Korean J Physiol Pharmacol.  2016 Mar;20(2):213-220. 10.4196/kjpp.2016.20.2.213.

Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

• Cuong DV
• Kim HK
• Marquez J
• Kim N
• Ko KS
• Rhee BD
• Han J

• KMID: 2388678
• DOI: http://doi.org/10.4196/kjpp.2016.20.2.213

Abstract

Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 microg/ml BG, 100 microg/ml peptidoglycan (PGN), or 10 microM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation.

Keyword

Beta-1,3-glucan; Mast cell degranulation; Mitochondrial calcium uniporter

MeSH Terms

Animals
Calcimycin
Calcium*
Cytosol
Exocytosis
Inflammation
Ion Transport*
Mast Cells*
Membrane Potentials
Mice*
Mitochondria
Peptidoglycan
Ruthenium Red
Shock
Calcimycin
Calcium
Peptidoglycan
Ruthenium Red

Figure

• 

  Fig. 1 Characterization of mouse bone marrow-derived mast cells (BMMCs) cultured for 4 weeks.(A) Microscopic image of BMMCs (×400 magnification). (B) Toluidine blue-stained image of BMMCs (×400 magnification). (C) Flow cytometry analysis of Fcε RI and c-Kit-positive populations in BMMCs. (D) Confocal microscopy images of cells with positive staining for c-Kit (green) and Fcε RI (red) (×200 magnification).

• 

  Fig. 2 Beta-1,3-glucan stimulation increases cytosolic and mitochondrial Ca2+ levels in BMMCs.(A) Confocal microscopy images of BMMCs (green, 5 µM Fluo4-AM; red, 5 µM Rhod2-AM). (B) Fluorescence intensity trace of mitochondrial Ca2+, cytosolic Ca2+, and mitochondrial membrane potential (ΔΨm) during BG stimulation of mast cell degranulation. (C) Normalized changes in mitochondrial Ca2+ levels monitored in BMMCs stained with Rhod2-AM. (D) Normalized changes in cytosolic Ca2+ levels monitored in BMMCs stained with Fluo4-AM. (E) Normalized changes in ΔΨm monitored in TMRE-stained BMMCs. a, b, and c represent 5, 30, and 50 min, respectively. (F) Normalized comparison of mitochondrial Ca2+ ele vation induced by BG or PGN treatment. (C~E) *p<0.05 vs. time point a, †p<0.05 vs. time point b. (F) *p<0.05 vs. Control

• 

  Fig. 3 BG-induced mast cell degra nulation was inhibited by ruthenium red (RuR), a mitochondrial calcium uniporter (MCU) blocker.(A) Repre sentative fluorescence images of BMMCs stained with LysoTracker Red (LTR). BG treatment gradually reduced LTR intensity. (B) Normalized changes in LTR fluorescence mediated by BG or BG+RuR treatment. (C) Two-dimensional fluorescence intensity image (a, b, and c represent 5, 30, and 50 min, res pectively). (D) Left: Illustration of two phases of degranulation. ① membrane proximal vesicle degranulation (<20 min), ② cytoplasmic vesicle to be tran slocated to membrane for degranulation (>20 min). Right: Cytoplasmic LTR- stained vesicle after BG-induced early phase degranulation of membrane proximal vesicles (>20 min). (E) Changes in the time course of LTR intensity during mast cell degranulation.

• 

  Fig. 4 Degranulation analysis by tryptase release measurement.The tryptase release ratio was monitored in the presence or absence of extracellular Ca2+ (1.8 mM) and/or ruthenium red (5 µM). The Ca2+ ionophore A23187 served as positive control. *p<0.05 vs CT, †p<0.05 vs indicated comparisons.

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