Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.     

Mechanisms of mouse mast cell activation and inactivation for IgE-mediated histamine release.

The IgE-mediated histamine release from mouse mast cells requires Ca++, is optimal at 37 degrees C, and is enhanced by phosphatidylserine. The rate of release is relatively slow. The mast cells can be activated to release histamine by either anti-IgE or anti-Fab antibodies and, in the case of cells from sensitized mice, by the immunizing antigen. The incubation of mast cells with antigen in the absence of Ca++ or phosphatidylserine fails to release histamine. Such cells are desensitized to the further addition under optimal conditions of the same antigen. Desensitization is antigen specific, requires optimal levels of antigen, and occurs at both 30 degrees and 37 degrees C. In contrast, anti-IgE desensitizes all IgE-mediated histamine release reactions.     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.     

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.     

Magainin-2 releases histamine from rat mast cells.

The magainins are basic 23 amino acid peptides with a broad spectrum of antimicrobial activity. Their bactericidal effect has been attributed to their capacity to interact with lipid bilayer membranes. We observed histamine release by magainin-2 amide from rat peritoneal mast cells (ED50 = 13 micrograms/ml) but not from human basophils. This histamine-releasing reaction from peritoneal mast cells was due to a secretory rather than cytolytic effect, i.e., release occurred without concomitant liberation of lactic dehydrogenase. Furthermore, the pretreatment of mast cells with magainin-2 amide did not desensitize cells against subsequent challenge with other secretagogues. Maximum histamine release occurred in less than a minute at 25 and 37 degrees C. The addition of Ca2+ was not required for histamine release, although release was enhanced by the addition of 0.3-1 mM Ca2+. The addition of 3 mM Ca2+ or Mg2+ was markedly inhibitory. The presence of Na+ or Cl- ions in the medium was not required for release. Therefore, histamine release is not due to the formation of anion-selective channels in the membrane of mast cells. The results indicated that the characteristics of histamine secretion induced by magainin-2 amide were unlike IgE-mediated release but were similar to the mechanism of release attributed to some other basic peptides and to compound 48/80.     

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.     

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.     

Effects of calcium and phosphatidyl serine in rat mast cell reaction to dextran.

Histamine release from Sprague-Dawley rat mast cells by dextran was completely inhibited by the absence of exogenous Ca2+ (in contrast to release from the same cells by antigen). Also, spontaneous leakage of histamine from the cells increased in the absence of Ca2+, and cell responsiveness was not completely restored by readding Ca2+. We found no effective substitute for Ca2+ in the release reaction. Ca2+ was not maximally effective immediately when added back to Ca-deficient cells, but almost the full effect of diluting Ca2+ in the medium (which decreased release) and of adding PS (which increased release) were very rapidly established, suggesting that both Ca2+ and PS might act (in part) at superficial cell sites. Release from activated cells could be stopped short by adding glucose or by diluting the cell-dextran mixture with normal buffer, as well as by adding EDTA, which deserves further study.     

The calcium signal and phosphatidylinositol breakdown in 2H3 cells

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.     

IgE-mediated release of histamine from human cutaneous mast cells

We investigated the ability of antigen-IgE interactions to stimulate histamine release from human infant cutaneous mast cells. Skin obtained at circumcision contained numerous perivascular mast cells, as assessed by light and electron microscopy. The histamine content of this tissue averaged 17.7 ng (+/- 1.5 SEM)/mg wet weight. Challenge of 200-microns thick sections of unsensitized skin with varying concentrations of monoclonal murine antibodies to human IgE caused no net release of histamine. After skin sections were incubated in the presence of 5 micrograms/ml of human myeloma IgE (S) for 120 min at 37 degrees C, monoclonal anti-IgE challenge resulted in 40.1% (+/- 6.0 SEM) histamine release. Similar passive sensitization with 1/20 dilutions of serum from humans expressing IgE to purified Juniperus sabinoides (JS) antigen rendered the tissue responsive to specific antigen challenge. Dose-related histamine release occurred over 30 min with optimal release of 12.6% (+/- 2.4 SEM) after stimulation with 100 ng/ml of JS antigen. This reaction required sensitization with serum containing IgE to JS and was antigen-specific. Optimal reactions to antigen occurred at 3 mM added Ca++, 34 degrees C to 37 degrees C, pH 7.2. Antigen-induced release was markedly influenced by the added Ca++ concentration; no release occurred in the absence of Ca++, 54% of the optimal response was observed at 2 mM Ca++, and 28% of the optimal response occurred at 4 mM Ca++. The addition of Mg++ did not influence antigen-induced release. The results of this study provide functional evidence that 1) human infant cutaneous mast cells express Fc-epsilon receptors; 2) these receptors are largely unoccupied in vivo; and 3) stimulation of passively sensitized infant mast cells with anti-IgE or specific antigen leads to immediate histamine release. This new system should permit detailed in vitro studies of immediate hypersensitivity reactions in human skin.     

Inhibitory effects of GMCHA-OPhBut on phospholipid methylation and histamine release in mast cells activated by concanavalin A, anti-IgE, and antigen

[3H]Methyl group incorporation and histamine secretion in rat mast cells induced by anti-IgE and con A were strongly inhibited by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a strong and specific inhibitor for pH 7 tryptase (Muramatsu et al. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625) which is present in rat mast cells. The IC50s for these events were of the order of 10(-6) M. Addition of GMCHA-OPhBut after the maximal increase in [3H]methyl group incorporation in rat mast cells activated by con A and anti-IgE induced rapid reduction of the methylated phospholipid, and the later histamine release was strongly suppressed. Mast cells were prepared with Mg2+-free Tyrode-HEPES solution, and challenged with anti-IgE with or without Mg2+. With Mg2+, [3H]methyl group incorporation was enhanced, and histamine was secreted time-dependently. Without Mg2+, [3H]methyl group incorporation fell to one-third, whereas histamine secretion was not affected. These results were incompatible with the above results. From these results it was strongly suggested that a trypsin-like protease, probably pH 7 tryptase, is involved not only in the early events, such as activation of phosphatidylethanolamine methyltransferase I and/or II, but also in the late events such as histamine release, and phospholipid methylation is not associated with histamine secretion.     

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.     

Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Localized mast cell degranulation induced by concanavalin A-sepharose beads. Implications for the Ca2+ hypothesis of stimulus-secretion coupling

Concanavalin A (Con A) covalently linked to Sepharose 4B beads induced localized degranulation of sensitized rat peritoneal mast cells in regions of contact between beads and cells. This degranulation was Ca2+ dependent and was not seen when sensitized mast cells bound to beads conjugated with a nonstimulating lectin, wheat germ agglutinin, or when unsensitized mast cells bound to Con A-Sepharose. The finding that sensitized mast cells which had adhered to Con A-Sepharose beads degranulated in regions of the cell away from the area of bead contact if exposed to soluble Con A excluded the possibility that the localized release was due to a redistribution of the IgE receptors or putative Ca2+ channels to the region of bead contact. The results suggest that, if an influx of Ca2+ is the mechanism for initiating mast cell degranulation, then the opening of Ca2+ channels in the plasma membrane of activated mast cells is a localized event and that Ca2+ acts locally within the cell to initiate exocytosis.     

Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

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.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Effects of membrane depolarization and divalent cations on anaphylactic histamine secretion

The effects of membrane depolarization and divalent cations on histamine release have been studied in sensitized mast cells. Membrane potential of these cells has been measured with intracellular microelectrodes. Our results show that mast cells have a large resting potential (-61 +/- 12 mV) however they do not generate active membrane electrical responses when are depolarized by passing current through the recording microelectrode. High external K+ does not increase histamine release. Histamine secretion is supported by alkali-earth divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) but strongly inhibited by transition metals. Ca2+ concentrations above 1 mM inhibit histamine release, however, this effect is not mimicked by Sr2+ and Ba2+.