methyl-2-5-dihydroxycinnamate and daidzein

The facilitative hexose transporter GLUT1 is a multifunctional protein that transports hexoses and dehydroascorbic acid, the oxidized form of vitamin C, and interacts with several molecules structurally unrelated to the transported substrates. Here we analyzed in detail the interaction of GLUT1 with a group of tyrosine kinase inhibitors that include natural products of the family of flavones and isoflavones and synthetic compounds such as the tyrphostins. These compounds inhibited, in a dose-dependent manner, the transport of hexoses and dehydroascorbic acid in human myeloid HL-60 cells, in transfected Chinese hamster ovary cells overexpressing GLUT1, and in normal human erythrocytes, and blocked the glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts. Kinetic analysis of transport data indicated that only tyrosine kinase inhibitors with specificity for ATP binding sites inhibited the transport activity of GLUT1 in a competitive manner. In contrast, those inhibitors that are competitive with tyrosine but not with ATP failed to inhibit hexose uptake or did so in a noncompetitive manner. These results, together with recent evidence demonstrating that GLUT1 is a nucleotide binding protein, support the concept that the inhibitory effect on transport is related to the direct interaction of the inhibitors with GLUT1. We conclude that predicted nucleotide-binding motifs present in GLUT1 are important for the interaction of the tyrosine kinase inhibitors with the transporter and may participate directly in the binding transport of substrates by GLUT1.

Topics: Adenosine Triphosphate; Animals; Binding, Competitive; CHO Cells; Cinnamates; Cricetinae; Enzyme Inhibitors; Flavonoids; Genistein; Glucose Transporter Type 1; Hexoses; HL-60 Cells; Humans; Isoflavones; meta-Aminobenzoates; Monosaccharide Transport Proteins; Phenols; Protein Binding; Protein-Tyrosine Kinases; Quercetin; Salicylates; Substrate Specificity; Tyrosine; Tyrphostins

We have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen.

Topics: Adenosine Triphosphate; Apyrase; Arachidonic Acid; Blood Platelets; Calcium; Cinnamates; Collagen; Enzyme Activation; Genistein; Humans; Indomethacin; Isoflavones; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Signal Transduction; Tetradecanoylphorbol Acetate; Thrombin

We have investigated the mechanism of Ca2+ entry in fura-2-loaded human platelets using the inhibitors of tyrosine kinases, genistein, and methyl-2,5-dihydroxycinnamate. Genistein (100 microM; 30 min) or methyl-2,5-dihydroxycinnamate (1 microgram/ml; 30 min) reduced ADP-evoked protein-tyrosine phosphorylation at specific bands as assessed by gel electrophoresis and Western blotting with a specific antiphosphotyrosine antibody. Both compounds also reduced ADP-evoked [Ca2+]i rises in the presence, but not the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. The inactive analogue of genistein, daidzein, was without effect on protein-tyrosine phosphorylation or ADP-evoked Ca2+ elevation in the presence or absence of external Ca2+. Methyl-2,5-dihydroxycinnamate (1 microgram/ml; 5 min) significantly reduced the Ca2+ influx evoked by depletion of the intracellular Ca2+ stores using the inhibitor of the endomembranous Ca(2+)-ATPase, thapsigargin. These results with tyrosine kinase inhibitors are unlikely to be the result of the inhibition of other protein kinases since kinases A, C, and G all inhibit agonist-evoked rises in [Ca2+]i in platelets. These data support a role for tyrosine kinases in the control of Ca2+ entry in human platelets.

Topics: Adenosine Diphosphate; Blood Platelets; Blood Proteins; Calcium; Calcium-Transporting ATPases; Cinnamates; Fluorescent Dyes; Fura-2; Genistein; Humans; In Vitro Techniques; Isoflavones; Kinetics; Phosphotyrosine; Protein-Tyrosine Kinases; Terpenes; Thapsigargin; Time Factors; Tyrosine

Platelet activation is associated with the phosphorylation of a number of platelet proteins at tyrosine residues. The significance of this is unknown. Here we have investigated the effects of two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, on thrombin-evoked protein tyrosine phosphorylation and Ca2+ signal generation in fura-2-loaded human platelets. Both compounds inhibited thrombin-evoked tyrosine phosphorylation and reduced the elevation of [Ca2+]i in the presence, but not the absence, of external Ca2+. This suggested a selective inhibition of thrombin-evoked Ca2+ entry but not release from internal stores. Both compounds also reduced thrombin-evoked Mn2+ entry. In contrast, selective blockade of protein kinase C with Ro 31/8220-002 potentiated the thrombin-evoked Ca2+ signal. These data are compatible with a role for protein tyrosine phosphorylation contributing to thrombin-evoked Ca2+ entry in human platelets.

Topics: Biological Transport; Blood Platelets; Blotting, Western; Calcium; Cells, Cultured; Cinnamates; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Genistein; Humans; Indoles; Isoflavones; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Thrombin; Tyrosine