the cells preincubated with cytochalasin D (300 nM) for 15 min. (c) Time between DNP-HSA stimulation and the first Ca²⁺ release (Lag period) in the cells preincubated without (Control) or with cytochalasin D (300 nM) for 15 min (CytD+). (d) Time between DNP-HSA stimulation and the [Ca²⁺]_i peak (Time to peak) in the cells preincubated without (Control) or with cytochalasin D (300 nM) for 15 min (CytD+). (e) Normalized-piles of AUC of the DNP-HSA-induced Ca²⁺ release in the cells preincubated without (Control) or with cytochalasin D (300 nM) (CytD+). The total AUC for a 5 min DNP-HSA stimulation was taken as 100%. Inset: AUC for 5 min after DNP-HSA stimulation of the cells preincubated without (Control) or with cytochalasin D (CytD+). Results are expressed as means±s.e.mean of 33–36 cells. ^**Significantly different with P<0.01. N.S. Not significantly different.

Figure 7

Augmentation of DNP-HSA-induced Ca²⁺ influx by actin disassembly. (a) The fura-PE3-loaded RBL-2H3 cells were treated with 300 nM cytochalasin D (CytD+), 100 nM mycalolide B (MLB+) or with neither agent (Control) in normal HEPES buffered solution for 15 min. After stimulation with DNP-HSA (10 ng ml⁻¹) for 5 min in the absence of Ca²⁺ (with 0.5 mM EGTA), 1.5 mM Ca²⁺ was added to the medium without EGTA. Typical recordings are shown in panel a. (b) AUC for 10 min after the addition of 1.5 mM Ca²⁺. Results are expressed as means±s.e.mean of 27–57 cells. Significantly different with ^*P<0.05, ^**P<0.01.

Figure 8

Augmentation of DNP-HSA-induced Ca²⁺ influx by actin disassembly and its inhibition by xestospongin C. (a) The fura-PE3-loaded RBL-2H3 cells were stimulated with DNP-HSA (10 ng ml⁻¹) in a Ca²⁺-free solution for 5 min. The cells were subsequently incubated without (Control) or with xestospongin C (10 μM) for 15 min (XestC+). Then 1.5 mM Ca²⁺ was added to external space. Typical recordings are shown in panel a. (b) The fura-PE3-loaded RBL-2H3 cells were stimulated with DNP-HSA (10 ng ml⁻¹) in the Ca²⁺-free solution for 5 min. The cells were subsequently incubated with cytochalasin D (300 nM), and without (CytD+) or with xestospongin C (10 μM) for 15 min (CytD+XestC). Then 1.5 mM Ca²⁺ was added to external space. Typical recordings are shown in panel b. (c) AUC for 10 min after the addition of Ca²⁺. Results are expressed as means±s.e.mean of 37–45 cells. Significantly different with ^**P<0.01.

Figure 9

Effect of actin disassembly on the thapsigargin-induced increase in [Ca²⁺]_i. (a) The fura-PE3-loaded RBL-2H3 cells were treated without (Control) or with 300 nM cytochalasin D for 15 min (CytD+) or 3 μM jasplakinolide for 45 min (JP+). The cells were subsequently stimulated with 100 nM thapsigargin (TG) for 5 min in the absence of Ca²⁺ (with 0.5 mM EGTA). Then 0.1 mM Ca²⁺ was added to the medium without EGTA. Note that 0.1 mM Ca²⁺, instead of 1.5 mM Ca²⁺, was applied to the medium in this series of experiments because capacitative Ca²⁺ entry (CCE) was strongly and maximally activated by thapsigargin. Typical traces are shown in panel a. (b) AUC for 5 min after stimulation with thapsigargin. (c) AUC for 10 min after the addition of 0.1 mM Ca²⁺. Results are expressed as means±s.e.mean of 29–37 cells. N.S. Not significantly different.