Evidence for the lack of involvement of free radicals in adriamycin-induced histamine release from rat peritoneal cells

The antineoplastic drug adriamycin, at a concentration of 100 micrograms/ml, caused a significant histamine release from rat peritoneal mast cells. This exocytotic process was unaffected by pretreatment with various concentrations of catalase, superoxide-dismutase, D-mannitol, alpha-tocopherol and reduced glutathione. Only very high concentrations of n-acetylcysteine significantly limited this release; this substance was also active in limiting histamine secretion induced by a classic mast cell secretagogue, compound 48/80.     

The intracerebroventricularly administered mast cells degranulator compound 48/80 increases the pituitary-adrenocortical activity in rats

The effect of brain mast cells degranulation by compound 48/80 on the pituitary-adrenocortical activity, measured indirectly through corticosterone secretion, and the involvement of a histaminergic mechanism in that stimulation was investigated in conscious rats. All the drugs were given intracerebroventricularly (icv), histamine antagonists 15 min prior to compound 48/80. Compound 48/80 induced a significant dose- and time-related increase in the serum corticosterone levels. That increase, measured 1 h after administration of compound 48/80, was moderately diminished by icv pretreatment of rats with mepyramine and cimetidine, histamine H1- and H2-receptor antagonists. Three hours after administration of compound 48/80 mast cells of the thalamus and the hypothalamus were completely degranulated. At the same time the thalamus and the whole brain histamine levels were substantially higher than in the saline-treated control rats. The above results suggest that histamine liberated from the brain mast cells and central histamine receptors play a moderate role in increasing the pituitary-adrenocortical activity by compound 48/80.     

Somatostatin-induced histamine secretion in mast cells. Characterization of the effect

Somatostatin, in concentrations up to 100 ng/ml, had no effect on mast cell secretion induced either by compound 48/80 or by the ionophore A23187. In higher concentration somatostatin induced mast cell secretion. Light and electron microscopic observations showed that the secretory response was identical with that induced by 48/80 and involved extrusion of secretory granules by exocytosis. As with 48/80, this response to somatostatin was inhibited by treating the mast cells with EDTA or EGTA or by exposing them briefly to A23187 in calcium-free media, all of which procedures seemingly deplete cellular calcium stores. Reintroduction of calcium (but not magnesium), restored secretory responsiveness. Somatostatin-induced secretion further resembled that induced by 48/80 or A23187 in its intensity, rapid time course, and dependence on albumin. Pretreatment of mast cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP substantially reduced the secretory responses to both somatostatin and 48/80 but had little effect on the response to A23187. Somatostatin, like 48/80, lowered intracellular cyclic AMP levels in a time and dose-dependent fashion. Finally, as earlier found for 48/80, somatostatin attached to Sepharose columns retarded the passage of mast cells and elicited histamine release indicating an action at the cell surface. The stimulant action of somatostatin is thus very similar to that of the classic mast-cell secretagogue compound 48/80.     

Pharmacological characterizations of the in vitro anaphylactic contraction of the guinea-pig esophageal muscularis mucosae

Characteristics of the antigen-induced contraction of the isolated esophageal muscularis mucosae of actively sensitized guinea-pigs to ovalbumin (OA) were examined in vitro, and they were compared with those of compound 48/80- or polymyxin B-induced contraction. OA, above 0.01 microgram/ml, produced a sustained contraction of the sensitized esophageal muscularis mucosae, the amplitude of which was about 80-100% of the maximum contraction induced by carbachol (10 microM), while compound 48/80 and polymyxin B (10-300 micrograms/ml) produced less potent contractions of the non-sensitized esophageal muscularis mucosae. Contractions to OA or compound 48/80, but not to polymyxin B, were diminished by their repetitive applications. The contractile responses to OA, compound 48/80 and polymyxin B depended on the external calcium concentrations, and were abolished in the calcium-free medium. Pretreatment with tetrodotoxin (0.3 microM), atropine (0.3 microM), diphenhydramine (30 microM) or DSCG (300 microM) did not modify any of these contractions, whereas BW755C (100 microM) and quercetin (10 microM) significantly inhibited them. Indomethacin (10 microM) largely prevented only the polymyxin B-induced contraction, while FPL55712 (10 microM) inhibited both contractions to OA and compound 48/80. These findings indicate that the OA-induced anaphylactic contraction of the esophageal muscularis mucosae taken from the OA-sensitized guinea-pig may be an indirect action via the stimulation of releases of some mast cell-derived spasmogens. The spasmogens may involve the lipoxygenase products of arachidonic acid in part, but not histamine, acetylcholine or the cyclooxygenase products.     

Inhibition of mast cell histamine release by flavonoids and biflavonoids

The effect of 11 flavonoids and 4 biflavonoids on the release of histamine from peritoneal rat mast cells induced by compound 48/80 and calcium ionophore A23187 was studied. Dihydroflavonoids (flavanones) and (+)-catechin did not modify histamine release induced by both secretagogues. Flavone, apigenin and cromoglycate inhibited the secretion elicited by compound 48/80 but did not modify the A23187-induced secretion. The effect of kaempferol on the compound 48/80-induced histamine release was biphasic. Low doses (10 (-6) to 10 (-5)M) of the compound potentiated secretion whereas higher doses inhibited histamine secretion. Some of the drugs tested revealed a higher potency as referred to quercetin. Luteolin, a tetrahydroxyflavone and amentoflavone, a biapigenin, exhibited the highest inhibitory effects of mast cell histamine secretion.     

The effect of concentration on the binding of compound 48/80 to rat mast cells: a fluorescence microscopy study

A fluorescent analog of the chemical histamine liberator, compound 48/80, has been synthesized by the covalent attachment of rhodamine to the 48/80 polymer (R-48/80). The histamine liberating characteristics of this analog were similar to those of the parent compound. The binding characteristics of R-48/80 to rat peritoneal mast cells were then studied using fluorescence microscopy. At concentrations that caused minimal secretory stimulation (less than 1.0 microgram/ml), R-48/80 bound to the mast cell surface in a diffuse manner, with no indication of patching or capping. When the cells were incubated at higher concentrations, where non-cytotoxic histamine secretion was stimulated, the drug bound heavily to the exposed granules, but not to unexposed granules or other cell organelles. At cytotoxic concentrations, R-48/80 caused extensive cell clumping, with the drug bound to masses of cell debris and released granules. Therefore, although R-48/80 binds initially to the cell membrane, its primary binding site at concentrations that induce secretion becomes the mast cell granule. The properties of these granules should thus be considered when studying the binding of compound 48/80 or other cationic drugs to rat peritoneal mast cells.     

Alterations in histamine levels in the rat induced by compound 48/80

Histamine release in the rat was induced in vivo either by a single dose of compound 48/80 injected i.v. or by four repeated, daily doses of the same compound injected i.p. After i.v. injection the levels of blood histamine were determined and after i.p. injections the changes in both tele-methylhistamine and histamine levels in different tissues were investigated. I.v. injection of 48/80 induced a very rapid and marked increase of blood histamine by 7.4 to 11-fold over the control levels within the first two minutes. After repeated i.p. injections of compound 48/80 most tissues showed higher than normal tele-methylhistamine/histamine ratios. The results suggest that agents known to induce release of histamine from mast cells may exert significant changes in blood and tissue histamine levels and that liberated histamine is thereafter extensively catabolized.     

Sodium-potassium ATPase inhibition potentiates compound 48/80-induced histamine secretion from mast cells.

The effect of ouabain on the histamine secretion induced by compound 48/80 has been studied using rat peritoneal mast cells. Ouabain did not modify histamine release in the presence of millimolar concentrations of extracellular calcium. However, when mast cells were previously washed with a calcium-free buffer, ouabain strongly potentiated histamine release elicited by compound 48/80. The full potentiation of mast cell secretion by ouabain required 30 min preincubation before adding compound 48/80. It was inhibited by lanthanum and EGTA. Potassium deprivation mimicked the effect of ouabain. A 30 min preincubation time without potassium was also required. Potassium concentrations below 2.7 mM increased the effect of ouabain whereas higher potassium concentrations reversed this effect. The potentiation of compound 48/80-induced histamine release by ouabain or potassium deprivation was not immediately reversed by washing away ouabain or by adding potassium, respectively. The data confirm that sodium-potassium ATPase is involved, through a calcium-dependent process, in the regulation of histamine release from mast cells.     

Inhibition of anaphylaxis like reaction and mast cell activation by Sitagliptin

Mast cells stimulation activates degranulation process resulting in releasing of mediators, such as histamine. In this study, the effect of aqueous extract of sitagliptin, a selective dipeptidylpeptidase-4 inhibitor, on the mast cell-mediated allergic response was studied with the possible mechanisms of action, focusing on the histamine release and pro-inflammatory cytokine secretion in mast cells. Sitagliptin produced dose dependent inhibition in compound 48/80-induced systemic reactions. In addition, sitagliptin attenuated IgE-mediated skin allergic reaction. Sitagliptin dose-dependently reduced compound 48/80- and IgE-induced histamine release from mast cells. Sitagliptin decreased the secretion of pro-inflammatory cytokines, tumor necrosis factor-α, in mast cells. So, the finding of this study provides evidence that sitagliptin inhibits mast cell derived allergic reactions, and involvement of pro-inflammatory cytokine secretion in such effects.     

Mechanism of histamine release induced by levofloxacin, a fluoroquinolone antibacterial agent

The present study was designed to clarify the mechanism of histamine release caused by levofloxacin, a fluoroquinolone antibacterial agent, using rat peritoneal mast cells. Levofloxacin induced a concentration-dependent histamine secretion from 300 microg/ml without lactate dehydrogenase leakage, and the release was rapidly completed within 30 s. This action was dependent on temperature, energy, pH and intracellular Ca(2+), similarly to the effect of compound 48/80, a basic compound. Unlike that with the calcium ionophore A23187, histamine secretion due to levofloxacin or compound 48/80 was prevented by pretreatment with either pertussis toxin or benzalkonium chloride, a selective inhibitor of G proteins of G(i) subtypes. Moreover, the histamine release elicited by levofloxacin or compound 48/80 was suppressed by hydrolysis of sialic acid residues on the cell surface brought about by neuraminidase. These results demonstrate that the mechanism by which levofloxacin exerts histamine release may be closely linked to activation of pertussis toxin-sensitive G proteins.     

The combination of rat mast cell and rabbit aortic smooth muscle is the simple bioassay for the screening of anti-allergic ingredient from methanolic extract of Corydalis tuber

We have assessed the release of histamine from mast cells by smooth muscle contraction. 0.3 microg/ml compound 48/80 showed no effect on concentration-response relationship of histamine in rabbit aorta. Compound 48/80 induced release of histamine from rat mast cells. When aorta was stimulated by compound 48/80 in the presence of mast cells, contraction was evoked in concentration-dependent manner. This mast cell-dependent contraction was completely blocked by H1 receptor antagonist, 1 microM diphenhydramine. When mast cells was treated with compound 48/80 inhibitor benzalkonium chloride, mast cell-dependent contraction was inhibited, although benzalkonium chloride itself showed no effect on concentration-response relationship of histamine in rabbit aorta. At high concentration of 10 microg/ml, benzalkonium chloride itself evoked histamine release from mast cells and indeed inhibitory effect of 10 microg/ml benzalkonium chloride on mast cell-dependent contraction was lower than that of 3 microg/ml. We have applied this bioassay to search anti-allergic ingredient from a total methanolic extract of Corydalis tuber (Corydalis turtschaninovii BESSER forma yanhusuo Y. H. CHOU et C. C. HSU). Successively, we have isolated five fractions. The fractions I-IV are identified to be corybulbine (1), tetrahydropalmatine (2), corydaline (3) and yuanhunine (4), respectively. Main component of fraction V is the mixture of 3 and canadine (5). Fractions II and V significantly inhibited mast cell-dependent contraction in rabbit aorta as well as inhibited histamine release from rat mast cells. Furthermore, fractions I, III and V inhibited histamine-induced contraction in rabbit aorta at non-competitive manner. From these results, combination of rat mast cells and rabbit aorta is good bioassay to search the anti-allergic ingredient, and we have obtained effective fractions from Corydalis tuber using this assay.     

Influence of histamine releasing agents on gastric acid secretion of isolated bullfrog gastric mucosa.

The influence of histamine releasing agents on gastric acid secretion was studied in isolated bullfrog gastric mucosa preparations. Maximum acid secretory responses in our preparations were obtained by stimulation with tetragastrin (5 X 10(-7) g/ml), histamine (1 X 10(-5) g/ml) and bethanechol (1 X 10(-6) g/ml). Compound 48/80 (1 X 10(-4) g/ml) showed a transient stimulatory action which was followed by a gradual depression of basal acid secretion. The stimulatory phase of compound 48/80 was completely antagonized by burimamide (1 X 10(-5) g/ml), a histamine H2-receptor antagonist. In gastric mucosa preincubated with compound 48/80, the secretagogue action of tetragastrin or bethanechol was not exerted, although this preparation continued to respond to histamine. The effects of Triton X-100, decylamine and polymixin B were quite similar to those of compound 48/80. After pretreatment with compound 48/80, the gastric mucosa preparation became refractory to the stimulatory action of compound 48/80 or Triton X-100. It is thus suggested that endogenous histamine may play an important role in the secretagogue action of tetragastrin and bethanechol.     

Suppression of anaphylactic reaction in murine bySiegesbeckia pubescens

The aqueous extract ofSiegesbeckia pubescens (SPAE) inhibited compound 48/80-induced systemic anaphylaxis 100% with the dose of 1.0, 0.5 mg/g body weight (BW) at 1 h before or 5 min, 10 min after intraperitoneal injection of compound 48/80. The passive cutaneous anaphylaxis (PCA) reaction also inhibited to 78.5% by oral administration of SPAE (1.0 mg/g BW). When SPAE pretreated on mice at concentrations ranging from 0.0001 to 1.0 mg/g BW, the serum histamine levels were reduced in a dose-dependent manner. Moreover, SPAE (100-800 mug/ml) dose-dependently inhibited the histamine release from peritoneal mast cells (RPMC) by compound 48/80 (5 mug/ml). Analysis by microscopic appearance observation revealed that SPAE (500 mug/ml) stabilized the RPMC membrane. Therefore, these findings indicate that SPAE inhibits anaphylactic reactions through stabilization of mast cell membrane.     

CHANGES IN HISTAMINE CONTENT FOLLOWING PHARMACOLOGICALLY-INDUCED MAST CELL DEGRANULATION IN THE RAT CONJUNCTIVA

Compound 48/80 was applied into one eye of male Wistar rats and a drop of vehicle into the contralateral eye. Another group of rats received sodium cromoglycate in both eyes every 6 h for a period of 48 h. One eye was challenged with compound 48/80 30 min after the end of treatment with sodium cromoglycate. The eyes were monitored clinically and the histamine content of the conjunctiva was determined fluorometrically. The basal histamine levels in rat conjunctival homogenates were quantified. Pharmacologically-induced mast cell degranulation by a single application of 0.1 g ml(-1)of compound 48/80 resulted in significant decreases of conjunctival histamine levels 1, 12 and 24 h after challenge. Sodium cromoglycate prevented the effect of compound 48/80 when administered into the eye prior to the challenge with the non-immunogenic histamine releaser. Upon termination of the application, the membrane stabilizer was unable to reverse the reduced histamine levels in the conjunctival homogenates.     

Rubus croceacanthus Leveille inhibits mast cell-mediated anaphylactic-like reaction and tumor necrosis factor-alpha secretion

This work aims at examining the effect of the concentrated methanol extract of Rubus croceacanthus Leveille (RCL) on mast cell-mediated anaphylactic-like reaction in a murine model. RCL inhibited compound 48/80-induced systemic anaphylactic-like reaction. When RCL was given as pre-treatment at concentrations ranging from 0.01 to 1 mg/ml, the histamine release from rat peritoneal mast cells induced by compound 48/80 or anti-dinitrophenyl (DNP) immunoglobulin E (IgE) was reduced in a dose-dependent manner. RCL also inhibited passive cutaneous anaphylaxis activated by anti-DNP IgE. In addition, RCL inhibited phorbol 12-myristate 13-acetate and A23187-induced tumor necrosis factor-alpha secretion from human mast cell line HMC-1 cells. These results indicate that RCL may possess a strong anti-anaphylactic activity.     

Mechanisms of histamine release by compound 48/80

1. Rat and guinea-pig lung tissues were incubated for 20 min at 37 degrees C in Krebs-Ringer phosphate buffer at pH 7.4, or in Tyrode-Tris buffer at pH 8.2, and the release of histamine produced by adding different concentrations of compound 48/80 to the incubation medium was determined.2. At pH 7.4, increasing concentrations of 48/80 increased the release of histamine from the rat lung, with a tendency towards a maximum. No release of histamine from guinea-pig lung was observed at this pH. At pH 8.2, histamine release occurred both from rat and guinea-pig lung, and was proportional to the logarithm of the concentration of compound 48/80.3. Histamine release from rat lung by 20 mug/ml. of 48/80 decreased when the pH was raised from 7.4 to 8.2; but the release caused by 1 mg/ml. of 48/80 increased both in rat and guinea-pig lung as the pH was raised.4. 2-4-Dinitrophenol (DNP) inhibited the release of histamine from rat lung by a concentration of 20 mug/ml. of 48/80; the inhibition was prevented by glucose. DNP did not affect histamine release from rat or guinea-pig lung by a concentration of 1 mg/ml. of 48/80 and enhanced the release when the pH was raised from 7.4 to 8.2.5. 1 mg/ml. of 48/80 did not inhibit the enhanced oxygen consumption produced by DNP in the isolated rat diaphragm.6. Iodoacetic acid (IAA) or a Ca/Mg-free medium inhibited the release of histamine by 20 mug/ml. of 48/80 from rat lung but not the release produced by 1 mg/ml. in either rat or guinea-pig lung.7. The degranulation of rat mesentery mast cells caused by 20 mug/ml. of compound 48/80 was inhibited by DNP. The degranulation evoked by 1 mg/ml. of 48/80 was also sensitive to this inhibitor; in this instance, however, the metachromatic staining reaction of the mesentery mast cells was greatly diminished.8. It is concluded that two processes of histamine release by compound 48/80 occur in rat lung. One, dependent on cell metabolism, involves, mast cell granule secretion. The other, independent of cell metabolism, seems to consist of a simple exchange reaction between histamine and compound 48/80, and this is the only one occurring in guinea-pig lung.     

Comparison of histamine release from peritoneal mast cells derived from diabetic and control rats.

Clinical and experimental diabetes are associated with an increased number of mast cells and elevated tissue histamine concentrations. This study compared histamine release from peritoneal mast cells derived from diabetic and control rats. Experimental diabetes was induced by a single i.v. injection of streptozotocin (50 mg/kg body weight). Measurement of plasma glucose levels confirmed the diabetic state. Peritoneal mast cells were stimulated for 10 min with the lectin concanavalin A (0.5-100 micrograms/ml) in the presence or absence of phosphatidylserine, clinical dextran (0.6-1200 micrograms/ml) in the presence of phosphatidylserine, the calcium ionophore A23187 (0.1-1 microM) or the basic releasing agent compound 48/80 (0.1-10 micrograms/ml). Histamine release induced by these agents was similar in both populations. Further studies will compare the differences in histamine release from mast cells isolated from different tissues, e.g. heart and lung. In addition, physiological stimuli which are altered in the diabetic state (e.g. hyperosmolalar solutions and free radical generating systems) are under investigation.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca2+ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 micrograms/ml of compound 48/80, a rapid increase in intracellular Ca2+ was observed. At concentrations higher than 10(-6) M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca2+ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca2+ release from the intracellular Ca store.     

The effect of different calcium antagonists on histamine- and carbachol-induced acid secretion in the isolated mouse stomach

Earlier experiments suggested that the action of muscarinic agonists in gastric acid secretion might be partly mediated by endogenous histamine release. This possibility could be tested by use of various calcium antagonists provided that they show different activity on histamine- and carbachol-induced secretion. In the present work, three calcium antagonists, verapamil, nifedipine and sodium nitroprusside were used on the isolated mouse stomach where acid secretion induced by histamine and by carbachol was measured. Verapamil (100 microM) did not affect either histamine- or carbachol-induced acid secretion. Nifedipine (100 microM) reduced histamine-induced acid secretion, whereas the effect of carbachol remained unchanged. Sodium nitroprusside (10-100 microM) reduced both histamine- and carbachol-induced acid secretion. The differences in action of calcium antagonists in histamine- and carbachol-induced acid secretion could exclude the possibility that the effect of carbachol is partly mediated by histamine release from mast cells present in the mouse stomach.     

Inhibition of mast cell-dependent anaphylaxis by succinic acid

We have examined the effect of succinic acid on anaphylaxis. Succinic acid (100 mM) significantly inhibited systemic anaphylaxis induced by compound 48/80 in mice and dose-dependently inhibited local anaphylaxis activated by anti-dinitrophenyl IgE. Further 10 and 100 mM significantly inhibited histamine release from rat peritoneal mast cells activated by compound 48/80 or anti-dinitrophenyl IgE. In addition succinic acid (0.1 and 1 mM) had a significant inhibitory effect on anti-dinitrophenyl IgE-induced tumour necrosis factor-alpha secretion from rat peritoneal mast cells. The level of cyclic AMP in rat peritoneal mast cells, when succinic acid (100 mM) was added, transiently and significantly increased about 4 times compared with that of basal cells. These results suggest a possible use of succinic acid in managing mast cell-dependent anaphylaxis.