phalloidine and fasudil

Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting.. Intracellular calcium concentration ([Ca(2+)](i)) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[(3)H]inositol- (0.5 μCi/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used.. Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca(2+)](i), cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the G(i) inhibitor pertussis toxin. Agonists potently stimulated migration of T24 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration.. G protein-coupled receptors potently stimulated cell migration in T24 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Amides; Bacterial Proteins; Bacterial Toxins; Calcium; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Movement; Fura-2; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Lysophospholipids; Mutation; Pertussis Toxin; Phalloidine; Protein Kinase Inhibitors; Pyridines; rho GTP-Binding Proteins; rho-Associated Kinases; Type C Phospholipases

To understand the role of Rho kinases in regulation of astrocyte morphology, we investigated the effects of Rho kinase inhibitors on the morphology of cultured rat cortical astrocytes. Cultured astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but changed into process-bearing stellate cells following treatment with the selective Rho kinase inhibitor Y-27632 (1-10 microM). The Y-27632-induced astrocyte stellation was abolished by treatment with colchicine, indicating that the response requires reorganization of cytoskeletal elements. The effect of Y-27632 was mimicked by another Rho kinase inhibitor HA1077, but not by the protein kinase C inhibitor GF-109203X or the protein kinase A inhibitor KT5720. These results suggest that Rho kinases are in an activated state in the absence of stimuli and contribute to the maintenance of polygonal morphology of cultured astrocytes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Amides; Animals; Astrocytes; Carbazoles; Cell Size; Cells, Cultured; Cerebral Cortex; Colchicine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Gout Suppressants; Indoles; Intracellular Signaling Peptides and Proteins; Maleimides; Phalloidine; Protein Serine-Threonine Kinases; Pyridines; Pyrroles; Rats; rho-Associated Kinases

Cell migration and cytokinesis require reorganization of the cytoskeleton, involving phosphorylation and dephosphorylation of proteins such as myosin II and moesin. Myosin and moesin bind directly to a regulatory subunit of myosin/moesin phosphatase (MMP) that contains a protein type-1 phosphatase (PP1) catalytic subunit. Here we examined the role of MMP in cytoskeletal dynamics using a phosphorylation-dependent inhibitor protein specific for MMP, called CPI-17. Fibroblasts do not express CPI-17, making them a null background to study effects of expression. Wild type CPI-17 in rat embryo fibroblasts caused (1) abnormal accumulation of cortical F-actin fibers, distinct from the stress fibers induced by expression of active RhoA; (2) progressive contraction of cell area, leaving behind filamentous extensions that stained for F-actin and moesin, but not myosin; and (3) significantly retarded spreading of fibroblasts on fibronectin with elevated myosin II light chain phosphorylation. A phosphorylation site mutant CPI-17(T38A) and inhibitor-2 (Inh2), another PP1-specific inhibitor protein, served as controls and did not elicit these same responses when expressed at the same level as CPI-17. Inhibition of myosin light chain kinase by ML-9 prevented the abnormal accumulation of cortical microfilaments by CPI-17, but did not reverse shrinkage in area, whereas kinase inhibitors HA1077 and H7 prevented CPI-17-induced changes in microfilament distribution and cell contraction. These results highlight the physiological importance of myosin/moesin phosphatase regulation to dynamic remodeling of the cytoskeleton.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actin Cytoskeleton; Actins; Animals; Cell Size; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Fibronectins; Gene Expression; Muscle Proteins; Myosin Light Chains; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase; Phalloidine; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Plasmids; Rats