Calcium requirement in compound 48/80 induced histamine release from rat mast cells in vitro

The effect of magnesium and EDTA on compound $\text{48}\text{80}$-induced histamine release and adenosine triphosphate (ATP) content of mast cells has been studied. Inhibition of histamine release after preincubation of the cells with or without EDTA in the absence of calcium and the reversal by calcium indicate that calcium is required for compound $\text{48}\text{80}$-induced histamine release. The presence of magnesium potentiate the inhibition caused by the lack of calcium. The inhibition of histamine release is not related to changes in cellular ATP content. The observations with EDTA suggest that calcium may be provided for the release process from intracellular sources.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

Permeability increase in black lipid membrane induced by compound 4880

When compound $\text{48}\text{80}$, a potent histamine liberator, was added in the aqueous phase facing the black lipid membrane, the conductivity of the membrane was remarkably increased. Although valinomycin displayed a distinct selectivity for K⁺ movement, such selection for ionic permeability was not observed in the case of compound $\text{48}\text{80}$.     

Compound 4880 is a selective and powerful inhibitor of calmodulin-regulated functions

Compound $\text{48}\text{80}$, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound $\text{48}\text{80}$ was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca²⁺-transport ATPase, with IC₅₀ values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca²⁺ transport into inside-out red blood cell vesicles by compound $\text{48}\text{80}$ follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca²⁺-transport ATPase induced by calmodulin is inhibited by compound $\text{48}\text{80}$ according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound $\text{48}\text{80}$ bind to calmodulin in a Ca²⁺-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound $\text{48}\text{80}$ is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca²⁺-transport ATPase that has been described hitherto. In addition, compound $\text{48}\text{80}$ was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca²⁺-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound $\text{48}\text{80}$, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca²⁺-transport ATPase or calf cardiac sarcolemma (Na⁺ + K⁺)-transport ATPase are far less influenced by compound $\text{48}\text{80}$ as compared with trifluoperazine and calmidazolium. Because of its high specificity compound $\text{48}\text{80}$ is proposed to be a promising tool for studying calmodulin-dependent processes.     

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-α, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-α, IL-6, and IL-8 release from mast cells.     

Mast cell histamine release induced by Portuguese man-of-war (Physalia) venom

Nematocyst venom from Portuguese Man-of War ($\text{Physalia}$ sp.) tentacles causes isolated rat peritoneal mast cells to release histamine. Extent of histamine release is dose-dependent (K_0.5 = 6.1 μg venom/ml) and attains 100% at high doses of venom. Release is independent of intra- and extracellular calcium levels and does not depend upon a cellular supply of ATP. The rate of histamine release is temperature-dependent and the extent of release is maximized broadly over the range of 10–30°C. The cytoplasmic marker lactate dehydrogenase, is released concomitantly with histamine but is more sensitive to the venom (K_0.5 = 2.1 μg/ml). Antimycin A, while it does not significantly affect venom-induced histamine release, increases the sensitivity of lactate dehydrogenase release (K_0.5 = 0.2 μg/ml). We conclude that $\text{Physalia}$ nematocyst venom induces the release of histamine from mast cells by a cytolytic mechanism and that this action is antagonized by an intracellular, energy-requiring process.     

Antigen-induced histamine release from sensitized tissue and the measurement of calcium ion fluxes

Antigen stimulated release of histamine from sensitized guineapig lung was inhibited by the exclusion of calcium ions from the incubation fluid. Subsequent addition of calcium ions induced release, but the magnitude of this release decreased with time. When the releasing potential had declined to zero, addition of an alternative antigen together with calcium ions induced further release. If the primary challenge was inhibited by the presence of an antiallergic agent, challenge by a second antigen was similarly inhibited, in contrast to the effect when there was no primary antigen challenge. Antigen challenge induced a flux of ${}^{45}\text{Ca}$ into cells, these fluxes were inhibited by compounds which inhibited histamine release. Inhibition of release did not correlate with inhibition of calcium flux with some agents, suggesting that the measured flux is the sum of at least two fluxes, one secondary to release. These results are explained in a scheme for antigen-induced histamine release.     

Effect of disodium cromoglycate on mast cell-mediated immediate-type allergic reactions

We investigated the effect of disodium cromoglycate (DSCG) on mast cell-mediated immediate-type hypersensitivity. DSCG inhibited systemic allergic reaction induced by compound 48/80 dose-dependently. Passive cutaneous anaphylaxis was inhibited by 71.6% by oral administration of DSCG (1 g/kg). When DSCG was pretreated at concentration rang from 0.01-1000 g/kg, the serum histamine levels were reduced in a dose dependent manner. DSCG also significantly inhibited histamine release from rat peritoneal mast cell (RPMC) by compound 48/80. We confirmed that DSCG inhibited compound 48/80-induced degranulation of RPMC by alcian blue/nuclear fast red staining. In addition, DSCG showed a significant inhibitory effect on anti-dinitrophenyl IgE-mediated tumor necrosis factor-alpha production. These results indicate that DSCG inhibits mast cell-mediated immediate-type allergic reaction.     

Effects of a Dictamnus dasycarpus T. extract on allergic models in mice

The anti-allergic effect of a 70% ethanol extract from Dictamnus dasycarpus Turcz (DDT) was studied in mice. DDT at doses of 200 and 500 mg/kg inhibited the systemic anaphylactic shock induced by compound 48/80 in a dose-dependent manner. It also inhibited dose-dependently the scratching behavior induced by compound 48/80, histamine and serotonin. An increase in the vascular permeability induced by compound 48/80, histamine and serotonin was also inhibited by DDT. In an in vitro study, DDT inhibited the histamine released from rat peritoneal mast cells induced by compound 48/80. It seems likely from these findings that DDT was effective in antagonizing certain pharmacological effects induced by compound 48/80 that occurred via both histamine and serotonin released from mast cells. In conclusion, DDT may be effective in the relief of symptoms of allergic atopic dermatitis and other allergy-related diseases.     

Effects of tannins and related polyphenols on superoxide-induced histamine release from rat peritoneal mast cells.

The effects of tannins and related polyphenols on KO2- and compound 48/80-induced histamine release from rat peritoneal mast cells were examined. Pretreatment with hydrolyzable tannins (1-100 microM) significantly inhibited KO2-induced histamine release. Dimeric ellagitannins, which have hexahydroxydiphenoyl (HHDP) and valoneoyl residues and/or a valoneoyl-related acyl unit in the molecule, showed more potent inhibitory effects than monomeric hydrolyzable tannins. The most effective inhibition was exhibited by agrimoniin and euphorbin C (IC50 0.68 and 0.80 microM), which have dehydrodigalloyl and euphorbinoyl groups, respectively, as well as the HHDP group. However, procyanidins, flavonoids and related polyphenols with small molecular weights, except for epigallocatechin gallate, exhibited negligible effects. Although clinically used antiallergic drugs, azelastine, astemizole, ketotifen and epinastine have been shown to prevent KO2-induced histamine release, their potencies were all less than those of ellagitannins. An inhibitory effect on compound 48/80-induced histamine release was also exhibited by higher molecular weight tannins. The inhibitory effect on histamine release caused by different stimulants suggested that ellagitannins act as cell membrane stabilizers as well as radical scavengers.     

Inhibitory effect of tea polyphenols on histamine and leukotriene B4 release from rat peritoneal exudate cells

Summary The effect of tea polyphenols on the release of chemical mediators, histamine and leukotriene B₄ (LTB₄), from rat peritoneal exudate cells (PEC) was studied. Among polyphenols, (−)-epigallocatechin gallate (EGCG) most strongly inhibited the histamine release from the cells stimulated with a calcium ionophore, A23187 or compound 48/80. Though (+)-catechin (C) and (−)-epicatechin (EC) had no effect, (−)-epigallocatechin (EGC) and (−)-epicatechin gallate (ECG) moderately inhibited the histamine release. Similarly, EGCG, ECG, and EGC inhibited LTB₄ release from PEC, whereas C and EC were not effective. The magnitude of the inhibitory effect on the release of these mediators of tea polyphenols was in the order of EGCG>ECG>EGC. These results indicated an important role of the triphenol structure in the inhibitory activity. Therefore, the possible antiallergic effect of tea polyphenols can be expected.     

Release of the chloride-dependent arginine aminopeptidase from PMN leukocytes and macrophaces during phagocytosis

The zymosan particles induced a time-dependent release of the chloride-dependent arginine aminopeptidase from rat peritoneal macrophages during $\text{in}$$\text{vitro}$ incubations. Intraperitoneal injections of zymosan, a streptococcal cell preparation and a $\text{Micrococcu}$-suspension caused the release of the chloride-activated arginine aminopeptidase into the peritoneal fluid. The arginine aminopeptidases obtained both from the cell cultivation media and the peritoneal washes were partly purified. The enzymes were similar with regard to the following properties: chloride activation with an optimum at physiological concentrations; strong inhibition by 10^?6M $\text{p}$-chloromercuribenzoate; similar elution properties and preferential hydrolysis of mainly the $\text{N}$-L-aminoacyl-2-naphthylamines of arginine and lysine. The chloride-activated arginine aminopeptidase released into the media in $\text{in}$$\text{vitro}$ conditions was inactivated in contrast to the enzyme released into the peritoneal fluid as a result of the intraperitoneal injections. The timing of the release of the chloride-activated arginine aminopeptidase both $\text{in}$ and $\text{in}$$\text{vitro}$ suggests that the enzyme plays a role in the initial phases of inflammation.     

Effect of interleukin-1 receptor antagonist (IL-1RA) on histamine and serotonin release by rat basophilic leukemia cells (RBL-2H3) and peritoneal mast cells

Recently, it has been appreciated that cultured mast cells are significant sources of cytokines. However, the role of interkeukin-1 (IL-1) on mast cells and/or basophil degranulation is still unclear. In this report we provide evidence that rat basophilic leukemia cells (RBLC) cultured with a natural inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1RA) (500 ng/ml) for 48 h, strongly inhibited the spontaneous release of serotonin (5HT) and histamine (from 22.50 to 43.49%), compared to untreated cells (control). When IL-1RA-treated and untreated RBLC were stimulated with a secretagogue (anti-IgE), no difference was found in the percent of 5HT and histamine release. Moreover, in another set of experiments using rat peritoneal mast cells (RPMC) treated and untreated with IL-1RA, we found that IL-1RA did not affect the release of 5HT or histamine, even when the secretagogue anti-IgE or compound 48/80 (C48/80) were used. The present studies describe an additional biological activity of IL-1RA, inhibiting histamine and 5HT release from RBLC cultures.Abbreviations IL-1 interleukin-1 - RA receptor antagonist - 5HT serotonin - RBLC rat basophilic leukemia cells - RPMC rat peritoneal mast cells - IgE immunoglobulin E - Fc immunoglobulin E receptor - CPM counts per minute - BSA bovine serum albumin - C48/80 compound 48/80 - TNF tumor necrosis factor     

Ethanol inhibition of antigen-induced histamine release from guinea pig lung: correlation with intracellular levels of cyclic nucleotides.

The effect of ethanol on histamine release from lungs of sensitized guinea pigs was studied in conjunction with measurements of tissue concentrations of cyclic AMP and cyclic GMP. Addition of antigen $\text{in vitro}$ elicited a rapid increase in cyclic AMP and cyclic GMP and stimulated release of histamine. Ethanol (2%) inhibited antigen-induced release of histamine over 95% and completely inhibited the increase in both cyclic nucleotides. The activity of cyclic AMP-dependent protein kinase was only slightly affected by ethanol.Metiamide blocked the ovalbumin stimulated increase in cyclic AMP but not cyclic GMP. Pyrilamine did not prevent the rise in either cyclic nucleotide. This suggests that the antigen-induced rise in cyclic AMP is an indirect result of histamine released from the tissue. The inability of H₁ and H₂ receptor antagonists to affect antigen-induced elevation of cyclic GMP in sensitized lung fragments suggests that an elevation in cyclic GMP might be either a primary event in the mediator release sequence or secondary to the release of a mediator other than histamine. The ability of ethanol to inhibit mediator release might be due to its capacity to attenuate the antigen-induced elevation of cyclic GMP in sensitized lung.     

Inhibition of non-cytotoxic histamine liberation from isolated rat mast cells by antihistaminic preparations]

Phenothiazines (chlorpromazine and promethazine) and antihistaminic quinuclidine derivatives [phencarol, quinuclidyl-3-di-(o-tolyl) carbinol, hydrochloride quinuclidyl-3-di-(o-methoxyphenyl) carbinol--HQMC] at concentrations preceding the histamine-releasing ones inhibited the compound 48/80-induced histamine release from the isolated rat mast cells. HQMC inhibited histamine release induced by selective liberators (compound 48/80, MCD-peptide, specific antigen), but potentiated histamine release induced by nonselective liberators (chlorpromazine, tryton X-100). The inhibition by HQMC of histamine release induced by compound 48/80 increased during 1 min and was reversible. The inhibitory effect of all the compounds tested was partially counteracted by glucose.     

Potentiation of histamine release by sodium cromoglycate.

Human lung slices passively sensitized with allergic serum released histamine when incubated with specific antigen and anti-IgE but anti-IgG had no effect. Sodium cromoglycate (SCG) inhibited antigen induced histamine release but the dose-response curve was bell-shaped. Inhibition of anti-IgE induced release was linearly related to dose, whereas that induced by anti-IgG was potentiated by increasing doses of SCG. After sensitization with allergic serum in which IgE had been inactivated by heating, specific antigen released little or no histamine but this was potentiated by SCG. It is concluded that SCG inhibits IgE mediated but potentiates IgG mediated allregic reactions thus explaining its characteristic dose-response curve $\text{in vitro}$.     

Activated macrophages digest the extracellular matrix proteins produced by cultured cells.

Activated mouse peritoneal macrophages were cultured directly on the extracellular matrix proteins produced by smooth muscle cells $\text{in}\text{vitro}$. The breakdown of the connective tissue proteins to the level of amino acids was followed by observing the release of radioactivity from matrices labelled with [³H]proline. These studies showed that macrophages produce enzymes capable of digesting the matrix and indicated a major role for the macrophage plasminogen activator in this digestion.     

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.     

Degradation of surface-labeled hepatoma membrane polypeptides: Effect of inhibitors

When their membrane proteins were labeled with ¹²⁵I by lactoperoxidase, dividing hepatomacells lost radio activity to the medium in a biphasic manner ($\text{T}\text{1}\text{2}\text{= 16–26}\text{h}\text{, > 40}\text{h}$). Lysosomotropic weak bases, chloroquine, and NH₄Cl inhibited the rapid phase by 59%. More than 50% of the radio activity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60–71% inhibited by lysosomotropic agents chloro quine and NH₄Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH₄Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-l-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by21–24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 °C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lyososomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to, agents which disrupt the cytoskeleton.     

Prostaglandins and cardiac anaphylaxis.

Manifestations of cardiac anaphylaxis include sinus tachycardia and arrhythmias, both of which result from histamine release. The marked decrease in coronary flow, which also occurs during cardiac anaphylaxis, cannot be attributed solely to histamine release.To indirectly assess the possible role of prostaglandins in cardiac anaphylaxis, hearts from sensitized guinea pigs were challenged $\text{in}$$\text{vitro}$ in the presence of indomethacin. This resulted in a marked increase in histamine release, which caused a greater tachycardia and an increase in the incidence of arrhythmias. Moreover, for the same degree of histamine release sinus rate increments were larger in the presence of indomethacin. However, despite the enhanced cardiac dysfunction, coronary flow rate did not decrease.The results suggest that, during cardiac anaphylaxis, prostaglandins modulate histamine release and the effects of released histamine. Furthermore, since we have found that PGF_2α is released from the anaphylactic heart, we tentatively ascribe the anaphylactic reduction in coronary flow to the elaboration of PGF_2α.