Inhibitory effect of nifedipine and cromolyn sodium on skin reactions and 45Ca uptake and histamine release in rat mast cells induced by various stimulating agents

The effect of nifedipine, one of the calcium channel antagonists, was examined in allergic and nonspecific reactions using rat skin and peritoneal mast cells. Rat passive cutaneous anaphylaxis response mediated by IgE antibody was inhibited by nifedipine (100 micrograms/rat) and also by cromolyn sodium (5 mg/rat). Skin reactions induced by histamine or methacholine were not inhibited, and skin reaction induced by serotonin was slightly inhibited by these drugs. The 45Ca uptake and histamine release in sensitized mast cells stimulated by ovalbumin were suppressed by nifedipine. In actively sensitized cells, nifedipine seems to be more effective on the inhibition of 45Ca uptake, but less effective on histamine release than cromolyn sodium. In mast cells, nonimmunologically stimulated by concanavalin A, compound 48/80, and calcium ionophore A23187, nifedipine exerted considerable inhibitory effects on both 45Ca and histamine release.     

The relationship between histamine secretion and 45calcium-uptake by mast cells

1. Unstimulated mast cells from the peritoneal cavity of the rat take up (45)Ca: the initial phase of rapid uptake being complete after 1 min incubation of the cells with the isotope. Stimulation of the mast cells with an antigen-antibody reaction, dextran or concanavalin A induces an increase in the uptake of (45)Ca which is accompanied by a release of granular material: this increase in (45)Ca uptake is also complete in 1 min. The majority of the stimulated (45)Ca uptake cannot be explained in terms of binding of Ca to released granular material, or to an enlargement in either the extracellular compartment or the cell surface area.2. The magnitude of the increase in (45)Ca uptake caused by stimulating the mast cells increases when the degree of histamine secretion increases.3. The increased (45)Ca uptake induced by stimulation of the mast cells and the degree of histamine secretion are both dependent on extracellular H ion concentration. Changes of pH cause similar changes in (45)Ca uptake and secretion with maxima at pH 7.5.4. Two thirds of the (45)Ca uptake induced by an antigen-antibody reaction or by the Ca ionophore A 23187 is unaffected by inhibiting glycolysis and oxidative phosphorylation. Histamine secretion on the other hand is practically abolished by this metabolic inhibition. Thus, (45)Ca uptake proceeds in the absence of the discharge of granules.5. Dibutyryl cyclic AMP or theophylline inhibit both the increase in (45)Ca uptake and the histamine secretion caused by stimulating mast cells with an antigen-antibody reaction. Cyclic AMP, cyclic GMP and dibutyryl cyclic GMP have no effect on uptake or secretion.6. The Ca ionophore, A 23187, induces uptake of (45)Ca and histamine secretion, neither effect being inhibited by either dibutyryl cyclic AMP or theophylline.7. Phosphatidyl serine increases both (45)Ca uptake and the histamine release induced by an antigen-antibody reaction, dextran or concanavalin A.     

The inhibition of histamine release by antiallergic drugs.

Three new antiallergic drugs, Doxantrazole, PRD-92 and N5', as well as disodium cromoglycate, inhibited the IgE-mediated PCA reaction in the rat triggered by the homologous antigen, but did not have an antagonistic effect on histamine itself. Moreover, all the drugs examined caused in vitro inhibition of antigen-mediated histamine release from peritoneal mast cells and chopped lung tissue of sensitized rats producing IgE antibodies. Doxantrazole had a synergistic effect on the inhibition of histamine release by isoproterenol, whereas the other drugs were devoid of this capacity. PRD-92 and N5' inhibited the ionophore A23,187 induced histamine release, but did not have any effect on the D2O-enhanced histamine release which was triggered by antigen.     

Binding of45Calcium to isolated rat mast cells in connection with histamine release

The time courses for histamine release and uptake of⁴⁵Ca were compared on isolated rat mast cells after stimulation under different experimental conditions with antigen, compound 48/80, ATP, or the ionophore A23187. Except for ATP the results did not show a time-dependent correlation between histamine release and⁴⁵Ca uptake.The uptake of⁴⁵Ca under conditions where membrane-bound calcium is utilized for optimal anaphylactic histamine release did not differ from that of controls, whereas the presence of⁴⁵Ca in the incubation medium led to a substantial uptake without influence on the histamine release.The uptake of⁴⁵Ca induced by antigen or compound 48/80 was completely inhibited by antimycin A as confirmed by the use of two different methods. In addition, an energy-dependent restitution of the permeability properties of the plasma membrane seemed to follow histamine release. Antimycin A partly reduced the uptake of⁴⁵Ca after stimulation with ATP, and did not affect binding following exposure of the cells to the ionophores A23187 or X537A.The ionophore A23187 was able to reduce the⁴⁵Ca content of mast cells previously loaded with the isotope. Mast cells pretreated with A23187 in the absence of extracellular calcium did, after washing, accumulate substantial amounts of⁴⁵Ca without release of histamine.The results suggest that only small amounts of calcium are required to trigger histamine release and that studies with⁴⁵Ca do not distinguish between specific uptake of calcium and nonspecific equilibration secondary to morphological secretory changes.The present results were in part presented at the Sixth Meeting of The Histamine Club, London, April 1977.     

The effect of phosphatidyl serine on the activation stage of histamine release induced by neutrophil cationic protein.

Histamine release from rat mast cells induced by cationic protein (band 2) from rabbit neutrophil lysosomes occurs in Ca++-deficient medium. At higher concentrations of Ca++ the release is inhibited. Strontium not only supports, but also enhances the release of histamine in the absence of Ca++. Progressive enhancement of release occurs between 1.8 and 14.4 mM Sr++. The release of histamine from mast cells, activated at low temperature (0-4 degrees C) in the presence of 14.4 mM Ca++ and then washed prior to incubation at 37 degrees C, is inhibited. However, if phosphatidyl serine (PS) (10 microgram) is present with 14.4 mM Ca++, the inhibition is reversed. There is also inhibition of release when cells, activated in the presence of 1.8 mM Ca++, are incubated in the second stage with 14.4 mM Ca++, but this inhibition is less pronounced than when the 14.4 mM Ca++ is in the activation stage. PS enhances the release in the presence of both Ca++ and Sr++. The presence of PS in the activation stage enhances the release, but there is no significant enhancement when cells activated in the absence of PS are washed and incubated in the presence of PS. This suggests that PS enhancement of histamine release occurs at the activation stage, probably through the efficient delivery of calcium to the membrane sites, thereby increasing the efficacy of the membrane perturbation by band a protein.     

Differential histamine release by dextran and the ionophore A23187: the actions of inhibitors.

Inhibitors of mast cell membrane activation reduced histamine release from rat mast cells induced by dextran and phosphatidyl serine but not that induced by the calcium ionophore A23187. Such inhibitors included cromoglycate, an orally-active anti-allergic agent 3-(5-tetrazolyl)thioxanthone 10,10-dioxide, dibutyryl cyclic 3':5'-AMP, theophylline and dicumarol. Inhibitors of mast cell metabolism reduced both types of release and these included oligomycin, papevevime, and the two uncouplers of oxidative phosphorylation-alpha2,4-dinitrophenol and CCCP. Inhibition of histamine release from rat isolated peritoneal mast cells by either a mixture of dextran and phosphatidyl serine or the ionophore A23187 thus allows inhibitors of mast cell membrane activation to be distinguished from those affecting cell metabolism or the later stages of the secretory process.     

Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors

Inhibition of IgE dependent histamine release from human mast cells by protease inhibitors has been observed in skin, tonsil and synovial tissues. However, little is known about the actions of protease inhibitors on tryptase release from human lung mast cells. We therefore examined the ability of protease inhibitors to modulate tryptase and histamine release from human lung mast cells. IgE dependent tryptase release from dispersed lung mast cells was inhibited to a maximum of approximately 53.8% and 44.5% by N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), respectively. A similar degree of inhibition of calcium ionophore A23187 (CI) induced tryptase release was also observed with these two inhibitors. Preincubation of TLCK or TPCK with the mast cells at 37 degrees C for 20 minutes before addition of anti-IgE or CI did not improve their ability to inhibit anti-IgE and CI induced tryptase release. At a concentration of 10 microg/ml, protamine inhibited anti-IgE or CI induced tryptase release; but at 100 microg/ml, it increased anti-IgE and CI induced release of tryptase from lung mast cells. A concentration dependent inhibition of anti-IgE and CI induced release of histamine from lung mast cells was also observed with TLCK, TPCK and protamine. The maximum inhibition of anti-IgE induced histamine release was approximately 40.7%, 40.2% and 33.4% with TLCK, TPCK and protamine, respectively. At the concentrations tested, TLCK and TPCK by themselves did not stimulate tryptase and histamine release from lung mast cells. A specific inhibitor of aminopeptidase, amastatin, had no effect on anti-IgE induced tryptase and histamine release and was used as control. In conclusion, it was demonstrated that protease inhibitors are able to inhibit IgE dependent tryptase and histamine release from human lung mast cells, which suggested that they could be developed to a novel class of anti-inflammatory drugs to treat allergic conditions in man.     

Effect of staurosporine on histamine release from rat serosal mast cells

Rat serosal mast cells were challenged with compound 48/80 or calcium ionophore A23187 and the effect of staurosporine, a new inhibitor of protein kinase C, on histamine release from the cells was investigated. Histamine release induced by compound 48/80 or calcium ionophore A23187 was inhibited by staurosporine in a concentration-dependent manner and 0.1 and 1 microM staurosporine inhibited the histamine release significantly. The inhibitory effect of K-252a, another novel protein kinase C-inhibitor, was significantly higher than that of staurosporine on calcium ionophore A23187-induced histamine release. These results suggest that protein kinases will be involved in the process during mediator release from rat serosal mast cells.     

Role of phospholipase A2 and pertussis toxin-sensitive G proteins in histamine secretion induced by substance P

Incubation of mast cells with substance P or ionophore A23187 resulted in histamine release and arachidonic acid liberation from purified rat peritoneal mast cells. The treatment of mast cells with 100 ng/ml of pertussis toxin for 2h inhibited the effect of substance P on both histamine release and arachidonic acid liberation, but the response to the ionophore A23187 was not affected. Para-bromophenacyl bromide, a selective inhibitor of phospholipase A₂, inhibited similarly histamine and arachidonate release induced by substance P but not that evoked by ionophore A23187. These results suggest that phospholipase A₂ plays a key role in the histamine secretion induced by substance P.

     

The influence of phosphatidyl serine on the release of histamine from isolated rat mast cells induced by different agents

The influence of phosphatidyl serine (PS) on histamine release from isolated rat mast cells induced by antigen, compound 48/80, adenosine-5′-triphosphate (ATP), the ionophore A23187, and decylamine was studied. PS enhanced antigen-induced release but inhibited the release caused by compound 48/80, A23187, and decylamine. PS did not influence the release induced by ATP. The different effects of PS on the action of the various histamine releasing agents do not conform to a unifying model for the action of PS on the release process. Possible interactions between PS and the agents in the incubation medium as well as at specific reactive sites on the plasma membrane might explain some of the effects of PS. Consequently, the results cannot be used as evidence for the existence of basic differences in the release process induced by various calcium-and energy-dependent releasing agents.     

The influence of hypertonicity on histamine release from isolated rat mast cells

Increasing the tonicity of the incubation medium gradually inhibited histamine release from isolated rat mast cells when induced by A-23187, ATP, Thapsigargin, sodium fluoride and compound 48/80 in the presence of extracellular calcium, as well as by the latter in the absence of calcium. In contrast, hypertonicity had but negligible effect on the release caused by the monovalent cation ionophore X-537A. The swelling action induced by both ATP and X-537A on rat mast cells was markedly depressed by hypertonicity. Thus, the swelling action seemed to be inherent to the mechanism by which ATP stimulates mast cells to a calcium-dependent histamine secretion, whereas this was not the case with X-537A. For Thapsigargin and sodium fluoride it was demonstrated that only the secretory step, induced by calcium, was inhibited by hypertonicity, and this could be reversed by adjusting the incubation medium to isotonicity. Therefore, the results suggest that the uptake of calcium in the rat mast cells after stimulation is a passive process.     

Effects of norepinephrine on transmembrane calcium transport in rat mast cells

The effect of norepinephrine on transmembrane passage of calcium in rat peritoneal mast cells, was studied in an in vitro system. It was found the histamine release from mast cells induced by the ionophore A 23 187 in normal calcium medium and compound 48/80 in a calcium-free medium was suppressed by 10(-3) M norepinephrine but not at concentrations in the range 10(-5)-10(-4) M. When the secretory process is totally dependent on the presence of calcium in the incubation medium, i.e. calcium-depleted cells, 10(-5) M norepinephrine suppresses the histamine release induced by low concentrations of compound 48/80. The effect of norepinephrine (10(-5) M) on strontium-induced "spontaneous' histamine release was also studied. It was found that norepinephrine (10(-5) M) totally inhibits the progressive histamine release induced by strontium. It is possible to evoke secretion in a calcium-free medium, and subsequent introduction of Ca2+ will result in optimal histamine release. This demonstrates a secretory process in which we can distinguish between utilization of endogenous versus exogenous calcium. The release that is dependent on extracellular calcium is inhibited by norepinephrine (10(-5) M). These data indicate that the suppressive effect of norepinephrine (concentrations less than 10(-3) M) on histamine release from rat mast cells is due to an interference in transmembrane passage of calcium.     

Antiallergic effect of epinastine (WAL 801 CL) on immediate hypersensitivity reactions: (I). Elucidation of the mechanism for histamine release inhibition.

Epinastine caused an inhibition of histamine release from rat peritoneal mast cells induced by both antigen-antibody reaction and compound 48/80. Epinastine was similarly effective in inhibiting compound 48/80-induced histamine release not only from isolated rat peritoneal mast cells but also from rat mesenterial pieces. Also, histamine release from lung pieces obtained from actively sensitized guinea pigs after exposure to antigen challenge was markedly inhibited by epinastine. The drug was effective in inhibiting not only Ca2+ uptake into lung mast cells in actively sensitized guinea pigs but also Ca2+ release from the intracellular Ca store of rat peritoneal mast cells exposed to both compound 48/80 and substance P. No significant changes were observed in phosphodiesterase activity in rat peritoneal mast cells treated with epinastine, while adenylate cyclase activity was augmented by epinastine. Epinastine has no inhibitory effect on histamine release induced by Ca2+ or IP3 from permeabilized mast cells. However, the drug significantly and dose-dependently suppressed calmodulin activity suggesting that histamine release inhibition due to epinastine may be partly attributable to Ca(2+)-calmodulin dependent process(es). The drug caused no visible changes in thermodynamic behavior of lipids, either in order parameter or in differential scanning calorimetry, indicating that the drug has no influence on membrane fluidity.     

The stimuli releasing histamine from murine bone marrow-derived mast cells. 2. Mechanisms involved in histamine release induced by extracellular ATP and its metabolites.

Extracellular ATP stimulated histamine release and generation of leukotrience C4 (LTC4) accompanied with the formation of inositol phosphates and a rapid increase in intracellular Ca2+ ([Ca2+]i) in mouse bone marrow-derived cultured mast cells (BMMC). The rank order of histamine-releasing potency of ATP and its metabolites is ATP greater than ADP greater than AMP greater than adenosine. Nonhydrolyzable ATP analog, adenosine-5'-O-[2-thiotriphosphate] (ATP-S) released more histamine from the cells than ATP. On the other hand, simultaneous addition of adenosine analogues at micromolar concentrations potentiated histamine release from the cells induced by ATP (50 microM) or DNP-HSA antigen (0.1 ng/ml) in the following rank order: adenosine greater than AMP much greater than ADP = ATP. Histamine release potentiated by adenosine was blocked by the treatment with pertussis toxin, whereas histamine release induced by ATP was not affected by the toxin, suggesting that extracellular ATP stimulate histamine release from BMMC probably via mechanisms independent of the potentiation of histamine release induced by adenosine.     

Protein kinase Cdelta functions downstream of Ca2+ mobilization in FcepsilonRI signaling to degranulation in mast cells

BACKGROUND: Mobilization of Ca 2+ plays an important role in the degranulation of mast cells. Although events upstream of Ca 2+ mobilization in the regulation of degranulation are relatively well characterized, the downstream mediators of Ca 2+ remain largely unknown. OBJECTIVE: We sought to characterize the downstream signaling mechanism by which Ca 2+ mobilization mediates degranulation in antigen-stimulated mast cells. METHODS: The effect of various inhibitors was examined in the antigen-induced or Ca 2+ ionophore A23187-induced degranulation process, and the effect of inhibitors on histamine release was tested in the mouse model of asthma. RESULTS: The delta isoform of protein kinase C (PKC) functions downstream of Ca 2+ in the signaling pathway from FcepsilonRI to degranulation in RBL-2H3 mast cells. Stimulation of cells with either antigen or the Ca 2+ ionophore A23187 induced a rapid translocation of PKC-delta from the cytosol to the cellular membranes, and either treatment with the PKC-delta-specific inhibitor rottlerin or infection with an adenovirus encoding a dominant negative mutant of PKC-delta markedly inhibited degranulation induced with antigen or A23187. Furthermore, both the translocation of PKC-delta and degranulation induced by A23187 were inhibited by prevention of the accumulation of reactive oxygen species normally elicited by the ionophore. Finally, intraperitoneal injection of rottlerin prevented the increase in the concentration of histamine in bronchoalveolar lavage fluid induced by means of antigen challenge in a mouse model of allergic asthma. conclusion: PKC-delta plays an essential downstream mediatory role in the degranulation elicited by Ca 2+ mobilization, and reactive oxygen species mediate the activation of PKC-delta by Ca 2+ in the regulation of degranulation.     

Calcium uptake in mast cells,energy metabolism and histamine secretion

The inhibition of energy metabolism of mast cells causes an inhibition of histamine secretion. As the secretion is generally initiated by the influx of calcium into the cell, we have made correlative studies of the effect of blocking the energy metabolism on calcium uptake and histamine secretion.When the influx of calcium is increased by exposing the cells to low concentrations of saponin or ionophore A23187, histamine release occurs, having the character of a secretory response. Brief incubation of the cells with antimycin A, 10^–9 M–10^–7 M, prior to exposure to saponin or the calcium ionophore gave similar dose-response curves for the inhibitory effect of antimycin A on calcium uptake and histamine release.The inhibition of calcium uptake in untreated mast cells by antimycin A, 10^–9 M–10^–7 M, showed good correlation to the inhibition of anaphylactic histamine release and the release induced by compound 48/80. The antigen-induced histamine release is dependent on extracellular calcium and an inhibition of its uptake by antimycin A could by itself inhibit the release. Compound 48/80 on the other hand induces histamine release both in the presence and absence of calcium, and both are similarly inhibited by 10^–9 M–10^–7 M antimycin A. This indicates that antimycin A has other sites of action apart from the inhibition of the influx of extracellular calcium. The inhibitory effect of antimycin A on compound 48/80-induced histamine secretion in the absence of extracellular calcium may be due to an inhibitio of energy requiring steps in the final phase of the secretory process. These steps could be phosphorylation and activation of enzymes involved in the secretion. Since calmodulin has been demonstrated in a high concentration in the cytosol of the mast cell, and histamine release induced by antigen, compound 48/80, saponin and the calcium ionophore, is inhibited by calmodulin-antagonists, it seems likely that calmodulin-dependent protein kinase plays an important role in the release process.It may then be tentatively concluded that the activation of calcium channels in the plasma membrane as well as the activation of the enzymes involved in the final phase of secretion are both are energyrequiring steps, dependent on ATP.     

Inhibition of rat peritoneal mast cell exocytosis by frusemide: A study with different secretagogues

It has been reported that the loop diuretic frusemide can prevent exercise induced asthma, and that this effect may be due to the inhibition of mast cells in the airway. By using various mast cell secretagogues which increase intracellular calcium via different routes, this study attempted to elucidate the mechanism of the mast cell stabilizing action of frusemide. As well as confirming that immunologically induced histamine release from rat peritoneal mast cells was dose dependently inhibited by frusemide (10⁻³–10⁻⁵ M), the present study has extended the observation to histamine release induced by compound 48/80. The inhibitory potency was however less in the case of compound 48/80 induced release. Frusemide induced inhibition by the two secretagogues was decreased by drug preincubation. In contrast, histamine release induced by ionophore A23187 and thapsigargin was not inhibited by frusemide. The prototype antiallergic compound disodium cromoglycate (DSCG) demonstrated a similar specificity pattern against the various secretagogues. Another loop diuretic, bumetanide, did not show the same results as frusemide on rat peritoneal mast cell degranulation. Hence it is concluded that frusemide does not inhibit immunological activation of mast cells via its diuretic Na⁺/K⁺/Cl⁻ co-transporter capacity. Instead, it protects mast cells in a similar manner to DSCG. accepted by W. Lorenz     

Influence of glucocorticoids on histamine release and45Calcium uptake by isolated rat mast cells

Hydrocortisone and prednisolone inhibited the histamine release from isolated rat mast cells induced by antigen, the ionophore A23187, and compound 48/80 in the absence and in the presence of calcium. Hydrocortisone reduced the response to the different releasing agents by 50% in the concentration range 2–6×10^–4 M, whereas prednisolone was about 1.5 times more potent.The inhibitory effect of hydrocortisone had a rapid onset of action and maximal inhibition was observed after preinculation for 20 minutes. The effect of hydrocortisone was reversed by including 2 mM glucose in the medium. The reversal was only partial with antigen and the ionophore A23187, indicating a greater energy requirement of these releasing agents compared with compound 48/80.The inhibition of the histamine release was accompanied by a concentration-related inhibition of the⁴⁵Ca uptake, ecept with the ionophore. The inhibition of the⁴⁵Ca uptake was also reversed by glucose, but differences were noted concerning the influence of preincubation time. The observations are explained by an association of⁴⁵Ca to cellular material, e.g. granules, secondary to the release process.The effects of hydrocortisone and prednisolone were qualitatively identical and were not observed with estriol. The glucocorticoid inhibition of histamine release does not seem to be caused by effects on phospholipase activity or cyclic AMP metabolism. The present observations are fully consistent with an impaired mitochondrial function as the mechanism for the mast cell effects of the glucocorticoids.     

Cyclosporin A inhibits rat mast cell activation.

To gain further insight into the mechanism of immunosuppression by cyclosporin A (CyA), the effect of CyA on activation of isolated rat mast cells was studied. CyA alone, up to a concentration of 80 microM, had no effect on histamine release from unstimulated mast cells in both calcium-supplemented and calcium-free media. However, in the presence of extracellular calcium CyA inhibited, in a dose-dependent manner (range from 8 nM to 80 microM), histamine release induced by three unrelated secretagogues, the compound 48/80, calcium ionophore A23187 and concanavalin A plus phosphatidylserine. In the absence of extracellular calcium no, or only a marginal, effect of CyA on histamine release induced by the secretagogues was observed. CyA also inhibited the uptake of radiolabeled calcium by the secretagogues-treated cells. However, CyA did not interfere with the activation-related early increase in the intracellular free calcium. Thus, CyA-mediated inhibition of mast cell activation is related to external calcium uptake. These results indicate that rat mast cells belong to the immune cell types whose activity can be modulated by physiologically relevant concentrations of CyA.