Anti-Allergic Potential of Cinnamaldehyde via the Inhibitory Effect of Histidine Decarboxylase (HDC) Producing Klebsiella pneumonia.

Allergy is an immunological disorder that develops in response to exposure to an allergen, and histamines mediate these effects via histidine decarboxylase (HDC) activity at the intracellular level. In the present study, we developed a 3D model of Klebsiella pneumoniae histidine decarboxylase (HDC) and analyzed the HDC inhibitory potential of cinnamaldehyde (CA) and subsequent anti-allergic potential using a bacterial and mammalian mast cell model. A computational and in vitro study using K. pneumonia revealed that CA binds to HDC nearby the pyridoxal-5'-phosphate (PLP) binding site and inhibited histamine synthesis in a bacterial model. Further study using a mammalian mast cell model also showed that CA decreased the levels of histamine in the stimulated RBL-2H3 cell line and attenuated the release of ß-hexoseaminidase and cell degranulation. In addition, CA treatment also significantly suppressed the levels of pro-inflammatory cytokines TNF-α and IL-6 and the nitric oxide (NO) level in the stimulated mast cells. A gene expression and Western blotting study revealed that CA significantly downregulated the expressions of MAPKp38/ERK and its downstream pro-allergic mediators that are involved in the signaling pathway in mast cell cytokine synthesis. This study further confirms that CA has the potential to attenuate mast cell activation by inhibiting HDC and modifying the process of allergic disorders.

Effects of the rhizomes of Atractylodes japonica and atractylenolide I on allergic response and experimental atopic dermatitis.

Although some anti-allergic activities of the rhizome of Atractylodes japonica have been previously reported, the active principle(s) for anti-allergic action is not fully elucidated and the effect of this plant material on atopic dermatitis (AD) is not known. In this study, the 70% ethanol extract of the rhizome of A. japonica was found to significantly inhibit 5-lipoxygenase (5-LOX)-catalyzed leukotrienes (LT) production from rat basophilic leukemia (RBL)-1 cells. From the extract of A. japonica, three major sesquiterpene derivatives including atractylenolide I, atractylenolide III and eudesma-4,7-dien-8-one were successfully isolated. Among these compounds, only atractylenolide I was shown to strongly inhibit 5-LOX from RBL-1 cells (IC(50) = 18.6 µM). To evaluate the effects of experimental AD, the ethanol extract of A. japonica (200 mg/day) was administered orally to hapten-treated NC/Nga mice which is an animal model of AD. It was firstly found that the extract significantly inhibited AD-like symptoms in mice, as judged by severity score and scratching behavior. Taken together, it is concluded that A. japonica possesses the inhibitory activity on 5-LOX and an animal model of AD, and atractylenolide I may contribute, at least in part, to these anti-allergic actions of A. japonica.

A detailed mathematical model predicts that serial engagement of IgE-Fc epsilon RI complexes can enhance Syk activation in mast cells.

The term serial engagement was introduced to describe the ability of a single peptide, bound to a MHC molecule, to sequentially interact with TCRs within the contact region between a T cell and an APC. In addition to ligands on surfaces, soluble multivalent ligands can serially engage cell surface receptors with sites on the ligand, binding and dissociating from receptors many times before all ligand sites become free and the ligand leaves the surface. To evaluate the role of serial engagement in Syk activation, we use a detailed mathematical model of the initial signaling cascade that is triggered when FcepsilonRI is aggregated on mast cells by multivalent Ags. Although serial engagement is not required for mast cell signaling, it can influence the recruitment of Syk to the receptor and subsequent Syk phosphorylation. Simulating the response of mast cells to ligands that serially engage receptors at different rates shows that increasing the rate of serial engagement by increasing the rate of dissociation of the ligand-receptor bond decreases Syk phosphorylation. Increasing serial engagement by increasing the rate at which receptors are cross-linked (for example by increasing the forward rate constant for cross-linking or increasing the valence of the ligand) increases Syk phosphorylation. When serial engagement enhances Syk phosphorylation, it does so by partially reversing the effects of kinetic proofreading. Serial engagement rapidly returns receptors that have dissociated from aggregates to new aggregates before the receptors have fully returned to their basal state.

alpha-Tocopherol enhances degranulation in RBL-2H3 mast cells.

Based on the observation that 3 months alpha-tocopherol supplementation caused an up-regulation of the mRNA of vesicular transport proteins in livers of mice, the functional relevance was investigated in RBL-2H3 cells, a model for mast cell degranulation. In total, 24 h incubation with 100 muM alpha-tocopherol enhanced the basal and phorbol-12-myristyl-13-acetate/ionomycin-stimulated release of beta-hexosaminidase and cathepsin D as measured by enzymatic analysis as well as Western blotting and immunocytochemistry, respectively. beta-Tocopherol exerted the same effect, whereas alpha-tocopheryl phosphate and trolox were inactive, indicating that both the side chain and the 6-OH group at the chroman ring are essential for activation of degranulation. alpha-Tocopherol did not induce mRNA expression of soluble NSF-attachment protein receptor (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins, such as N-ethylmaleimide sensitive fusion protein, complexin-2, SNAP23 or syntaxin-3, in the RBL-2H3 cell model. In view of the well known alpha-tocopherol-mediated activation of protein phosphatases, which regulate soluble NSF-attachment protein receptor activities by dephosphorylation, underlying mechanisms are discussed in terms of preventing oxidative inactivation of protein phosphatases and so far unknown functions in certain membrane domains.

Cbl-b promotes chemotherapy-induced apoptosis in rat basophilic leukemia cells by suppressing PI3K/Akt activation and enhancing MEK/ERK activation.

The ubiquitin ligase Cbl-b is a negative regulator of the PI3K/Akt pathway, the survival pathway implicated in chemotherapy resistance. However, it remains unclear whether Cbl-b can regulate chemosensitivity through modulating Akt activation. In this study, VP-16-induced RBL-2H3 cells apoptosis was accompanied by the activation of Akt and ERK. The PI3K inhibitor LY294002, not the ERK inhibitor PD98059, enhanced the apoptosis. In addition, down-regulation of Cbl-b was also detected. Over expression of Cbl-b significantly enhanced VP-16-induced cell apoptosis with inhibition of Akt activity, while a dominant negative (DN) RING Finger domain mutation completely abolished this enhancement. On the other hand, ERK activity was enhanced by Cbl-b, and the ERK inhibitor PD98059 reversed Cbl-b-enhanced apoptosis. The consistent results were also showed in the process of Ara-c treatment. These observations indicate that Cbl-b promotes RBL-2H3 apoptosis induced by VP-16 or Ara-c, probably through inhibition of Akt and activation of ERK.

Killing of rat basophilic leukemia cells by lethal toxin from Clostridium sordellii: critical role of phosphatidylinositide 3'-OH kinase/Akt signaling.

Clostridium sordellii lethal toxin (TcsL) belongs to the family of clostridial glucosylating toxins. TcsL exhibits glucosyltransferase activity to inactivate Rho and Ras proteins. On cultured cells, TcsL causes actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect"). This study is based on the concept that the cytotoxic effects of TcsL depend on the glucosylation of critical substrate proteins rather than on the glucosyltransferase activity per se. The cytotoxic effects of TcsL depend on the glucosyltransferase activity of TcsL, as neither chemically inactivated TcsL nor a glucosyltransferase-deficient mutant version of TcsL caused it. The TcsL homologous toxin B from Clostridium difficile serotype F strain 1470 (TcdBF) also failed to cause cytotoxic effects. Correlation of the toxins' respective protein substrate specificities highlighted (H/K/N)Ras as critical substrate proteins for the cytotoxic effects. (H/K/N)Ras are critical upstream regulators of phosphatidylinositide 3'-OH kinase (PI3K)/Akt survival signaling. Tauroursodeoxycholic acid (TUDCA) classified to activate PI3K/Akt signaling downstream of apoptosis-inducing stimuli prevented the cytotoxic effects of TcsL. In conclusion, (H/K/N)Ras glucosylation and subsequent inhibition of PI3K/Akt signaling are critical for the cytotoxic effects of TcsL.

Inhibitory effect of various Tunisian olive oils on chemical mediator release and cytokine production by basophilic cells.

Tunisian olive oils have been traditionally used as a medicinal food for chronic inflammation. To investigate the antiallergic effect of virgin olive oil samples from five principal olive varieties grown in various regions of Tunisia, we used the type I allergy reaction model using rat basophilic leukemia (RBL-2H3) cells and different dilutions of olive oil samples to determine beta-hexosaminidase release inhibition at two different response stages. Results showed that the Sayali olive oil significantly inhibited beta-hexosaminidase release by the IgE antibody-sensitized, BSA antigen-stimulated RBL-2H3 cells at the antibody-antigen binding stage. The result of our experiment shows that the anti-allergic effect of olive oil at this binding stage may be dependent on their flavone content. The Zarrazi olive oil significantly inhibited beta-hexosaminidase release at the antigen-receptor binding stage. Moreover, we investigated the effect of olive oil samples on histamine release and production of cytokines by activated human basophilic (KU812) cells. Different dilutions of Sayali olive oil dose-dependently inhibited the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4), and different dilutions of Zarrazi olive oil dose-dependently inhibited histamine release and IL-4 production by calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA)-stimulated KU812 cells.

Kinetics of the ceramide kinase inhibitor K1, a suppressor of mast-cell activation.

In a previous study, we synthesized a novel inhibitor of ceramide kinase, K1. In this study, we determined that inhibition by K1 is non-competitive and that four intact six-membered rings are important to the inhibitory activity. Furthermore, we identified an effective in vivo concentration for K1, at which it did not influence any cellular lipid synthesis other than that of ceramide 1-phosphate (C1P) using RBL-2H3 cells, and found that K1 suppressed the activation of mast cells.

Interaction of ligands for the peroxisome proliferator-activated receptor gamma with the endocannabinoid system.

BACKGROUND AND PURPOSE: There is good evidence that agents interacting with the endocannabinoid system in the body can also interact with the peroxisome proliferator-activated receptor gamma. The present study was designed to test whether the reverse is true, namely whether peroxisome proliferator-activated receptor gamma ligands have direct effects upon the activity of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase. EXPERIMENTAL APPROACH: Fatty acid amide hydrolase activity was measured in rat brain homogenates, C6 glioma and RBL2H3 basophilic leukaemia cells. Cellular uptake of anandamide was also assessed in these cells. KEY RESULTS: Peroxisome proliferator-activated receptor gamma activators inhibited the metabolism of the endocannabinoid anandamide in rat brain homogenates with an order of potency MCC-555 > indomethacin approximately ciglitazone approximately 15-deoxy-Delta(12,14)-prostaglandin J(2) approximately pioglitazone > rosiglitazone > troglitazone. The antagonists BADGE, GW9662 and T0070907 were poor inhibitors of anandamide hydrolysis. The inhibition by ciglitazone was competitive and increased as the pH of the assay buffer was decreased; the K(i) value at pH 6.0 was 17 microM. In intact C6 glioma cells assayed at pH 6.2, significant inhibition of anandamide hydrolysis was seen at 3 microM ciglitazone, whereas 100 microM was required to produce significant inhibition at pH 7.4. Ciglitazone also interacted with monoacylglycerol lipase as well as with cannabinoid CB(1) and CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: Ciglitazone may be useful as a template for the design of novel dual action anti-inflammatory agents which are both inhibitors of fatty acid amide hydrolase and agonists at the peroxisome proliferator-activated receptor gamma.

Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells.

Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca(2+) mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)(2) rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca(2+) mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the gamma-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.

N-cyclohexanecarbonylpentadecylamine: a selective inhibitor of the acid amidase hydrolysing N-acylethanolamines, as a tool to distinguish acid amidase from fatty acid amide hydrolase.

Anandamide ( N-arachidonoylethanolamine) and other bioactive N-acylethanolamines are degraded to their corresponding fatty acids and ethanolamine. This hydrolysis is mostly attributed to catalysis by FAAH (fatty acid amide hydrolase), which exhibits an alkaline pH optimum. In addition, we have identified another amidase which catalyses the same reaction exclusively at acidic pH values [Ueda, Yamanaka and Yamamoto (2001) J. Biol. Chem. 276, 35552-35557]. In attempts to find selective inhibitors of this acid amidase, we screened various derivatives of palmitic acid, 1-hexadecanol, and 1-pentadecylamine with N-palmitoylethanolamine as substrate. Here we show that N-cyclohexanecarbonylpentadecylamine inhibits the acid amidase from rat lung with an IC50 of 4.5 microM, without inhibiting FAAH at concentrations up to 100 microM. The inhibition was reversible and non-competitive. This compound also inhibited the acid amidase in intact alveolar macrophages. With the aid of this inhibitor, it was revealed that rat basophilic leukaemia cells possess the acid amidase as well as FAAH. Thus the inhibitor may be a useful tool to distinguish the acid amidase from FAAH in various tissues and cells and to elucidate the physiological role of the enzyme.

Induction of leukotriene C4 synthase after the differentiation of rat basophilic leukemia cells with retinoic acid and a low dose of actinomycin D and its suppression with methylprednisolone.

Leukotriene C(4) synthase (LTC(4) S) is a pivotal enzyme for generation of cysteinyl-leukotrienes (cysLTs). LTC(4) S activity in rat basophilic leukemia-1 (RBL-1) cells increased after culture in the presence of retinoic acid (RA) analogues, which was inhibited by cycloheximide or actinomycin D (ACD). Unexpectedly, the co-addition of a low dose of ACD with RA further potentiated the upregulation of the LTC(4) S activity. Daunorubicin and mitomycin C also had a similar effect. When stimulated with calcium ionophore A23187, control cells did not produce cysLTs, but RA-treated cells generated cysLTs and the co-addition of ACD further increased. While LTC(4) S mRNA and protein increased in the cells treated with RA, the co-addition of ACD further potentiated both in proportion to the LTC(4) S activity. The effect of ACD was considered to enhance the transcription rate of LTC(4) S gene, but not the mRNA-stability. The addition of methylprednisolone (MP) inhibited generation of cysLTs from the cells with A23187-stimulation and also did LTC(4) S activity, but did not inhibit 5-lipoxygenase (5-LOX). The suppression of LTC(4) S with MP showed a dependent manner on the time-point and duration of MP-treatment after RA-addition which was correlated with reduction in LTC(4) S mRNA and protein. The cells cultured with RA plus ACD contained more histamine, chymase activity, and granules in the cytoplasm than the control cells, suggesting differentiation to mature mast cells. In consideration of RA-differentiation therapy, it may be of pathophysiological relevance that the antineoplastic agents potentiate RA-induced, steroid-sensitive, induction of LTC(4) S in RBL-1 cells.

Co-localization of leukotriene a4 hydrolase with 5-lipoxygenase in nuclei of alveolar macrophages and rat basophilic leukemia cells but not neutrophils.

The synthesis of leukotriene B(4) from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A(4) hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A(4) hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A(4) hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A(4) hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A(4) hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situ and ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A(4) hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A(4) hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A(4) hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A(4) hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A(4) hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B(4).

Activation of store-operated Ca2+ entry in RBL cells without the contribution of protein kinases.

The role of protein kinases on store-operated Ca2+ entry in rat basophilic leukaemia cells (RBL) has been studied using the whole-cell configuration of the patch-clamp technique and Ca2+ imaging with fura-2. Specific inhibitors of tyrosine kinase (lavendustin A), mitogen-activated protein (MAP) kinase (SB 203580, PD 98059), Ca2+/calmodulin-dependent kinase (CaMK, KN-62, KN-93) and protein kinase C (PKC, bisindolylmaleimide I) had no significant effect on peak current amplitude and time constant of activation. Likewise, the broad spectrum kinase blockers H-7 and staurosporine did not alter Ca2+ entry compared to control recordings. Store-mediated Ca2+ entry was unaffected if intracellular ATP was substituted by either adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) or adenylyl-imidodiphosphate (AMP-PNP). Similarly, buffering intracellular Mg2+, an essential cofactor for protein kinases, had no effect on Ca2+ influx. These results indicate that protein phosphorylation by various kinases is not required for the activation of the store-operated Ca2+ current in RBL cells.

Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines.

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

Phospholipases stimulate secretion in RBL mast cells.

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.

Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells.

Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P(3) to activate the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca(2+) buffering. Dialysis with Ins(1,4,5)P(3) in weak Ca(2+) buffer did not activate any macroscopic I(CRAC) even after precautions had been taken to minimize the extent of Ca(2+) entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P(3) for >150 s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) pump blocker thapsigargin activated I(CRAC), and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P(3). This indicates that the Ins(1,4,5)P(3) receptors had not inactivated much over this timecourse. When external Ca(2+) was replaced by Ba(2+), Ins(1,4,5)P(3) still failed to generate any detectable I(CRAC) even though Ba(2+) permeates CRAC channels and is not taken up into the intracellular Ca(2+) stores. In strong Ca(2+) buffer, I(CRAC) could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P(3), stimulation of these receptors with carbachol did not activate I(CRAC) even after inhibition of PKC. The inability of Ins(1,4,5)P(3) to activate macroscopic I(CRAC) in weak Ca(2+) buffer was not altered by inhibition of Ca(2+)-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P(3) to activate I(CRAC) under conditions of weak intracellular Ca(2+) buffering is not due to strong inactivation of the Ins(1,4,5)P(3) receptors. Instead, a futile Ca(2+) cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca(2+) to ensure that the threshold for activation of macroscopic I(CRAC) has not been exceeded.

Role of phospholipase D-derived phosphatidic acid as a substrate for phospholipase A2 in RBL-2H3 cells.

The implications of phospholipase D (PLD) in cytosolic phospholipase A2 (cPLA2) activation were studied in a mast cell line, RBL-2H3, upon stimulation with antigen. Antigen-stimulated prostaglandin D2 generation was apparently suppressed by ethanol with a concomitant decrease in phosphatidic acid (PA) formation. The prostaglandin D2 generation was also inhibited almost completely by methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA2, but not by diacylglycerol lipase inhibitor. Furthermore, stimulation with antigen resulted in an increase in lysophosphatidic acid formation, which was suppressed by MAFP in parallel with an increase in PA formation. These results suggest that PA formed by the catalytic action of PLD is used as a substrate for cPLA2, thus PLD regulates cPLA2 activation in antigen-stimulated RBL-2H3 cells.

Magnolol inhibits leukotriene synthesis in rat basophilic leukemia-2H3 cells.

We have observed an inhibitory action of magnolol on the production of leukotriene (LT) C4 and LTB4, important lipid mediators in allergy and inflammation. IgE- and A23187-stimulated production of LTC4 and LTB4 was measured by radio-immunoassay (RIA) in the absence or presence of various concentrations of magnolol in intact rat basophilic leukemia (RBL)-2H3 cells. Magnolol dose-dependently inhibited synthesis of LTC4 and LTB4. Magnolol inhibited the IgE-mediated increase of intracellular calcium ion concentration, resulting in the inhibition of cytosolic phospholipase A2 (cPLA2) and possibly 5-lipoxygenase (5-LO), both calcium ion-dependent enzymes. In cell-free studies magnolol inhibited LTC4 synthase activity. LTA4 hydrolase activity was only inhibited at the higher concentration (2.5 x 10(-5)M). These results indicate that magnolol inhibits production of LTs by inhibiting PLA2, 5-LO, LTC4 synthase and LTA4 hydrolase which are essential for LT-synthesis. Magnolol may have anti-allergic effect by blocking LT-synthesis.