A quantitative electron histochemical estimation of ruthenium red binding to heparin in mast cell granules

Summary Mast cell granules were shown to contain an apparently branched meshwork of fibers, which had a diameter of about 240 Å and a denser core of about 20–30 Å. Mast cell granules from rats were found to have a median weight of 39×10^–14 g after critical-point drying. Their dry mass increased 23% when stained with ruthenium red at pH 5.0. The staining reaction was found to be stoichiometric, and the bulk of the stain appeared to be taken up by heparin in a molar relationship of one ruthenium red cationic complex per sulfate group of heparin. The uptake of the stain was largely localized to the cores of the granule fibers, which after staining appeared double contoured. These findings suggest that mast cell heparin is integrated into the fibrous structure of the mast cell granules.This project was supported in part by Grant No. P-259-H from the American Cancer Society.The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Mouse mast cell secretory granules can function as intracellular ionic oscillators

Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB/J) and beige (Bg(j)/Bg(j)) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25 +/- 4.2 microM (mean +/- SD, n = 68). Exposure to 3 microM inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of approximately 10 microM amplitude and a period around 8-10 s. The [Ca2+]L oscillations were accompanied by a corresponding oscillatory release of [Ca2+]L to the extraluminal space. Control experiments using ruthenium red (2 microM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca2+]L and Ca2+ release result from a Ca2+/K+ exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K+ into the granular lumen via an apamin-sensitive Ca2+-sensitive K+ channel (ASK(Ca)), whereas Ca2+ release takes place via an InsP3-receptor-Ca2+ (InsP3-R) channel. These results are consistent with previous observations of [Ca2+]L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca2+]L oscillations and quantal periodic Ca2+ release in/from intracellular Ca2+ storage compartments.     

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky’s fixative and OsO₄ and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.     

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.     

Evidence of an adriamycin binding site in the secretory granules of the mast cell

Adriamycin induced significant non-cytotoxic histamine release from rat peritoneal mast cells to which the drug showed a very high affinity. The relationship between adriamycin-induced exocytosis and its uptake by purified rat peritoneal mast cells was studied. Adriamycin induced histamine release and was highly concentrated in mast cells at 37 degrees C but not at 0 degrees C. However, if exocytosis was provoked by other secretagogues like compound 48/80, protamine, concanavalin A, and ionophore A23187, and cells were then treated with adriamycin at 0 degrees C, the concentrations of the antineoplastic drug significantly increased. Adriamycin binding to purified granular material was similar to that of intact cells treated at 37 degrees C, but was not modified by metabolic inhibitors, extremes of temperature (0 or 45 degrees C) or by the carboxylic ionophore monensin. On the contrary, sodium cromoglycate limited adriamycin binding to granular materials as well. In addition, sodium cromoglycate, but not monensin, displaced the antineoplastic drug from mast cells, even when added after adriamycin. We conclude that the high affinity of adriamycin for mast cells is ascribable to the externalization of a granular binding site, as a consequence of the exocytotic process. The experiments with sodium cromoglycate suggest that this binding site could be in common with the antiallergic drug.     

Changes in the distribution of anionic constituents in secretory granules of mouse pancreatic acinar cells after pilocarpine-induced degranulation.

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.     

Conjugated avidin binds to mast cell granules

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.     

Polyamines are present in mast cell secretory granules and are important for granule homeostasis

Background

Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules.

Methodology/Principal Findings

Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system.

Conclusions/Significance

Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.     

The structure of mast cell secretory granules in the blind mole rat (Spalax ehrenbergi)

Eosinophils, basophils, and mast cells produce and secrete active substances whose role is to attack invading parasites and protect the host. In this study we use morphometric methods to study mast cells in the blind mole rat (Spalax ehrenbergi). The subterranean and solitary way of life of this species has led to the evolutionary development of special anatomical, morphological, behavioral, and physiological adaptations. Because of its particular lifestyle, the mole rat is less exposed to parasites than other rodents. This could provide a unique model for research into the pathobiology of mast cells. The paracrystalline structure of the mast cell granule content is composed of parallel plates. Diffraction analysis of electron micrographs of thin sections of araldite-embedded tissues indicated that each crystal line plate is a periodic array of parallelograms. The crystal unit cell volume is approximately 930 nm(3), suggesting that each unit cell is composed of one heparin molecule and one to three additional adsorbed proteins. Morphometric data show that characteristics of the secretory granules of mast cells of the blind mole rat resemble those of other rodents. The mast cell unit granule volume in the present study was calculated to be 0.055 microm(3), similar to that of rat peritoneal mast cells.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Isolation of membrane-bound rat mast cell granules

A technique for obtaining membrane-bound rat peritoneal mast cell granules in high yield is described. Mast cells purified by centrifugation into 38% BSA gradients were sonicated in Ca²⁺, Mg²⁺-free Tyrode's-gelatin buffer, incubated in EDTA for 15 min at 37 °C, and differentially centrifuged through a 0.34 M sucrose cushion to yield a granular preparation with >80% of the granules bound by perigranular membranes. The perigranular membranes were demonstrated morphologically by light and electron microscopy and functionally by histamine distribution.     

A phosphatidylinositol kinase in rat mast cell granules

Intact granules were isolated from sonicated purified rat serosal mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase that catalyzes the formation of diphosphoinositide from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg2+ or Mn2+ for activity; Ca2+, fluoride and cyclic AMP are inhibitory. The Km for ATP is 25 microM. The initial reaction is rapid, but the response ceases within a few minutes. A comparison of the rate of phosphorylation of intact and broken membrane granules suggests that the phosphorylation occurs on the outer (cytoplasmic) surface of the granules.     

Histochemical characterization of anionic constituents in oocyte-cumulus complex of rats

Fertilization is a process that involves the recognition and adhesion of negatively charged spermatozoa to the oocyte investments. It is not known, however, if charge properties of the interacting gametes play a role in fertilization. The present study evaluates the content and distribution of anionic constituents in the oocyte-cumulus complex of rats. Polycationic colloidal gold (PCG), ruthenium red (RR) and wheat germ agglutinin (WGA) were used as cytochemical markers of anionic sites at the light (LM) and electron microscopical (EM) levels. Isolated oocyte-cumulus complexes were fixed with glutaraldehyde (GA) and OsO₄ containing RR, or with GA without RR, and embedded in araldite or LR-gold. For LM, deresined, semi-thin, araldite-embedded sections were labelled with PCG intensified by silver, or with biotinylated lectins visualized by avidinperoxidase. For EM, thin LR-gold sections were labelled with PCG, whereas RR labelling was examined in araldite sections. The zona pellucida (ZP) failed to bind any of the polycationic markers used, but intensely bound neutralized WGA. In contrast, cumulus cell membranes bound PCG but not RR, whereas the oolemma bound RR but not PCG. The results indicate that the ZP is practically devoid of negatively charged constituents, and tends to repel positively charged ligands possibly due to the presence of cationic determinants. The binding of PCG to cumulus cells probably reflects a high content of membrane-bound heparan sulphate, whereas the binding of RR to the oolema indicates the presence of membrane sialoglycoconjugates. We suggest that during sperm-egg interaction the negatively charged spermatozoon may be electrostatically repelled by the cumulus cells, yet at the same time attracted by the ZP.     

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80–100nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.