cytochalasin-d and fasudil

Norepinephrine transporter (NET) regulates noradrenergic synaptic transmission by controlling extracellular levels of norepinephrine (NE). The small GTPase, RhoA, and its downstream effector Rho kinase (ROCK) are involved in the regulation of actin cytoskeleton and focal adhesion/stress fiber formation, which may play an important role in various functions of the sympathetic nervous system. We report here the effect of fasudil, a ROCK inhibitor, on the functions of NET in cultured bovine adrenal medullary cells as a model of sympathetic neurons. Treatment of bovine adrenal medullary cells with fasudil caused an increase in [(3)H]NE uptake in time (8-120 h) and concentration (10-100 μM)-dependent manner. Another ROCK inhibitor, Y-27632 (10-100 μM, 1 day), also increased [(3)H]NE uptake by the cells. Kinetics analysis of the effect of fasudil on NE transport showed a significant increase in the V (max) of NE transport with little change in K (m). When both extracellular and intracellular Ca(2+) were removed by the deprivation of extracellular Ca(2+) and BAPTA-AM, a cell-permeable Ca(2+) chelator, [(3)H]NE uptake induced by fasudil was completely abolished. Nocodazole, an inhibitor of microtubule polymerization, but not cytochalasin D, an inhibitor of actin polymerization, suppressed the stimulatory effect of fasudil on [(3)H]NE uptake. The present findings suggest that the ROCK inhibitor fasudil up-regulates NET function in a Ca(2+)-dependent and/or nocodazole-sensitive pathway in adrenal medullary cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenal Medulla; Amides; Animals; Calcium; Cattle; Cells, Cultured; Cytochalasin D; Dose-Response Relationship, Drug; Nocodazole; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Protein Kinase Inhibitors; Pyridines; rho-Associated Kinases; Time Factors; Up-Regulation

Disruption of the actin cytoskeleton elicits profound changes in cell survival and function. The actin cytoskeleton is regulated in a hierarchical manner by Rho GTPases. Rho kinase, a downstream effector of RhoA, regulates the formation of stress fibers and focal adhesions. Disruption of the actin cytoskeleton causes apoptosis in airway epithelial cells. To examine further the relation of cytoskeletal integrity and apoptosis, we tested whether inhibition of Rho kinase would elicit apoptosis in airway epithelial cells. Inhibition with either Y-27632 or HA1077 induced membrane ruffling and loss of actin stress fibers, and apoptosis in airway epithelial cells that was blocked by inhibiting caspase function or by inhibiting protein synthesis. Cells overexpressing constitutively active Rho kinase, but not native Rho kinase, were resistant to Rho kinase inhibitor-induced stress fiber disruption and apoptosis. Inhibition of Rho kinase disrupted actin stress fibers but did not induce apoptosis in 3T3 cells. We demonstrate that Rho kinase inhibition induces airway epithelial cell apoptosis associated with changes in actin filament integrity. Our data suggest that Rho kinase may be a regulator of early initiation of apoptosis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Actins; Amides; Animals; Apoptosis; Caspase Inhibitors; Cell Membrane; Cells, Cultured; Cytochalasin D; Cytoskeleton; Enzyme Inhibitors; Epithelial Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice; Protein Serine-Threonine Kinases; Pyridines; Respiratory Mucosa; rho-Associated Kinases; Stress Fibers

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (<10 nM); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Animals; Bombesin; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Crk-Associated Substrate Protein; Cytochalasin D; Cytoskeletal Proteins; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Immunoblotting; Indoles; Maleimides; Marine Toxins; Mice; Microscopy, Fluorescence; Myosin-Light-Chain Phosphatase; Nucleic Acid Synthesis Inhibitors; Oxazoles; Paxillin; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Retinoblastoma-Like Protein p130; Thiazoles; Thiazolidines; Time Factors

The role of actin organization in occupancy-induced receptor internalization remains poorly defined. Here we report that treatment of mouse Swiss 3T3 cells with latrunculin A, a potent inhibitor of actin polymerization (including cortical actin), inhibited the internalization of the endogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judged by uptake of (125)I-labeled GRP or fluorescent Cy3-labeled bombesin. In contrast, cells pretreated with cytochalasin D showed minimal inhibition of bombesin/GRP receptor internalization. Similarly, pretreatment of Swiss 3T3 cells with the potent Rho-kinase inhibitor HA-1077, at concentrations (10-20 microM) that abrogated bombesin-mediated stress fiber formation, did not significantly alter receptor-mediated internalization of (125)I-GRP. These results indicate that bombesin/GRP receptor internalization depends on latrunculin A-sensitive cortical actin rather than on rapidly turning over actin stress fibers that are disrupted by either cytochalasin D or HA-1077. The rates and total levels of internalization of the endogenously expressed endothelin A receptor and epidermal growth factor receptor were also markedly reduced by latrunculin A in Swiss 3T3 cells. The potency of latrunculin A for inhibiting G protein-coupled receptor endocytosis was comparable to that for reducing internalization of the epidermal growth factor tyrosine kinase receptor. We conclude that cortical actin structures, disrupted by latrunculin A, are necessary for occupancy-induced receptor internalization in animal cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Actins; Animals; Bombesin; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; GTP-Binding Proteins; Iodine Radioisotopes; Kinetics; Mice; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Receptors, Bombesin; Receptors, Endothelin; Signal Transduction; Thiazoles; Thiazolidines