st-638 and herbimycin

To investigate the role of tyrosine kinases (TK) in IgE-mediated signal transduction in human lung mast cells (HLMC) and basophils.. Peripheral blood basophils (n > or = 4) and human lung mast cells (n > or = 6).. Cells were preincubated with TK inhibitor for 15 min at 37 degrees C, before the addition of anti-IgE.. Histamine release (HR) was assayed using a fluorimetric technique. Results were compared using nonparametric statistics.. Piceatannol and ST638 significantly (p < or = 0.05) inhibited anti-IgE induced HR from HLMCs and basophils whilst lavendustin C had no effect in either cell type. Herbimycin A also significantly (p < or = 0.05) inhibited anti-IgE induced HR from both cell types, an effect which was dose dependent but did require a 16 h preincubation with drug.. In summary, HLMCs and basophils exhibit distinct inhibitory profiles in the presence of various inhibitors of TK.

Topics: Basophils; Benzoquinones; Cell Count; Cinnamates; Enzyme Inhibitors; Histamine Release; Humans; Immunoglobulin E; Lactams, Macrocyclic; Lung; Mast Cells; Phenols; Platelet Aggregation Inhibitors; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Stilbenes; Sulfides

The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells.

Topics: Animals; Benzoquinones; Blotting, Western; Calcimycin; Calcium; Cinnamates; Enzyme Activation; Enzyme Inhibitors; Genistein; Hydrogen Peroxide; Isoflavones; Lactams, Macrocyclic; PC12 Cells; Phosphatidylinositol 4,5-Diphosphate; Phospholipase D; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Sulfides; Tetradecanoylphorbol Acetate; Type C Phospholipases

We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Topics: Animals; Antibodies, Monoclonal; Benzoquinones; Calcium; Cell Line; Cinnamates; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fura-2; Histamine Release; Inositol 1,4,5-Trisphosphate; Kinetics; Lactams, Macrocyclic; Leukemia, Basophilic, Acute; Molecular Weight; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Receptors, IgG; Rifabutin; Sulfides; Tumor Cells, Cultured; Tyrosine

We have investigated the relationship between tyrosine phosphorylation and respiratory-burst activity in mouse bone-marrow-derived macrophages (BMM). We demonstrate that zymosan, an agent known to trigger the macrophage respiratory burst, also triggers the activation of tyrosine kinase activity, resulting in rapid tyrosine phosphorylation on numerous proteins, and provide evidence for the role of tyrosine phosphorylation in the triggering of the BMM respiratory burst. Agents, such as tumour necrosis factor alpha (TNF alpha), interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which prime the macrophage for an enhanced zymosan-triggered respiratory burst, increase tyrosine phosphorylation triggered by zymosan. The zymosan-triggered tyrosine phosphorylation and respiratory-burst activity were partially suppressed by the tyrosine kinase inhibitors alpha-cyano-3-ethoxy-4-hydroxy-5-phenylmethylcinnamide (ST638) and herbimycin A. In addition, pre-exposure of BMM to vanadate, a phosphotyrosine phosphatase inhibitor, greatly enhanced the ability of zymosan to induce tyrosine phosphorylation and trigger the respiratory burst. These data highlight the importance of the balance between tyrosine kinase and phosphotyrosine phosphatase activity in determining the ultimate level of tyrosine phosphorylation in BMM and suggest that zymosan-triggered tyrosine phosphorylation is an important biochemical signal for triggering of the respiratory burst.

Topics: Animals; Benzoquinones; Bone Marrow Cells; Cells, Cultured; Cinnamates; Enzyme Activation; In Vitro Techniques; Interferon-gamma; Lactams, Macrocyclic; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred CBA; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Respiratory Burst; Rifabutin; Sulfides; Tumor Necrosis Factor-alpha; Tyrosine; Vanadates; Zymosan

An isoflavone compound, genistein, which is known as a protein tyrosine kinase inhibitor, concentration-dependently (0.1-30 micrograms/ml) suppressed human platelet aggregation, serotonin secretion, and protein tyrosine phosphorylation induced by collagen or stable thromboxane A2 analogs [U46619 and 9,11-epithio-11,12-methano-thromboxane A2 (STA2)]. However, genistein did not inhibit these thrombin (0.1 unit/ml)-induced platelet responses. Although thrombin induced an increase in the platelet phosphotyrosine content, genistein at 100 micrograms/ml only slightly attenuated thrombin-induced protein tyrosine phosphorylation. Genistein competitively inhibited [3H]U46619 binding to washed platelets, in a concentration-dependent fashion. Daidzein (another isoflavone compound), which does not have a hydroxyl group at the 5-position of genistein and lacks inhibitory activity for protein tyrosine kinase, was found to suppress [3H]U46619 binding, leading to the inhibition of collagen- or STA2-induced platelet responses. These results indicate that the blockage by genistein of platelet responses induced by collagen or thromboxane A2 is due to its preventive action on thromboxane A2 binding to the receptor, rather than via inhibition of protein tyrosine phosphorylation, and that the drug does not appear to be a particularly good inhibitor of tyrosine phosphorylation in intact platelets.

Topics: Anti-Bacterial Agents; Benzoquinones; Blood Platelets; Cinnamates; Genistein; Humans; Isoflavones; Lactams, Macrocyclic; Phospholipids; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Protein-Tyrosine Kinases; Quinones; Rifabutin; Serotonin; Signal Transduction; Sulfides; Thromboxane A2; Tyrosine