thapsigargin and methyl-2-5-dihydroxycinnamate

We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets.

Topics: Actins; Blood Platelets; Calcium; Cinnamates; Cytochalasin D; Cytoskeleton; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Humans; Ions; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins pp60(c-src); ras Proteins; Thapsigargin; Thrombin; Time Factors; Tyrosine

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation.

Topics: Aldosterone; Angiotensin II; Animals; Calcium; Calcium Channel Blockers; Cattle; Cells, Cultured; Cinnamates; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Genistein; Phenols; Protein-Tyrosine Kinases; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Zona Glomerulosa

The effect of two structurally distinct tyrosine kinase inhibitors, genistein (100 microM) and methyl-2, 5-dihydroxycinnamate (25 microM) on ATP- and thapsigargin-induced Ca2+ signals in Fura-2-loaded rat peritoneal macrophages was investigated. Both compounds were shown to inhibit ATP-evoked Ca2+ entry but not to release from internal stores. Both compounds also inhibit the store-dependent or "capacitative" Ca2+ influx stimulated by emptying the intracellular Ca2+ stores with endoplasmic Ca(2+)-ATPase inhibitor thapsigargin (100 nM). Genistein and methyl-2, 5-dihydroxycinnamate have no effect on Ca2+ release from intracellular stores. Tyrosine phosphatase inhibitor orthovanadate Na (50 microM) increases ATP-induced Ca2+ entry but does not prevent the inhibitory effect of genistein. These data are compatible with the role played by tyrosine phosphorylation in the control of Ca2+ entry in rat peritoneal macrophages.

Topics: Adenosine Triphosphate; Animals; Calcium; Cinnamates; Enzyme Inhibitors; Genistein; Isoflavones; Macrophages, Peritoneal; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Thapsigargin; 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

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