calcimycin and Leukemia--Basophilic--Acute

Mast cells play a central role in allergic and chronic inflammation. Extracts from Clausena lansium (Lour.) Skeels (Rutaceae) possess many pharmacological effects including anti-inflammatory, anti-oxidant, anti-cancer, and anti-trichomonal activities. In addition, the leaves and fruit are used in Chinese folk medicine. We have isolated and identified four known cinnamamides from this plant: lansiumamide C, lansamide I, lansiumamide B, and SB-204900. However, the biological activities of these compounds are not yet understood. The purpose of this paper is to clarify the pharmacological effects of these compounds on mast cells.. We measured inflammatory molecules in A23187-stimulated rat basophilic leukemia cells (RBL-2H3) treated with these compounds using HPLC, ELISA, and immunoblotting methods. In addition, some signaling molecules were investigated by immunoblotting.. Lansamide I, lansiumamide B, and SB-204900 significantly decreased histamine release. Furthermore, lansiumamide B- and SB-204900-treated cells also reduced the protein and/or mRNA levels of TNF-α. SB-204900 markedly suppressed the phosphorylation of p38 MAPK.. Our findings suggest that lansiumamide B and SB-204900 attenuate mast-cell-induced inflammation.

Topics: Animals; Calcimycin; Calcium Ionophores; Cell Line; Cell Survival; Cinnamates; Clausena; Cyclooxygenase 2; Disease Models, Animal; Histamine; Interleukin-6; Leukemia, Basophilic, Acute; Mast Cells; p38 Mitogen-Activated Protein Kinases; Plant Extracts; Plant Leaves; Rats; Tumor Necrosis Factor-alpha

Type I allergy is characterized by the release of granule-associated mediators, lipid-derived substances, cytokines, and chemokines by activated mast cells. To evaluate the anti-allergic effects of macelignan isolated from Myristica fragrans Houtt., we determined its ability to inhibit calcium (Ca(2+)) influx, degranulation, and inflammatory mediator production in RBL-2 H3 cells stimulated with A23187 and phorbol 12-myristate 13-acetate. Macelignan inhibited Ca(2+) influx and the secretion of β-hexosaminidase, histamine, prostaglandin E(2), and leukotriene C(4); decreased mRNA levels of cyclooxygenase-2, 5-lipoxygenase, interleukin-4 (IL-4), IL-13, and tumor necrosis factor-α; and attenuated phosphorylation of Akt and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. These results indicate the potential of macelignan as a type I allergy treatment.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; beta-N-Acetylhexosaminidases; Calcimycin; Calcium; Cell Degranulation; Cell Line, Tumor; Cyclooxygenase 2; Dinoprostone; Extracellular Signal-Regulated MAP Kinases; Histamine Release; Hypersensitivity; Inflammation Mediators; Interleukin-13; Interleukin-4; JNK Mitogen-Activated Protein Kinases; Leukemia, Basophilic, Acute; Leukotriene C4; Lignans; Mast Cells; Myristica; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Proto-Oncogene Proteins c-akt; Rats; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Immediate-type hypersensitivity is characterized by elevated levels of immunoglobulin E (IgE) and activated mast cell plays a crucial role by releasing granule contents, lipid-derived mediators, cytokines, and chemokines. To evaluate the antiallergic effects of panduratin A isolated from Boesenbergia pandurata Roxb., we determined its effects on calcium (Ca(2+)) influx, degranulation, and inflammatory mediators in calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA)-stimulated rat basophilic leukemia (RBL-2H3) cells. Panduratin A (20 μM) inhibited secretion of β-hexosaminidase (46.69 ± 9.6 %), histamine (34.32 ± 2.1 %), and Ca(2+) influx (43.84 %). Panduratin A reduced the production of prostaglandin E(2) (PGE(2), 47.58 ± 3.4 %), leukotriene B(4) (LTB(4), 98.15 ± 1.6 %), and the mRNA expression of cyclooxygenase-2, 5-lipoxygenase, interleukin (IL)-4, IL-13, and tumor necrosis factor-α. Furthermore, panduarin A attenuated phosphorylation of Akt, the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. These results indicate that panduratin A might be useful as an agent against immediate-type hypersensitivity.

Topics: Animals; Arachidonate 5-Lipoxygenase; beta-N-Acetylhexosaminidases; Calcimycin; Calcium; Cell Degranulation; Cell Line, Tumor; Chalcones; Cyclooxygenase 2; Extracellular Signal-Regulated MAP Kinases; Histamine; Histamine Release; Hypersensitivity, Immediate; Immunoglobulin E; Inflammation Mediators; Interleukin-13; Interleukin-4; JNK Mitogen-Activated Protein Kinases; Leukemia, Basophilic, Acute; Leukotriene B4; Mast Cells; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Prostaglandins E; Proto-Oncogene Proteins c-akt; Rats; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Zingiberaceae

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.

Topics: Animals; Anti-Allergic Agents; Antigen-Antibody Complex; Antigens; Basophils; Benzopyrans; beta-N-Acetylhexosaminidases; Calcimycin; Calcium; Cell Line, Tumor; Dose-Response Relationship, Drug; Fluorescent Dyes; Formazans; Histamine Release; Humans; Immunoglobulin E; Ionophores; Leukemia, Basophilic, Acute; Leukemia, Experimental; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nuclear Magnetic Resonance, Biomolecular; p-Methoxy-N-methylphenethylamine; Plant Extracts; Rats; Spectroscopy, Fourier Transform Infrared; Sphagnopsida; Tetradecanoylphorbol Acetate; Tetrazolium Salts

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.

Topics: Animals; Calcimycin; Carbon Radioisotopes; Cell Line, Tumor; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Ionophores; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Rats

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca(2+) (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca(2+) as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca(2+)-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca(2+). The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM.CERK complexes. An in vitro CaM binding assay also demonstrated Ca(2+)-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca(2+) sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca(2+)-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca(2+)-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells.

Topics: Amino Acid Sequence; Animals; Binding Sites; Calcimycin; Calcium; Calcium Chloride; Calmodulin; Cell Degranulation; Cell Line, Tumor; Ceramides; CHO Cells; Cricetinae; Cricetulus; Egtazic Acid; Enzyme Activation; Gene Deletion; Immunosorbent Techniques; Leukemia, Basophilic, Acute; Mast Cells; Mice; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Phosphotransferases (Alcohol Group Acceptor); Point Mutation; Rats; Sulfonamides; Transfection

Proteinase 3 (PR3) and human neutrophil elastase (HNE) are serine proteinases stored in the azurophilic granules of neutrophils. In contrast to HNE, PR3 is the target of antineutrophil cytoplasm antibodies (ANCA) in Wegener's granulomatosis. The mechanisms leading to the membrane expression of PR3 and HNE are still unclear and appear to be critical to understand the pathophysiological role of ANCA. Stably transfected rat basophilic cell lines (RBL) with PR3 or HNE were used to analyze the PR3 and HNE secretion mechanisms and differentiate between them. RBL cells were lacking endogenous PR3 and HNE. They were stably transfected with HNE or PR3 or an inactive mutant of PR3 (PR3S203A). Using the calcium ionophore A23187 as a secretagogue, higher serine proteinase activity was secreted in the supernatant of RBL/HNE than in RBL/PR3. It is interesting that PR3 and PR3/S203A were also expressed at the plasma membrane, thus demonstrating that serine protease activity was not required for plasma membrane expression. In contrast, no expression of plasma membrane HNE could be detected in RBL/HNE. Apoptosis induced by etoposide was evaluated by DNA fragmentation, the presence of cytoplasmic histone-associated DNA fragments, and annexin V labeling. No membrane HNE was detected in RBL/HNE. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 and PR3S203A increased with etoposide concentrations and appeared closely related to annexin V labeling. Our data suggest that membrane PR3 originates from two distinct pools, the granular pool mobilized following degranulation or a plasma membrane pool mobilized upon apoptosis.

Topics: Animals; Annexin A5; Antibodies, Antineutrophil Cytoplasmic; Apoptosis; Calcimycin; Calcium; Cell Membrane; Cytoplasmic Granules; DNA Fragmentation; Etoposide; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Leukemia, Basophilic, Acute; Leukocyte Elastase; Myeloblastin; Rats; Recombinant Proteins; Serine Endopeptidases; Transfection; Tumor Cells, Cultured

Antigen-induced aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells induces degranulation to release chemical mediators, leading to acute allergic inflammation. We have demonstrated that the treatment of rat mast cells, RBL-2H3, with a phenoxazine derivative Phx-1 (2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one) suppresses the antigen-induced degranulation. Biochemical analysis reveals that the complementary signaling pathway through Gab2 and Akt is inhibited by this compound in mast cells. These findings suggest that phenoxazine derivatives may have a therapeutic potential for allergic diseases by inhibiting mast cell degranulation.

Topics: Adaptor Proteins, Signal Transducing; Animals; beta-N-Acetylhexosaminidases; Calcimycin; Cell Degranulation; Cell Line; Dose-Response Relationship, Drug; Forecasting; Immunoglobulin E; Leukemia, Basophilic, Acute; Mast Cells; Oxazines; Phosphoproteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Receptors, IgE; Signal Transduction

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.

Topics: Animals; Calcimycin; Cell Differentiation; Cell Line; Cell Size; Chymases; Cycloheximide; Dactinomycin; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutathione Transferase; Histamine; Interleukin-4; Leukemia, Basophilic, Acute; Lipoxygenase; Methylprednisolone; Rats; RNA Stability; RNA, Messenger; Serine Endopeptidases; Solubility; Time Factors; Transcription, Genetic; Tretinoin

Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke.. HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments.. We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation.. Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.

Topics: Animals; beta-N-Acetylhexosaminidases; Calcimycin; Cell Degranulation; Cell Division; Flow Cytometry; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hot Temperature; HSP70 Heat-Shock Proteins; Hypersensitivity; Immunohistochemistry; Ionophores; Leukemia, Basophilic, Acute; Nicotiana; Oxidative Stress; Rats; Tumor Cells, Cultured

The synthesis of platelet-activating factor (PAF) by -stimulated RBL-2H3 cells was significantly suppressed by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx). When the cells overexpressing PHGPx (L9 cells) were pretreated with diethyl maleate, which reduces PHGPx activity, PAF synthesis upon stimulation rose to levels seen in mock-transfected cells (S1 cells). Hydroperoxide levels, which are reduced in L9 cells, are involved in regulating PAF synthesis, because the addition of hydroperoxyeicosatetraenoic acid increased PAF production in -stimulated L9 cells to control cell levels. The activity of acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, which is involved in the last step of PAF synthesis, is also reduced in L9 cells. p38 kinase inhibitors block acetyltransferase activity in normal -stimulated cells, suggesting that p38 kinase is involved in regulating acetyltransferase activity. Recombinant active p38 kinase activates acetyltransferase, whereas alkaline phosphatase reverses this, suggesting p38 kinase directly phosphorylates acetyltransferase. p38 kinase phosphorylation is blocked in L9 cells, indicating that high hydroperoxide levels are needed for the activation of p38 kinase. Thus, intracellular hydroperoxide levels participate in regulating p38 kinase phosphorylation, which in turn controls the activation of acetyltransferase and thus the synthesis of PAF. These observations suggest that PHGPx is an important component of the mechanisms regulating inflammation.

Topics: Acetyl Coenzyme A; Acetyltransferases; Alkaline Phosphatase; Animals; Calcimycin; Glutathione Peroxidase; Hydrogen Peroxide; Kinetics; L Cells; Leukemia, Basophilic, Acute; Mice; Mitochondria; Mitogen-Activated Protein Kinases; Phospholipid Hydroperoxide Glutathione Peroxidase; Phosphorylation; Platelet Activating Factor; Rats; Reactive Oxygen Species; Recombinant Proteins; Transfection; Tumor Cells, Cultured

A simple and sensitive method, applicable to quantification of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) produced in cells has been developed using high-performance liquid chromatography on a silica gel column with chemiluminescence detection. 5-HPETE was clearly separated from other positional isomers of HPETEs and hydroxyeicosatetraenoic acids with hexane-isopropanol-acetic acid (97:3:0.01, v/v) as the mobile phase. The lower limit of detection was about 100 pg. 5-HPETE produced in 10(7) cells of RBL-2H3 cells stimulated with A23187 was determined as 480+/-30 pg. In the present study, 5-HPETE, which occurs naturally, was detected and quantitated for the first time in intact cells.

Topics: Animals; Calcimycin; Calibration; Chromatography, High Pressure Liquid; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotrienes; Luminescent Measurements; Malates; Rats; Tumor Cells, Cultured

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 5-Lipoxygenase; Biomarkers; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Micellar Electrokinetic Capillary; Culture Media, Conditioned; Electrophoresis, Capillary; Enzyme Activation; Evaluation Studies as Topic; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotriene B4; Lipoxygenase Inhibitors; Prostaglandins B; Quercetin; Quinolones; Rats; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

Leukotriene (LT) synthesis is initiated by the enzyme 5-lipoxygenase (5-LO). Prolonged cell stimulation causes the translocation of 5-LO to the nuclear envelope and the synthesis of LT, with subsequent inactivation and persistent membrane association of 5-LO. In this study, we examined whether persistent membrane association of 5-LO, as well as the inactivation of 5-LO, could be prevented by shortening the length of cell stimulation or by blocking LT synthesis. As expected, stimulation of rat basophilic leukaemia (RBL) cells, a mast cell model, or alveolar macrophages (AMs) with calcium ionophore for 15 min caused 5-LO translocation, LT generation and the inactivation and persistent membrane association of 5-LO. When RBL cells or AMs instead were stimulated for 0.5-5 min, translocation of 5-LO and synthesis of LT still occurred. However, after washing and resting, the 5-LO enzyme returned to its original intracellular distribution. Furthermore these cells showed a retained capacity for LT synthesis on subsequent re-stimulation. Similar results were obtained when cells were stimulated with either formyl peptide or zymosan, instead of ionophore. In contrast, blockade of LT synthesis during the initial stimulation, with the selective inhibitors zileuton or MK-886, did not inhibit 5-LO translocation, inactivation or persistent membrane association resulting from prolonged cell stimulation. We conclude that, in long-lived immune cells, 5-LO translocation is reversible when cell stimulation is short, but persistent after prolonged stimulation. In addition 5-LO remains active and LT synthetic capacity is retained after transient stimulation, whereas significant inactivation of 5-LO occurs after prolonged stimulation. Finally, results with LT synthesis inhibitors indicate that inactivation and persistent membrane association of 5-LO can result independently of 5-LO activation.

Topics: Animals; Arachidonate 5-Lipoxygenase; Biological Transport; Calcimycin; Cell Membrane; Enzyme Activation; Female; Hydroxyurea; Interphase; Leukemia, Basophilic, Acute; Leukotrienes; Lipoxygenase Inhibitors; Macrophages, Alveolar; Mast Cells; Rats; Rats, Wistar; Tumor Cells, Cultured

When RBL-2H3 rat basophilic leukemia cells were stimulated by antigen or the Ca2+ ionophore A23187, the activity to increase histamine production by rat bone marrow cells in the conditioned medium increased time-dependently. To characterize the histamine-production-increasing factor (HPIF) produced by RBL-2H3 cells, the conditioned medium was collected 8 h after stimulation by A23187, and the factor was purified by three-step chromatography, the specific activity being increased by 9000-fold. The partial amino acid sequence of the peptide obtained by S. aureus V8 protease digestion was identical to the internal amino acid sequence of rat granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, GM-CSF mRNA levels in RBL-2H3 cells were increased by A23187 with a peak at 4 h. Furthermore, recombinant rat GM-CSF increased histamine production by rat bone marrow cells. These findings suggested that HPIF produced by the stimulated RBL-2H3 cells is GM-CSF. Possible significant roles of HPIF at the late phase of allergic inflammation are discussed.

Topics: Amino Acid Sequence; Animals; Antigens; Bone Marrow Cells; Calcimycin; Culture Media, Conditioned; Dinitrophenols; Granulocyte-Macrophage Colony-Stimulating Factor; Histamine; Ionophores; Leukemia, Basophilic, Acute; Male; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Serum Albumin, Bovine; Tumor Cells, Cultured

The mast cell lines rat basophilic leukemia (RBL) and mouse C57 cells respond to IgE/antigen complexes by degranulation. Treatment of these cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), (10-100 nM) for 24-48 h enhanced IgE/antigen-induced exocytosis as monitored by release of hexosaminidase. A short term incubation with the hormone did not affect exocytosis, ruling out a rapid non genomic mechanism. The presence of vitamin D receptors, demonstrated by immunoblotting and the lack of effect of 24,25(OH)2D3 suggest a role for these receptors in the enhancing effect. 1,25(OH)2D3 also enhanced exocytosis induced by the calcium ionophore A23187 in the presence or absence of phorbol ester indicating modulation of events distal to signal transduction. 1,25(OH)2D3 enhanced exocytosis in the presence of cytochalasin D, indicating that the action of the hormone is not due to effects on microfilament structure. The results of this study suggest that 1,25(OH)2D3 may affect the allergic or pro-inflammatory potential of mast cells.

Topics: Animals; Antigens; beta-N-Acetylhexosaminidases; Calcimycin; Calcitriol; Cell Degranulation; Cytochalasin D; Dinitrophenols; Exocytosis; Immunoglobulin E; Leukemia, Basophilic, Acute; Mast Cells; Mice; Nucleic Acid Synthesis Inhibitors; Rats; Receptors, Calcitriol; Tumor Cells, Cultured

Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calcium-Binding Proteins; Cells, Cultured; Exocytosis; Leukemia, Basophilic, Acute; Mast Cells; Membrane Glycoproteins; Mice; Nerve Tissue Proteins; Rats; Subcellular Fractions; Synaptotagmin I; Synaptotagmins; Transfection; Tumor Cells, Cultured

We studied the effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells. Among tea polyphenols, (-)- epigallocatechin gallate (EGCG) most strongly and dose-dependently inhibited histamine release from cells stimulated with a calcium ionophore, A23187. (-)-Epigallocatechin (EGC) and (-)-epicatechin gallate (ECG) with a triphenol residue moderately inhibited histamine release, whereas diphenolic (+)-catechin (C) and (-)-epicatechin (EC) did not. The magnitude of the inhibitory effect was in the order EGCG > ECG > EGC. Among simple polyphenols, the triphenol compounds, pyrogallol (PG) and gallic acid (GA) exerted inhibitory activity, but the diphenols, pyrocatechol, hydroquinone, and resorcinol did not. In addition, the mixture of PG and GA inhibited histamine release as strongly as EGCG with two triphenol residues. Similarly, they inhibited histamine release induced by IgE-antigen complex stimulation more efficiently than that induced by A23187 stimulation. EGCG did not inhibit the increase of intracellular Ca2+ in RBL-2H3 cells stimulated with A23187 or IgE antigen. These results indicate that the triphenol structure plays an important role in the inhibitory activity of tea polyphenols. Their activity seemed to be exerted through the metabolic events occurring after the elevation of intracellular Ca2+ concentration.

Topics: Animals; Calcimycin; Calcium; Flavonoids; Histamine Antagonists; Histamine Release; Immunoglobulin E; Intracellular Fluid; Leukemia, Basophilic, Acute; Phenols; Polymers; Polyphenols; Rats; Structure-Activity Relationship; Tea; Tumor Cells, Cultured

We examined the action of Shinpi-To (Formula divinita; TJ-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC4 and LTB4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C4 and LTB4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A2 (PLA2), 5-lipoxygenase (5-LO). LTC4 synthase, and LTA4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA2 activity. It also suppressed production of LTC4 and LTB4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC4 and LTB4 when LTA4-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca2+ ([Ca2+]i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA2 and 5-LO activities without affecting the mobilization of [Ca2+]i.

Topics: Analysis of Variance; Animals; Arachidonic Acid; Asthma; Bronchodilator Agents; Calcimycin; Calcium; Cell Membrane; Drugs, Chinese Herbal; Ephedrine; Immunoglobulin E; Ionophores; Isotope Labeling; Leukemia, Basophilic, Acute; Leukotriene A4; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Phospholipases A; Phospholipases A2; Radioimmunoassay; Rats; Tritium; Tumor Cells, Cultured

The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo.. In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively.. 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA.. Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models.. Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Chemotactic Factors; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Immunoenzyme Techniques; Indoles; Ionophores; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotriene E4; Lipoxygenase Inhibitors; Neutrophil Activation; Neutrophils; Oxindoles; Rabbits; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Thromboxane B2; Zymosan

Rat basophilic leukemia cells have previously been described to undergo striking cell surface changes after IgE-mediated stimulation of histamine secretion, whereby the dorsal surface loses its microvilli and acquires characteristic wavy ruffles. We have found using scanning electron microscopy, phase contrast and immunofluorescence, that a proportion of these cells also exhibit the formation of circular membrane ruffles on their dorsal surface after exposure to an IgE-directed secretagogue; some cells also develop circular membrane ruffles following stimulation by phorbol myristate acetate or by calcium ionophore A23187. A flattened morphology appears to be linked to circular membrane ruffle formation in that these ruffles were found in areas of presumed cell spreading which are largely devoid of intermediate filaments and displaced to one side of the cell's nucleus, and they were not observed on rounded cells. This is in contrast to the wavy ruffles which are found on the entire cell surface including the region overlying the nucleus, and which are observed in rounded cells as well as spread cells. Circular ruffle formation and secretion are triggered by similar concentrations of antigen, but the circular ruffles are formed more slowly and only become abundant at times after most of the histamine has been released. The circular membrane ruffles showed no obvious association with endocytosis, as detected using fluorescein isothiocyanate-dextran as a fluid phase marker. The position of accumulation of endocytotic vesicles occurring subsequent to secretion was not found to be related to the circular membrane ruffles, but was observed around the nucleus. Circular membrane ruffles contain F-actin, and their formation is prevented by cytochalasin D. At least three types of myosin, types I, II and V are present and presumably play a role in circular ruffle formation.

Topics: Actin Cytoskeleton; Actins; Animals; Antigens; Calcimycin; Cell Membrane; Intermediate Filaments; Leukemia, Basophilic, Acute; Mast Cells; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microtubules; Myosins; Rats; Receptors, IgE; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Ceramide, a product of sphingomyelin hydrolysis by sphingomyelinase, elicits various cellular functions and has recently been regarded as a second messenger. To investigate the role of ceramide in rat basophilic leukemia (RBL-2H3) cells, the effects of a cell-permeable analogue, N-acetylsphingosine (C2-ceramide), on Ag-mediated cellular responses were examined. C2-Ceramide inhibited Ag- or PMA-induced activation of phospholipase D (PLD), whereas Ca2+ ionophore A23187-induced PLD activation was not affected. C2-Ceramide failed to inhibit PLD activity in two different in vitro assay systems. Since PLD activity is known to be regulated by several factors, the effects of C2-ceramide on these factors were examined. We have previously reported the possible involvement of protein tyrosine kinase in Ag-mediated PLD activation. However, C2-ceramide had no effect on Ag-induced protein tyrosine phosphorylation, including mitogen-activated protein (MAP) kinases. In fura-2-loaded RBL-2H3 cells, C2-ceramide suppressed Ag-induced Ca2+ influx, leaving initial Ca2+ increase and inositol phosphate production unaffected. Western blot analysis revealed that Ag caused translocation of protein kinase C (PKC) alpha, beta 1, beta 2, delta and epsilon isozymes from cytosol to membrane fraction. Translocation of alpha, beta 1, and beta 2 isozymes was specifically prevented by C2-ceramide. Moreover, C2-ceramide suppressed Ag-induced serotonin release. In the absence of extracellular Ca2+, Ag-induced PLD activation and release reaction were greatly reduced. The inhibitory profile was nearly the same as that obtained in C2-ceramide-treated cells. These results suggest that C2-ceramide inhibits Ag-induced PLD activation and serotonin release, probably through the blockage of Ca2+ influx and translocation of Ca(2+)-dependent PKC isozymes in RBL-2H3 cells.

Topics: Animals; Antigens; Biological Transport; Calcimycin; Calcium; Enzyme Activation; Enzyme Induction; Extracellular Space; Hydrolysis; Immunoglobulin E; Leukemia, Basophilic, Acute; Phosphatidylinositols; Phospholipase D; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Type C Phospholipases

Azelastine, oxatomide, and ketotifen are used for patients with allergic diseases. These drugs inhibit the release of chemical mediators including the leukotrienes; however, the mechanism involved is unclear.. To clarify the mechanism of inhibition, we investigated the effects of three drugs on the function of phospholipase A2, 5-lipoxygenase, leukotriene C4 synthase, and leukotriene A4 hydrolase, which are all catabolic enzymes involved in synthesizing leukotriene C4 and leukotriene B4 in rat basophilic leukemia (RBL)-1 cells.. The production of leukotriene C4 and leukotriene B4 was measured by high performance liquid chromatography (HPLC). All three drugs inhibited the production of leukotriene C4 and leukotriene B4 when cells were stimulated with A23187. All three drugs also inhibited the A23187-stimulated release of 3H-arachidonic acid from membrane phospholipids. Azelastine inhibited the production of leukotriene C4, but not leukotriene B4, when either arachidonic acid or leukotriene A4 free acid was used as the substrate in our cell free system. Oxatomide and ketotifen did not inhibit the synthesis of either leukotriene C4 or leukotriene B4 in the same cell free study.. Results indicated that oxatomide and ketotifen inhibit the production of leukotriene C4 and leukotriene B4 by inhibiting phospholipase A2 activity, whereas, azelastine inhibits the leukotriene C4 production by inhibiting phospholipase A2 and leukotriene C4 synthase.

Topics: Animals; Anti-Allergic Agents; Calcimycin; Epoxide Hydrolases; Glutathione Transferase; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Phospholipases A; Phospholipases A2; Phthalazines; Rats; Tumor Cells, Cultured

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

Topics: 2,4-Dinitrophenol; Adenosine Diphosphate Ribose; Androstadienes; Animals; Bacterial Proteins; Bacterial Toxins; Calcimycin; Carbachol; Cattle; Cell Line; Clostridioides difficile; Cytoplasmic Granules; Dinitrophenols; Enzyme Inhibitors; Glucosyltransferases; Intercellular Signaling Peptides and Proteins; Kinetics; Leukemia, Basophilic, Acute; Peptides; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, IgE; Serotonin; Serum Albumin, Bovine; Tritium; Tumor Cells, Cultured; Wasp Venoms; Wortmannin

Exocytosis of secretory granules, including histamine, in rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen or Ca(2+)-ionophore (A23187), was observed under a video-enhanced light microscope. Exocytotic events of individual granules including fusion, extrusion, and membrane retrieval were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in its time courses. The earlier one of the peaks was inhibited by wortmannin ( > 100 nM), as an inhibitor of myosin light chain kinase (MLCK), and the other was not. Exocytosis by antigen-stimulation, however, showed only one peak, which was inhibited by low concentration of wortmannin ( < 50 nM) as an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). These results indicate that quantitative analysis of exocytosis visualized by video-enhanced light microscope reveals two different pathways, through P13-kinase and MLCK, of inhibitory effects on exocytosis by wortmannin in RBL-2H3 cells.

Topics: Androstadienes; Animals; Antigens; Calcimycin; Cell Line; Cytoplasmic Granules; Dinitrophenols; Enzyme Inhibitors; Exocytosis; Kinetics; Leukemia, Basophilic, Acute; Membrane Fusion; Microscopy, Video; Myosin-Light-Chain Kinase; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Rats; Serum Albumin, Bovine; Time Factors; Tumor Cells, Cultured; Wortmannin

Effects of fatty acids on accumulation and secretion of histamine in rat basophilic leukemia RBL-2H3 cells and leukotriene release from peritoneal exudate cells isolated from Wistar rats were examined in relation to the manifestation of type I allergic reactions. When RBL-2H3 cells were cultured for 24 h in the presence of 1 mM short chain fatty acids, a marked increase in histamine accumulation was observed, especially with butyric acid. In addition, Ca-ionophore A23187-stimulated histamine release was enhanced in the cells treated with 0.1 mM mono to hexa unsaturated fatty acids with 18 to 22 carbon-chains. On the other hand, LTB4 release from rat peritoneal exudate cells was inhibited in the presence of polyunsaturated fatty acids, both n-6 and n-3, having more than 3 double bonds. Inhibitory activity was enhanced by an increase in the number of double bonds, and docosahexaenoic acid (DHA) exerted the highest activity with complete inhibition at 0.1 mM and 35.5% inhibition even at 10 microM. A hydrophobic radical scavenger (9,10-diphenylanthracene) and two antioxidants (butyrated hydroxytoluene and alpha-tocopherol) inhibited the production of LTB4, but hydrophilic counterparts (mannitol and ascorbic acid) did not. These results suggest that lipophilic anti-oxidative agents, as well as PUFA, inhibit the production of LTB4.

Topics: Animals; Antioxidants; Ascitic Fluid; Calcimycin; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids; Fatty Acids, Unsaturated; Free Radical Scavengers; Histamine; Histamine Release; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotrienes; Rats; Rats, Wistar; Tumor Cells, Cultured

Rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca2+-dependent secretion of granules when stimulated with antigen or the Ca2+-ionophore A23187, were observed under a video-enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31-8425, Ro31-8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL-2H3 cells.

Topics: Animals; Antigens; Calcimycin; Dinitrophenols; Enzyme Inhibitors; Exocytosis; Histamine; Image Enhancement; Indoles; Leukemia, Basophilic, Acute; Maleimides; Microscopy, Fluorescence; Microscopy, Video; Naphthalenes; Phospholipase D; Protein Kinase C; Rats; Serotonin; Serum Albumin, Bovine; Time Factors; Tumor Cells, Cultured

The cDNA encoding the rat equivalent of the human hematopoietic tyrosine phosphatase, also known as leukocyte phosphatase, was isolated from a rat basophilic leukemia mast cell cDNA library. By two-dimensional electrophoresis, the protein expressed in the mast cells was of a size (40 kDa) and pI (6.9) predicted from the deduced amino acid sequence. Thus, although previously shown to be preferentially expressed in T cells and B cells, the phosphatase is also found in mast cells. By immunofluorescence microscopy, rat hematopoietic tyrosine phosphatase localized to discrete, globular compartments within the cytoplasm and was not found either in the nucleus or associated with the cell surface membrane. Aggregation of high affinity IgE receptors in the mast cells induced tyrosine phosphorylation of the phosphatase. The tyrosine phosphorylation was mimicked by stimulation with calcium ionophore A23187 but not by direct activation of protein kinase C. Since phosphorylation of the phosphatase was dramatically reduced when the cells were activated in Ca(2+)-free media, it is dependent on a rise in intracellular Ca2+. These data strongly suggest that hematopoietic tyrosine phosphatase may be involved in the IgE receptor-mediated signaling cascade.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Brain; Calcimycin; Calcium; Cell Line; Cloning, Molecular; Cytosol; DNA Primers; Gene Expression; Immunoblotting; Intracellular Signaling Peptides and Proteins; Leukemia, Basophilic, Acute; Mice; Molecular Sequence Data; Organ Specificity; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein Tyrosine Phosphatases, Non-Receptor; Rats; Receptors, IgE; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine

Exposure to ozone has been reported to cause increased immediate bronchial reactivity to inhaled allergen in asthmatics. The purpose of these studies was to determine whether ozone induces either spontaneous physiological degranulation or enhanced immunoglobulin E (IgE)-mediated degranulation of mast cells, thus accounting for the in vivo effects noted in asthmatics. A rat mast cell line (RBL-2H3) was exposed to different levels of ozone (0.1, 0.3, 0.5, and 1.0 ppm), covered by different amounts of buffer, and both cytotoxic and nontoxic exposure conditions were determined. In addition to cytotoxicity, spontaneous release of granule products and prostaglandin D2 (PGD2) associated with ozone exposure were assessed. RBL-2H3 cells were also exposed to ozone under noncytotoxic conditions followed by stimulation with alpha-IgE to cross-link membrane-bound IgE and A23187 so that the effect of ozone on stimulated degranulation could be examined. Only exposure conditions associated with cytotoxicity were associated with spontaneous release of mast cell serotonin, indicating no physiologic degranulation due to ozone exposure. Data presented herein also demonstrate that ozone substantially inhibited both IgE- and A23187-induced degranulation. Neither catalase nor superoxide dismutase protected cells from the inhibitory effect of ozone, indicating that ozone does not act through generation of H2O2 or superoxide. Additionally, ozone caused a modest increase in spontaneous PGD2 generation only under cytotoxic conditions. Thus ozone appears to inhibit mast cell degranulation after IgE- or A23187-mediated stimulation and causes direct release of mast cell granule products and PGD2 only under conditions associated with membrane cytotoxicity.

Topics: Animals; Asthma; Calcimycin; Catalase; Cell Line; Cell Survival; Cytoplasmic Granules; Dose-Response Relationship, Drug; Humans; Immunoglobulin E; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Ozone; Prostaglandin D2; Rats; Superoxide Dismutase; Tumor Cells, Cultured

The effects of cyclosporin A (CSA) on arachidonic acid (AA) metabolism were investigated in intact rat basophilic leukemia-1 (RBL-1) cells and cell lysates. Calcium ionophore (A23187)-stimulated synthesis of cysteinyl leukotrienes (LTC4, LTD4, and LTE4), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) in intact cells in the absence or presence of CSA was measured by reversed-phase high-performance liquid chromatography (HPLC). CSA inhibited the production of cysteinyl LTs, LTB4, and 5-HETE in intact cells in a dose-dependent manner. The synthesis of cysteinyl LTs, LTB4, and 5-HETE was also measured after the incubation of cell lysates with free AA in the absence or presence of CSA. CSA did not inhibit synthesis of cysteinyl LTs, but rather stimulated production of LTB4 and 5-HETE in cell lysate. A23187-stimulated release of incorporated [3H]AA from intact cells was not inhibited by CSA. CSA did not inhibit the synthesis of cysteinyl LTs and LTB4 when cells incubated with LTA4 as the substrate. These results indicate that the inhibitory effects of CSA on the synthesis of LTs and 5-HETE in intact cells are attributable to a modulatory action on a step in the series of intracellular events that includes the activation of 5-lipoxygenase, which are initiated by Ca2+ influx and end in the release of metabolites from the cell membrane, rather than to a direct inhibitory action on enzymes in the LT biosynthetic pathway.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Chromatography, High Pressure Liquid; Cyclosporine; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotrienes; Phospholipases A; Rats; Tumor Cells, Cultured

To determine the regulatory mechanism of Leukotriene (LT) B4 synthesis by cytokines, we investigated the regulation of LTB4 generation by short-term (30 min) priming and long-term (15 h) enzyme-inducing actions of the four cytokines interleukin (IL)-3, IL-5, tumor necrosis factor alpha (TNF-alpha), and transforming growth factor alpha (TGF-alpha) in rat basophilic leukemia-1 (RBL-1) cells. Pretreatment of cells with IL-3 or IL-5 for 30 min increased A23187- (5x10(-9)M) stimulated synthesis of LTB4 by three to four times over control levels. However, IL-3 or IL-5 lacked this effect when stimulated with exogenous arachidonic acid A at 10(-4)M. TNF-alpha and TGF-alpha had no priming effect on LTB4 synthesis following stimulation with either A23187 (5x10(-9)M) or AA(10(-4)M). Stimulation with the calcium ionophore (A23187)(10(-5)M) or AA(10(-4)M) following 15-h exposure to these cytokines had no effect. These results suggest that IL-3 and IL-5 increase the production of LTB4 by priming the activity of phospholipase A2(PLA2) without inducing enzymes in the arachidonate 5-lipoxygenase pathway. Such a priming effect may be important in regulating the development of allergic and other diseases involving the inflammatory reaction.

Topics: Animals; Arachidonic Acid; Calcimycin; Calcium; Dose-Response Relationship, Drug; Interleukin-3; Interleukin-5; Leukemia, Basophilic, Acute; Leukotriene B4; Rats; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Leukotriene (LT) synthesis involves the translocation of enzymatically active 5-lipoxygenase (5-LO) from a soluble site to a bound site, where it interacts with 5-lipoxygenase-activating protein (FLAP). In human polymorphonuclear leukocytes (PMNs), 5-LO moves from the cytosol to the nuclear envelope (NE) to interact with FLAP. However, 5-LO has recently been found within the nucleus, as well as the cytosol, of rat basophilic leukemia (RBL) cells and alveolar macrophages (AMs). To assess whether nuclear 5-LO can contribute to LT synthesis in these cells, we investigated whether this enzyme pool 1) translocates upon cell activation, 2) colocalizes with FLAP, and 3) is enzymatically active. By cell fractionation followed by immunoblotting, both cytosolic and nuclear soluble 5-LO decreased dramatically in RBL cells following activation with the calcium ionophore A23187. Concurrently, 5-LO increased in the pelletable nuclear pool, where FLAP was also detected. The loss of both cytosolic and nuclear soluble 5-LO, with concomitant increase exclusively at the NE, as well as co-localization with FLAP, were confirmed by indirect immunofluorescent and confocal microscopy. In AMs, the nuclear soluble pool of 5-LO moved to the NE, where FLAP was also found; however, the cytosolic 5-LO pool did not translocate. Application of these methods to PMNs confirmed that cytosolic 5-LO moved to the nuclear envelope and co-localized with FLAP. By cell-free assay, nuclear soluble proteins from both RBL cells and AMs, but not PMNs, were able to generate 5-LO products from arachidonate, and this was inhibited by the direct 5-LO inhibitor zileuton. Cytosolic proteins from all cell types also showed cell-free 5-LO activity. These results demonstrate three distinct patterns of 5-LO translocation that are specific for each cell type: translocation of only a cytosolic pool in PMNs, of only a nuclear pool in AMs, and of both cytosolic and nuclear pools in RBL cells. By virtue of its enzymatic activity and ability to translocate, nuclear 5-LO has the potential to contribute to LT synthesis in RBL cells and AMs. Finally, these results provide a foundation for considering the individual functions of discrete pools of 5-LO in future studies.

Topics: Animals; Arachidonate 5-Lipoxygenase; Calcimycin; Cell Fractionation; Cell Line; Cell Nucleus; Cytosol; Enzyme Activation; Fluorescent Antibody Technique; Humans; Kinetics; Leukemia, Basophilic, Acute; Leukotrienes; Macrophages, Alveolar; Microscopy, Confocal; Neutrophils; Rats; Tumor Cells, Cultured

15-Hydroxyeicosatetraenoic acid [15-(S)-HETE], a major arachidonic acid metabolite produced from the 15-lipoxygenase pathway, has been characterized as an antiinflammatory cellular mediator since it can inhibit the in vivo and in vitro formation of the proinflammatory leukotrienes via the 5-lipoxygenase pathway in various cells. 15-HETE has been confirmed to inhibit the 5-lipoxygenase in rat basophilic leukemia cell (RBL-1) homogenates with an I50 = 7.7 microM. The I50 of the 12-HETE isomer was 6 microM whereas prostaglandin F2 alpha was ineffective. In order to examine the mechanistic basis underlying the inhibitory action of 15-HETE, association assays of [3H]-15-HETE with RBL-1 subcellular fractions were carried out. The presence of the zwitterionic detergent CHAPS enhanced specific [3H]-15-HETE binding in the membrane fractions three-fold and specific 15-HETE binding was distributed among the nuclear (32%)-, granule (19%)-, plasma membrane (35%)-, and cytosol (14%)-enriched fractions. Studies using combined granule and plasma membrane enriched-, CHAPS treated-fractions showed that [3H]-15-HETE binding was time-dependent, specific and reversible, sensitive to pertussis toxin treatment, and indicated a single class of binding sites with a Kd = 460 +/- 160 nM and Bmax = 5.0 +/- 1.1 nM. Competition experiments showed that the order of 15-HETE or analogs in inhibiting the binding of [3H]-15-HETE was: 15(S)-HETE > or = 12-(S)-HETE = 5-(S)-HETE > 15-(R)-HETE > arachidonic acid. Prostaglandin F2 alpha and lipoxin B4 were ineffective as competitors. The similar profiles of the binding assays and inhibition of the 5-lipoxygenase suggest that 15-HETE binding sites may mediate this inhibitory action of 15-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Binding Sites; Binding, Competitive; Calcimycin; Cell Line; Cholic Acids; Dinoprost; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Lipoxygenase Inhibitors; Rats; Subcellular Fractions; Tumor Cells, Cultured

Western blot analysis shows the presence of protein kinase C (PKC) alpha, beta1, beta2, delta, epsilon, zeta isozymes in rat basophilic leukemia (RBL-2H3) cells. Antigen stimulation caused preferential translocation of protein kinase C (PKC) delta and epsilon to membranes. On the other hand, when the cells were stimulated by phorbol 12-myristate 13-acetate (PMA) or ionophore (A23187), PKCalpha and beta were predominantly translocated. Phospholipase D (PLD) was activated by three stimulants in order: A23187 > PMA >> antigen. Therefore, PKCalpha and beta seem to be involved in the activation of PLD in RBL cells. The translocation of all PKC isozymes in A23187 stimulation was weak. Therefore, PLD activation in A23187 stimulation may require some other factors than PKC which are associated with the increase of Ca2+ in the cells. The cells stimulated by antigen secreted serotonin to the same level as the cells stimulated by A23187. In both stimulation, PKCalpha and epsilon were translocated to almost similar level, suggesting that PKCalpha and epsilon are involved in the secretion. All PKC isozymes except zeta were markedly translocated in PMA stimulation, but secretion did not occur. These results indicate that translocation of PKC alpha and beta may be associated with PLD activation and also that both translocation of PKCalpha and epsilon and intracellular calcium increase are required for serotonin secretion in RBL cells.

Topics: Animals; Calcimycin; Calcium; Enzyme Activation; Ionophores; Isoenzymes; Leukemia, Basophilic, Acute; Phospholipase D; Protein Kinase C; Rats; Serotonin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Rat basophilic leukemia (RBL-2H3) cells undergo morphological and cytoskeletal changes during antigen-induced secretion of allergic mediators. The exact role these changes play in the process of secretion is unclear. Using confocal microscopy we now show that PMA+A23187 causes extensive F-actin rearrangements during secretion of [3H] 5-HT. We also describe for the first time the association of myosin with F-actin during this secretory process. In unstimulated cells, myosin and F-actin are concentrated at the plasma membrane with no evidence of stress fibres. Upon addition of PMA or A23187, both F-actin and myosin are rearranged into membrane ruffles and discrete aggregations (foci), followed by the formation of parallel stress fibres located on the ventral membrane. This is in contrast to reports in other cell types in which PMA has been described as causing the disruption of F-actin stress fibres. The time course of secretion coincides with the formation of the foci and ruffles whilst the stress fibres form after the majority of secretion has occurred. These changes are accompanied by a 40% decrease in cell height and a two-fold increase in cell spreading and they occur in the absence of extracellular calcium but are inhibited by the protein kinase C inhibitor, Bisindolylmaleimide, which also inhibits secretion. The formation of myosin-decorated stress fibres, foci, and ruffles is not sufficient to cause secretion, as PMA alone induces these changes without any secretion. The relevance of actin and myosin rearrangements for the regulation of secretion is discussed.

Topics: Actins; Animals; Calcimycin; Calcium; Cell Membrane; Cell Size; Histamine Release; Leukemia, Basophilic, Acute; Maleimides; Mast Cells; Microscopy, Fluorescence; Myosins; Protein Kinase C; Rats; Serotonin; Tetradecanoylphorbol Acetate; Tritium; Tubulin; Tumor Cells, Cultured; Vimentin

We examined the effect of alpha-linolenic acid (18:3 (n-3)) pretreatment on the metabolism of omega-3 and omega-6 polyunsaturated fatty acids and histamine content and release of RBL-2H3 cells. RBL-2H3 cells grew without reduction in number when incubated with subculture media for 3 d and then placed again in serum-free medium with bovine serum albumin (BSA) and epidermal growth factor (EGF). Cholesterol pullulan (10 micrograms/ml) emulsified alpha-linolenic acid (20 micrograms/ml) was recommended as an additional form serum free medium. We determined the fatty acid composition in all neutral lipids, free fatty acids and all phospholipids in alpha-linolenic acid-treated cells. In all cases the concentration of alpha-linolenic acid and docosahexenoic acid (DHA, 22:6 (n-3)) was increased, while linolenic acid (18:2 (n-6)) was slightly and arachidonic acid (20:4 (n-6)) was markedly decreased. Content of histamine in alpha-linolenic acid-treated cells was remarkably lower than that of untreated cells. Accordingly, net histamine release stimulated by antigen or A23187 was also markedly decreased in the alpha-linolenic acid-treated cells, as was the percent histamine release stimulated by antigen. Results from our in vitro experiment suggest that the anti-allergic effect of alpha-linolenic acid may be caused either by the decrease in histamine content or by inhibition of the release of chemical mediator resulting from changes in the fatty acid composition.

Topics: alpha-Linolenic Acid; Animals; Basophils; Calcimycin; Cell Division; Cholesterol; Chromatography, High Pressure Liquid; Dietary Fats, Unsaturated; Dinitrophenols; Dose-Response Relationship, Drug; Emulsions; Epidermal Growth Factor; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Glucans; Haptens; Histamine H1 Antagonists; Histamine Release; Leukemia, Basophilic, Acute; Lipid Metabolism; Phospholipids; Rats; Serum Albumin, Bovine; Tumor Cells, Cultured

We investigated inhibitory actions of dexamethasone (DEX) on retinoic acid (RA)-induced enhancement of leukotriene C4 (LTC4) synthesis in rat basophilic leukemia-1 (RBL-1) cells. Cultured cells were preincubated with RA for 16 h with or without DEX, and generation of LTC4 was measured by high performance liquid chromatography in cell-free and intact cell systems. RA (0.1 microgram/ml) significantly potentiated calcium ionophore-stimulated production of LTC4 synthesis. DEX inhibited the RA-induced enhancement of LTC4 synthesis by up to approximately 95% in intact cells when stimulated with calcium ionophore. RA-induced LTC4 synthase activity, which was determined by enzyme assay, was also inhibited by DEX by 65% in a cell-free system. This discrepancy of inhibition between the intact and cell-free systems was due to a partial inhibition of phospholipase A2 activity by DEX in the intact cells. These results indicate that the production of LTC4 is predominantly regulated at a level of LTC4 synthase. The induction of new LTC4 synthase activity by RA and inhibition of the RA-induced activity by DEX are important regulatory mechanisms of LTC4 synthesis.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Dexamethasone; Glutathione Transferase; Kinetics; Leukemia, Basophilic, Acute; Leukotriene C4; Phospholipases A; Phospholipases A2; Rats; Tretinoin; Tumor Cells, Cultured

Ag-induced cross-linking of IgE bound to its high affinity receptor (Fc epsilon RI) at the surface of basophils or mast cells triggers a number of biochemical events culminating in the release of several inflammatory mediators. In rat basophilic leukemia (RBL-2H3) cells expressing the G protein-coupled m1 muscarinic receptor, Ag/IgE-induced cross-linking of Fc epsilon RI, calcium ionophore A23187, and carbachol through M1 receptors stimulated tyrosine phosphorylation of several proteins, including two of 42 and 44 kDa. Proteins of identical molecular masses were recognized by anti-MAP-kinase antibodies, and these immunoreactive proteins exhibited in part a slightly increased molecular mass on SDS polyacrylamide gels after incubation of cells with secretory stimuli. All stimuli led to the activation of MAP kinase, which co-purified on Mono Q chromatography with 42- and 44-kDa proteins, which were tyrosine phosphorylated in response to secretory stimuli and reacted with anti-(MAP kinase) antibodies. Finally, 42- and 44-kDa proteins immunoprecipitated by anti-MAP-kinase antibodies and anti-phosphotyrosine antibodies were recognized by anti-phosphotyrosine and anti-MAP-kinase antibodies, respectively. Primarily threonine and tyrosine residues were found to be phosphorylated in 42- and 44-kDa proteins immunoprecipitated from [32P]phosphate-labeled cells that had been treated with secretory stimuli. The dose dependence of secretagogue-induced MAP kinase activation correlated with that of increases in serotonin release from activated cells, and the maximum of MAP kinase activation coincided with the maximum rate of secretion. Down-regulation or inhibition of protein kinase C as well as incubation of cells with the tyrosine kinase inhibitor genistein markedly inhibited MAP kinase activation in parallel with serotonin release. Taken together, these findings demonstrate that 42- and 44-kDa MAP kinases are activated in response to secretory stimuli and provide some evidence for a functional link between MAP kinase activation and signaling events leading to mediator release in RBL cells.

Topics: Animals; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinases; Cell Degranulation; Enzyme Activation; Leukemia, Basophilic, Acute; Mitogen-Activated Protein Kinase 1; Mitogens; Molecular Weight; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Receptors, IgE; Serotonin; Signal Transduction; Tumor Cells, Cultured; Tyrosine

Several inhibitors of tyrosine phosphatases, which included vanadate/H2O2, phenylarsine oxide, and diamide, blocked exocytosis in basophilic RBL-2H3 cells that had been transfected with the gene for the muscarinic m1 receptor. Because this block was observed whether the secretagogue acted through receptors (i.e. antigen and the muscarinic agonist, carbachol) or by direct activation of intracellular mechanisms (i.e. A23187, A23187 in combination with phorbol 12-myristate 13-acetate, and thapsigargin), the inhibitors appeared to act at a step distal to the mobilization of Ca2+ and activation of protein kinase C. All secretagogues caused the tyrosine phosphorylation of a 40-kDa protein, whereas the inhibitors caused a hyperphosphorylation of this protein. Therefore, both tyrosine kinase and phosphatase activities appear to regulate this phosphorylation which may, in turn, regulate secretion. The 40-kDa protein was identified as a mitogen-activated protein kinase-like protein on the basis of its reactivity to anti-mitogen-activated protein kinase antibodies. In addition, when cells were stimulated the tyrosine phosphorylated and the immunoreactive protein comigrated as a doublet on one-dimensional and as multiple phosphorylated forms on two-dimensional gel-electrophoretic systems.

Topics: Actins; Animals; Arachidonic Acid; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Cell Line; Electrophoresis, Polyacrylamide Gel; Exocytosis; Hexosaminidases; Hydrogen Peroxide; Inositol Phosphates; Kinetics; Leukemia, Basophilic, Acute; Phospholipids; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Rats; Tumor Cells, Cultured; Tyrosine; Vanadates

Quantal Ca2+ release is a novel motif for the mediation of signal transduction in which the amplitude of a biological response following multiple stepwise increases in agonist concentration is retained. The release of Ca2+ from permeabilized cells in response to the second messenger inositol 1,4,5-trisphosphate (InsP3) proceeds in this fashion. The mechanisms leading to quantal Ca2+ release are unknown. InsP3 releases 50-90% of the Ca2+ sequestered within the intracellular stores of mammalian cells permeabilized with saponin. However, preparation of microsomes results in the loss of this sensitivity. In this report, functionally intact intracellular Ca2+ stores were isolated from rat basophilic leukemia (RBL) cells by osmotic lysis followed by differential and sucrose density gradient centrifugation. From this preparation, 64% of the stored Ca2+ is released by InsP3. We demonstrate that quantal Ca2+ release is retained by isolated Ca2+ stores and is identical to that observed in permeabilized cells. Addition of a subsaturating (28 nM) concentration of InsP3 to permeabilized cells at 37 degrees C results in the release of only a small fraction of the sequestered Ca2+. When the cells are cooled to 11 degrees C, the remaining Ca2+ is rapidly released. Hence, the mechanism leading to the quantal nature of Ca2+ release is reversible and is thus not likely to be the result of a covalent modification of the channel protein or of the Ca2+ store.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcimycin; Calcium; Computer Simulation; DNA, Neoplasm; Inositol 1,4,5-Trisphosphate; Kinetics; L-Lactate Dehydrogenase; Leukemia, Basophilic, Acute; Microsomes; Quantum Theory; Rats; Second Messenger Systems; Structure-Activity Relationship; Subcellular Fractions; Tumor Cells, Cultured

The effect of the stable cAMP analogue 8-Br-cAMP on leukotriene D4 (LTD4)-, 5'-N-ethyl-carboxamidoadenosine (NECA)-, antigen- and Ca2+ ionophore-induced inositol phosphate (IP) production was studied in RBL-1 cells. The cAMP analogue significantly inhibited LTD4- and antigen induced-IP production, thus supporting the hypothesis of a negative interaction between cAMP and phosphoinositide breakdown in blood cells. Ionophore-induced IP release, which was blocked by a 5-lipoxygenase inhibitor and by a LT-receptor antagonist, and therefore is probably mediated by LTs, was also inhibited by 8-Br-cAMP. NECA-induced IP release was not significantly inhibited by the cyclic nucleotide, thus showing that the effect described herein is not a general action on receptor-activated phospholipase C. 8-Br-cAMP did, however, inhibit GTP gamma S-induced IP release in permeabilised RBL-1 cells, thus suggesting that the inhibition does not occur at the receptor level but might be due, at least in part, to an effect on some receptor-coupled G proteins.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Antigens; Calcimycin; Inositol Phosphates; Leukemia, Basophilic, Acute; Lipoxygenase Inhibitors; Rats; Receptors, Immunologic; Receptors, Leukotriene; SRS-A; Tumor Cells, Cultured; Vasodilator Agents

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.

Topics: Alkaloids; Androstadienes; Animals; Basophils; Calcimycin; Calcium; Carbazoles; Dose-Response Relationship, Drug; Histamine Release; Humans; Immunoglobulin E; Indoles; Leukemia, Basophilic, Acute; Myosin-Light-Chain Kinase; Rats; Tumor Cells, Cultured; Wortmannin

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.

Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Basophils; Biological Transport; Blotting, Western; Calcimycin; Calcium; Electrophoresis, Polyacrylamide Gel; Genistein; Histamine Release; Immunoglobulin E; Isoflavones; Leukemia, Basophilic, Acute; Mast Cells; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Rats; Receptors, Fc; Receptors, IgE; Signal Transduction; Tumor Cells, Cultured; Tyrosine

The effects of neutrophil-derived histamine-releasing activity (HRA-N) on arachidonic acid (AA) metabolism is unknown. Human basophils exposed to HRA-N released 25% of total histamine but no leukotriene C4 (LTC4). To confirm this phenomenon, rat basophilic leukemia (RBL) cells were exposed to HRA-N as well as anti-IgE, or calcium ionophore A23187. RBL cells incubated with A23187 released 44% of available serotonin and 59 and 124 pmol/10(6) cells of prostaglandin D2 (PGD2) and LTC4, respectively. Anti-IgE stimulation resulted in 34% serotonin release and the generation of 34 pmol PGD2 per 10(6) cells and 72 pmol LTC4 per 10(6) cells. In contrast, HRA-N (2 U/ml) induced 20% serotonin release, 4 pmol PGD2 per 10(6) cells, and 0.6 pmol LTC4 per 10(6) cells. Neither increasing the dose nor the incubation time of HRA-N enhanced the generation of AA metabolite. Additionally, the spectrum of AA metabolites generated by RBL cells in response to those agents was examined by reverse-phase high-performance liquid chromatography. RBL cells stimulated with A23187 released PGD2, LTB4, and its isomers, LTC4, and 5-hydroxyeicosatetraenoic acid. In contrast, HRA-N stimulation resulted in only minimal PGD2 generation and no other discernable AA metabolites. Thus, HRA-N causes selective release of serotonin without inducing AA metabolites. These data suggest that HRA-N activates mast cells through a unique pathway.

Topics: Animals; Antibodies, Anti-Idiotypic; Arachidonic Acid; Basophils; Biomarkers, Tumor; Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Histamine Release; Humans; Immunoglobulin E; Leukemia, Basophilic, Acute; Lymphokines; Neutrophils; Radioimmunoassay; Rats; Serotonin; Tumor Cells, Cultured; Tumor Protein, Translationally-Controlled 1

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonate 5-Lipoxygenase; Basophils; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Compartmentation; Cell Membrane; Egtazic Acid; In Vitro Techniques; Inositol Phosphates; Ionomycin; Leukemia, Basophilic, Acute; Leukotrienes; Rats; Receptors, Immunologic; Receptors, Leukotriene; SRS-A; Terpenes; Thapsigargin; Tumor Cells, Cultured

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.

Topics: Animals; Antigens; Antigens, Differentiation, B-Lymphocyte; Calcimycin; Calcium; Catechols; Cell Line; Dinitrophenols; Immunoblotting; Immunoglobulin E; Kinetics; Leukemia, Basophilic, Acute; Magnesium; Nitriles; Peptide Mapping; Phosphopeptides; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Rats; Receptors, Fc; Receptors, IgE; Serotonin; Serum Albumin, Bovine; Signal Transduction; Tyrosine; Tyrphostins

Overnight incubation of rat basophilic leukemia-1 (RBL-1) cells with retinoic acid enhanced calcium ionophore-stimulated syntheses of LTC4 by more than 28-times (from 5.91 +/- 0.31 to 168.31 +/- 22.66 ng/2.10(6) cells) nd LTD4 by more than 7-times (from 5.27 +/- 0.12 to 39.38 +/- 14.89 ng/2.10(6) cells). The stimulatory action first appeared after a 10 h incubation with retinoic acid and was completely abolished by concomitant presence of low concentration cycloheximide (0.5 micrograms/ml) in the medium. However, LTB4 synthesis was dose-dependently inhibited by incubation with retinoic acid. Reduced form of glutathione significantly increased the synthesis of LTC4, but showed no action on the syntheses of LTD4 or LTB4. These results indicate a new enzyme synthesis of LTC4 synthetase.

Topics: Animals; Calcimycin; Cycloheximide; Glutathione; Leukemia, Basophilic, Acute; Leukotriene B4; Rats; SRS-A; Tretinoin; Tumor Cells, Cultured

Short-term stimulation (up to 16 hours) of interleukin-3 (IL-3)-dependent mouse bone marrow-derived mast cells, Abelson transformed mouse liver-derived mast cells, or rat basophilic leukemia cells by either IgE-Ag or calcium ionophore A23187 resulted in inhibition of their proliferation as measured by 3H-thymidine incorporation and MTT (tetrazolium) assays, and in accumulation of the mRNAs of c-fos, c-jun, junB and slightly of junD proto-oncogenes. The involvement of protein kinase C (PKC) in these responses was investigated by using several approaches of enzyme activity regulation. Direct activation of the PKC was achieved by short-term exposure of the cells to the PKC-specific activator phorbol 12-myristate-13-acetate (PMA). Inhibition of PKC activity was obtained by either prolonged treatment of the cells with PMA or by exposure of the cells to the PKC inhibitors H-7 and staurosporine. The results showed the following: (1) Short-term exposure of mast cells to PMA was sufficient to induce inhibition of proliferation. (2) An increase in PKC activity was associated with a decrease in the proliferation of IgE-dinitrophenol (DNP) or calcium ionophore A23187-stimulated cells. (3) A direct correlation was found between the increase in PKC activity and the increase in the level of the mRNAs of the jun proto-oncogenes in cells activated by both stimuli mentioned. (4) While an increase in PKC activity was associated with the upregulation of the level of c-fos mRNA during calcium ionophore A23187 stimulation, it showed the opposite effect on the expression of the mRNA of this proto-oncogene when the cells were triggered by IgE-DNP. Therefore, we concluded that PKC plays various roles in the expression of the mRNA of c-fos in activated mast cells depending on the stimulus involved. In addition, the expression of the mRNA of c-jun and junB proto-onogenes is not coordinately regulated with that of c-fos during immunologic stimulation. This discordancy, which is associated with the increase in PKC activity in mast cells, may play a role in the regulation of the transcription of AP-1-responsive genes, and therefore could be associated with the regulation of proliferation of these cells.

Topics: Animals; Calcimycin; Cell Division; Cell Line, Transformed; Dinitrophenols; Gene Expression Regulation; Genes, fos; Genes, jun; Immunoglobulin E; Leukemia, Basophilic, Acute; Liver; Mast Cells; Mice; Protein Kinase C; Rats; RNA, Messenger; Serum Albumin, Bovine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 micrograms/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2(+)-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.

Topics: Alkaloids; Animals; Antigens; Arachidonic Acids; Calcimycin; Carbazoles; Cell Line; Cell Membrane; Cells, Cultured; Dexamethasone; Immunoglobulin E; Indole Alkaloids; Ionomycin; Kinetics; Leukemia, Basophilic, Acute; Leukemia, Experimental; Macrophages; Phorbol 12,13-Dibutyrate; Phospholipases A; Phospholipases A2; Protein Kinase C; Rats; Serum Albumin, Bovine; Staurosporine; Tetradecanoylphorbol Acetate

Exposure to antigen (Ag) caused a biphasic 1,2-diacylglycerol (DG) production in [3H]myristic acid-labeled RBL-2H3 cells; the early, small transient phase and the second large sustained phase. The accumulation of phosphatidic acid (PA) or phosphatidylethanol (PEt) in the presence of ethanol was paralleled by the second-phase DG generation. Ag-induced formation of phosphocholine and choline in [3H]choline-labeled cells suggested the hydrolysis of phosphatidylcholine (PC) by phospholipases C and D. Treatment with phorbol myristate (PMA) or A23187 caused increases in [3H]DG and water-soluble [3H]choline metabolites. In protein kinase C (PKC) down-regulated cells, PEt formation was markedly reduced. In these cells DG production induced by Ag and A23187 was largely suppressed, thus indicating that PKC would play an important regulatory role for PC hydrolysis. However, because the A23187 treatment showed significant accumulation of water-soluble choline metabolites in PKC down-regulated cells, an increase in intracellular Ca2+ is another factor regulating PC hydrolysis. Taken together, these results may indicate that PC hydrolysis in response to Ag is dependent on PKC and Ca2+.

Topics: Animals; Antigens; Calcimycin; Cell Line; Choline; Diglycerides; Hydrolysis; Kinetics; Leukemia, Basophilic, Acute; Myristic Acid; Myristic Acids; Phosphatidylcholines; Phospholipase D; Protein Kinase C; Tetradecanoylphorbol Acetate; Type C Phospholipases

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.

Topics: Animals; Calcimycin; Cell Membrane; Histamine Release; Immunoglobulin E; Leukemia, Basophilic, Acute; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Rats; Tumor Cells, Cultured

As a continuation of our efforts to understand leukotriene biosynthesis mechanisms, we have studied the effect on RBL-1 cells of a series of phospholipase C (PLC) and phospholipase A2 (PLA2) activators including calcium ionophore (A23187), leukotriene D4 (LTD4), PAF, fMLP and bradykinin. LTD4 and A23187 (the latter only at 20 microM concentration and only after a 45 min incubation time) were shown to induce phosphoinositide (PI) breakdown, whilst A23187 induced leukotriene biosynthesis. For the first time it was shown that cAMP analogues markedly inhibit LTD4-induced IP formation. Moreover, the 5-lipoxygenase inhibitor AA861 abolished the ionophore-induced PI breakdown, thus suggesting that this effect is a novel example of PLC activation following PLA2 activation and 5-lipoxygenase-derived metabolite production.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Cyclic AMP; Enzyme Activation; Inositol Phosphates; Leukemia, Basophilic, Acute; Leukotrienes; N-Formylmethionine Leucyl-Phenylalanine; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Rats; Second Messenger Systems; SRS-A; Tumor Cells, Cultured; Type C Phospholipases

Rat basophilic leukemia (RBL-1) cells (1.5 x 10(9) were incubated with (15S), 15-hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HPETE) (100 microM) and calcium ionophore A23187 (5 microM) for 30 min at 37 degrees C. The reaction products were extracted and separated on reverse-phase high performance liquid chromatography (RP-HPLC). The fraction of interest which showed the characteristic UV spectrum of lipoxins (LXs) (lambda max at 301 nm, shoulders at 289 nm and 317 nm) was further purified using a second RP-HPLC. This step produced four distinct peaks, three of which co-eluted with synthetic lipoxin A4 (LXA4), lipoxin B4 (LXB4), and authentic 6S-LXA4 standards, respectively. These fractions were analyzed by capillary gas chromatography and mass spectrometry (GC/MS) for structural identity. Based on these data (chromatographic profile, UV spectrum, mass spectra) and the reported criteria (Serhan/Samuelsson, 1984-88), these three fractions were identified with LXA4[(5S,6R,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetra enoic acid)], LXB4[(5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-eic osa-tetraenoic acid)] and 6S-LXA4[(5S,6S,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosate traenoic acid)]. It is concluded that RBL-1 cells have the capacity to generate LXs by metabolizing arachidonic acid (AA) derivatives (i.e., 15-HPETE) and that LXs produced by RBL-1 cells are indistinguishable from those derived from other cells.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotrienes; Lipid Peroxides; Lipoxins; Tumor Cells, Cultured

The human leukemic cell line KU-812-F is known to differentiate into mature basophil-like cells under serum-free culture conditions. In the present study, the activity of histidine decarboxylase (HDC), a histamine-forming enzyme, in KU-812-F cells was found to be high, ranging from 10 to 57 pmol/min/mg protein. The great variation in HDC activity appeared to be due to different percentages and degrees of maturity of basophil-like cells during differentiation of this cell line. The enzyme was inhibited by alpha-fluoromethylhistidine but not by carbidopa, was unable to form dopamine from L-3,4-dihydroxyphenylalanine, and had a Km value for histidine of 0.27 mM, indicating that it was HDC and not aromatic amino acid decarboxylase. The HDC activity increased 1.8-fold when the cells were stimulated by phorbol myristate acetate, which is known to activate protein kinase C, and this increase was blocked by staurosporine, a potent inhibitor of protein kinase C.

Topics: Alkaloids; Calcimycin; Carboxy-Lyases; Cycloheximide; Enzyme Induction; Histidine Decarboxylase; Humans; Leukemia, Basophilic, Acute; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Biological Transport; Calcimycin; Calcium; Cell Membrane; Cytosol; Edetic Acid; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotrienes; Mast Cells; Rats; SRS-A; Tumor Cells, Cultured

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Drug Synergism; Leukemia, Basophilic, Acute; Phospholipases; Phospholipids; Prostaglandin D2; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Topics: Animals; Calcimycin; Cell Line; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotrienes; Lipid Peroxides; Lipoxins; N-Formylmethionine Leucyl-Phenylalanine; Rats; Tetradecanoylphorbol Acetate