Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Histamine release by pharmacological agents in the absence of external free Ca2+

The involvement of extracellular free Ca2+ in histamine release was investigated in rat peritoneal mast cells. Incubation of non-antigenized cells in a media with high extracellular potassium did not increase histamine release. Secretion induced by A23187 and compound 48/80 in the presence of Ca2+ requires metabolic energy. In the absence of external free Ca2+ (2.5 microM) histamine release induced by A23187 is reduced but not abolished. Secretion induced by compound 48/80 is independent of extracellular Ca2+. These results lead us to suggest that mast cell plasma membranes probably lack voltage-gated Ca2+ channels and that external Ca2+ may not be an absolute requisite for histamine secretion.     

The effect of calmodulin on histamine release in the rat peritoneal mast cell

To investigate the role of the Ca2+-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound 48/80, polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca2+. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release.     

Strain differences in histamine release from mouse peritoneal mast cells induced by compound 48/80 or A23187

Strain differences among BALB/c, BDF1, CDF1, C3 H/He, C57 BL/6, DBA/2, ddy and ICR mice were investigated with respect to the ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80, a Ca2+ dependent histamine releaser, and the Ca2+ ionophore A23187. The ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80 were found to be high in BALB/c, ddY and ICR mice, but low in BDF1, CDF1, C3 H/He, C57 BL/6 and DBA/2 mice. Those induced by Ca2+ ionophore A23187 were high in BALB/c, BDF1, CDF1, C3 H/He, DBA2, ddy and ICR mice but low in C57 BL/6 mice. These results indicate that differences in histamine release from mouse peritoneal mast cells are strain dependent.     

Inhibition of histamine liberation in mast cells from the lung and the intestine of the dog by isoproterenol]

Enzymatically isolated dog lung and gut mast cells were stimulated with compound 48/80, ionophore A23187, concanavalin A and FNa-Ca. Cell response elicited by A23187, concanavalin A or 48/80 is almost completely inhibited by isoproterenol. Concanavalin A induced histamine release on gut mast cells is high, indicating an elevated degree of sensitization of these cells. Results point to the existence of beta adrenergic inhibitory activity on dog lung and gut mast cells.     

No direct involvement of plasma membrane divalent cation-activated adenosine triphosphatase in histamine release from rat mast cells

Showdomycin, a very slowly penetrating SH reagent, hardly affected the histamine release induced by any of secretagogues tested, suggesting no exposure of sulfhydryl groups involved in the granule secretion process on the cell surface. N-ethylmaleimide(NEM), a considerably penetrating SH reagent, almost completely inhibited histamine release induced by secretagogues such as compound 48/80, polymyxin B, concanavalin A or digitonin at 100 microM and by A23187 at 500 microM. However, (Ca2+-Mg2+)-ATPase activity was hardly inhibited by NEM modification at 500 microM. These findings suggest that plasma membrane divalent cation-activated ATPase is not involved directly in the granule secretion process of mast cells.     

Evidence for de novo synthesis of phosphatidylinositol coupled with histamine release in activated rat mast cells

Phospholipid metabolisms in rat mast cells activated by ionophore A23187 and compound 48/80 were examined with reference to 'phosphatidylinositol (PI) cycle'. The addition of A23187 to [3H]glycerol-prelabeled mast cells induced a marked accumulation of the radioactivity in 1,2-diacylglycerol(DG) and phosphatidic acid(PA) within 10 to 30 sec. A great enhancement of [3H]glycerol incorporation into PA and PI was also detected during histamine release. On the other hand, 48/80 was far less effective than A23187 both in producing 1,2- DG and PA and in accerelating [3H]glycerol incorporation into PA and PI, despite the comparable ability of histamine release. The activity of Ca2+ uptake into mast cells, as measured by pulse-labeling with 45Ca2+, was increased when exposed to both of two agents. These data provide circumstantial evidence that phospholipid metabolisms, mainly de novo PI synthesis, may be a part of the triggering events for Ca2+ mobilization and secretory process. The PI metabolism induced by two different stimulants appears to behave in a different manner.     

Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 48/80

Rat peritoneal mast cells respond to various secretagogues, such as ionophore A23187, concanavalin A (Ig E receptor cross-bridging) and compound 48/80 (membrane perturbing), to secrete histamine and to liberate arachidonic acid. Arachidonic acid release was made identifiable by pretreatment with BW755C, an inhibitor of both lipoxygenase and cyclo-oxygenase. The extent of arachidonic acid release varied among these three secretagogues. A23187 appeared to be most potent, whereas compound 48/80 was weakest. The sources of released arachidonic acids may be different depending on the types of stimulants. The stimulation with A23187 released arachidonic acid mainly from phosphatidylcholine and triacylglycerol. After treatment with concanavalin A and compound 48/80, in addition to phosphatidylcholine, phosphatidylinositol also appeared to serve as a donor of arachidonic acid.     

Amomum xanthiodes inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

In this study, we investigated the effect of Amomum xanthiodes (Zingiberaceae) extract (AXE) on the mast cell-mediated allergy model and studied the possible mechanism of action. We found that AXE inhibited compound 48/80-induced systemic reactions and plasma histamine release in mice. Additionally, AXE decreased immunoglobulin E (IgE)-mediated local allergic reactions and passive cutaneous anaphylaxis (PCA), and AXE dose-dependently attenuated the release of histamine from rat peritoneal mast cells (RPMC) activated by compound 48/80 or IgE. The amounts of AXE needed for inhibition of compound 48/80-induced plasma histamine release and PCA were similar to disodium cromoglycate, the known anti-allergic drug. We found that AXE increased the cAMP levels and decreased the compound 48/80-induced intracellular Ca2+. Furthermore, AXE attenuated the phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (A23187)-stimulated tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 secretion in human mast cells. The inhibitory effect of AXE on the proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB)-dependent. In addition, AXE decreased PMA plus A23187-induced degradation of IkappaBalphaand the nuclear translocation of NF-kappaB. Our findings provide evidence that AXE inhibits mast cell-derived immediate-type allergic reactions, and that cAMP, intracellular Ca2+, proinflammatory cytokines, and NF-kappaB are involved in these effects.     

Effect of isoproterenol on the liberation of histamine from rat lung mast cells]

Rat lung mast cells were stimulated with drugs with distinct mechanisms of action, namely concanavalin A, compound 48/80 ionophore A23187, in the presence of the beta adrenergic agonist (-)isoproterenol. Cells show a high response when they are stimulated with FNa-calcium. Isoproterenol does not inhibit histamine release induced by any stimuli, but enhances the response to concanavalin A and compound 48/80. Results point to the lack of beta activity on rat lung mast cells.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Ca2+ influx,phosphoinositide hydrolysis,and histamine release induced by lysophosphatidylserine in mast cells

We have previously demonstrated that snake venom phospholipases A₂ (PLA₂s) and mammalian PLA₂s induced inflammatory processes. This effect was correlated with the activity of the enzymes and the release of lipid mediators. We have now determined the role of lysophosphatidylserine (LysoPS) as an inflammatory lipid mediator. Thus, we have studied the possibility that intracellular calcium concentration, phosphoinositide hydrolysis, and the subsequent histamine release in mast cells is due to the action of lysophosphatidylserine. Lysophosphatidylserine-stimulated release of histamine was significantly higher than release by other lysophospholipids. The contribution of increased phospholipase C activity and the intracellular Ca²⁺ influx were therefore examined. LysoPS increased mast cell calcium concentration, and this increment was associated with phospholipase C activation and release of inositol phosphates. The increase in intracellular calucium and histamine degranulation induced by LysoPS were inhibited by apomorphine. Pretreatment of mast cells with pertussis toxin decreased the secretagogic effect of LysoPS and compound 48/80 without modifying the effect of the ionophore A23187. These results suggest that pertussis toxinsensitive G-protein might be involved in the mast cell degranulation produced by lysophosphatidylserine and allow the increase in phospholipase C activity, thus enhancing intracellular calcium concentration, which then induces exocytosis of histamine. © 1995 Wiley-Liss Inc.     

Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells

Human cultured mast cells (HCMC) release histamine and sulfidoleukotrienes (LTs) upon IgE-FcepsilonRI-mediated mast cell activation. We analyzed the Ca2+ and PKC signaling in HCMC and compared it to that in rodent mast cells. In HCMC, after IgE-mediated stimulation, an elevation of [Ca2+]i and PKC translocation to the membrane fraction was observed. As concerns Ca2+ signaling, 1) IgE-mediated histamine and LTs release was abolished after Ca2+ depletion, and the reconstitution of Ca2+ recovered the release of histamine and LTs. As regards PKC signaling, 1) staurosporine inhibited IgE-mediated mediator release. 2) PKC-downregulated mast cells did not release histamine and LTs. A23187 and PMA synergistically potentiated the activation of extracellular-regulated kinase and synergistically induced histamine and LTs release. These results demonstrated that HCMC might be useful for analysis of the signal transduction pathway for mediator release, such as histamine and LTs.     

Evidence of an adriamycin binding site in the secretory granules of the mast cell

Adriamycin induced significant non-cytotoxic histamine release from rat peritoneal mast cells to which the drug showed a very high affinity. The relationship between adriamycin-induced exocytosis and its uptake by purified rat peritoneal mast cells was studied. Adriamycin induced histamine release and was highly concentrated in mast cells at 37 degrees C but not at 0 degrees C. However, if exocytosis was provoked by other secretagogues like compound 48/80, protamine, concanavalin A, and ionophore A23187, and cells were then treated with adriamycin at 0 degrees C, the concentrations of the antineoplastic drug significantly increased. Adriamycin binding to purified granular material was similar to that of intact cells treated at 37 degrees C, but was not modified by metabolic inhibitors, extremes of temperature (0 or 45 degrees C) or by the carboxylic ionophore monensin. On the contrary, sodium cromoglycate limited adriamycin binding to granular materials as well. In addition, sodium cromoglycate, but not monensin, displaced the antineoplastic drug from mast cells, even when added after adriamycin. We conclude that the high affinity of adriamycin for mast cells is ascribable to the externalization of a granular binding site, as a consequence of the exocytotic process. The experiments with sodium cromoglycate suggest that this binding site could be in common with the antiallergic drug.     

Characterization of the effects of phorbol esters on rat mast cell secretion

When applied to the skin, phorbol esters (PEs) elicit signs of acute inflammation, suggesting they may induce the release of mediators from mast cells. Therefore, we have studied the effects of PEs on purified rat peritoneal and thoracic mast cells both alone and in conjunction with the calcium ionophore, A23187, and various other secretagogues that interact with immunoglobulin E (e.g., anti-IgE and Con A) or other cell surface receptors, e.g., somatostatin and compd 48/80. PEs alone caused little or no release of histamine. However, the PE 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) tremendously potentiated release induced by the calcium ionophore A23187, reducing the EC50 for A23187 from 832 ng/ml to 56 ng/ml. In the presence of suboptimal A23187 (50 ng/ml), only active tumor promoting PEs elicited histamine release. The EC50 values of the various active PEs were: TPA 5 ng/ml; 4 beta-PDD, 83 ng/ml; and 4-O-methyl-TPA, 807 ng/ml, with maximal histamine release ranging from 54 to 80%. TPA synergistically enhanced stimulation of histamine release by anti-IgE and Con A over the entire concentration-response range. In contrast, this synergism was absent when cells were stimulated with somatostatin and compd 48/80. Phorbol esters may act by increasing the activity of a calcium/phospholipid-dependent protein kinase (Ca/PL-PK). Mast cells do have Ca/PL-PK activity, and TPA in the presence of suboptimal A23187 induces protein phosphorylation comparable with other secretagogues. These results suggest that in the purified mast cell, PE-induced mediator release increases the sensitivity of release mechanisms for calcium, acts syngergistically with secretagogues interacting with IgE, and as suggested from structure-activity relationships, occurs via a specific mechanism of action perhaps involving the Ca/PL-PK.     

Inhibition of compound 48--80 induced histamine liberation from mast cells by triton X-100]

Triton X-100 at concentrations preceding those which liberated histamine, produced dose-dependent inhibition of compound 48/80-induced histamine release from rat mast cells. Triton X-100 (0.00002 1/1) depleted ATP content in the mast cells and blocked compound 48/80-induced histamine release. The inhibition of compound 48/80-induced histamine release and depletion of the ATP content in the mast cells was reversed by glucose (10 mmole). It is concluded that inhibition by Triton X-100 of histamine release induced by compound 48/80 is dependent on inhibition of energy production.     

Release of 14-kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process.

Group II phospholipase A2 was detected in appreciable amounts in rat peritoneal mast cells. The effect of several inhibitors specific to 14-kDa group-II phospholipase A2, including two proteinaceous inhibitors and a product of microorganisms with a low molecular mass, on mast-cell activation was examined. When rat peritoneal mast cells were sensitized with IgE and then challenged with antigen, the specific phospholipase-A2 inhibitors suppressed histamine release in a concentration-dependent manner. By contrast, these inhibitors showed no effect on prostaglandin generation under the same conditions. Histamine release from rat peritoneal mast cells subjected to non-immunochemical stimuli, such as concanavalin A, the Ca2+ ionophore A23187, compound 48/80 and substance P was also suppressed. When rat peritoneal mast cells were treated with 14-kDa-group-II-phospholipase-A2-specific inhibitors, washed and stimulated, histamine release was not affected appreciably. Similar suppressive effects of the inhibitors on histamine release were observed with mouse cultured bone-marrow-derived mast cells. When bone-marrow-derived mast cells were activated, they secreted both a soluble and an ecto-enzyme form of 14-kDa group-II phospholipase A2, although appearance of the enzyme associated with the external surface of cells was observed transiently. An appreciable amount of membrane phospholipids was degraded during activation of mast cells, which was decreased by treatment with 14-kDa-group-II-phospholipase-A2 inhibitor. These observations suggest that degranulation and eicosanoid generation in mast cells are regulated independently by discrete phospholipases A2 and that the 14-kDa group-II phospholipase A2 released from mast cells during activation may play an essential role in the progression of the degranulation process.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using 1H NMR.

1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.