Replenishment of the cellular calcium required for non-immunologic stimulation of mast cell histamine secretion: temperature sensitivity and inhibition by manganese and sodium-free conditions

Mast cells depleted of cellular calcium (Ca) by a 3 hr exposure to Ca-free conditions and then bathed in Ca-free Locke failed to release histamine when stimulated by compound 48/80 or peptides. The cellular Ca required for histamine release could be replenished by a 5 sec exposure to extracellular Ca at 37 degrees C. To inhibit this replenished cellular Ca dependent histamine secretion required an additional 3 hr exposure to Ca-free conditions. When cellular Ca was replenished at 4 degrees C, an additional 2 min incubation at 37 degrees C was required to restore stimulated secretion to a maximum. During this 2 min incubation period the replenished cellular Ca is suggested to be "processed" so that it can be used for secretion. Manganese (Mn) or cobalt added during (but not after) this 2 min incubation period prevented the restoration of histamine release. Preincubation of cellular Ca depleted mast cells in Mn (0.1-1 mM) blocked the effect of subsequent Ca replenishment at 37 degrees C while cobalt and barium were less inhibitory. Neither magnesium nor strontium were inhibitory. Extracellular sodium (Na) was required for the restoration of cellular Ca dependent histamine secretion. Lithium could substitute for Na but rubidium and potassium were ineffective.     

Actions and cross-reactivity of antiallergic agents and a calcium channel antagonist on rat peritoneal mast cells. Difference in the action mechanisms and cross-reactivity among the agents.

The actions of the antiallergic agents, disodium chromoglycate (DSCG), tranilast and ketotifen, and of a calcium channel antagonist, nicardipine, and cross-reactivity among the agents were examined by observing the inhibition of 45Ca uptake and histamine release in rat mast cells stimulated by antigen and compound 48/80 (comp. 48/80). 1) All agents inhibited 45Ca uptake and histamine release in mast cells stimulated by antigen. The inhibition of 45Ca uptake by the antiallergic agents paralleled the inhibition of histamine release, while nicardipine inhibition of 45Ca uptake was stronger than its inhibition of histamine release. 2) The action of DSCG on 45Ca uptake and histamine release was significantly decreased in cells stimulated with antigen and phosphatidylserine (PS), while tranilast inhibition of histamine release was not affected by the addition of PS despite a significant decrease in the inhibition of 45Ca uptake. 3) The inhibitory effect of DSCG and tranilast was significantly lower in mast cells stimulated by comp. 48/80 than in the cells stimulated by antigen. 4) Tachyphylaxis was observed in cells re-exposed to DSCG and tranilast following previous exposure to the agents. 5) Cross-reactivity was found between DSCG and tranilast.     

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.     

Histamine release induced from rat mast cells by the ionophore A23187 in the absence of extracellular calcium

Isolated rat mast cells were used to study whether ionophore A23187 could induce histamine release by mobilizing cellular calcium. The histamine release was a slow process which was completed after about 20 min incubation with A23187. The A23187-induced histamine release was inhibited after incubation of the cells with EDTA for 1 h in a 37°C water bath in calcium-free medium. Reintroduction of calcium in excess of EDTA induced the release of histamine. The observations suggest that A23187 can induce histamine release by mobilizing a cellular pool of calcium.     

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.     

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.     

The action of amlodipine on voltage-operated calcium channels in vascular smooth muscle.

1. The activity of the Na+/K(+)-pump in rat peritoneal mast cells was measured at various time intervals after induction of cellular histamine release by compound 48/80 or by the antigen-antibody reaction. The Na+/K(+)-pump activity was assessed as the ouabain-sensitive potassium uptake of the cells using 86Rb+ as a tracer for potassium (K+(86Rb+)-uptake). 2. Stimulation of the cells with compound 48/80 induced a time and concentration dependent increase of the Na+/K(+)-pump activity. The pump activity was maximal 2 min after stimulation of the cells. Then, the activity gradually decreased and reached a level not significantly different from the controls after 2 h of incubation. 3. When the cells were stimulated by the antigen-antibody reaction, there was also a rapid (within 5 min) stimulation of the Na+/K(+)-pump. In contrast to the stimulation with compound 48/80, the pump activity returned to the control level after 60 min of incubation with antigen. 4. The ouabain-resistant potassium uptake of the cells was increased after stimulation of the cells, regardless of the secretagogue used. This probably reflects the increased surface area of the cells present after the histamine release. 5. On the basis of the present results, we suggest a role for the Na+/K(+)-pump in the recovery process of the mast cell following histamine release.     

Prolonged exposure to a K-rich medium makes the rat mast cell membrane permeable to external calcium ions

Rat mast cells, pretreated with a Ca-free, high KCl medium for more than 30 min, released histamine when subsequently exposed to a medium containing 0.1-2 mM Ca, despite the absence of voltage-dependent Ca channels. Morphological studies showed that high KCl-treated cells became swollen and that the addition of Ca caused degranulation (exocytosis). Ca-stimulated histamine release was inhibited when the high KCl treatment medium contained dinitrophenol and 2-deoxyglucose. The response to Ca was also observed when KCl in the treatment medium was replaced by RbCl, CsCl, KBr or KNO3. When the high K-treated cells were incubated with 0.5 mM 45Ca at 0-1 degree C, 45Ca uptake by these cells was much larger than that by untreated cells, suggesting that the membrane permeability to Ca of these cells is increased. Although prolonged (90 min) incubation of the cells with an isotonic KCl medium increased the rate of leakage of lactate dehydrogenase possibly reflecting extensive cell swelling, most cells recovered the ability of responding to stimulation with compound 48/80 or with simultaneous removal of Na and Ca when subsequently incubated with a Na-based medium containing Ca. Raising the KCl concentration above normal osmolarity in the treatment medium reduced the extents of cell swelling, without reducing the response to Ca addition. Under various other experimental conditions, a poor correlation was also found between the extents of swelling and the magnitude of the response to Ca. These results raised the possibility that intracellular accumulation of a particular species of anions increased membrane permeability to Ca.     

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.     

Histamine release from isolated rat mast cells induced by opiates: effect of sterical configuration and calcium

The stereospecificity of the action of opiates on rat mast cells was investigated by means of the l- and d-isomers levorphanol and dextrorphan. The dose-response curves for histamine release induced by the 2 drugs were of a similar shape with a maximum at 2 X 10(-3) M and a pronounced minimum at 5 X 10(-3) M. At concentrations below 5 X 10(-3) M the effect of both drugs resembled that of morphine, i.e. the release occurred rapidly and inhibition was observed with naloxone, codeine, and antimycin A. Levorphanol, dextrorphan, and the antagonist levallorphan at concentrations above 5 X 10(-3) M seemed to be toxic to mast cells. The uptake of 45Ca in connection with histamine release induced by pethidine, levorphanol, and dextrorphan was lower than that of control cells, whereas the uptake induced by morphine did not differ from that of controls. The inhibition of compound 48/80-induced histamine release by morphine was paralleled by a reduced 45Ca uptake. The time course for the inhibitory effect of preincubation with morphine was similar for the histamine released induced by morphine and by compound 48/80. Washing of the cells after preincubation with morphine was without effect on the inhibition of morphine-induced histamine release, whereas the inhibition of compound 48/80 was reduced. The present observation with morphine and compound 48/80 support our previous impression of similarities in their mode of action, but a mechanism implying an interference by morphine with the disposition of calcium could also account for the findings. The observed antagonism between morphine and calcium resembles that of opiate receptors, but histamine release induced by opiates does not involve stereospecific opiate receptors.     

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.     

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.     

Effect of the carboxylic ionophore monensin on histamine release from rat peritoneal mast cells.

The effect of the monovalent carboxylic ionophore monensin, which mediates a one-for-one exchange of intracellular H+ for extracellular Na+, was investigated in purified rat peritoneal mast cells. Monensin inhibited histamine secretion induced by compound 48/80, adriamycin and the calcium ionophore A23187; the inhibitory effect was maximal when the compound was added at least 10 min before the secretagogues. Washing of cells before addition of the secretagogues did not abolish the inhibitory effect of monensin. On the contrary the carboxylic ionophore was completely ineffective in preventing concanavalin A-induced histamine release. When rat peritoneal mast cells were incubated in the presence of monensin for longer period (up to 5 hours), the substance induced a slow, progressive and dose dependent histamine release, which, at least for lower doses was noncytotoxic. The secretory effect of monensin was still present if the ionophore was washed away after 10 min of incubation, and the incubation continued in drug-free medium. Monensin stimulated histamine secretion was strictly dependent on extracellular Na+ concentrations, and independent on extracellular Ca++.     

Effect of TMB-8 on calcium transport in mast cells in relation to histamine secretion

We have previously reported an inhibition of histamine release by TMB-8 both in the presence and absence of calcium and with glucose in the medium. In the present investigation we have studied the effect of TMB-8 on calcium transport. The observations show that TMB-8 inhibits calcium uptake and enhances calcium efflux in mast cells. As antigen-induced histamine release from sensitized mast cells is primarily dependent on extracellular calcium, the inhibition of anaphylactic histamine release by TMB-8 is probably mainly due to an inhibition of calcium influx into the mast cells. We have shown an increased calcium efflux during histamine release from mast cells induced by compound 48/80 in the absence of calcium in the medium, suggesting the release of intracellular calcium stores. The increased calcium efflux was not inhibited by TMB-8. On the contrary, the enhanced calcium efflux caused by compound 48/80, was added to that by TMB-8. TMB-8 thus had no effect on the calcium release from intracellular stores by compound 48/80 but the enhanced calcium efflux by TMB-8 would tend to inhibit histamine release.     

Influence of aminoglycoside antibiotics, streptomycin and kanamycin on histamine secretion in mast cells.

Effects of kanamycin and streptomycin on histamine release from rat mast cells were examined in response of the cells to concanavalin A(Con A) plus phosphatidylserine (PS), phytohemagglutinin (PHA) plus PS or a mixture of low-molecular-weight polymers of P-methoxy-N-methylphenethylamine (compound 48/80). In the response to each of the above stimuli, kanamycin (20 mM) or streptomycin (20 mM) caused a decrease in the histamine release elicited in the presence of extracellular Ca2+ (1 mM), although streptomycin showed the much higher inhibitory potency than kanamycin. Similarly, streptomycin was much more effective in suppressing compound 48/80-triggered histamine release in the absence of external Ca2+. Histamine release in the absence of external Ca2+ in the response to the lectin plus PS diminished with increasing concentration of kanamycin, and in this respect streptomycin was much less effective. In the response to the lectin plus PS, external Ca2+ possessed potency to antagonize kanamycin (10 mM)- or streptomycin (10 mM)-caused inhibition of the histamine release, although more markedly the kanamycin-caused one. Streptomycin and kanamycin inhibit histamine release from mast cells challenged with IgE-directed secretagogue or compound 48/80, and the responsible mechanisms seem to implicate the Ca(2+)-antagonistic action on the stimulus-provoked influx of Ca2+ and impairment of the cellular events linked to exocytosis.     

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.     

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 experiments in 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.     

Histamine release from the isolated guinea-pig heart by compound 48/80

Histamine release was observed in isolated guinea-pig heart perfused by the constant flow method, after bolus injection of 50-200 micrograms of compound 48/80. Practically all the histamine is released within the first 3 min after 48/80. This effect is inhibited by the addition of sodium cyanide (300 microM) to a glucose-free perfusion fluid and also by the removal of calcium of the medium in presence or absence of EDTA (100 microM). This dependence on metabolism and calcium is highly suggestive of a non-cytotoxic action of 48/80 on the mast cells of guinea-pig heart.     

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.     

The effect of adrenocorticotropin on histamine and 5-hydroxytryptamine secretion from rat mast cells

Characteristics of histamine (Hi) and 5-hydroxytryptamine (5-HT) release from rat peritoneal mast cells in response to the polypeptide adrenocorticotropin (ACTH) were studied. During a 15 min incubation at 37 degrees C, ACTH evoked Hi as well as 5-HT release from rat mast cells at concentrations of 1 X 10(-4) M-1 X 10(-3) M. The release was dose-dependent and very rapid. After 15 sec the amount of the amines released was the same as after 4.5 min. In most experiments, the percentage of Hi release was slightly but significantly higher than the percentage of 5-HT release. Hi and 5-HT release induced by ACTH also took place in a calcium-free medium. However, the release of the amines was decreased when calcium was omitted. Comparison of the effects of ACTH, compound 48/80 and substance P on mast cell secretion showed that ACTH is about 100 times less active then substance P which was in turn about 100 times less active than compound 48/80. When both ACTH and compound 48/80 were used together as liberators , the release was significantly higher than with either liberator alone. Our results indicate that there are receptor sites for the endogenous polypeptide ACTH on the mast cell membrane which mediate Hi and 5-HT release. This release was found to resemble that evoked by the basic secretogogue compound 48/80 but in some aspects to be different from that evoked by substance P.