Isolated Rat Peritoneal Mast Cells Research Articles

Isolation of rat mast cell granules with intact membranes

A method for controlled sonication of isolated rat peritoneal mast cells and separation of the membrane-bound secretory granules on a self-generating density gradient of Percoll (polyvinyl-pyrrolidone-coated silica gel particles) is described. Ruthenium red, which binds exclusively to the matrix of the membrane-free granules, proved to be an excellent agent for the quantitative assessment of the state of the perigranular membranes in the various preparations. The percentage of intact granules, as observed in the electron microscope, increased from 49% in the primary granule pellet to 75% after separation on Percoll gradient, whereas the corresponding figures as revealed by the ruthenium red method were 69 and 93% respectively. The observed discrepancies were due to difficulties involved in sampling the granule pellets for electron microscopy.

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells.

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37 degrees C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0 degrees C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9 degrees C above ambient levels. Alternatively, the heat stress produced at 44 degrees C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.

An automated fluorimetric assay for the simultaneous determinations of histamine and 5-hydroxytryptamine released from rat mast cell preparations

An automated assay suitable for the extraction and simultaneous fluorimetric determination of histamine and 5-hydroxytryptamine (5-HT) released from rat isolated peritoneal mast cells is described. The method gave good reproducibility and accuracy in use and required a sample volume of 0.5 ml for assay at a sampling rate of 30/h. The system was evaluated by assessment of its performance in the determination of histamine and 5-HT released from rat mast cells in response to compound 48/80. The detection limit for the procedure was 2 ng 5-HT base/ml and 20 ng histamine base/ml.

Slow Reacting Substance (SRS) from Ionophore A23187-Stimulated Peritoneal Mast Cells of the Normal Rat

Abstract When rat peritoneal mast cells were exposed to the ionophore A23187, a principle was released that possessed the biologic properties of slow reacting substance (SRS) from various sources. The response was dose, time, and temperature dependent with no activity being demonstrated in unstimulated cells. Supporting evidence that the mast cell product was similar or identical to SRS obtained from other sources include: 1) appropriate differential bioassay profile, 2) resistance to lipolysis and proteolysis, 3) acid lability and base stability, 4) inactivation by limpet arylsulfatase, and 5) inhibition by low concentrations FPL 55712. These data demonstrate that the isolated rat peritoneal mast cell contains the biosynthetic capacity to produce a bioreactive substance with the properties of SRS.

Xanthine oxidase-induced histamine release from isolated rat peritoneal mast cells: involvement of hydrogen peroxide

Xanthine oxidase-induced histamine release from isolated rat peritoneal mast cells: involvement of hydrogen peroxide

Inhibition and stimulation of histamine release by oxatomide

Oxatomide, a histamine H 1-antagonist, was also found to inhibit histamine secretion stimulated by allergen and other ligands in a variety of in vitro models: 1. Isolated rat peritoneal mast cells, stimulated to secrete by specific antigen, dextran and concanavalin A; 2. Antigen-stimulated histamine secretion by basophil leucocytes from patients with allergic asthma; and 3. Anti-IgE-induced histamine release from chopped human lung passively sensitized with IgE from allergic asthmatics. Although oxatomide was active in inhibiting histamine secretion stimulated by these various means, it possessed a histamine-releasing effect on its own when used in high concentration, particularly in rat peritoneal mast cells. This effect was less pronounced in human basophils, and was not observed in human lung mast cells. Thus, the inhibition and stimulation of histamine release by oxatomide seemed to be independent actions.

INHIBITORY ACTION OF 6-ETHYL-3-(lH-TETRAZOL-5-YL)CHROMONE (AA-344) ON IgE-, IgGa- OR CHEMICAL AGENT-INDUCED HISTAMINE RELEASE FROM ISOLATED RAT MAST CELLS

The antiallergic action of 6-ethyl-3-(lH-tetrazol-5-yl)chromone (AA-344) was studied on isolated rat peritoneal mast cells. AA-344 clearly inhibited the IgE- mediated release of histamine caused by various concentrations of antigen and the 50% inhibitory concentration was 0.1 pM. On the IgGa-mediated release of histamine, a peak inhibition of AA-344 was observed at 10 pM. The histamine release induced by chemical agents such as concanavalin A, dextran and compound 48/80 was depressed by AA-344 at the range of 0.1-1 mM. The results obtained in this study indicate that the antiallergic action of AA-344 is due to selective inhibition on the immunological release of the chemical mediator from mast cells.

Inhibition of Compound 48/80-Mediated Histamine Release from Isolated Rat Mast Cells by Oosponol-Related Compounds (4-Acyl-Isocoumarins).

Oosponol (4-hydroxymethylketone-8-hydroxyisocoumarin) is a metabolic product isolated from Oospora astringens which originated from house dust in a room of an asthmatic patient. The compound and the structurally related isocoumarins were studied to determine the inhibition of histamine release induced by compound 48/80 from isolated rat peritoneal mast cells. The released histamine was assayed by fluorometry. The compounds tested were not observed to release histamine. Some of 4-acyl-isocoumarins inhibited the histamine release at doses less than 10 µM, whereas the 3-acyl and the 4-alkyl-compounds were not effective at doses over 100 µM. The pretreatment of mast cell with the compound for 15 min before the application of compound 48/80 was more effective than the simultaneous administration. The mode of inhibitory action of KIT-302, 4-(4’-carboxy-benzoyl)-isocoumarin, was non-competitive antagonism to compound 48/80 on the mast cells.

Calcium and stimulus‐secretion coupling in the mast cell: stimulant and inhibitory effects of calcium‐rich media on exocytosis

1. Isolated rat peritoneal mast cells incubated in Ca-free media for 2 h, with or without EDTA, and observed by phase-contact microscopy, became ;bubbled' in appearance when subsequently exposed to media rich in calcium (16-110 mM).2. Electron microscopy showed the response to be ;compound' exocytosis of the sort elicited by conventional mast cell secretagogues such as antigen (in sensitized cells) and 48/80.3. The response to Ca was inhibited by withdrawing glucose and adding dinitrophenol and was thus energy-dependent.4. Mg in similarly high concentration had no such stimulant effect on Ca-deprived cells, and excess Ca stimulated only after Ca deprivation.5. It is suggested that Ca deprivation may increase the permeability of the plasma membrane of the mast cell thereby allowing some Ca, when subsequently introduced in high concentration, to penetrate and activate exocytosis; and the results are considered further support for the postulated mediator function of Ca in stimulus-secretion coupling.6. Two inhibitory effects of calcium in high concentration were detected: (a) suppression of migration or expulsion of granules from the exocytotic pits within the cellular domain; and (b) diminished sensitivity to 48/80.

Functional Characterization of Rat Mast Cell Arylsulfatase Activity

Extracts of isolated rat peritoneal mast cells were demonstrated to contain appreciable quantities of arysulfatase activity. The enzyme was inhibited by both phosphate and sulfate ions and demonstrated a pH optimum of 5.0. The enzyme was recovered in the eluate of DE-52 columns and appeared to have a m.w. of 150,000 of Sephadex G-200 gel filtration. These findings and the anomalous kinetic behavior of the enzyme suggest that at least part of the enzymatic activity is of the arylsulfatase IIA type. While spontaneous release of the enzyme was observed, challenge of isolated rat mast cells with a goat anti-rat IgE serum resulted in a significant increase in release of the enzyme. The arylsulfatase activity extracted from isolated rat mast cells demonstrated comparable activity in inactivating slow reacting substance of anaphylaxis (SRS-A) to that described for human eosinophil and lung arylsulfatase.

Cyclic AMP independent inhibition by papaverine of histamine release induced by compound 48/80

Compound 48/80-induced histamine release from isolated rat peritoneal mast cells was inhibited in a dose-dependent manner by papaverine ( ic 50 approx 20 μM). This effect of papaverine was not influenced by PGE 1 (14–140 μM), even though PGE 1 markedly increased must cell cAMP levels. Papaverine (0.5 mM) completely inhibited histamine release without causing any change in cAMP levels. Theophylline (0.1 and 0.5 mM) potentiated histamine release induced by submaximal concentrations of compound 48/80, while cAMP levels were increased. IBM X was as potent as papaverine in causing inhibition of mast cell phosphodiesterase. IBM X (0.14–0.7 mM) had no effect on histamine release but caused a 6–20 fold increase in mast cell cyclic AMP. Papaverine inhibition of histamine release was gradual at the onset and was parallelled by a depletion of mast cell ATP content. The inhibition of 48/80-induced histamine release and depletion of mast cell ATP levels was reversed by glucose. It is concluded that papaverine induced inhibition of 48/80-induced histamine release is independent of cAMP, is unrelated to phosphodiesterase inhibition but is dependent upon inhibition of energy production.

Release of chymotrypsin-like esterase from isolated rat peritoneal mast cells by arginine-rich calf thymus histone.

Release of chymotrypsin-like esterase from isolated rat peritoneal mast cells by arginine-rich and lysine-rich calf thymus histones was investigated. The arginine-rich histone released the esterase, but the lysine-rich histone did not release the esterase from the mast cells. The isolated mast cells spontaneously released the esterase gradually. The chymotrypsin-like esterase released from the mast cells is considered to digest the destructed material in tissues in a state of chronic destruction, such as chronically inflamed tissues and malignant tumours.

Differential histamine release by dextran and the ionophore A23187: the actions of inhibitors.

Inhibitors of mast cell membrane activation reduced histamine release from rat mast cells induced by dextran and phosphatidyl serine but not that induced by the calcium ionophore A23187. Such inhibitors included cromoglycate, an orally-active anti-allergic agent 3-(5-tetrazolyl)thioxanthone 10,10-dioxide, dibutyryl cyclic 3':5'-AMP, theophylline and dicumarol. Inhibitors of mast cell metabolism reduced both types of release and these included oligomycin, papevevime, and the two uncouplers of oxidative phosphorylation-alpha2,4-dinitrophenol and CCCP. Inhibition of histamine release from rat isolated peritoneal mast cells by either a mixture of dextran and phosphatidyl serine or the ionophore A23187 thus allows inhibitors of mast cell membrane activation to be distinguished from those affecting cell metabolism or the later stages of the secretory process.

The effect of surfactants upon mammalian cells in vitro

A series of highly pure surfactants were examined in vitro for their capacity to inhibit DNA synthesis in guinea-pig kidney fibroblasts, to release histamine from isolated rat peritoneal mast cells, and to effect the cytolytic release of cytoplasmic proteins from guinea-pig kidney fibroblasts. The surfactants studied included a homologous series of carboxylic acid sodium soaps of chain length C 8-C 16 and a series of chain length C 12 with different chemical head groups. Of the carboxylic acid soaps, sodium laurate (chain length C 12) was more potent at inhibiting DNA synthesis, at releasing histamine and at lysing fibroblasts than either the higher or lower homologues. Exchange of the carboxylic acid head group on the alkyl chain for a sulphate or isethionate moiety increased the potency of the surfactant and introducing three or six ethoxy groups further increased the potency to living cells.

No role for zinc in the storage of histamine in rat peritoneal mast cells.

The histamine in the granules of mast cells has been proposed to be stored as a heparin-zinc-histamine complex. Due to its chelating action zinc should increase (double) the histamine binding capacity of heparin. We have determined the zinc content of isolated rat peritoneal mast cells to 2.4-4.1 nmol/10-6 cells and of their granules to 13.3-21.4 nmol/mg dry weight. The corresponding amounts of histamine were 150 and 680 nmol respectively. The zinc content found is far (20 times) too low to allow for an adequate binding of histamine in a heparin-zinc-histamine complex. In vitro the granules take up zinc in the same manner as they take up other cations and zinc competes with histamine for granule storage sites. Consequently, Hi-uptake is reduced and not enhanced in the presence of zinc in the suspension medium. In summary no evidence was found for a storage function of zinc in a heparin-zinc-histamine complex.

Incorporation of imipramine into isolated rat peritoneal mast cells in vitro.

AbstractThe incorporation of imipramine into isolated rat peritoneal mast cells was fluorometrically studied in vitro. The binding of imipramine was characterized by the following features: 1. it took place in two phases, the first of which was very rapid, 2. it increased with increasing exogenous concentration in a sigmoidal fashion, 3. it increased with increasing exogenous pH, 4. it was dependent on neither temperature nor calcium ions in the medium nor did the binding occur specifically to mast cells but also to leucocytes, 5. it was reduced by several imipramine derivatives, tricyclic antidepressants and 5‐hydroxytryptamine at high concentrations, 6. about 40 % of the cell‐bound drug was easily washed out; the remaining 60 % represented a level approached after prolonged washing and seemed to be fairly tightly bound, 7. the subcellular location of imipramine in mast cells was different from that of 5‐hydroxytryptamine, indicating a nongranular binding of the drug. It is concluded that imipramine binds to mast cells in a passive manner and is associated with the cell membrane where it seems to be bound to at least 2 different sites. At low concentrations imipramine is probably bound to sites specifically influencing the operativeness of the amine carrier. Higher concentrations of imipramine results in binding to nonspecific sites resulting in impairment of the structure and function of the cell membrane.

Inhibition by nicotinamide of a homologous PCA reaction and antigen-induced histamine release from isolated rat peritoneal mast cells.

Inhibition by nicotinamide of a homologous PCA reaction and antigen-induced histamine release from isolated rat peritoneal mast cells.

The effect of zinc ions on selective and nonselective histamine release in vitro

The effect of different concentrations of zinc ions on the in vitro release of histamine from isolated rat peritoneal mast cells induced by compound 48/80 and Triton X-100, as well as the influence of the in vivo administration of zinc ions on the subsequent in vitro release of histamine from isolated peritoneal mast cells and lung tissue, have been investigated. The results show that intraperitoneal administration of zinc for 20 days significantly inhibited the spontaneous release of histamine from lung tissue as well as that induced by a low dose of compound 48/80.

The inhibitory effect of nicotinamide on asthma-like symptoms and eosinophilia in guinea pigs, anaphylactic mast cell degranulation in mice, and histamine release from rat isolated peritoneal mast cells by compound 48-80.

The prior treatment of guinea pigs with nicotinamide diminished the symptoms of experimental bronchial asthma and the intensity of anaphylactic shock. Nicotinamide was also found to inhibit anaphylactic mast cell degranulation in mice and histamine release from rat-isolated peritoneal mast cells by compound 48/80. The role of nicotinamide in bronchial asthma is discussed.

Incorporation of guanethidine into isolated rat peritoneal mast cells and mast cell granules in vitro.

AbstractThe incorporation of guanethidine into isolated rat peritoneal mast cells was studied in vitro. The incorporation of the drug is dependent upon 1. temperature, 2. the pH of the medium, 3. the presence of Ca‐ions in the medium, it 4. shows saturation characteristics and is 5. inhibited by 5‐hydroxytryptamine and tricyclic antidepressants. Of the metabolic inhibitors tested, only FCCP reduced incorporation of guanethidine. The intracellular location of guanethidine in mast cells is mainly in the amine‐storing organelles. It is concluded that the incorporation of guanethidine is partly due to an active transport mechanism located in the cell membrane, while the incorporation of the drug into isolated membrane‐free mast cell granules is a passive process resulting in binding to sites in the granule matrix.