Receptors for C3 on rat peritoneal mast cells.

Purified rat peritoneal mast cells adhere to schistosomula of Schistosoma mansoni which have been pre-incubated in fresh normal rat serum. This cytoadherence reaction is dependent on complement and in particular on components of the alternative pathway. Since antibodies to rat C3 but not IgG block the attachment of the cells to the complement-treated larvae, it appears that C3-specific receptors on the mast cell surface are responsible for the adherence phenomenon. These receptors can also be demonstrated by the rosetting of mast cells with rat complement-treated zymosan particles or fluoresceinated bacteria. The key properties of the receptors are their specificity for homologous (rat) complement, their sensitivity to digestion with trypsin, and their functional dependence on Mg++ ions. Thus, the rat mast cell receptors share many of the characteristics of the C3 receptors previously identified on monocytes, macrophages, and polymorphonuclear leukocytes.     

Immunologic release of heparin from purified rat peritoneal mast cells.

High m.w. [35S]heparin, labeled in vivo or in vitro, was released from purified rat mast cells by challenge with rabbit anti-rat F(ab')2, guinea pig anti-rat IgE, or calcium ionophore. The released and the residual heparin were isolated by Dowex 1 chromatography and were of comparable size by Sepharose 4B gel filtration. The majority of the released heparin was found by differential centrifugation to be granule-associated. Net percentage of mast cell heparin release, quantitated by metachromasia after isolation on Dowex 1 chromatography, correlated in a linear fashion with net percentage of histamine release, with heparin exhibiting a threshold requirement for onset of release. The correlation of histamine and high m.w. heparin release provides chemical support for the conclusion of others from ultrastructural studies that mast cell activation by immunologic means or by the calcium ionophore results in secretion of the whole granule.     

Mycophenolic acid inhibits the degranulation of rat peritoneal mast cells.

The effect of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase, on the degranulation of rat peritoneal mast cells (RMC) was studied. RMC were pretreated for 48 hr with 0.1-10 microM MPA before the cells were sensitized with IgE and triggered with specific Ag. The net amount of [3H]5-HT released from granules was decreased by 44 and 32% with 1 and 10 microM MPA treatment, respectively. MPA inhibition of degranulation was completely reversed by the addition of 30 microM guanosine to the incubation medium. There was no difference in the apparent number or affinity of IgE binding sites between control and MPA-treated RMC. MPA pretreatment also had no effect on the IgE receptor-mediated production of PGD2 in RMC. These results suggest that depletion of intracellular GTP pools by MPA can disrupt the signaling between the IgE receptor and the secretory granules and that, under these same conditions, the release and metabolism of arachidonic acid are unaffected.     

Secretory responses of rat peritoneal mast cells to high intracellular calcium

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.     

The presence of ANP in rat peritoneal mast cells

Atrial natriuretic peptide （ANP） is an important component of the natriuretic peptide system. A great role in many regulatory systems is played by mast cells. Meanwhile involvement of these cells in ANP activity is poorly studied. In this work, we have shown the presence of ANP in rat peritoneal mast cells. Pure fraction of mast cells was obtained by separation of rat peritoneal cells on a Percoll density gradient. By Westem blotting, two ANP-immunoreactive proteins of molecular masses of 2.5 kDa and 16.9 kDa were detected in lysates from these mast cells. Electron microscope immunogold labeling has revealed the presence of ANP-immunoreactive material in storage, secreting and released granules of mast cells. Our findings indicate the rat peritoneal mast cells to contain both ANP prohormone and ANP. These both peptides are located in mast cell secretory granules and released by mechanism of degranulation. It is discussed that many mast cell functions might be due to production of natriuretic peptides by these cells.     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.     

Rapid separation of rat peritoneal mast cells with Percoll

Rat peritoneal mast cells were separated by using density gradients of PVP-coated silica particles (Percoll). Mast cells were either isolated on preformed Percoll gradients or cell separation was made simultaneously with the gradient formation. Both procedures resulted in mast cell suspensions of 95 to 99 per cent purity. As tested by Ruthenium red staining and electron microscopy, the isolated mast cells showed a very good preservation of cell structure and reacted easily to the degranulating agent Compound 48/80. Practically all mast cells could be recovered from the peritoneal cell suspension. Percoll was found to be superior to earlier isolation procedures by giving a practically pure and intact mast cell suspension and by avoiding cell aggregation.     

Alterations of the electrophoretic mobility distribution of rat mast cells after immunologic activation.

Changes in the electrophoretic mobility distributions of rat serosal mast cells after immunologic activation have been measured using the laser Doppler technique of electrophoretic light scattering. Rat serosal mast cells of 98% purity isolated by isopycnic and velocity gradient sedimentation had a highly negative electrophoretic mobility which was unaffected by incubation with normal rabbit serum or, at 4 degrees C or in the absence of Ca+2, with rabbit anti-rat E(ab')2 antiserum. Immunologic activation of the cells with this antiserum in the presence of Ca+2 at 37 degrees C resulted in a dose- and time-dependent increase in the electrophoretic mobility. Thus at a 1:25 dilution of anti-F(ab')2 the mean and mode electrophoretic mobilities of the mast cell population increased 25 and 21%, respectively. The width of the electrophoretic mobility distribution also increased with activation, indicating a heterogeneous response of the mast cells in the population. The increase in electrophoretic mobility after immunologic activation is not diminished by treatment of the cells with 1 M NaCl to solubilize adsorbed mast cell granule or heparin.     

Histamine H-1 receptors on rat peritoneal mast cells

In order to characterize the receptor subtype involved in histamine stimulation of increased cyclic AMP levels in rat mast cells with consequent impairment of anaphylactically induced mediator release, the binding of the H-1 receptor antagonist [³H] pyrilamine to mast cells was examined. Pyrilamine bound rapidly, in a saturable and reversible fashion, and with increased binding at 4°C as compared with 21°C and 37°C. [³H] Pyrilamine binding was displaced by H-1 antagonists (tripelnnamine > yrilamine ≧ iphenhydramine) > histamine > the H-2 antagonist, cimetidine. H-1 agonists displaced pyrilamine binding less efficiently than histamine but better than H-2 agonists. Rat mast cells have a single homogeneous population of low affinity (K_D = 222 ± 33 nM) H-1 receptors with a Bmax of 9.7 ± 2.3 pm/10⁶ mast cells and 5.4 ± 0.92 × 10⁶ binding sites per mast cell. Thus, the mast cell has an H-1 type histamine receptor which is probably involved in histamine-induced cyclic AMP increases.     

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

The calcium concentration, [Ca]i, in single rat peritoneal mast cells was measured by means of the new Ca indicator dye fura-2. Upon stimulation with antigen or compound 48/80, [Ca]i rose for seconds to values greater than 5 microM. These Ca transients did not depend on the presence of extracellular Ca, and they sometimes occurred spontaneously, especially in the presence of exogenous phosphatidylserine. Calcium transients did not necessarily lead to degranulation. Degranulation usually occurred during periods of somewhat elevated [Ca] (0.5-1 microM) following transients but was sometimes observed at [Ca]i less than or equal to 250 nM. We found no evidence that an antigen-induced Ca influx is required for degranulation.     

Metabolism of leukotriene D4 by rat peritoneal mast cells

Metabolism of sulfidopeptide leukotrienes, leukotrienes (LT) C4 and D4 by rat peritoneal mast cells was studied. Rat peritoneal mast cells converted LTD4 to LTE4 but not LTC4 to LTD4. The LTD4-metabolizing activity was equally distributed on the cell surface and inside cells, but not released to the extracellular milieu even when a considerable portion of histamine and the secretory granule enzymes were released. Among various enzyme inhibitors examined, o-phenanthroline, a metal chelator, and dithiothreitol significantly suppressed the LTD4-metabolizing activity of mast cell. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of LTD4 to LTE4 by rat peritoneal mast cells.     

Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.     

Properties of [3H] diazepam binding to rat peritoneal mast cells

Benzodiazepine binding to rat peritoneal mast cells was investigated using [³H] diazepam as the radioactive probe. The specific binding of [³H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a K_D = 90 ± 10 nM and B_max of 261 ± 60 fmoles/10⁶ cells. The binding of [³H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [³H] diazepam is reversible with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2 ± 0.2}\text{min.}$ Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [³H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.     

Membrane retrieval in non-exocytic and exocytic rat peritoneal mast cells

Mast cells were incubated with the membrane marker cationized ferritin (CF) in order to study the fate of cell membrane in both non-exocytic and in exocytic cells. Non-exocytic mast cells took up CF by a macropinocytic process by fusion of microfolds. CF particles occurred in lysosomal-like structures, in mast cell granules, and eventually in Golgi vesicles. Exocytic cells took up CF mainly by micropinocytosis and after 2 h CF appeared in Golgi vesicles and in progranules. The different uptake routes of CF in non-exocytic and in exocytic mast cells probably reflect that the former cells constantly take up material by macropinocytosis, perhaps for nutrition. The latter cells undergo a process of exocytosis-endocytosis in order to re-use retrieved surface membrane in new granule formation and in order to regain their ordinary size, which is greatly increased upon secretion.     

Acetaldehyde induces histamine release from purified rat peritoneal mast cells

Acetaldehyde is a widely distributed compound in the human environment and it is also formed in the human body from various endogenous and exogenous sources, exogenous ethanol being the most important one. Many alcohol-associated hypersensitivity reactions, e.g. Oriental flushing reaction, appear to be attributable to acetaldehyde rather than to ethanol itself. The pathogenetic mechanism behind such hypersensitivity reactions has been suggested to be histamine release from mast cells or blood basophils. However, the direct effects of acetaldehyde on mast cells, the main source of histamine in a mammalian body, have not been studied. The aim of the present study was, thus, to evaluate whether physiological concentrations of acetaldehyde could release histamine from purified rat peritoneal mast cells. The effects of ethanol were studied similarly. The results show that acetaldehyde, already at a concentration of 50 microM, significantly increases the release of histamine from mast cells. Ethanol has a similar effect but only at molar concentrations. These results indicate that acetaldehyde may contribute to the development of various hypersensitivity reactions by directly increasing histamine release from mast cells.     

Purification and partial characterization of an alpha-chymotrypsin-like protease of rat peritoneal mast cells.

An alpha-chymotrypsin-like enzyme was isolated from mast cells of the rat peritoneal cavity by extraction with 0.8 M potassium phosphate, 2 per cent protamine sulfate followed by affinity chromatography on hen ovoinhibitor-agarose and adsorption on barium sulfate. This procedure yielded over 9 mg of protease from the peritoneal lavage fluid of 100 rats, equivalent to 44 per cent of the initial activity. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical isoelectric focusing, and amino-terminal sequence analysis. The protease contains no covalently bound carbohydrate and has a molecular weight of approximately 26,000. The enzyme molecule is a single polypeptide chain with an amino-terminal sequence homologous to that of the B chain of bovine alpha-chymotrypsin. The kinetic parameters, Km and kcat, for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester were determined at pH 8.0 and 25 degrees C as 1.1 X 10(-3) M and 84 sec-1, respectively. The value of the second-order rate constant for inactivation of mast cell protease by diisopropylphosphofluoridate was 300 times lower than for bovine alpha-chymotrypsin.