Estimating the Time Course of Pore Expansion During the Spike Phase of Exocytotic Release in Mast Cells of the Beige Mouse

2002 ◽  
Vol 64 (5) ◽  
pp. 979-1010 ◽  
Author(s):  
B Farrell
Keyword(s):  
Mast Cells ◽  
Time Course ◽  
Beige Mouse ◽  
Pore Expansion

scholarly journals Ionic control of the size of the vesicle matrix of beige mouse mast cells.

1991 ◽  
Vol 98 (4) ◽  
pp. 771-790 ◽  
Author(s):  
M J Curran ◽  
M S Brodwick
Keyword(s):  
Mast Cells ◽  
Ionic Composition ◽  
Matrix Material ◽  
Bathing Solution ◽  
Initial Shape ◽  
Beige Mouse ◽  
The Matrix ◽  
Matrix Expansion ◽  
Ph Buffer ◽  
Matrix Contraction

Isolated matrices of the giant secretory vesicles of mast cells of the beige mouse were reliably produced by the osmotic lysis of isolated vesicles. These matrices maintained their form, and their sizes were easily measured using Nomarski optics. The size of the matrix depended on the ionic composition of the bathing solution. The physiologically relevant ions, histamine and serotonin, contracted the matrix. Multivalent cations condensed the matrix relative to univalents. Ag+, acid pH (below 5), and basic pH (above 9) expanded the matrix. In the presence of 10 mM histamine, lowering the pH from 9 to 5 contracted the matrix more than can be attributed to the pH-dependent matrix contraction in zero histamine. The nontitratable organic cation, dimethonium, contracts the matrix with little effect of pH in the range of 5-9. These results suggest that histamine acts as a matrix contractor in the divalent form. The dose-response (contraction) relation for histamine was gradual from micromolar to 316 mM (millimolar) histamine. Experiments with mixtures of histamine and sodium show antagonistic effects on the matrix but are inconsistent with either a model where ions compete for identical sites or a parallel model where ions interact with separate independent sites. In vigorous histamine washoff experiments, the half time for vesicle expansion in 10(-4) M pH buffer was approximately 4 s; in isotonic NaCl solution, it was 0.5 s. When 1 M histamine was presented to closely apposed matrices, fusion resulted. The matrix material returned to its initial shape after being mechanically deformed with a glass probe. These results suggest that the matrix size is controlled by its ion exchange properties. The matrix expansion can quantitatively account for the vesicular size increase observed upon exocytosis (as a postfusional event) and the osmotic nonideality of intact vesicles. The mechanical expansion is probably significant in the widening of the exocytotic pore and the dispersal of the vesicular contents.

scholarly journals A Highly Temperature-sensitive Proton Current in Mouse Bone Marrow–derived Mast Cells

1997 ◽  
Vol 109 (6) ◽  
pp. 731-740 ◽  
Author(s):  
Miyuki Kuno ◽  
Junko Kawawaki ◽  
Fusao Nakamura
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Intracellular Ph ◽  
Time Course ◽  
Reversal Potential ◽  
Outward Current ◽  
Temperature Sensitive ◽  
Mouse Bone Marrow ◽  
Bafilomycin A1 ◽  
Ph Homeostasis

Proton (H+) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q10 of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q10 was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K+, Cl−, and Na+), but depended on the pH gradient, suggesting that H+ (equivalents) is a major ion species carrying the current. The H+ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H+ current was reversibly inhibited by Zn2+ but not by bafilomycin A1, an inhibitor for a vacuolar type H+-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn2+, and depletion of extracellular K+, but not by bafilomycin A1. These results suggest that the H+ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H+ conductance.

scholarly journals Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2α activation

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 561-567 ◽  
Author(s):  
Eriya Kikawada ◽  
Joseph V. Bonventre ◽  
Jonathan P. Arm
Keyword(s):  
Mast Cells ◽  
Immune Responses ◽  
Time Course ◽  
Gene Knockout ◽  
Peritoneal Macrophages ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Group V ◽  
Signaling Mechanisms ◽  
Gene Knockout Mice

Abstract Mast cells may be activated through Toll-like receptors (TLRs) for the dose- and time-dependent release of eicosanoids. However, the signaling mechanisms of TLR-dependent rapid eicosanoid generation are not known. We previously reported a role for group V secretory phospholipase A2 (PLA2) in regulating phagocytosis of zymosan and the ensuing eicosanoid generation in mouse resident peritoneal macrophages, suggesting a role for the enzyme in innate immunity. In the present study, we have used gene knockout mice to define an essential role for MyD88 and cytosolic PLA2α in TLR2-dependent eicosanoid generation. Furthermore, in mast cells lacking group V secretory PLA2, the time course of phosphorylation of ERK1/2 and of cPLA2α was markedly truncated, leading to attenuation of eicosanoid generation in response to stimulation through TLR2, but not through c-kit or FcεRI. These findings provide the first dissection of the mechanisms of TLR-dependent rapid eicosanoid generation, which is MyD88-dependent, requires cPLA2α, and is amplified by group V sPLA2 through its regulation of the sequential phosphorylation and activation of ERK1/2 and cPLA2α. The findings support the suggestion that group V sPLA2 regulates innate immune responses.

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