ag-213 and methyl-2-5-dihydroxycinnamate

Deoxygenation of sickle (SS) cells causes cationic alterations leading to cell dehydration by various mechanisms, including activation of Ca2+-sensitive K channels and possibly of K-Cl cotransport. Since an abnormal tyrosine kinase (TK) activity exists in SS cells we investigated the possible role of tyrosine phosphorylation in SS cell dehydration. In density-fractionated SS reticulocytes and discocytes, but not in normal red cells, deoxygenation increased membrane and cytosolic TK activities and tyrosine phosphorylation of band 3, independently of external Ca2+. These effects were abolished by the TK inhibitors methyl 2, 5-dihydroxycinnamate (DiOH) or tyrphostin 47 (T47). Deoxygenation-induced Ca2+ uptake was not affected by the inhibitors and Na+ gain was reduced by T47 and not by DiOH. Both inhibitors decreased the loss of K+ and cellular dehydration. The effect of the inhibitors on K+ efflux was still observed in the absence of external Ca2+. These data indicate that the TK inhibitors do not interfere with deoxygenation-induced membrane permeabilization, but affect Ca2+-independent K+ efflux. It cannot be excluded, however, that the TK inhibitors also attenuate Ca2+-sensitive K+ efflux. Based on recent evidence from the literature, it is suggested that the diminution of K+ efflux results in part from inhibition of K-Cl cotransport activity.

Topics: Anemia, Sickle Cell; Biological Transport; Calcium; Catechols; Cinnamates; Enzyme Inhibitors; Erythrocytes; Humans; In Vitro Techniques; Nitriles; Oxygen; Phosphorylation; Potassium; Protein-Tyrosine Kinases; Tyrosine; Tyrphostins

Protein tyrosine kinases (PTKs) of the src family are thought to play an important role in platelet signal transduction, but little is known about the targets of these enzymes in platelets. We determined that exposure of human platelets to pervanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in the activity of phospholipase D (PLD), an enzyme that might be involved in signaling events leading to aggregation or secretion. To further investigate whether tyrosine phosphorylation is a step in the pathway of activation of PLD in response to thrombin, we tested the effects of a series of PTK inhibitors on the activity of platelet PLD. PLD was activated in response to 0.3 U/ml thrombin, and this activation was reduced by several of the PTK inhibitors, especially genistein, methyl 2,5-dihydroxycinnamate (MDHC), ST271, and the tyrphostins A25 and A47. In saponin-permeabilized platelets, we observed a marked inhibition of GTP-gamma S-stimulated PLD by many of the PTK inhibitors, consistent with the possibility that PTKs are involved in the regulation of PLD activity by a G-protein or small GTP-binding protein. MDHC did not affect PLD activity in permeabilized cells, which suggests that this compound might inhibit PLD in intact platelets via another pathway. The inhibitors were also tested for their effects on the phosphorylation of a peptide substrate of src-family PTKs in a platelet membrane preparation and in permeabilized platelets. Several of the compounds partially inhibited peptide phosphorylation in the membrane preparation and in permeabilized platelets, most notably ST271, ST638, and tyrphostin A25.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Blood Platelets; Catechols; Cinnamates; Enzyme Activation; Genistein; GTP-Binding Proteins; Humans; In Vitro Techniques; Isoflavones; Molecular Sequence Data; Nitriles; Peptides; Phospholipase D; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; Styrenes; Substrate Specificity; Thrombin; Tyrphostins; Vanadates

The effect of several tyrosine kinase inhibitors was tested on Ca2+ influx mediated by thapsigargin-and CCh-induced intracellular store depletion. Genestein inhibited Ca2+ influx in a concentration dependent manner without affecting Ca2+ release or Ca2+ pumping activity. A measureable effect was observed at 3 microM with total inhibition of influx seen at 100 microM. Tyrphostin A25 (300 microM; 78% inhibition) and methyl 2,5 dihydroxycinnamate (10 microM; 51% inhibition) also inhibited Ca2+ influx. The degree of attenuation was not markedly altered by preincubation of the inhibitors. Genestein also inhibited Ca2+ influx induced by CCh. These data indicate that inhibition of Ca2+ influx could in part underlie the previously reported inhibition of enzyme secretion by these agents.

Topics: Animals; Barium; Calcium; Carbachol; Catechols; Cinnamates; Genistein; Ion Transport; Isoflavones; Male; Nitriles; Pancreas; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Terpenes; Thapsigargin; Tyrphostins