Inhibitory effect of lysophosphatidylcholine on the histamine release from rat peritoneal mast cells

Histamine release from isolated rat peritoneal mast cells induced by compound 48/80 (0.5 microgram/ml) or antigen-antibody reaction was inhibited by lysophosphatidylcholine in a dose-dependent fashion at concentrations up to 4 microM. Within the same range of concentration, lysophosphatidylcholine exhibited a membrane-stabilizing action on the model membrane systems decreasing the permeability of lipid bilayer and the fluidity of liposomal membrane in the liquid crystalline state. At concentrations higher than 8 microM, lysophosphatidylcholine damaged the cell membrane and subsequently histamine was released. It was assumed that lysophosphatidylcholine may act as an endogenous membrane stabilizer inhibiting histamine release in normal mast cells.     

Histamine release by compound 48/80: evidence for the depletion and repletion of calcium using chlortetracycline and 45calcium

Selective release of histamine from rat peritoneal mast cells in vitro was induced by compound 48/80 (1 microgram/ml). Most of the release (75-80%) occurred in a calcium(Ca)-free medium but optimum release was obtained in the presence of 0.9 mM Ca. The release in Ca-free medium still occurred after 180 min incubation. However, prolonged incubation (180 min) in a medium containing chelating agents (EDTA or EGTA) resulted in complete inhibition of histamine release, loss of fluorescence seen with chlortetracycline (CTC) and loss of previously loaded 45Ca from the mast cells. Addition of Ca to these cells resulted in rapid restoration of fluorescence with chlortetracycline. There was also a rapid uptake of 45Ca. Partial depletion of cellular Ca (60 min incubation with EDTA) reduced the rate as well as the amount of histamine release by compound 48/80. These data provide direct evidence for the depletion of cellular Ca which is utilized by compound 48/80 to induce histamine release.     

The role of membrane bound sialic acid of rat mast cells in histamine release induced by compound 48/80 and derivatives as well as calcium

Histamine release induced by compound 48/80 from rat mast cells is not dependent on extracellular Ca2+. Preincubation of mast cells with trypsin has only little effects on histamine release induced by this polycation. This work also demonstrates that histamine release induced by compound 48/80 and its analogues in the absence of extracellular Ca2+ depends on membrane bound sialic acid of the mast cell. Neuraminidase treatment of the cells in the presence of extracellular Ca2+ leads to histamine liberation. These findings suggest that sialic acid residues of the mast cell membrane constitute the site at which polycations exert their stimulatory actions of histamine liberation.     

The utilization of adenosine triphosphate in rat mast cells during histamine release induced by anaphylactic reaction and compound 48/80

Summary The ATP content of rat peritoneal mast cells has been studied in relation to histamine release induced by compound 48/80 and antigen-antibody (anaphylactic) reaction in vitro. When the ATP content of actively sensitized mast cells was reduced to different levels by oligomycin, a good correlation was obtained between the ATP levels and the amounts of histamine released by the anaphylactic reaction. A similar linear relation has previously been demonstrated between the ATP levels of mast cells and histamine release induced by compound 48/80. The ATP content of mast cells was also studied at different intervals after the exposure of the cells to antigen or compound 48/80. No significant change in the ATP content was observed in untreated mast cells during the short period when histamine release occurs. If, however, the mast cells were preincubated with oligomycin or 2-deoxyglucose to reduce the rate of ATP synthesis while a large part of the histamine release remained unaffected—a decrease in the ATP content could be demonstrated in close time relation to both anaphylactic and compound 48/80-induced histamine release. The observations indicate an increased utilization of ATP in mast cells during the release process.     

Influence of aging on the histamine release and membrane fluidity of rat peritoneal mast cells

The maturational changes in the degree of homologous passive cutaneous anaphylaxis (PCA) and the histamine release from peritoneal mast cells induced by several secretagogues were studied using Wistar rats (4-40 weeks old). Although the increase in vascular permeability of the rat skin induced by intradermal injection of histamine did not change significantly from one maturation period to the next, 6- to 8-weeks old rats were both the most susceptible to PCA reactions and the most responsive to histamine-releasing stimuli. Among rats in this age group (6-8 weeks), the fluidity of the resting cell membrane and the extent of membrane fluidity increase in response to compound 48/80 were greatest. Analysis of the lipid composition of mast cells indicated that the ratio of cholesterol to phospholipids was lowest at the age of 6-8 weeks. From the present study, we concluded that the maturational changes in the extent of histamine release seem to be related to membrane fluidity, which has a profile similar to that of maturation.     

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 (ic₅₀ approx 20 μM). This effect of papaverine was not influenced by PGE₁ (14–140 μM), even though PGE₁ 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.     

Mechanism of histamine release from rat isolated peritoneal mast cells by dextran: the role of immunoglobulin E

In vivo passive sensitization of rat peritoneal mast cells with Nippostrongylus braziliensis antiserum or rat monoclonal myeloma IgE greatly enhanced histamine release in vitro by dextran or anti IgE, but did not alter release by compound 48/80 or A23187. Conversely, removal of IgE from the cells by acid pH abolished histamine release by dextran and anti IgE but did not impair release by compound 48/80. Whereas, histamine release from cells isolated from rats genetically resistant to dextran (NR rats) by anti IgE was potentiated by passive sensitization, dextran was unable to stimulate secretion from control or sensitized NR cells. The results suggest that dextran releases histamine by interaction with cell-fixed IgE and that the NR mast cell membrane lacks the ability to interpret this stimulus.     

Calcium dependency of inhibition by arachidonic acid of compound 48/80-induced histamine release from mast cells

Compound 48/80 ( compd 48/80)-induced histamine secretion from rat mast cells was inhibited almost completely by pretreatment of the cells at 37 degrees with 25 microM arachidonic acid in the presence of 1.8 mM Ca2+. As the Ca2+ concentration was reduced below 1.8 mM, 25 microM arachidonic acid became less inhibitory and, then, progressively more stimulatory for histamine release with or without compd 48/80. No additive effect on histamine release was obtained by combining compd 48/80 and arachidonic acid. Pretreatment of mast cells with lidocaine, an inhibitor of Ca2+ binding to phospholipid, or with nordihydroguaiaretic acid, an inhibitor of Ca2+ flux and lipoxygenase, stimulated arachidonic acid-induced histamine release. Arachidonic acid also inhibited a compd 48/80-induced spike increment of intracellular 45Ca2+ uptake and a decrease of total 45Ca2+ uptake by 45Ca2+-preloaded mast cells. Arachidonic acid and Ca2+ also suppressed melittin-induced histamine release and compd 48/80-induced release of radioactivity from mast cells preloaded with [3H]arachidonic acid. These results suggest that exogenous arachidonic acid or its metabolite(s) may interact with membrane-associated Ca2+, disturbing Ca2+ availability for the trigger mechanism of compd 48/80-induced histamine release or inhibiting the subsequent metabolism of arachidonic acid via the lipoxygenase pathway to form active metabolites involved in the histamine liberating mechanism.     

Mechanism of action of disodium cromoglycate--mast cell calcium ion influx after a histamine-releasing stimulus.

Rat peritoneal mast cells release histamine and accumulate ⁴⁵Ca in a dose-dependent manner when concentrations of compound 48/80 ranging from 0·1 to 1·0 μg/ml are incubated with suspensions of the cells for 5 min at 37°. Influx of ⁴⁵Ca stimulated by compound 48/80 can be inhibited to varying degrees by prior addition of disodium cromoglycate. Inhibition was dependent on the concentration of both disodium cromoglycate and compound 48/80. The electrokinetic properties of intact rat mast cells are described; disodium cromoglycate caused a plasma membrane alteration possibly related to Ca²⁺ influx. Cromoglycate increased mast cell electrophoretic mobility but decreased the electrophoretic mobility of rat erythrocytes. The net electrophoretic mobility was a function of terminal sialic acid residues, ionic strenght, and pH. Binding of disodium cromoglycate to Ca²⁺ could not be demonstrated by a variety of sensitive physical techniques. The data support the theory that secretion of mast cell histamine is coupled to Ca²⁺ influx. It is suggested that disodium cromoglycate prevents mast cell histamine release by a plasma membrane alteration which prevents an increase in membrane permeability to Ca²⁺ stimulated by compound 48/80.     

Histamine release by phosphatidic acid from isolated mast cells of two types of genetically different rats

Phosphatidic acid produced a similar dose-dependent release of histamine from isolated peritoneal mast cells of two types of rat, one of which did not release histamine when dextran was present in the incubation mixture. This release induced by phosphatidic acid resembled that evoked by a basic secretagogue like compound 48/80, being independent of exogenous calcium and unaffected by either phosphatidyl serine or its lyso derivative. The non-cytotoxic release of histamine induced by phosphatidic acid is therefore distinct from that by dextran and does not involve dextran receptors residing on the plasma membrane of mast cells of rats which show the dextran anaphylactoid reaction.     

Mechanisms of histamine release by compound 48/80

1. Rat and guinea-pig lung tissues were incubated for 20 min at 37 degrees C in Krebs-Ringer phosphate buffer at pH 7.4, or in Tyrode-Tris buffer at pH 8.2, and the release of histamine produced by adding different concentrations of compound 48/80 to the incubation medium was determined.2. At pH 7.4, increasing concentrations of 48/80 increased the release of histamine from the rat lung, with a tendency towards a maximum. No release of histamine from guinea-pig lung was observed at this pH. At pH 8.2, histamine release occurred both from rat and guinea-pig lung, and was proportional to the logarithm of the concentration of compound 48/80.3. Histamine release from rat lung by 20 mug/ml. of 48/80 decreased when the pH was raised from 7.4 to 8.2; but the release caused by 1 mg/ml. of 48/80 increased both in rat and guinea-pig lung as the pH was raised.4. 2-4-Dinitrophenol (DNP) inhibited the release of histamine from rat lung by a concentration of 20 mug/ml. of 48/80; the inhibition was prevented by glucose. DNP did not affect histamine release from rat or guinea-pig lung by a concentration of 1 mg/ml. of 48/80 and enhanced the release when the pH was raised from 7.4 to 8.2.5. 1 mg/ml. of 48/80 did not inhibit the enhanced oxygen consumption produced by DNP in the isolated rat diaphragm.6. Iodoacetic acid (IAA) or a Ca/Mg-free medium inhibited the release of histamine by 20 mug/ml. of 48/80 from rat lung but not the release produced by 1 mg/ml. in either rat or guinea-pig lung.7. The degranulation of rat mesentery mast cells caused by 20 mug/ml. of compound 48/80 was inhibited by DNP. The degranulation evoked by 1 mg/ml. of 48/80 was also sensitive to this inhibitor; in this instance, however, the metachromatic staining reaction of the mesentery mast cells was greatly diminished.8. It is concluded that two processes of histamine release by compound 48/80 occur in rat lung. One, dependent on cell metabolism, involves, mast cell granule secretion. The other, independent of cell metabolism, seems to consist of a simple exchange reaction between histamine and compound 48/80, and this is the only one occurring in guinea-pig lung.     

Increased Utilization of Endogenous ATP in Isolated Rat Mast Cells during Histamine Release Induced by Compound 48/80

Abstract: In order to investigate whether the energy dependence of histamine release induced by compound 48/80 from isolated rat mast cells reflects an increased utilization of ATP during the release process, the ATP content in samples of mast cells was determined 5 sec. before and 25 sec. after the addition of compound 48/80. No change in the ATP content was found when histamine release was induced from cells with an intact energy metabolism. However, when the cells had been preincubated for 1 min. with antimycin A to inhibit oxidative production of ATP, histamine release was accompanied by a greater reduction (P < 0.005) in the ATP content than that observed in the control samples. In contrast, when corresponding experiments were performed with chlorpromazine or n-decylamine as releasing agent, the ATP content was not lower in the samples in which histamine release had been induced than in the controls, regardless of whether antimycin A was present or not. The results indicate that histamine release from rat mast cells induced by compound 48/80, but not by chlorpromazine or n-decylamine, is accompanied by an increased utilization of ATP in the cells.     

The action of local anaesthetics on histamine release.

The effect of the local anaesthetics lidocaine, procaine and tetracaine on compound 48/80-induced histamine release from isolated rat mast cells has been investigated. They inhibited histamine release in a dose-dependent manner; at a concentration of 20 mM there was almost total inhibition of histamine release by lidocaine and about 75% inhibition by procaine. Tetracaine exerted a biphasic effect: at concentrations below 1 mM it inhibited, but at concentrations above 1 mM it potentiated histamine release. The inhibitory effect of lidocaine on compound 48/80-evoked histamine release was dependent upon the time of preincubation of mast cells with this anaesthetic and it persisted after washing the cells and resuspension in a lidocaine-free medium. An increase of calcium ions antagonized the inhibitory action of lidocaine. These results can be explained by (1) blockade of membrane receptors for calcium binding which leads to a decrease in intracellular calcium concentration and (2) increase of cellular cyclic AMP content which subsequently inhibits the releasing process.     

Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1).

Rapid intravenous administration of the glycopeptide antibiotic, vancomycin, may cause a hypotensive reaction which can usually be prevented by infusing vancomycin in dilute solutions. The release of histamine from circulating cells such as basophils and tissue mast cells has been implicated in hypotensive reactions since the effects can be prevented by antihistamine pretreatment. The direct effects of vancomycin on histamine release were therefore investigated in rat peritoneal mast cells and rat leukemic basophils (RBL-1 cells). Suspension cultures of mast cells or RBL-1 cells were exposed to vancomycin for 30-60 minutes at concentrations comparable to those infused clinically (2.28 or 4.56 mg/ml). Vancomycin induced a time- and dose-dependent release of histamine into the culture media from both cell types. The reference degranulating agent, Compound 48/80 (CP 48/80), was also shown to induce histamine release from mast cells and RBL-1 cells. Mast cells were significantly more sensitive to vancomycin and CP 48/80 than RBL-1 cells and, unlike RBL-1 cells, were responsive to the inhibitory effects of cromolyn sodium on histamine release. Cromolyn sodium did not inhibit vancomycin-induced histamine release in RBL-1 or mast cells. Morphologically, mast cells exposed to either vancomycin or CP 48/80 exhibited dose-related degranulation. On the other hand, treatment-related degranulation effects of either vancomycin or CP 48/80 on RBL-1 cells could not be reliably distinguished from controls by qualitative evaluation. Based upon these findings it is concluded that mast cells may represent a more useful model to evaluate the potential of investigational agents to release histamine and to study mechanisms of histamine release than RBL-1 cells.     

Role of microtubules on Ca2+ release from the endoplasmic reticulum and associated histamine release from rat peritoneal mast cells.

In order to study the role of cytoskeletons on histamine release from mast cells, the effects of cytoskeleton-inhibiting agents were investigated. Since neither colchicine, vinblastine nor cytochalasin D was effective in inhibiting the IP3 formation, it is possible that neither microtubules nor microfilaments of rat peritoneal mast cells participate in the initial membrane events of the histamine release. However, both colchicine and vinblastine, but not cytochalasin D, were effective in inhibiting Ca2+ release from the intracellular Ca store. It was accordingly suggested that the microtubules, rather than microfilaments, are intimately related to the Ca2+ releasing process from the endoplasmic reticulum. The fluorescence intensity of the mast cells stained with FITC-labeled anti-tubulin antibody reflects the amount of tubulin polymers inside the cell, and colchicine treatment decreased the fluorescence intensity, indicating that colchicine is effective in depolymerizing the microtubules of rat mast cells. By contrast, the amount of tubulin polymer in the mast cells increased by compound 48/80, indicating that the rearrangement of microtubules took place in the mast cells, leading to histamine release. When permeabilized mast cells were exposed to potassium antimonate solution, microtubules attached themselves to the endoplasmic reticulum and many Ca antimonate dots were observed. From the present results, it was concluded that microtubules play an important role in the processes leading to Ca2+ release from the intracellular Ca store and subsequent histamine release.     

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 micrometers, whereas the 3-acyl- and the 4-alkyl-compounds were not effective at doses over 100 microns. 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.     

Labelling of the inner side of granules membrane during exocytosis. A method to differentiate secretion processes

A fluorescence method to monitor quantitatively exocytosis phenomena in suspensions of intact cells was investigated, on the basis of the particular incorporation properties of a specific plasma membrane fluorescent label trimethylammonium-diphenylhexatriene (TMA-DPH). The method was tested with purified peritoneal mast cells stimulated by compound 48/80. Kinetics and dose-dependent response to compound 48/80 could be described accurately by the TMA-DPH fluorescence increase accompanying the exocytosis phenomenon, as controlled by conventional titrations of the released histamine. TMA-DPH bound to the outer side of the plasma membrane of resting cells and additively to the inner side of granules connected by pores to the exterior of stimulated mast cells.     

Inhibition by calcium antagonists of histamine release and calcium influx of rat mast cells: Difference between induction of histamine release by concanavalin A and compound 48/80

The relation between calcium influx and histamine release from rat mast cells was investigated. When purified mast cells pretreated with a calcium antagonist (MnCl₂ or methoxyverapamil (D-600)) were exposed to concanavalin A or compound 48/80 in Tyrode solution (pH 7.4) at 37°C, the calcium antagonists inhibited the extracellular calcium-dependent component of concanavalin A-induced histamine release. MnCl₂ also inhibited the extracellular calcium-dependent component of compound 48/80-induced histamine release, whereas D-600 did not inhibit the release. D-600 inhibited the ⁴⁵Ca uptake induced by concanavalin A, but did not inhibit the ⁴⁵Ca uptake induced by compound 48/80. It was found that the inhibitory action of calcium antagonists depended on the uptake of extracellular calcium. These observations suggest that concanavalin A and compound 48/80 stimulate different mechanisms of calcium influx. Studies on inactivation of the mechanisms of calcium influx showed that calcium influx into cells activated by concavalin A stopped when concanavalin A was washed out, whereas the influxactivated by compound 48/80 was still operative after compound 48/80 had been washed out.     

Sodium-potassium ATPase inhibition potentiates compound 48/80-induced histamine secretion from mast cells.

The effect of ouabain on the histamine secretion induced by compound 48/80 has been studied using rat peritoneal mast cells. Ouabain did not modify histamine release in the presence of millimolar concentrations of extracellular calcium. However, when mast cells were previously washed with a calcium-free buffer, ouabain strongly potentiated histamine release elicited by compound 48/80. The full potentiation of mast cell secretion by ouabain required 30 min preincubation before adding compound 48/80. It was inhibited by lanthanum and EGTA. Potassium deprivation mimicked the effect of ouabain. A 30 min preincubation time without potassium was also required. Potassium concentrations below 2.7 mM increased the effect of ouabain whereas higher potassium concentrations reversed this effect. The potentiation of compound 48/80-induced histamine release by ouabain or potassium deprivation was not immediately reversed by washing away ouabain or by adding potassium, respectively. The data confirm that sodium-potassium ATPase is involved, through a calcium-dependent process, in the regulation of histamine release from mast cells.     

The effect of concentration on the binding of compound 48/80 to rat mast cells: a fluorescence microscopy study

A fluorescent analog of the chemical histamine liberator, compound 48/80, has been synthesized by the covalent attachment of rhodamine to the 48/80 polymer (R-48/80). The histamine liberating characteristics of this analog were similar to those of the parent compound. The binding characteristics of R-48/80 to rat peritoneal mast cells were then studied using fluorescence microscopy. At concentrations that caused minimal secretory stimulation (less than 1.0 microgram/ml), R-48/80 bound to the mast cell surface in a diffuse manner, with no indication of patching or capping. When the cells were incubated at higher concentrations, where non-cytotoxic histamine secretion was stimulated, the drug bound heavily to the exposed granules, but not to unexposed granules or other cell organelles. At cytotoxic concentrations, R-48/80 caused extensive cell clumping, with the drug bound to masses of cell debris and released granules. Therefore, although R-48/80 binds initially to the cell membrane, its primary binding site at concentrations that induce secretion becomes the mast cell granule. The properties of these granules should thus be considered when studying the binding of compound 48/80 or other cationic drugs to rat peritoneal mast cells.