Inhibition of mast cell disruption and histamine release in rat anaphylaxis in vitro. Comparison with compound 48/80

In vitro anaphylactic reaction causes mast cell damage and histamine release from rat tissue. Histamine release is correlated with mast cell damage and both phenomena are simultaneously inhibited by various metabolic inhibitors, antipyretics, calcium lack and previous heating of the tissue at 45°. The mast cell damage produced by antigen in sensitized rat tissues is morphologically similar to that caused by compound 48/80, both agents causing extrusion of granules. Mast cell damage and histamine release induced by antigen or by compound 48/80 are inhibited alike by several substances and conditions. It is suggested that in rats the histamine-releasing mechanism of the antigen-antibody reaction in anaphylaxis is very similar to that of compound 48/80.     

Comparison of histamine release induced by synthetic polycations with that by compound 48/80 from rat mast cells.

We compared the histamine release induced by polyethylenimines and polyallylamines with that induced by compound 48/80. Lidocaine inhibited the histamine release induced by polyethylenimine with a molecular weight of 600 (PEI6), but disodium cromoglycate did not. The histamine releases induced by all polyethylenimines and polyallylamines tested were inhibited by lidocaine, but not by disodium cromoglycate. Islet activating protein inhibited the histamine release induced by PEI6. Its effects on the release by other polyethylenimines and polyallylamines were less than that on PEI6. It is likely that the inhibition of G proteins by islet activating protein resulted in a decrease of the histamine release. This possibility was supported by the finding that guanyl-5'-(beta, gamma-imino) triphosphate enhanced the histamine release. An inhibitor of polyphosphoinositide phosphodiesterase, neomycin, did not affect the histamine releases induced by these polymers. The effect of PEI6 seemed to resemble that of compound 48/80. After pretreatment of mast cells with wheat germ agglutinin and with Limax flavus agglutinin, releases of histamine induced by PEI6 and compound 48/80 decreased, suggesting that the binding sites of PEI6 and compound 48/80 had sialic acid and/or N-acetyl glucosamine residues. The binding site for PEI6 seemed to especially overlap those of compound 48/80.     

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.     

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.     

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.     

The role of cell-fixed calcium in histamine release by compound 48/80.

1. Histamine release by compound 48/80 was substantially reduced in a time-dependent manner (maximum at 30 min) by pre-incubating mast cells in calcium-free medium at 37 degrees C but not at 2 degrees C. This effect was optimal at pH 7.0 to 7.5. 2. The re-introduction of calcium (0.1 to 3 mmol/l) restored histamine release to the control value; this effect was independent of temperature. 3. Strontium (1 to 30 mmol/l) partially reversed the effect of calcium deprivation but the same concentrations of barium and magnesium depressed histamine release even further. Magnesium (3 to 15 mmol/l) antagonized the effect of calcium replacement. 4. Results suggest that the level of cell-fixed calcium involved in compound 48/80-induced histamine release may be controlled by the combination of rapid passive influx and slow active efflux.     

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.     

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 an in vitro histamine release inhibitor.     

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.     

Interaction between barium, strontium and calcium in histamine release by compound 48/80.

Pre-incubating rat peritoneal and pleural mast cells for 30 min at 2 degrees C in Ca2+-free Tyrode containing Ba2+ (30 mM) markedly reduced histamine release by compound 48/80 (0.5 microgram/ml). Evoked secretion was not significantly altered by pre-incubation with Sr2+ (30 mM) at this temperature whilst Mg2+ (30 mM) and La3+ (0.1 mM) slightly inhibited and potentiated release respectively. The effect of Ba2+ was concentration related and progressively reversed by Ca2+ (1--10 mM). Pre-incubation at 37 degrees C in Ca2+-free medium alone substantially reduced histamine release by compound 48/80. This reduction was unaltered by either Mg2+ (30 mM) or La3+ (0.1 mM), prevented by either Sr2+ (30 mM) or Ca2+ (1 mM) and augmented by Ba2+ (30 mM) which also markedly increased spontaneous histamine release during pre-incubation at 37 degrees C. Results suggest that Ba2+ and Sr2+ interact with cell-fixed calcium to modulate histamine release by compound 48/80.     

Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80

The kinetics of Ca2+ uptake and efflux have been measured in rat peritoneal mast cells stimulated with compound 48/80 using rapid mixing and a silicone oil centrifugation technique. Responses at one-second time intervals were resolved beginning as early as three seconds after initial stimulation. The results clearly demonstrate that Ca2+ uptake occurs after the initiation of histamine release. Ca2+ efflux occurs simultaneously with histamine release. The implications of these findings are discussed and the technique is described.     

Histamine release induced from mast cells by active components of compound 48/80

Compound 48/80 and 14C-labeled compound 48/80 were synthesized, and fractionated by thin-layer chromatography into 14 components (A-N) with various histamine releasing activities and different Ca2+ 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 enhanced by extracellular Ca2+, 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 Ca2+ -free medium than in Ca2+-containing medium, and was partially reduced by pretreatment of mast cells with neurotensin or substance P, Ca2+-independent releasers. Apparently both fractions D and H are useful reagents for investigating the role of Ca2+ in histamine release and releaser binding in mast cells.     

Inhibitory activity of indolin-2-one derivatives on compound 48/80-induced histamine release from mast cells

Four alkaloids, previously identified in Isatis species, were tested for their inhibitory effect on histamine release. Whereas tryptanthrin, indirubin and deoxyvasicinone did not inhibit histamine release, the effect of indolin-2-one exceeded that of the mast cell stabilizing drug disodium chromoglycate.     

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.