thapsigargin and herbimycin

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.

Topics: Benzoquinones; Bradykinin; Calcium; Calcium Channels; Enzyme Inhibitors; Fibroblasts; Gingiva; Histamine; Humans; Lactams, Macrocyclic; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Receptors, Histamine H1; Rifabutin; Thapsigargin; Time Factors; Tyrosine; Tyrphostins

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

Topics: Antigens, CD; Antigens, Surface; Benzoquinones; Calcium; Calcium Signaling; CD146 Antigen; Cells, Cultured; Chelating Agents; Cytoskeletal Proteins; Egtazic Acid; Endothelium, Vascular; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Lactams, Macrocyclic; Membrane Glycoproteins; Neural Cell Adhesion Molecules; Paxillin; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Quinones; Rifabutin; Signal Transduction; Thapsigargin; Umbilical Veins

Although there is evidence that protein tyrosine kinase inhibitors (PTKIs) suppress bone resorption activity, the mechanism of action of these compounds on osteoclastic bone resorption remains obscure. In the present study, we investigated the effect of PTKIs on cytosolic Ca(2+) concentration ([Ca(2+)](i)) and on the cytoskeleton in rat osteoclasts. The PTKIs, genistein and herbimycin A, reversibly elevated [Ca(2+)](i) measured by fura-2 microfluorimetry. The PTKI-induced increase was abolished by omission of extracellular Ca(2+), but was not attenuated by depletion of Ca(2+) stores. The PTKI-induced increase was inhibited by addition of La(3+) and Ni(2+), but not abolished by dihydropyridine (DHP) Ca(2+) channel blockers. Genistin, an inactive analogue of genistein, had no effect on [Ca(2+)](i). In the cytoskeleton assay, genistein rapidly disrupted the actin ring formation that serves as a marker for the resorbing state of osteoclasts. Disruption of the actin ring formation was also diminished in Ca(2+)-free extracellular solution. These results suggest that PTKIs in rat osteoclasts elevate [Ca(2+)](i) via activation of a DHP-insensitive, nonspecific Ca(2+) entry pathway and disrupt the formation of actin rings, resulting in suppression of bone resorption activity. The regulation of this Ca(2+)-influx by PTKIs is likely to contribute to inhibition of bone resorption by these compounds.

Topics: Actin Cytoskeleton; Actins; Animals; Animals, Newborn; Benzoquinones; Bone Resorption; Calcium; Calcium Channel Blockers; Cells, Cultured; Cytosol; Enzyme Inhibitors; Genistein; Kinetics; Lactams, Macrocyclic; Lanthanum; Microscopy, Fluorescence; Nickel; Osteoclasts; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Wistar; Rifabutin; Thapsigargin

We investigated the effects of vascular endothelial growth factor (VEGF(165)) on [Ca(2+)](i)-transient in cultured lymphatic endothelial cells (LEC) and mechanical activity of isolated dog thoracic ducts. VEGF (0.1-10 ng/ml) caused a dose-dependent increase of the [Ca(2+)](i) in LEC. Pretreatment with 10(-5) M genistein or 5x10(-6) M herbimycin A produced a significant reduction of the VEGF-induced [Ca(2+)](i)-transient. In the presence of 10(-6) M thapsigargin, VEGF caused no significant effect on the [Ca(2+)](i)-transient. Pretreatment with Ca(2+)-free solution containing 0.1 mM EGTA produced no significant effect on the peak increase of [Ca(2+)](i) induced by 0.1 or 10 ng/ml VEGF, but significantly depressed the sustained part of [Ca(2+)](i) observed at the higher concentration of VEGF. The VEGF (0.1-10 ng/ml) caused a significant dilation of the isolated lymph vessels with intact endothelium, which were precontracted with U46,619. The 10 ng/ml VEGF-induced dilation was significantly reduced by 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME). The action of L-NAME was inhibited by the simultaneous application of 10(-3) M L-arginine. Mechanical rubbing of the endothelium also caused significant inhibition of the VEGF-induced dilation. The findings suggest that VEGF(165) may activate the receptor-related tyrosine kinase and cause the release of Ca(2+) from the inositol 1,4, 5-triphosphate-sensitive intracellular Ca(2+) stores in LEC. VEGF(165) also produces endothelium-dependent nitric oxide-mediated dilation of the precontracted isolated lymph vessels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Antibiotics, Antineoplastic; Arginine; Benzoquinones; Biological Transport; Calcium; Cells, Cultured; Chelating Agents; Dogs; Egtazic Acid; Endothelial Growth Factors; Endothelium, Lymphatic; Enzyme Inhibitors; Female; Genistein; Growth Inhibitors; In Vitro Techniques; Lactams, Macrocyclic; Lymph; Lymphokines; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Potassium; Quinones; Rifabutin; Stress, Mechanical; Thapsigargin; Thoracic Duct; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasoconstrictor Agents; Vasodilator Agents

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.

Topics: Benzoquinones; Calcium Signaling; Cell Adhesion; Cell Movement; Estrenes; Humans; Hydrolysis; Immunologic Capping; Inositol 1,4,5-Trisphosphate; Lactams, Macrocyclic; Leukotriene B4; Macrophage-1 Antigen; Monocytes; Phosphatidylinositols; Protein-Tyrosine Kinases; Pyrrolidinones; Quinones; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Rifabutin; Thapsigargin; Type C Phospholipases; U937 Cells

The neutralization of the polyanionic surface of the podocyte by perfusion of kidneys with polycations, such as protamine sulfate, leads to a retraction of podocyte foot processes and proteinuria. This study investigates the effects of protamine sulfate or anionic, neutral, or cationic dextrans on the cytosolic calcium activity ([Ca2+]i) in podocytes.. [Ca2+]i was measured in single cultured differentiated mouse podocytes with the fluorescence dye fura-2/AM.. Protamine sulfate caused a concentration-dependent and partially reversible increase of [Ca2+]i (EC50 approximately 1.5 micromol/liter). Pretreatment of the cells with heparin (100 U/liter) inhibited the protamine sulfate-mediated increase of [Ca2+]i. Like protamine sulfate, diethylaminoethyl dextran (DEAE-dextran) concentration dependently increased [Ca2+]i in podocytes (EC50 approximately 20 nmol/liter), whereas dextran sulfate or uncharged dextran (both 10 micromol/liter) did not influence [Ca2+]i. A reduction of the extracellular Ca2+ concentration (from 1 mmol/liter to 1 micomol/liter) partially inhibited the protamine sulfate and the DEAE-dextran-induced [Ca2+]i response. Flufenamate (100 micromol/liter) or Gd3+ (10 micromol/liter), which are known to inhibit nonselective ion channels, did not influence the [Ca2+]i increase induced by protamine sulfate. In the presence of thapsigargin (50 nmol/liter), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, both protamine sulfate and DEAE-dextran increased [Ca2+]i.. The data indicate that polycations increase podocyte [Ca2+]i. The increase of [Ca2+]i may be an early event in the pathogenesis of protamine sulfate-mediated retraction of podocyte foot processes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Animals; Benzoquinones; Calcium Signaling; Cells, Cultured; DEAE-Dextran; Heparin; Kidney Glomerulus; Lactams, Macrocyclic; Marine Toxins; Mice; Muramidase; Oxazoles; Protamines; Quinones; Rifabutin; Staurosporine; Thapsigargin

The insulin receptor is a tyrosine kinase receptor that is found in mammalian brain and at high concentrations in the bag cell neurons of Aplysia. We show here that insulin causes an acute rise in intracellular Ca2+ concentration ([Ca2+]i) in these neurons and triggers release of neuropeptide. The insulin-sensitive intracellular Ca2+ pool differs pharmacologically from previously described Ca2+ stores that are sensitive to inositol trisphosphate and from mitochondrial Ca2+ stores. Insulin, but not thapsigargin, stimulates Ca2+ release at the distal tips of neurites, the presumed site of neuropeptide secretion. The effects of insulin on intracellular Ca2+ release and neuropeptide secretion occur without triggering spontaneous action potentials. The insulin-sensitive rise in [Ca2+]i moves into the distal tips of neurites after exposure to a cyclic AMP analogue, a treatment that causes a similar translocation of neuronal vesicles. Our data indicate that Ca2+ release from a distinct intracellular pool associated with secretory vesicles may contribute to secretion of neuropeptide in the absence of neuronal discharge.

Topics: Animals; Benzoquinones; Calcium; Calcium Channels; Cells, Cultured; Cyclic AMP; Endoplasmic Reticulum; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Insulin; Invertebrate Hormones; Lactams, Macrocyclic; Neurons; Neuropeptides; Protein-Tyrosine Kinases; Quinones; Receptors, Cytoplasmic and Nuclear; Rifabutin; Thapsigargin

Leukotriene D4 (LTD4) has been found to induce calcium signalling in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the gamma-subtype (PLC-gamma). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD4 receptor. The tyrosine kinase inhibitors genistein and herbimycin. A both reduced the LTD4-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. In accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular mobilisation of calcium, which was supported by the findings that LTD4 was able to induce an increase in the tyrosine phosphorylation of PLC-gamma even when one of the tyrosine kinase inhibitors was present. Of possible interest regarding the effect of genistein on LTD4-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-gamma antibodies from LTD4-stimulated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD4-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD4. Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD4-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs downstream of PLC-gamma activation and is directly implicated in the regulation of agonist-mediated calcium influx.

Topics: Benzoquinones; Calcium; Cell Line; Cytosol; Enzyme Activation; Epithelium; Genistein; GTP-Binding Proteins; Humans; Ileum; Intestinal Mucosa; Isoenzymes; Isoflavones; Jejunum; Lactams, Macrocyclic; Leukotriene D4; Phospholipase C gamma; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Terpenes; Thapsigargin; Type C Phospholipases

Perturbation of the T cell antigen-specific receptor leads to a series of signaling events that includes a rapid increase in phosphoinositide hydrolysis, intracellular Ca2+, and tyrosine phosphorylation. We have examined the function of tyrosine phosphorylation in isolation by introducing the v-src tyrosine kinase into a T cell hybridoma. T cell receptor-mediated increases in phosphoinositide hydrolysis and, in particular the generation of inositol 1,4,5-trisphosphate, were comparable between v-src+ and v-src- cells. Unexpectedly, the v-src+ cells exhibited spontaneously elevated intracellular Ca2+ and exaggerated Ca2+ increases when stimulated via the T cell receptor. The enhanced Ca2+ response was not due to tyrosine phosphorylation of the T cell receptor itself, since the phenotype was evident in T cell receptor zeta chain-/v-src+ cells as well. These results demonstrate that an active protein tyrosine kinase can markedly affect intracellular Ca2+ handling by a process independent of inositol 1,4,5-trisphosphate production and T cell receptor tyrosine phosphorylation and raise the possibility that tyrosine kinases may directly regulate T cell receptor-mediated changes in intracellular Ca2+.

Topics: Animals; Benzoquinones; Calcium; Cations, Divalent; Columbidae; Fluorescent Antibody Technique; Hybridomas; Hydrolysis; Inositol Phosphates; Lactams, Macrocyclic; Oncogene Protein pp60(v-src); Phosphorylation; Protein-Tyrosine Kinases; Quinones; Receptors, Antigen, T-Cell; Rifabutin; Signal Transduction; T-Lymphocytes; Terpenes; Thapsigargin