cytochalasin-d and mevastatin

Salmonella Typhimurium DT104 is an emerging enteric pathogen in swine of increasing medical importance. In this study, the time course and the actin-dependent host signaling processes necessary for invasion of a S. Typhimurium DT104 field isolate were investigated in IPEC J2 epithelial cells derived from porcine small intestine. Internalized bacteria were quantified by a gentamicin resistance assay. DT104 internalization into epithelial monolayers increased steadily between 15 and 120min after apical inoculation. Internalization was reduced by the Rho GTPase inhibitor mevastatin, the N-WASP inhibitor wiskostatin and the actin-disrupting agent cytochalasin D, but not the Rac1 GTPase inhibitor NSC-23766. Early DT104 invasion of porcine enterocytes appears to be mediated by Rac1 GTPase-independent changes in epithelial actin assembly.

Topics: Animals; Carbazoles; Cell Line; Colony Count, Microbial; Cytochalasin D; Electric Impedance; Epithelial Cells; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intestine, Small; Lovastatin; Nucleic Acid Synthesis Inhibitors; Propanolamines; Salmonella Infections, Animal; Salmonella typhimurium; Swine; Swine Diseases; Time Factors

The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine-M to SH-SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t(1/2) = 2 min) and paxillin that were independent of integrin-extracellular matrix interactions, cell attachment, and the production of phosphoinositide-derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY-294002, under conditions in which the synthesis of phosphatidylinositol 4-phosphate was markedly attenuated. These results indicate that muscarinic receptor-mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH-SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.

Topics: Actins; Androstadienes; Cell Adhesion; Cell Adhesion Molecules; Chromones; Cytochalasin D; Cytoskeletal Proteins; Cytoskeleton; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; GTP-Binding Proteins; Humans; Lovastatin; Morpholines; Muscarinic Agonists; Neuroblastoma; Nucleic Acid Synthesis Inhibitors; Paxillin; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Protein-Tyrosine Kinases; Receptor, Insulin; Receptors, Muscarinic; Second Messenger Systems; Tumor Cells, Cultured; Tyrosine; Wortmannin

We have previously shown that the leukotriene D4 (LTD4)-induced mobilization of intracellular Ca2+ in epithelial cells is mediated by a G-protein that is distinctly different from the pertussis toxin-sensitive G-protein that regulates the subsequent influx of Ca2+. In the present study, we attempted to gain further knowledge about the mechanisms involved in the LTD4-induced mobilization of intracellular Ca2+ in epithelial cells by investigating the effects of compactin, an inhibitor of the isoprenylation pathway, on this signalling event. In cells preincubated with 10 microM compactin for 48 h, the LTD4-induced mobilization of intracellular Ca2+ was reduced by 75% in comparison with control cells. This reduction was reversed by co-administration of mevalonate (1 mM). The effect of compactin occurred regardless of whether or not Ca2+ was present in the extracellular medium, suggesting that isoprenylation must occur before Ca2+ is released from intracellular stores. In accordance with this, we also found that both the LTD4-induced formation of inositol 1,4,5-trisphosphate and the LTD4-induced phosphorylation of phospholipase C gamma 1 (PLC gamma 1) on tyrosine residues were significantly reduced in compactin-pretreated cells. These results open up the possibility that the activation of PLC gamma 1 is related to a molecule that is sensitive to impaired activity of the isoprenylation pathway, such as a small monomeric G-protein. This idea was supported by the observation that Clostridium botulinum C3 exoenzyme-induced inhibition of Rho proteins abolished the LTD4-induced intracellular mobilization of Ca2+. A regulatory role of Rho proteins in the LTD4-induced activation of PLC gamma 1 is unlikely to be indirectly mediated via an effect on the cytoskeleton, since cytochalasin D had no major effect on the LTD4-induced mobilization of Ca2+. Although the mechanism of interaction remains to be elucidated, the present findings indicate an important role of an isoprenylated protein such as Rho in the LTD4-induced Ca2+ signal.

Topics: ADP Ribose Transferases; Botulinum Toxins; Calcium; Cell Line; Cytochalasin D; Cytosol; Enzyme Inhibitors; Epithelium; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Intestines; Isoenzymes; Kinetics; Leukotriene D4; Lovastatin; Membrane Proteins; Mevalonic Acid; Protein Prenylation; rhoB GTP-Binding Protein; Signal Transduction; Type C Phospholipases