methyl-2-5-dihydroxycinnamate and herbimycin

Herbimycin A, lavendustin A, and methyl 2,5-dihydroxycinnamate were used to study the role of protein tyrosine kinases in collagen-platelet interaction. All three compounds produced a concentration dependent inhibition of platelet aggregation induced by collagen type I, characterized by values of IC50 equaled to 0.9, 10.0, and 5.0 microM, respectively. This effect was accompanied by strong inhibition of phosphorylation of p125FAK, p90, p72syk, p60c-arc, and p56lyn. In the absence of the inhibitors, phosphorylation of these proteins is evoked by aggregation of platelets. In addition to the antiaggregatory effect, the tyrosine kinase inhibitors reduced adhesion of platelets to collagen although to much lower extent than aggregation. Platelets which adhered to collagen showed also the presence of phosphorylated p125FAK, p90, p72syk, p60c-arc, and p56lyn. Of these proteins, the extent of phosphorylation of p90 was particularly high. Adhesion of platelets was associated with inhibition of phosphorylation of p125FAK, p60c-arc, and p56lyn only when high concentration of lavendustin A and methyl 2,5-dihydroxycinnamate were used. Herbimycin A did not affect adhesion-evoked protein tyrosine phosphorylation. Phosphorylation of p90 and p72syk was not affected by inhibitors. This study indicates that collagen type I can induce different transmembrane signalling dependent upon whether platelet aggregates formation or adhesion of platelets to this protein occurs.

Topics: Benzoquinones; Cinnamates; Collagen; Cross-Linking Reagents; Drug Synergism; Humans; Lactams, Macrocyclic; Phenols; Platelet Adhesiveness; Platelet Aggregation; Protein-Tyrosine Kinases; Quinones; Rifabutin

The signal transduction events which govern major histocompatibility complex-unrestricted tumour cell destruction by nonspecific killer T lymphocytes induced with anti-CD3 antibody have not yet been determined. In this study we used pharmacologic inhibitors to investigate the role of protein tyrosine kinases (PTK) and protein kinase C (PKC) in this process. The PTK-inhibitors herbimycin A, genistein, and methyl 2,5-dihydroxycinnamate blocked anti-CD3-activated killer T (AK-T) lymphocyte-mediated killing of tumour target cells. The PKC-inhibitors staurosporine, calphostin C, and myristoylated PKC pseudosubstrate peptide, as well as PKC desensitization by phorbol 12-myristate 13-acetate pretreatment, also suppressed the cytolytic effector function of AK-T lymphocytes. Lack of tumoricidal activity was not due to reduced AK-T lymphocyte binding to tumour target cells but was associated with the abrogation of granule exocytosis, indicating that PTK and PKC are involved in the postbinding process which results in delivery of the 'lethal hit' by AK-T lymphocytes.

Topics: Animals; Antibody-Dependent Cell Cytotoxicity; Benzoquinones; Cinnamates; Female; Genistein; Granzymes; Immunosuppressive Agents; Isoflavones; Killer Cells, Natural; Lactams, Macrocyclic; Mice; Mice, Inbred C57BL; Muromonab-CD3; Neoplasms; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Serine Endopeptidases; Signal Transduction

Three sialagogues, isoproterenol (IPR), carbachol, and methoxamine, caused induction of ornithine decarboxylase (ODC) in cultured rat parotid explants. All the protein tyrosine kinase inhibitors tested suppressed this ODC induction but enhanced sialagogue-dependent amylase secretion. Sodium orthovanadate showed the reverse effects as the kinase inhibitors. Immunoblot analysis with anti-phosphotyrosine antibody revealed that herbimycin A depresses IPR-stimulated tyrosine phosphorylation of parotid proteins. Herbimycin A did not affect the IPR- or dibutyryl cAMP-induced surge of the parotid cAMP level but inhibited these agonist-dependent ODC inductions. These results suggest that sialagogue-induced ODC induction and amylase secretion are mediated by different signal transduction pathways and that protein tyrosine kinase participates in IPR-dependent ODC induction and amylase secretion in the process subsequent to the cAMP surge.

Topics: Amylases; Animals; Benzoquinones; Bucladesine; Carbachol; Chamomile; Cholecystokinin; Cinnamates; Cyclic AMP; Enzyme Induction; Enzyme Inhibitors; Flavonoids; Genistein; Isoflavones; Isoproterenol; Lactams, Macrocyclic; Male; Methoxamine; Oils, Volatile; Organ Culture Techniques; Ornithine Decarboxylase; Parotid Gland; Phenols; Plants, Medicinal; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Wistar; Rifabutin; Sialic Acids; Vanadates

Protein tyrosine kinase (PTK) inhibitors have been reported to inhibit proliferation of vascular smooth muscle cells (SMC). To elucidate the mode of this inhibition, the effects on the cell cycle of cultured vascular SMC of three PTK inhibitors with different modes of action (methyl 2,5-dihydroxycinnamate, genistein, and herbimycin A) were studied. Rat aortic SMC were synchronized to the G0 phase of the cell cycle and then released to proceed through the cell cycle by the addition of platelet-derived growth factor (PDGF), and [3H]thymidine incorporation into DNA was measured. The three PTK inhibitors all inhibited PDGF-induced DNA synthesis in a dose-dependent fashion, with IC50 values of 4.7 +/- 1.4 microM for methyl 2,5-dihydroxycinnamate, 6.7 +/- 2.5 microM for genistein, and 0.17 +/- 0.07 microM for herbimycin A. Time course studies suggested that the agents inhibited early G1 phase but not the G0-G1 transition. the lack of effect on the G0-G1 transition was also supported by the finding that the agents did not inhibit the ligand-induced autophosphorylation of PDGF receptor nor the induction of c-fos mRNA at concentrations which were sufficient to inhibit DNA synthesis. PTK inhibitors inhibited progression of the S phase when they were added to SMC that had been arrested at the G1-S border with hydroxyurea. Methyl 2,5-dihydroxycinnamate also blocked the M phase when it was added to SMC cultured in the presence of 10% fetal calf serum, while genistein and herbimycin A did not inhibit the M phase under the same experimental conditions. In accordance with our previous observation, methyl 2,5-dihydroxycinnamate impaired microtubule networks and formation of the mitotic spindle during the M phase. Our findings indicated that PTK inhibitors inhibit multiple steps of the vascular SMC cell cycle.

Topics: Animals; Aorta; Benzoquinones; Cell Cycle; Cells, Cultured; Cinnamates; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; G1 Phase; Genistein; Isoflavones; Kinetics; Lactams, Macrocyclic; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Sprague-Dawley; Resting Phase, Cell Cycle; Rifabutin; Time Factors

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.

Topics: 3T3 Cells; Animals; Benzoquinones; Cell Adhesion; Cell Adhesion Molecules; Cinnamates; Enzyme Inhibitors; Enzyme Stability; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Kinetics; Lactams, Macrocyclic; Mice; Mice, Inbred BALB C; Naphthalenes; Phosphorylation; Phosphoserine; Phosphotyrosine; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Rifabutin

Studies of human lung mast cells have usually focused on histamine release, although the enzymes stored in the granules may also contribute to the pathophysiology of the allergic response. We have used a simple colorimetric assay for tryptase to follow the release of proteolytic enzymes from human lung mast cells in vitro. Either human lung mast cell supernatants or authentic mast cell tryptase were mixed with benzoyl-DL-arginine-p-nitroaniline and incubated for up to 72 h at 37 degrees C. The appearance of nitroaniline was then measured at 410 nm in an ELISA plate reader. Cells were sonicated in H2O to measure total tryptase and histamine. Human lung mast cells contained the equivalent of 11.2 +/- 0.7 pg tryptase per cell and 3.2 +/- 0.3 pg of histamine. The amount of tryptase measured colorimetrically correlated with the level of tryptase measured by radioimmunoassay (Pharmacia), r = 0.92, P < 0.01. The inhibition profile of the proteolytic enzyme measured by the cleavage of BAPNA, was found to be identical to that of authentic lung mast cell tryptase. Over 90% of the maximum tryptase release was complete within 15 min whilst histamine release occurred within 5 min. In cells stimulated with 10 micrograms/ml anti-IgE we found a strong correlation between the release of tryptase and histamine, r = 0.95, P < 0.005. Finally, investigations with various pharmacological agents have supported our initial hypothesis that tryptase would mimic histamine release and provide an alternative marker for mast cell activation. In summary, we have utilised a simple enzymic assay as an indicator of human lung mast cell degranulation. In washed lung mast cells this assay appears be specific for granule tryptase and release of this activity into the supernatants of challenged cells correlates well with the presence of histamine. This assay offers several advantages over current methods of measuring mediator release from human lung mast cells in vitro and should provide an inexpensive and sensitive technique for following mast cell degranulation.

Topics: Benzoquinones; Benzoylarginine Nitroanilide; Cell Degranulation; Chymases; Cinnamates; Colorimetry; Cytoplasmic Granules; Genistein; Histamine Release; Humans; Immunologic Techniques; In Vitro Techniques; Isoflavones; Lactams, Macrocyclic; Lung; Mast Cells; Protein-Tyrosine Kinases; Quinones; Rifabutin; Sensitivity and Specificity; Serine Endopeptidases; Trypsin Inhibitors; Tryptases

Incubation of mouse thymocytes with the protein tyrosine kinase inhibitors herbimycin A and methyl-2,5-dihydroxycinnamate induced a decreased and altered profile of nuclear phosphotyrosine proteins in parallel with an increase in internucleosomal DNA fragmentation and cell death dose-dependently. No change in the profile of cytoplasmic phosphotyrosine proteins was observed. DNA fragmentation was dependent on the synthesis of RNA and protein, suggesting that the inhibition of tyrosine phosphorylation of the nuclear proteins induces apoptosis. DNA fragmentation was enhanced by simultaneous incubation with phorbol esters capable of activating protein kinase C. Genistein, another inhibitor of protein tyrosine kinase, induced DNA fragmentation more rapidly than herbimycin A, but there was no predominant alteration of phosphotyrosine proteins in early incubation, suggesting that genistein may induce apoptosis by a mechanism other than direct inhibition of protein tyrosinekinase activity.

Topics: Animals; Apoptosis; Benzoquinones; Cinnamates; DNA; Dose-Response Relationship, Drug; Genistein; Isoflavones; Lactams, Macrocyclic; Male; Mice; Mice, Inbred BALB C; Nuclear Proteins; Phorbol Esters; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Thymus Gland