calcimycin and mastoparan

Exposure of tri-n-butyl tin chloride (TBTCl) as a stressor to Euglena gracilis Z causes rapid alteration of cell morphology followed by deflagellation. The present study was undertaken to reveal the mechanism of the cell response at a molecular level. Chlamydomonas reinhardtii, in this study E. gracilis Z and its achlorophyllous mutant SM-ZK, gave similar results when subject to the same stressor. Indeed, similar results were obtained with both strains. Next, assuming that the morphological alteration caused by TBTCl is mediated by the inositide phosphate-lipid signaling pathway, the effects of signal transduction and Ca2+ release reagents (mastoparan as a G-protein activator, neomycin as a phospholipase C inhibitor, verapamil as a Ca2+ channel blocker, and A23187 as a Ca2+ ionophore) on morphology and intracellular Ca2+ levels were examined with or without TBTCl. The data strongly suggest that the morphological alteration is mediated by an increase in Ca2+ linked to the inositol phosphatide pathway. The cellular response to signal transduction inducing reagents was compared between the E. gracilis chlorophyllous Z strain and its achlorophyllous mutant SM-ZK strain. Significant differences were observed between the Z and SM-ZK strains in terms of the stress response and intracellular Ca2+ level.

Topics: Animals; Calcimycin; Calcium; Chlorophyll; Cytosol; Enzyme Inhibitors; Euglena gracilis; Flagella; Intercellular Signaling Peptides and Proteins; Mutation; Neomycin; Peptides; Phosphatidylinositols; Signal Transduction; Trialkyltin Compounds; Verapamil; Wasp Venoms; Water Pollutants, Chemical

The Ascidiacea, the invertebrate chordates, includes three orders; the Stolidobranchia is the most complex. Until the present study, the onset of oocyte maturation (germinal vesicle breakdown) had been investigated in only a single pyurid (Halocynthia roretzi), in which germinal vesicle breakdown (GVBD) begins when the oocyte contacts seawater (SW); nothing was known about internal events. This study strongly suggests the importance of protein phosphorylation in this process. Herdmania pallida (Pyuridae) functions like H. roretzi; GVBD occurs in SW. Oocytes of Cnemidocarpa irene (Styelidae) do not spontaneously undergo GVBD in SW but must be activated. Herdmania oocytes are inhibited from GVBD by pH 4 SW and subsequently activated by mastoparan (G-protein activator), A23187 (Ca2+ ionophore) or dimethylbenzanthracene (tyrosine kinase activator). This requires maturation promoting factor (MPF) activity; cyclin-dependent kinase inhibitors roscovitine and olomoucine are inhibitory. It also entails dephosphorylation as demonstrated by the ability of the phosphatase inhibitor vitamin K3 to inhibit GVBD. GVBD is also inhibited by the tyrosine kinase inhibitors tyrphostin A23 and genistein, and LY-294002, a phosphatidylinositol-3-kinase inhibitor previously shown to inhibit starfish GVBD. LY-294002 inhibits strongly when activation is by mastoparan or ionophore but not when activated by dimethylbenzanthracene (DMBA). The DMBA is hypothesized to phosphorylate a phosphatase directly or indirectly causing secondary activation, bypassing inhibition.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcimycin; Chromones; Enzyme Activation; Female; Hydrogen-Ion Concentration; Intercellular Signaling Peptides and Proteins; Maturation-Promoting Factor; Morpholines; Oocytes; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Seawater; Signal Transduction; Trypsin; Urochordata; Wasp Venoms

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.

Topics: Acetates; Calcimycin; Calcium; Cells, Cultured; Cholera Toxin; Cupressus; Cyclopentanes; Egtazic Acid; Fungi; GTP Phosphohydrolases; GTP-Binding Proteins; Hydrogen Peroxide; Intercellular Signaling Peptides and Proteins; Lanthanum; Lipoxygenase; Monoterpenes; Oxylipins; Peptides; Plant Growth Regulators; Signal Transduction; Suramin; Tropolone; Verapamil; Wasp Venoms

The mitochondrion has emerged as a key regulator of apoptosis, a form of animal programmed cell death (PCD). The mitochondrial permeability transition (MPT), facilitated by a pore-mediated, rapid permeability increase in the inner membrane, has been implicated as an early and critical step of apoptosis. Victorin, the host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats, has been demonstrated to bind to the mitochondrial P-protein and also induces a form of PCD. Previous results suggest that a MPT may facilitate victorin's access to the mitochondrial matrix and binding to the P-protein: (i) victorin-induced cell death displays features similar to apoptosis; (ii) in vivo, victorin binds to the mitochondrial P-protein only in toxin-sensitive genotypes whereas victorin binds equally well to P-protein isolated from toxin-sensitive and insensitive oats; (iii) isolated, untreated mitochondria are impermeable to victorin. The data implicate an in vivo change in mitochondrial permeability in response to victorin. This study focused on whether oat mitochondria can undergo a MPT. Isolated oat mitochondria demonstrated high-amplitude swelling when treated with spermine or Ca2+ in the presence of the Ca2+-ionophore A23187, and when treated with mastoparan, an inducer of the MPT in rat liver mitochondria. In all cases, swelling demonstrated size exclusion in the range 0.9-1.7 kDa, similar to that found in animal mitochondria. Further, MPT-inducing conditions permitted victorin access to the mitochondrial matrix and binding to the P-protein. In vivo, victorin treatment induced the collapse of mitochondrial transmembrane potential within 2 h, indicating a MPT. Also, the victorin-induced collapse of membrane potential was clearly distinct from that induced by uncoupling respiration, as the latter event prevented the victorin-induced PCD response and binding to P-protein. These results demonstrate that a MPT can occur in oat mitochondria in vitro, and are consistent with the hypothesis that an MPT, which allows victorin access to the mitochondrial matrix and binding to the P-protein, occurs in vivo during victorin-induced PCD.

Topics: Alamethicin; Apoptosis; Avena; Biological Transport; Calcimycin; Calcium; Cytochrome c Group; Fungal Proteins; Intercellular Signaling Peptides and Proteins; Ion Channels; Ionophores; Magnesium; Manganese; Membrane Potentials; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mycotoxins; Peptides; Protein Binding; Spermine; Wasp Venoms

Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A(2) activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Cell Polarity; Cyclic AMP-Dependent Protein Kinases; Cyclohexanones; Cyclooxygenase Inhibitors; Dogs; Endocytosis; Enzyme Activation; Enzyme Inhibitors; Heterotrimeric GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Ionomycin; Lipoprotein Lipase; Lipoxygenase Inhibitors; Organophosphonates; Peptides; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Kinase C; Ricin; Signal Transduction; Tetradecanoylphorbol Acetate; Wasp Venoms

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

Topics: 2,4-Dinitrophenol; Adenosine Diphosphate Ribose; Androstadienes; Animals; Bacterial Proteins; Bacterial Toxins; Calcimycin; Carbachol; Cattle; Cell Line; Clostridioides difficile; Cytoplasmic Granules; Dinitrophenols; Enzyme Inhibitors; Glucosyltransferases; Intercellular Signaling Peptides and Proteins; Kinetics; Leukemia, Basophilic, Acute; Peptides; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, IgE; Serotonin; Serum Albumin, Bovine; Tritium; Tumor Cells, Cultured; Wasp Venoms; Wortmannin

To elucidate the regulatory pathway through which pancreastatin inhibits insulin secretion, RINm5F insulinoma cells were challenged with physiological and pharmacological probes known to stimulate insulin release through different mechanisms. Utilizing the electrophysiological technique of capacitance measurements as a correlate to exocytosis, pancreastatin was found to significantly diminish maximum capacitance changes evoked by glyceraldehyde, an effect which was attenuated in pertussis toxin-treated cells. In static incubations of this cell line, pancreastatin significantly inhibited insulin secretion stimulated by glyceraldehyde, carbachol and A23187, secretagogues known to directly elevate beta-cell cytosolic Ca2+. This peptide also inhibited insulin secretion stimulated by phorbol myristate acetate (PMA), but only at incubation times < or = 15 min. It was without effect on insulin secretion stimulated by mastoparan and longer incubations (30 min) with PMA, where the secretory mechanisms are not necessarily Ca(2+)-dependent. Additionally, pancreastatin had no effect on carbachol-generated inositol phosphate accumulation but inhibited simultaneously stimulated insulin secretion. All inhibitory effects of pancreastatin were pertussis toxin sensitive. These results suggest that pancreastatin inhibits insulin secretion in RINm5F cells through a G-protein regulated mechanism at a control point involved in the Ca(2+)-directed exocytotic machinery, a feature shared by other physiologic inhibitors of insulin secretion.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Carbachol; Chromogranin A; Exocytosis; Glyceraldehyde; GTP-Binding Proteins; Inositol Phosphates; Insulin; Insulin Secretion; Insulinoma; Intercellular Signaling Peptides and Proteins; Pancreatic Hormones; Pancreatic Neoplasms; Peptides; Pertussis Toxin; Phorbol Esters; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

The acrosome reaction is a specialized exocytotic process. In the mouse there is compelling evidence that receptor-mediated activation of GTP-binding proteins by factors in the zona pellucida of oocytes is a central event in the acrosome reaction. Several reagents are able to affect GTP-binding proteins directly, bypassing the receptor-ligand step for activation. We have assessed the effect of several of these compounds on human spermatozoa, monitoring cell vitality and the acrosome reaction simultaneously using the triple-stain technique. GTP gamma S and aluminium fluoride complexes promote sperm activation very efficiently; amphiphilic peptides capable of activating G(o) and Gi, also elicit the acrosome reaction. The results indicate that activation of heterotrimeric GTP-binding proteins is sufficient to trigger acrosome exocytosis in human spermatozoa.

Topics: Acrosome; Aluminum Compounds; Amino Acid Sequence; Calcimycin; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intercellular Signaling Peptides and Proteins; Male; Molecular Sequence Data; Peptides; Reference Values; Spermatozoa; Wasp Venoms

Desensitization induced by challenge of mast cells with antigen is specific for IgE-dependent signals. During the secretory process mast cells release adenosine, which can induce a desensitization of adenosine receptors. To determine whether adenosine receptors may be desensitized from a previous antigen challenge, mast cells were sensitized with anti-DNP IgE antibody, challenged with DNP-BSA antigen, returned to culture overnight, resensitized, and rechallenged. Previously challenged cells exhibited increased spontaneous beta-hexosaminidase release, but adenosine retained its ability to augment beta-hexosaminidase release. Adenosine enhanced A23187-stimulated release of beta-hexosaminidase in control and previously challenged cells. Leukotriene C4 generation followed a similar pattern. Mastoparan, a direct G protein activator and mast cells secretagogue, produced a doubling of beta-hexosaminidase release in previously challenged cells. Results using other G protein activators were equivocal. Degranulation alone is insufficient to induce adenosine receptor hyposensitization. Whether the hyperresponsiveness to mastoparan is a consequence of uncoupling of IgE receptors from G proteins remains uncertain.

Topics: Adenosine; Alkaloids; Animals; beta-N-Acetylhexosaminidases; Bone Marrow Cells; Calcimycin; Cells, Cultured; Desensitization, Immunologic; GTP-Binding Proteins; Immunoglobulin E; Indicators and Reagents; Intercellular Signaling Peptides and Proteins; Leukotriene C4; Mast Cells; Mice; Mice, Inbred BALB C; Peptides; Protein Kinase C; Receptors, Purinergic P1; Serum Albumin, Bovine; Staurosporine; Wasp Venoms

Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.

Topics: Alkaloids; Antibodies, Anti-Idiotypic; B-Lymphocytes; Burkitt Lymphoma; Calcimycin; Cyclic AMP; Diglycerides; Herpesvirus 4, Human; Humans; Immunoglobulin G; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Peptides; Protein Kinase C; Receptors, Antigen, B-Cell; Signal Transduction; Staurosporine; Trifluoperazine; Tumor Cells, Cultured; Virus Replication; Wasp Venoms