Effects of prolonged incubation of rat peritoneal mast cells with compound 48/80.

Rat peritoneal mast cells (MC) co-cultured on a monolayer of 3T3 fibroblasts (MC/3T3) were continuously exposed to compound 48/80 for 14 days. As early as 2 days following continuous exposure to compound 48/80, the MC/3T3 appeared as a heterogeneous population, with various MC appearing partially or fully degranulated or intact; this morphological pattern continued throughout the duration of the experiment. MC/3T3 remained functionally active as demonstrated by their ability to secrete histamine 15 min after each replacement with fresh medium containing compound 48/80, although this capacity diminished towards the end of the 14-day experiment. Concomitant with the histamine release, a significant increase in cellular histamine pools was observed. When MC/3T3 continuously exposed to compound 48/80 for 7 or 14 days were acutely challenged with anti-IgE antibodies, they were able to secrete histamine and prostaglandin D2 in amounts similar to those produced by control MC. In contrast, when these cells were challenged on day 7 or 14 with a higher dose of compound 48/80 or with substance P, the release of histamine was partially inhibited. Our results indicate that continuous in vitro exposure to compound 48/80, and the resulting MC degranulation product histamine, does not adversely affect the ability of MC/3T3 to synthesize histamine and to respond to activation stimuli of a related secretagogue for 7 days and a non-related one for at least 14 days.     

Comparison of histamine and serotonin release from rat peritoneal mast cells induced by nerve growth factor and compound 48/80

Inflammation Research -     

Oxatomide protects against degranulation of rat peritoneal mast cells during in vitro challenge with antigen or compound 48/80. Ultrastructural aspects

The ultrastructure of isolated rat peritoneal mast cells was evaluated after in vitro degranulation and treatment with oxatomide, a new anti-allergic compound.In a first series of experiments, mast cells of rats infected withTrichinella spiralis larvae were incubated withTrichinella larvae somatic antigen to produce histamine release. The release was visualized in the electron microscope by exocytosis of the peripheral amine-containing granules, which resulted from fusion between several perigranular membranes and fusion of these membranes with the plasma membrane.A more drastic degranulation was provoked in a second series by incubation of unsensitized mast cells in the presence of the amine liberator compound 48/80. This treatment led to a complete extrusion of the granules in most of the cells, while in a smaller number of cells, only large vacuoles containing remnants of several granules were seen. The plasma membrane of these cells however was intact and there were no signs of exocytosis.The effect of oxatomide against mast cell degranulation was dose-dependent and comparable for the two types of histamine release. After incubation with high doses (10^–4 M, 5.10^–5 M) granule liberation was rarely observed in antigen-challenged and compound 48/80-challenged cells. Protection was apparently situated at the level of the plasma membrane which seemed to be unable to fuse with the perigranular membranes while fusion of perigranular membranes of individual granules was still possible. None of the tested concentrations of oxatomide induced spontaneous degranulation. High doses, however, led in a number of cells to some ultrastructural alterations such as partial disappearance of plasmalemmal folds, slight cytoplasmic oedema and the appearance of intranuclear microtubules. The latter were also seen in oxatomide-treated challenged mast cells.     

Microfilament-associated, local degranulation of rat peritoneal mast cells

When compound 48/80 was applied by means of microelectrophoresis to the surface of a rat peritoneal mast cell, localized degranulation was observed in the area close to the microelectrode tip. The extruded granules were connected to the cell surface by filaments. The filaments were elongated radially and, in some occasions, projected to a length of 5 microns. A few minutes later, the length of the protruded filaments became shorter and shorter and, finally, the extruded granules were reincorporated into the cell. When rhodamine-phalloidin, an F-actin-specific dye, was perfused the extruded granules and filaments were stained by this dye. This indicates that actin filaments or fragments of them exist on the granule surface and on the cell surface at the site of degranulation. These actin filaments bound to the mast cell granules may play an important role, not only for the extrusion of the granules, but also for the reuptake of extruded granules into the cytoplasm.     

Broncho-Vaxom® inhibits histamine release from rat mast cells induced by compound 48/80 and ionophore A23187

Broncho-Vaxom (BV) inhibited in dose-dependent manner the release of histamine from and degranulation of isolated rat peritoneal mast cells stimulated with compound 48/80 and the ionophore A23187. Inhibition persisted after removal of BV from the incubation medium before stimulation, but did not occur when bovine serum albumin (BSA) was used instead of BV. Binding of BV to mast cells was observed by electron microscopy on cells that had been incubated with colloidal-gold labelled BV. There was no significant difference between the binding of BV gold and BSA gold to the mast cells. Washing before fixation removed most of the BV gold from the cells. This study establishes BV as anin vitro histamine release inhibitor.     

Compound 48/80 and substance P induced release of histamine and serotonin from rat peritoneal mast cells

The effect of substance P and compound 48/80 on histamine and serotonin release from not isolated and isolated mast cells have been compared in experimentsin vitro. The response of not isolated and isolated mast cells were virtually identical. The release of both amines, in response to 48/80 and substance P, was dose-dependent. The percentage of histamine released by 48/80 was significantly higher than the percentage of serotonin, the difference being higher at lower concentrations of compound 48/80 after 15 min of incubation. Substance P also showed a tendency to higher efficiency for histamine than for serotonin release. In contrast to 48/80, the dose-response curves for histamine and serotonin release were parallel. These results support the view that the ratio between histamine and serotonin release depends on the liberator used. They also showed that this ratio can depend on the concentration of the agent inducing secretion. The results indicate that substance P as well as 48/80 act rather selectively as histamine liberators and that there is some difference in releasing properties of 48/80 and substance P.     

Platelet activation by endogenous 5-hydroxytryptamine and histamine released by mast cell degranulation with compound 48/80 in the rat

The intravenous injection of the mast cell degranulator C 48/80 (1 mg/kg) in rats did not produce thrombocytopenia nor circulating platelet aggregates but sensitized the platelets to aggregate upon turbulence challenge. Such turbulence-induced platelet aggregation was not accompanied by formation of thromboxane B2. Electron microscopy revealed absence of platelet degranulation. Turbulence-induced platelet aggregation was completely prevented by pre-treatment of the rats with cyproheptadine, dipyridamole and VK 774, partially with ketanserin (5HT₂-receptor antagonist), but not with methysergide (anti-serotonergic drug), pyrilamine (antihistaminic drug), suprofen, aspirin (cyclo-oxygenase inhibitors), phentolamine, propranolol, flunarizine, lidoflazine, oxycoumarin or Trasylol®. Combined treatment with the anti-histaminic drug pyrilamine and the 5HT₂-receptor antagonist ketanserin resulted in a dose-related inhibition for ketanserin of the turbulence-induced platelet aggregation. These experiments point to an interaction between histamine and 5-hydroxytryptamine in the platelet activation by mast cell released mediators.     

Binding of compound 48/80 by isolated rat mast cells in connection with histamine release

The binding of compound 48/80 in connection with histamine release from isolated rat mast cells was investigated by a two-step incubation procedure. After incubation of mast cells with known concentrations of compound 48/80, the supernatants were collected and subsequently exposed to fresh mast cells. The response in the second incubation step provided a measure of the amount of compound 48/80 remaining in the supernatants. At noncytotoxic concentrations of the releasing agent, binding capacities for compound 48/80 of up to 4 micrograms/10(5) mast cells (4% w/v) were observed. The binding of compound 48/80 was of high affinity, giving mast cell concentrations of the compound exceeding that in the incubation medium by four orders of magnitude. The binding occurred rapidly with a substantial proportion bound within the first minute. These findings indicate that quantitation of exocytotic events by means of basic dyes may be compromised by competition for granular binding sites by basic releasing agents.     

Plasma membrane fluidity studies by fluorescence polarization in rat mast cells stimulated by compound 48/80

Plasma membrane fluidity measurements were performed on purifled living mast cells using a novel non-permeant fluorescence polarization probe TMA-DPH, upon stimulation by compound 48/80. The fluorescence anisotropy increased rapidly after treatment by 48/80 in a dose-dependent way. The effect was found to be specific for mast cells; it was inhibited by the histamine release antagonist FR 7534 in a correlative manner. The role of calcium was examined. The results brought evidence for a plasma membrane fluidity decrease induced by 48/80; a biphasic mechanism was inferred for the histamine release process.     

Ca-dependent and Ca-independent histamine release from mast cells induced by active components of compound 48/80

Compound 48/80 and 14C-labelled compound 48/80 were synthesized, and fractionated by thin-layer chromatography into 14 components with various histamine-releasing activities and different Ca++ requirements for their actions. The histamine release induced from rat mast cells in vitro by the most active component, fraction D (molecular weight = 2280, a tridecamer composed of 13 monomer units), was greatly elevated by extracellular Ca++, and was partially reduced by pretreatment of the cells with dinitrophenylated Ascaris antiserum, an IgE. In contrast, the histamine release induced by fraction H (molecular weight = 1580, a nonamer composed of 9 monomer units), was higher in Ca-free medium than in Ca-containing medium, and partially suppressed by pretreatment of mast cells with neurotensin or substance P, both Ca-independent releasers. The binding potencies of the 14C-labelled components estimated at 4 degrees C in the presence of Ca++, where no degranulation of the cells occurs, generally paralleled their histamine-releasing activities. However, Ca++ was inhibitory for the binding of 14C-fraction H. The binding of fraction D to [3H]arachidonic-acid-preloaded mast cells induced a rapid accumulation of the labelled arachidonic acid into phosphatidic acid, phosphatidylinositol and phosphatidylcholine, with concomitant decrease of the labelled arachidonic acid from phosphatidylethanolamine prior to the detectable histamine release.     

In vitro uptake of 5-hydroxytryptamine and dopamine by rat mast cells after exocytosis induced by antigen or compound 48/80.

Mast cells from the peritoneal and pleural cavities of actively sensitized rats were isolated and incubated with biogenic amines (5-hydroxytryptamine and dopamine) with or without pretreatment with specific antigen. An anaphylactic reaction resulting in the release of 20-25% of the histamine in the cells led to a slightly reduced amine uptake. At concentrations which induced histamine release comparable to that during the anaphylactic reaction compound 48/80 had a similar effect on the uptake of the two amines. Histamine release induced by higher concentrations of compound 48/80 led to a more pronounced reduction in the uptake of the amines, the reduction being roughly proportional to the extent of the histamine release. It is concluded that the reduction in the in vitro amine uptake after anaphylactic and compound 48/80-induced histamine release is due to the fact that there are a fewer intact granules capable of storing histamine and not primarily due to a damage to the mechanisms by which mast cells take up biogenic amines in vitro. The observations further strengthen the view that anaphylactic and compound 48/80-induced histamine release are non-cytolytic processes.     

Effect of low temperatures on compound 48/80-induced intracellular Ca2+ changes and exocytosis of rat peritoneal mast cells

It has been well documented that compound 48/80-induced exocytosis of mast cells is accompanied by changes in intracellular Ca2+ concentration ([Ca2+]i) showing a biphasic pattern: an initial phase which constitutes an abrupt increase, followed by a plateau phase. The former is caused by Ca2+ release from intracellular Ca2+ stores, and the latter is the result of secondary Ca2+ influx. Low temperatures lead to the inhibition of exocytosis, but the precise mechanism remains unclear. The present study aims to reveal whether [Ca2+]i changes are affected by the environmental temperature. To this end, we developed a novel imaging method to record [Ca2+]i changes and exocytotic processes simultaneously. Rat peritoneal mast cells were loaded by Indo-1/AM or Fluo-3/AM for measuring [Ca2+]i, and the exocytosed granule matrices were stained by sulforhodamine-B. Cells were stimulated by compound 48/80, and [Ca2+]i changes and exocytosis were recorded by means of a real-time confocal microscope. At 37 degrees C, [Ca2+]i changes in stimulated mast cells showed a sustained plateau phase. Granule discharge was observed at the cell surface, and, in addition, most of the intracellular granule matrices were involved in compound exocytosis. The granule discharge and compound exocytosis proceeded over a period of a few minutes. At 4 degrees C, the plateau phase of [Ca2+]i changes declined rapidly, although the initial phase was not suppressed. Granule discharge occurred at the cell surface, but compound exocytosis ceased within a few minutes. These findings indicate that a low temperature inhibits compound exocytosis which can be caused by Ca2+ influx. The present imaging method represents a powerful tool for investigating the stimulus-secretion coupling of mast cells.     

Glucose metabolism in rat mast cells. Stimulation of the pentose phosphate pathway by compound 48/80

The glycogen content of rat peritoneal mast cells (mean: 3 nmoles/10⁶ cells) was increased 15% by incubation with glucose (1 mM) and reduced 35% when incubated without glucose at 37°C for 15 min. The storage capacity for glycogen is thus low. Lactate production at 37°C in a substrate-free medium was low (2.5–6.3 nmoles/10⁶ cells in 40 min), but was stimulated 5-fold in the aerobic medium and 10–15 fold in the anaerobic medium by glucose. Both aerobic and anaerobic glycolysis in presence of glucose can thus provide energy for histamine secretion.The initial enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, have been demonstrated in mast cells. The enzyme activity in mast cells was, however, low compared to the high activity in the other peritoneal cells. The extent of the pentose cycle activity was determined from the conversion of¹⁴C₁ ^–- and¹⁴C₆-glucose to¹⁴CO₂, expressing the specific¹⁴CO₂ yields as fractions of the total glucose utilization. The normal pentose cycle activity with 1 mM glucose was 0.4% of the glucose metabolism. This was remarkably simulated by an electron acceptor, phenazine methosulfate. The pentose cycle was enhanced to 0.71% (80% stimulation) after exposure of the mast cells to compound 48/80, causing 68% histamine release. The stimulation of the pentose cycle by compound 48/80 seems to be due to the enhancement of biosynthetic processes during the regenerative phase. We have previously reported stimulation of CO₂ and lactate production from glucose in mast cells by secretagogues. The present experiments also show a stimulation of total glucose utilization and increased incorporation of¹⁴C from glucose to lipids.