st-638 and Leukemia--Basophilic--Acute

Previously, we have demonstrated that tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic leukemia cells (RBL-2H3) is involved in a signal transduction system for high-affinity IgE receptor (Fc epsilon RI)-mediated histamine secretion. However, it is not clarified whether the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells is regulated by activation of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3-kinase). In this study, therefore, the effect of depletion of PKC in RBL-2H3 cells, or the influence of PKC, PI3-kinase and tyrosine kinase inhibitors on histamine release from RBL-2H3 cells was examined. The elimination of PKC in RBL-2H3 cells induced significant suppression of histamine release, although the tyrosine phosphorylation of 78 and 92 kDa proteins was not inhibited. The inhibition of histamine release was also observed by the treatment with a PKC inhibitor such as H-7, calphostin C, a PI3-kinase inhibitor such as wortmannin or a tyrosine kinase inhibitor such as ST638, genistein, hervimycin A, although the tyrosine phosphorylation of both proteins was inhibited by only ST638. These results suggest that the 78 kDa protein in RBL-2H3 cells is not identical to the protein-tyrosine kinase PTK72 and the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells occurs upstream of PKC and PI3-kinase activation or is regulated independently of the PKC- and PI3-kinase-dependent signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cells, Cultured; Cinnamates; Histamine Release; Leukemia, Basophilic, Acute; Molecular Weight; Naphthalenes; Phosphorylation; Protein Kinase C; Proteins; Rats; Receptors, IgE; Sulfides; Tyrosine

It is well known that rat basophilic leukemia cells (RBL-2H3) express high-affinity IgE receptors (Fc epsilon RI) and that the aggregation of these receptors causes the release of chemical mediators. When RBL-2H3 cells are sensitized with IgE antibody and subsequently stimulated by an antigen, significant histamine release and the tyrosine phosphorylation of several proteins are observed. In this study, we examined the effects of a synthetic naphthalene derivative, (7E)-N-(2-carboxyphenyl)-8-(2-naphthyl)-5,6-trans-5,6-methano-7-++ +octenamide (TEI-6472), on the Fc epsilon RI-mediated histamine release from RBL-2H3 cells. Preincubation for 10 min with 100 microM TEI-6472 caused significant inhibition of Fc epsilon RI-mediated histamine release from RBL-2H3 cells. Furthermore, Western blotting analysis using anti-phosphotyrosine antibody showed that Fc epsilon RI-mediated tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells was also significantly inhibited. Tyrosine phosphorylation of these 78 and 92 kDa proteins was not induced by direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) and the calcium ionophore A23187. However, the inhibition of histamine release from TEI-6472-treated RBL-2H3 cells was restored by direct activation of PKC. Taken together, these results suggest that tyrosine phosphorylation of the 78 and 92 kDa proteins in RBL-2H3 cells is involved in a signal transduction system for histamine secretion, and that these tyrosine phosphorylations may occur upstream of PKC activation.

Topics: Animals; Basophils; Cinnamates; Dinitrophenols; Haptens; Histamine Release; Immunoblotting; Immunoglobulin E; Immunosuppressive Agents; Leukemia, Basophilic, Acute; Naphthalenes; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Receptors, IgE; Serum Albumin; Signal Transduction; Sulfides; Time Factors; Tumor Cells, Cultured; Tyrosine

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

The effect of protein tyrosine phosphorylation on phospholipase D (PLD) activation measured by the formation of radiolabeled phosphatidylbutanol (PBut) was examined in rat basophilic leukemia (RBL-2H3) cells stimulated with antigen. The PLD activation elicited by antigen was attenuated dose-dependently by pretreatment with protein tyrosine kinase inhibitors, genistein and ST638. In parallel, tyrosine phosphorylation of 72 kDa protein was inhibited by the same pretreatment. These results, taken together with little effect of genistein on phosphoinositide hydrolysis, suggest that tyrosine kinase may be implicated in the IgE-mediated PLD activation which is regulated by a protein kinase C-independent process.

Topics: Animals; Antigens; Basophils; Cinnamates; Enzyme Activation; Genistein; Immunoglobulin E; In Vitro Techniques; Isoflavones; Leukemia, Basophilic, Acute; Phospholipase D; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Receptors, IgE; Signal Transduction; Sulfides; Tumor Cells, Cultured; Tyrosine