Effects of Peanut-Skin Procyanidin A1 on Degranulation of RBL-2H3 Cells

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m?z 599 [M+Na]⁺. Based on the results of ¹H-NMR, ¹³C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC₅₀ of 20.3 μM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca²⁺ influx from an internal store in RBL-2H3 cells.     

Polymeric grape-seed procyanidins, but not monomeric catechins and oligomeric procyanidins, impair degranulation and membrane ruffling in RBL-2H3 cells

Grape-seed proanthocyanidins (GSPs) are catechin polymers that are predicted to form helices in their global minimum-energy conformation and to have a mean degree of polymerization of seven (mDP = 7). The highly polymerized GSP-H fraction (mDP = 10) was found to impair degranulation in RBL-2H3 cells after stimulation with an antigen (Ag) and treatment with the Ca-ATPase inhibitor thapsigargin (Tg). In addition, GSP-H affected actin cytoskeleton and inhibited membrane ruffling in these cells, resulting in the suppression of exocytosis. By contrast, monomeric epicatechin, the dimeric procyanidins PA-1, PA-2, and PB-2, and the oligomerized GSP-L (mDP = 3) had no effect on membrane ruffling and degranulation. These findings indicate that the molecular size and length of GSP-H are needed for the inhibition of membrane ruffling and degranulation in RBL-2H3 mast-cell lines.     

Inhibitory effects of thunberginols A and B isolated from Hydrangeae Dulcis Folium on mRNA expression of cytokines and on activation of activator protein-1 in RBL-2H3 cells

Previously, thunberginols A and B from the processed leaves of Hydrangeae macrophylla var. thunbergii (Hydrangea dulcis folium) substantially inhibited the degranulation caused by antigen and calcium ionophore A23187, and the release of tumor necrosis factor (TNF)-α and interleukin (IL)-4 by antigen in RBL-2H3 cells. In the present study, we examined the effect of thunberginol B on the expression of mRNA of several cytokines [ILs-2, 3, 4 and 13, TNF-α and granulocyte/macrophage-colony stimulating factor (GM-CSF)] and effects of thunberginols A and B on activator protein (AP)-1 composed of c-jun and c-fos, which is essential for the expression of the cytokine mRNA, in RBL-2H3 cells. Thunberginol B inhibited up-regulated genes of all cytokines, and thunberginols A and B (30 μM) inhibited the phosphorylation of c-jun and expression of c-fos mRNA and phosphorylation of extracellular signal-regulated kinases (ERK1/2). In addition, the profile of gene expression by thunberginol B was similar to that by luteolin, a natural flavone with a potent anti-allergic effect.     

Ceramide kinase is a mediator of calcium-dependent degranulation in mast cells

Ceramide kinase (CERK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P) and is known to be activated by calcium. Although several groups have examined the functions of CERK and its product C1P, the functions of C1P and CERK are not understood. We studied the RBL-2H3 cell line, a widely used model for mast cells, and found that CERK and C1P are required for activation of the degranulation process in mast cells. We found that C1P formation was enhanced during activation induced by IgE/antigen or by Ca(2+) ionophore A23187. The formation of C1P required the intracellular elevation of Ca(2+). We generated RBL-2H3 cells that stably express CERK, and when these cells were treated with A23187, a concomitant C1P formation was observed and degranulation increased 4-fold, compared with mock transfectants. The cell-permeable N-acetylsphingosine (C(2)-ceramide), a poor substrate of CERK, inhibited both the formation of C1P and degranulation, indicating that C1P formation was necessary for degranulation. Exogenous introduction of CERK into permeabilized RBL-2H3 cells caused degranulation. We identified a cytosolic localization of CERK that provides exposure to cytosolic Ca(2+). Taken together, these results indicate that C1P formation is a necessary step in the degranulation pathway in RBL-2H3 cells.     

Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.     

Inhibitory effect of Japanese black vinegar on IgE-mediated degranulation of RBL-2H3 cells and a murine model of Japanese cedar pollinosis

Japanese black vinegar (JBV) is a traditional vinegar manufactured with steamed unpolished rice. After screening, beneficial effects of JBV on IgE-mediated allergic responses were found. In this study, acetic acid-free JBV was used to evaluate its antiallergic effects. JBV suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The inhibitory effect of JBV on the degranulation seemed to be caused by the bioactive ingredients other than proteins, because the activity was not affected by heat treatment or protease digestion. JBV inhibited the elevation in the intracellular Ca²⁺ concentration induced by antigen. Immunoblot analysis revealed that JBV suppresses degranulation of RBL-2H3 cells by downregulated phosphorylation of PI3K, Akt, and PLCγ1. In addition, oral administration of JBV significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-induced pollinosis model mice. Thus, JBV has a potential as a health-promoting food with the antiallergy effect.     

High-affinity IgE receptor-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells induces early and late protein-tyrosine phosphorylations.

We reported previously that stimulation of RBL-2H3 cells through the high-affinity IgE receptor resulted in tyrosine phosphorylation of a 72-kDa protein (pp72) that was coupled to signal transduction. In the present study, although pp72 tyrosine phosphorylation was induced only by antigen triggering, stimulation of RBL-2H3 cells by either antigen or the calcium-ionophore A23187 led to increased tyrosine phosphorylation of a 110-kDa protein (pp110). This tyrosine phosphorylated protein was also observed when RBL-2H3 cells were transfected with the G protein-coupled m3 muscarinic receptor and then stimulated to secrete with carbachol. In contrast to tyrosine phosphorylation of pp72, antigen-induced pp110 tyrosine phosphorylation required extracellular calcium, was absent in cells depleted of protein kinase C, and was detected between 1 and 5 min after stimulation. The protein-tyrosine kinase inhibitor genistein blocked both histamine release and tyrosine phosphorylation induced by A23187. Altogether, the data suggest a role for pp110 in secretion. However, protein kinase C activation induced pp110 tyrosine phosphorylation but not histamine release demonstrating that pp110 tyrosine phosphorylation alone is not sufficient for degranulation. We conclude that tyrosine phosphorylation of pp72 is associated with the early steps of IgE receptor-generated signaling, whereas pp110 tyrosine phosphorylation occurs secondary to calcium influx and protein kinase C activation.     

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).     

Inhibitory Effects of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and Rat Mast Cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC₅₀ values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

Inhibition of antigen-induced degranulation by aryl compounds isolated from the bark of Betula platyphylla in RBL-2H3 cells

The methanolic extract of the bark of Betula platyphylla was found to suppress antigen mediated degranulation of RBL-2H3 cells. Four arylbutanoids (1–4) and eight diarylhepatonoids (5–12) were isolated from the methanolic extract using bioassay-guided fractionation. Among them, compounds 4 and 12 were isolated and assigned for the first time. Compounds 1, 2, 3, 5, 10, and 12 showed remarkable inhibitory activity against the degranulation of RBL-2H3 by antigen stimulation in a dose dependent manner at the concentrations ranging from 10 μM to 100 μM.     

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin. When RBL-2H3 cells were pretreated with a MLCK inhibitor, ML-7, or a CaMKs inhibitor, KN-93, the ionomycin-dependent release of beta-hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin-dependent release of beta-hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+-induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+ concentrations in RBL-2H3 cells.     

Distinct phosphoinositide 3-kinases mediate mast cell degranulation in response to G-protein-coupled versus FcepsilonRI receptors

Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110beta and p85/p110delta but not p85/p110alpha are required for antigen-mediated calcium flux in RBL-2H3 cells (Smith, A. J., Surviladze, Z., Gaudet, E. A., Backer, J. M., Mitchell, C. A., and Wilson, B. S. et al., (2001) J. Biol. Chem. 276, 17213-17220). We now examine the role of Class IA PI 3-kinases isoforms in degranulation itself, using a single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110delta and p110beta blocked FcepsilonR1-mediated degranulation in response to FcepsilonRI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110alpha, despite the fact that these antibodies have no effect on antigen-induced calcium flux. These data suggest that p110alpha mediates a calcium-independent signal during degranulation. In contrast, only p110beta was required for enhancement of antigen-stimulated degranulation by adenosine, which augments mast cell-mediated airway inflammation in asthma. Finally, we examined carbachol-stimulated degranulation in RBL2H3 cells stably expressing the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly, carbachol-stimulated degranulation was blocked by antibody-mediated inhibition of the Class III PI 3-kinase hVPS34 or by titration of its product with FYVE domains. Antibodies against Class IA PI 3-kinases had no effect. These data demonstrate: (a) a calcium-independent role for p110alpha in antigen-stimulated degranulation; (b) a requirement for p110beta in adenosine receptor signaling; and (c) a requirement for hVPS34 during M1 muscarinic receptor signaling. Elucidation of the intersections between these distinct pathways will lead to new insights into mast cell degranulation.     

Synthesis of norlignans and in vitro inhibitory activity of antigen-induced degranulation

The synthesis and biological evaluation of a series of novel norlignans are described. Norlignans were evaluated for their inhibitory activity on the release of β-hexosaminidase, a marker of degranulation, from RBL-2H3 cells induced by the IgE-antigen complex. The results showed that norlignans 4c and 4e potently inhibited degranulation, with IC(50) values of 18.3 and 17.9 μM, respectively.     

Protective effect of chitosan oligosaccharides against FcɛRI-mediated RBL-2H3 mast cell activation

The activation of mast cells by immunoglobulin E-mediated stimuli is considered as a central event in allergic responses. In this regard, chitosan oligosaccharides (COS) of two different molecular weight ranges (1–3 kDa and 3–5 kDa) were investigated for their capabilities against the activation of RBL-2H3 mast cell sensitized with dinitrophenyl-specific immunoglobulin E antibody and stimulated by antigen dinitrophenyl-bovine serum albumin. It was found that COS significantly inhibited RBL-2H3 cell degranulation via attenuating the releases of histamine and β-hexosaminidase. Moreover, the inhibitory activity of COS was accompanied by a reduction in intracellular Ca²⁺ elevation. Notably, the expression of immunoglobulin Fc epsilon receptor I (Fc?RI) in RBL-2H3 cells was down-regulated by COS treatment in a dose-dependent manner. The suppressive effect of COS on RBL-2H3 cell activation suggested that COS may be potential candidates of novel inhibitors against allergic reactions.     

Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells

The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.     

Mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl)decyltriphenylphosphonium bromide) inhibits mast cell degranulation <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

The therapeutic effect of mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium bromide (SkQ1) in experimental models of acute inflammation and wound repair has been shown earlier. It was suggested that the antiinflammatory activity of SkQ1 is related to its ability to suppress inflammatory activation of the vascular endothelium and neutrophil migration into tissues. Here, we demonstrated that SkQ1 inhibits activation of mast cells (MCs) followed by their degranulation and histamine release in vivo and in vitro. Intraperitoneal injections of SkQ1 in the mouse air-pouch model reduced the number of leukocytes in the air-pouch cavity and significantly decreased the histamine content in it, as well as suppressing MC degranulation in the air-pouch tissue. The direct effect of SkQ1 on MCs was studied in vitro in the rat basophilic leukemia RBL-2H3 cell line. SkQ1 inhibited induced degranulation of RBL-2H3 cells. These results suggest that mitochondrial reactive oxygen species are involved in the activation of MCs. It is known that MCs play a crucial role in regulation of vascular permeability by secreting histamine. Suppression of MC degranulation by SkQ1 might be a significant factor in the antiinflammatory activity of this mitochondria-targeted antioxidant.     

Inhibition by dexamethasone of interleukin 13 production via glucocorticoid receptor-mediated inhibition of c-Jun phosphorylation

The antigen stimulation of RBL-2H3 cells induced interleukin 13 (IL-13) production, which was inhibited by the steroidal anti-inflammatory drug dexamethasone and by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Dexamethasone did not inhibit the antigen-induced phosphorylation of JNK but inhibited that of c-Jun. In a cell-free system, the phosphorylation of glutathione S-transferase-fused c-Jun by recombinant JNK was not inhibited by dexamethasone but was inhibited by the addition of recombinant glucocorticoid receptor (GR). These findings suggest that the inhibition of antigen-induced IL-13 production by dexamethasone is due to the GR-mediated inhibition of c-Jun phosphorylation induced by JNK.     

Inhibitory effects of water-soluble low-molecular-weight β-(1,3-1,6) D-glucan isolated from Aureobasidium pullulans 1A1 strain black yeast on mast cell degranulation and passive cutaneous anaphylaxis

We investigated the effects of water-soluble low-molecular-weight β-(1,3-1,6) D-glucan isolated from Aureobasidium pullulans 1A1 strain black yeast (LMW-β-glucan) on mast cell-mediated anaphylactic reactions. Although it is known that LMW-β-glucan has anti-tumor, anti-metastatic and anti-stress effects, the roles of LMW-β-glucan in immediate-type allergic reactions have not been fully investigated. We examined whether LMW-β-glucan could inhibit mast cell degranulation and passive cutaneous anaphylaxis (PCA). LMW-β-glucan dose-dependently inhibited the degranulation of both rat basophilic leukemia (RBL-2H3) and cultured mast cells (CMCs) activated by calcium ionophore A23187 or IgE. However, LMW-β-glucan had no cytotoxicity towards RBL-2H3 cells and CMCs. Furthermore, orally administered LMW-β-glucan inhibited the IgE-induced PCA reaction in mice. These results show LMW-β-glucan to be a possible compound for the effective therapeutic treatment of allergic diseases.     

Inhibitory effect of fulvic acid extracted from Canadian sphagnum peat on chemical mediator release by RBL-2H3 and KU812 cells

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and (13)C-nuclear magnetic resonance ((13)C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001-10.0 microg/ml of CP-FA and determined the beta-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca(2+)](i) level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001-10.0 microg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the beta-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca(2+)](i) level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.     

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).