Cyclic AMP independent inhibition by papaverine of histamine release induced by compound 48/80.

Compound 48/80-induced histamine release from isolated rat peritoneal mast cells was inhibited in a dose-dependent manner by papaverine (ic₅₀ approx 20 μM). This effect of papaverine was not influenced by PGE₁ (14–140 μM), even though PGE₁ markedly increased must cell cAMP levels. Papaverine (0.5 mM) completely inhibited histamine release without causing any change in cAMP levels. Theophylline (0.1 and 0.5 mM) potentiated histamine release induced by submaximal concentrations of compound 48/80, while cAMP levels were increased. IBM X was as potent as papaverine in causing inhibition of mast cell phosphodiesterase. IBM X (0.14–0.7 mM) had no effect on histamine release but caused a 6–20 fold increase in mast cell cyclic AMP. Papaverine inhibition of histamine release was gradual at the onset and was parallelled by a depletion of mast cell ATP content. The inhibition of 48/80-induced histamine release and depletion of mast cell ATP levels was reversed by glucose. It is concluded that papaverine induced inhibition of 48/80-induced histamine release is independent of cAMP, is unrelated to phosphodiesterase inhibition but is dependent upon inhibition of energy production.     

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 compound 48/80 on the carbachol-evoked acid secretion of the isolated mouse stomach

The aim of the present work was to determine in the isolated mouse stomach whether the depletion of mast cell histamine by compound 48/80 influences the acid secretion, evoked by carbachol. Each effect of carbachol was compared to the effect of histamine (10(-6) M), applied after each carbachol application. After carbachol and histamine, compound 48/80 was applied twice or three times successively; the second and the third application were not able to evoke any secretion. After 48/80, the applications of carbachol and histamine were repeated and their effects compared to those before the applications of compound 48/80. Three concentrations of the compound were used: 20, 100 and 500 micrograms/ml. The lowest concentration of the histamine liberator did not significantly change the response to carbachol, The highest one reduced it but the effect of histamine was reduced, too. Compound 48/80 in concentration 100 micrograms/ml significantly augmented the secretory effect of carbachol. The possible explanation for this effect is that histamine, liberated from mast cells, is taken up by some other cells in the tissue from where it is liberated by carbachol stimulation.     

Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1).

Rapid intravenous administration of the glycopeptide antibiotic, vancomycin, may cause a hypotensive reaction which can usually be prevented by infusing vancomycin in dilute solutions. The release of histamine from circulating cells such as basophils and tissue mast cells has been implicated in hypotensive reactions since the effects can be prevented by antihistamine pretreatment. The direct effects of vancomycin on histamine release were therefore investigated in rat peritoneal mast cells and rat leukemic basophils (RBL-1 cells). Suspension cultures of mast cells or RBL-1 cells were exposed to vancomycin for 30-60 minutes at concentrations comparable to those infused clinically (2.28 or 4.56 mg/ml). Vancomycin induced a time- and dose-dependent release of histamine into the culture media from both cell types. The reference degranulating agent, Compound 48/80 (CP 48/80), was also shown to induce histamine release from mast cells and RBL-1 cells. Mast cells were significantly more sensitive to vancomycin and CP 48/80 than RBL-1 cells and, unlike RBL-1 cells, were responsive to the inhibitory effects of cromolyn sodium on histamine release. Cromolyn sodium did not inhibit vancomycin-induced histamine release in RBL-1 or mast cells. Morphologically, mast cells exposed to either vancomycin or CP 48/80 exhibited dose-related degranulation. On the other hand, treatment-related degranulation effects of either vancomycin or CP 48/80 on RBL-1 cells could not be reliably distinguished from controls by qualitative evaluation. Based upon these findings it is concluded that mast cells may represent a more useful model to evaluate the potential of investigational agents to release histamine and to study mechanisms of histamine release than RBL-1 cells.     

D-galactose-specific sea urchin lectin sugar-specifically inhibited histamine release induced by datura stramonium agglutinin: differences between sugar-specific effects of sea urchin lectin and those of D-galactose- or L-fucose-specific plant lectins

A new sea urchin lectin from Toxopneustes pileolus, is D(+)galactose (Gal)-, D(+)fucose (Fuc)-specific. Incubation of rat peritoneal mast cells with the lectin in the presence of 0.3 mM CaCl2 for 10 min significantly and dose-dependently inhibited the histamine release induced by N-acetyl glucosamine (GlcNAc)-specific Datura stramonium agglutinin (DSA), an activator of the Gi-protein-dependent pathway in mast cells. This inhibition by the sea urchin lectin was sugar-specifically reversed in the presence of D(+)Gal or D(+)Fuc but not L(-)Fuc. The sea urchin lectin had no effect on the histamine release induced by compound 48/80, slightly inhibited the histamine release induced by substance P and mastoparan, and slightly enhanced the histamine release induced by melittin, but these effects were not dose-dependent. Compound 48/80, substance P, mastoparan and melittin are mast cell activators without sugar residues. It is suggested that the lectin binds to D(+)Gal residues of DSA to interfere with mast cell activation induced by DSA, a glycoprotein with arabinose and Gal residues. The effects of plant lectins with affinity to D(+)Gal, N-acetyl galactosamine and/or sialic acid and L(-)Fuc on the histamine release induced by DSA, compound 48/80 and substance P were also examined.     

A method for determining whether hypotension caused by novel compounds in preclinical development results from histamine release

INTRODUCTION: Many therapeutic agents stimulate histamine release from mast cells, which results in a decrease in blood pressure. The purpose of this study is to establish a method to determine if the mechanism of action, or one of the mechanisms, of hypotensive compounds is related to the release of histamine. The method was developed using a novel hypotensive compound, SC-372. METHODS: In Inactin anesthetized rats, after intravenous administration of SC-372 (0.3-7 mg/kg), the 2 and 7 mg/kg resulted in a dose-dependent decrease in blood pressure. Histamine (0.1 and 1 mg/kg) was injected intravenously to establish whether histamine release was the mechanism of action for the hypotension induced by SC-372. Compound 48/80 (0.1 mg/kg, promotes histamine release) and Cromolyn (1 mg/kg/min, [5 min], prevents histamine release from mast cells) were characterized and used intravenously in combination with/or compared to SC-372. RESULTS: Histamine resulted in a decrease in blood pressure that was unaffected by Cromolyn (1 mg/kg). Administration of Compound 48/80 resulted in a rapid reduction of systemic blood pressure. Intravenous infusion of Cromolyn prior to the injection of Compound 48/80 significantly attentuated the hypotensive response and the increase in histamine levels in the plasma. Intravenous administration of SC-372 resulted in a rapid reduction in blood pressure with a profile similar to that of Compound 48/80. When the rats were treated with Cromolyn prior to the administration of SC-372, both the blood pressure and plasma histamine levels were maintained at their pretreatment control levels. DISCUSSION: These data indicate that Compound 48/80 and Cromolyn can be used in rats to screen for histamine release-dependent drug-induced hypotension and suggest that the rapid decrease in blood pressure caused by SC-372 may result from histamine release from mast cells.     

Calcium dependency of inhibition by arachidonic acid of compound 48/80-induced histamine release from mast cells

Compound 48/80 ( compd 48/80)-induced histamine secretion from rat mast cells was inhibited almost completely by pretreatment of the cells at 37 degrees with 25 microM arachidonic acid in the presence of 1.8 mM Ca2+. As the Ca2+ concentration was reduced below 1.8 mM, 25 microM arachidonic acid became less inhibitory and, then, progressively more stimulatory for histamine release with or without compd 48/80. No additive effect on histamine release was obtained by combining compd 48/80 and arachidonic acid. Pretreatment of mast cells with lidocaine, an inhibitor of Ca2+ binding to phospholipid, or with nordihydroguaiaretic acid, an inhibitor of Ca2+ flux and lipoxygenase, stimulated arachidonic acid-induced histamine release. Arachidonic acid also inhibited a compd 48/80-induced spike increment of intracellular 45Ca2+ uptake and a decrease of total 45Ca2+ uptake by 45Ca2+-preloaded mast cells. Arachidonic acid and Ca2+ also suppressed melittin-induced histamine release and compd 48/80-induced release of radioactivity from mast cells preloaded with [3H]arachidonic acid. These results suggest that exogenous arachidonic acid or its metabolite(s) may interact with membrane-associated Ca2+, disturbing Ca2+ availability for the trigger mechanism of compd 48/80-induced histamine release or inhibiting the subsequent metabolism of arachidonic acid via the lipoxygenase pathway to form active metabolites involved in the histamine liberating mechanism.     

Experimental anxiety induced by histaminergics in mast cell-deficient and congenitally normal mice

To clarify the effect of mast cell-derived histamine release in the brain on anxiety, histaminergics-induced anxiety-like behaviors were examined by a light/dark test in mast cell-deficient (W/Wv) and congenitally normal (+/+) mice. In +/+ mice, when cimetidine (an H2 receptor antagonist) was coadministered with thioperamide (a neuronal histamine releaser acting via inhibition of H3 autoreceptors) or Compound 48/80 (C48/80, a selective histamine releaser from mast cells), the time spent in the light zone and the number of crossings between light and dark zones in a light/dark test decreased significantly, suggesting induction of anxiety. In W/Wv mice, however, experimental anxiety was induced by coadministration of thioperamide-cimetidine, but not C48/80-cimetidine. These results suggest that both nonneuronal mast cell-derived histamine and neuronal histamine play an important role in inducing experimental anxiety.     

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.     

Effect of nociceptin on histamine and serotonin release in the central nervous system

Role in pain sensation of both nociceptin (NC), the bioactive heptadecapeptide sequence of preproorphaninFQ and of histamine has been widely evidenced in the central nervous system (CNS). In the current series of experiments effect of intracerebroventricularly (i.c.v.) administered NC (5.5 nmol/rat) on histamine and serotonin levels in blood plasma, CSF and brain areas (hypothalamus and hippocampus) was studies and compared to the effect of the mast cell degranulator Compound 48/80(100microg/kg, i.c.v.) and the neuroactive peptide Substance P (50nmol/rat, i.c.v.). It was found that all the three compounds increased the histamine level in the CNS, however their activity concerning the mast cell-, and neuronal histamine release is different. NC could release histamine from both the mast cells and the neurons and it decreased CNS serotonin levels. Substance P was found the most potent in increasing CNS histamine levels. Compound 48/80 treatment resulted in elevated histamine levels both in the CNS and blood plasma. It is concluded that the histamine releasing effects of i.c.v. administered NC and SP are limited to the CNS, but in the effect of Compound 48/80 its blood-brain barrier impairing activity is also involved. Data also demonstrate that NC has significant effect on both the histaminergic and serotonergic neurotransmission in the CNS.     

Compound 48/80-induced secretion of histamine from rat peritoneal mast cells depends on a tryptase controlled step also leading to chymase activity.

Compound 48/80 caused activation of rat mast cell tryptase and chymase along with the release of histamine. Following low speed centrifugation, tryptase remained essentially in the supernatant while chymase sedimented. Enzyme activities as well as histamine release were inhibited by phenylmethyl-sulfonyl fluoride and tosyl-lysine chloromethyl ketone, which are respectively unspecific and specific inhibitors of trypsin-like enzymes. Chymostatin inhibited chymase, but not the release of histamine by 48/80. Tryptase activity thus seems to be essentially involved in the mechanism of histamine release, while chymase activity may be merely one of its accompanying events.     

Potassium-induced release of tritiated histamine from rat brain tissue slices.

The release of exogenous histamine was studied by superfusing brain slices following incubation with the radiolabeled amine. Histamine was released in a calcium-dependent way by 40 mM potassium. This release was high in hypothalamus and striatum and low in hippocampus and cortex. Compound 48/80, a mast cell histamine releasing agent, also induced histamine release, but only from hypothalamic tissue slices. It is suggested that the potassium-induced release of accumulated exogenous histamine is mainly from glial cells.     

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.     

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.     

Mechanism of histamine release from rat isolated peritoneal mast cells by dextran: the role of immunoglobulin E

In vivo passive sensitization of rat peritoneal mast cells with Nippostrongylus braziliensis antiserum or rat monoclonal myeloma IgE greatly enhanced histamine release in vitro by dextran or anti IgE, but did not alter release by compound 48/80 or A23187. Conversely, removal of IgE from the cells by acid pH abolished histamine release by dextran and anti IgE but did not impair release by compound 48/80. Whereas, histamine release from cells isolated from rats genetically resistant to dextran (NR rats) by anti IgE was potentiated by passive sensitization, dextran was unable to stimulate secretion from control or sensitized NR cells. The results suggest that dextran releases histamine by interaction with cell-fixed IgE and that the NR mast cell membrane lacks the ability to interpret this stimulus.     

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.     

Calcium uptake in mast cells, energy metabolism and histamine secretion

The inhibition of energy metabolism of mast cells causes an inhibition of histamine secretion. As the secretion is generally initiated by the influx of calcium into the cell, we have made correlative studies of the effect of blocking the energy metabolism on calcium uptake and histamine secretion. When the influx of calcium is increased by exposing the cells to low concentrations of saponin or ionophore A23187, histamine release occurs, having the character of a secretory response. Brief incubation of the cells with antimycin A, 10(-9) M-10(-7) M, prior to exposure to saponin or the calcium ionophore gave similar dose-response curves for the inhibitory effect of antimycin A on calcium uptake and histamine release. The inhibition of calcium uptake in untreated mast cells by antimycin A, 10(-9) M-10(-7) M, showed good correlation to the inhibition of anaphylactic histamine release and the release induced by compound 48/80. The antigen-induced histamine release is dependent on extracellular calcium and an inhibition of its uptake by antimycin A could by itself inhibit the release. Compound 48/80 on the other hand induces histamine release both in the presence and absence of calcium, and both are similarly inhibited by 10(-9) M-10(-7) M antimycin A. This indicates that antimycin A has other sites of action apart from the inhibition of the influx of extracellular calcium. The inhibitory effect of antimycin A on compound 48/80-induced histamine secretion in the absence of extracellular calcium may be due to an inhibition of energy requiring steps in the final phase of the secretory process.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The role of the Annexin-A1/FPR2 system in the regulation of mast cell degranulation provoked by compound 48/80 and in the inhibitory action of nedocromil

• 1.We investigated the role of Annexin (ANX)-A1 and its receptor, ALX/FPR2, in the regulation of mast cell degranulation produced by compound 48/80.
• 2.Both human cord-blood derived mast cells (CBDMCs) and murine bone marrow derived mast cells (BMDMCs) release phosphorylated ANX-A1 during treatment with glucocorticoids or the mast cell ‘stabilising’ drugs ketotifen and nedocromil.
• 3.Compound 48/80 also stimulated ANX-A1 phosphorylation and release and this was also potentiated by nedocromil. Anti-ANX-A1 neutralising monoclonal antibodies (Mabs) enhanced the release of pro-inflammatory mediators in response to compound 48/80.
• 4.Nedocromil and ketotifen potently inhibited the release of histamine, PGD₂, tryptase and β-hexosaminidase from mast cells challenged with compound 48/80. Anti-ANX-A1 neutralising Mabs prevented the inhibitory effect of these drugs.
• 5.BMDMCs derived from Anx-A1^−/− mice were insensitive to the inhibitory effects of nedocromil or ketotifen but cells retained their sensitivity to the inhibitory action of hu-r-ANX-A1.
• 6.The fpr2/3 antagonist WRW4 blocked the action of nedocromil on PGD₂, but not histamine, release. BMDMCs derived from fpr2/3^−/− mice were insensitive to the inhibitory effects of nedocromil on PGD₂, but not histamine release.
• 7.Compound 48/80 stimulated both p38 and JNK phosphorylation in CBDMCs and this was inhibited by nedocromil. Inhibition of p38 phosphorylation was ANX-A1 dependent.
• 8.We conclude that ANX-A1 is an important regulator of mast cell reactivity to compound 48/80 exerting a negative feedback effect through a mechanism that depends at least partly on the FPR receptor.

     

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.