cytochalasin-d and 1-2-dioctanoylglycerol

Organic anion transport in intact renal proximal tubule cells in animal model systems is downregulated by treatments that activate protein kinase C (PKC). How this downregulation is achieved is not yet known. Stimulation of PKC with sn-1,2-dioctanoylglycerol resulted in strong inhibition of p-aminohippurate transport mediated by the cloned human organic anion transporter 1 (hOAT1) expressed in Xenopus oocytes and HEK293 cells, as well as hOAT1 internalization in both expression systems. The sn-1,2-dioctanoylglycerol-induced transport inhibition was partially prevented by staurosporine. It was independent of the conserved canonical PKC consensus sites in hOAT1, however, and was unaffected by agents that destabilize actin filaments or microtubules, which altered baseline hOAT1-mediated p-aminohippurate uptake activity in oocytes. It is concluded that PKC-induced hOAT1 downregulation is achieved through carrier retrieval from the cell membrane and does not involve phosphorylation of the predicted classic hOAT1 PKC consensus sites.

Topics: Animals; Binding Sites; Biological Transport; Cell Line; Cell Membrane; Cloning, Molecular; Colchicine; Cytochalasin D; Cytoskeleton; Diglycerides; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Humans; Immunohistochemistry; Kinetics; Mutagenesis, Site-Directed; Oocytes; Organic Anion Transport Protein 1; p-Aminohippuric Acid; Phosphorylation; Polymerase Chain Reaction; Protein Kinase C; RNA, Complementary; Staurosporine; Temperature; Time Factors; Xenopus

Spinal cord explants from embryonic day seven (E7) chicken embryos were cultured without serum and in the presence of aprotinine, in a three-dimensional fibrin matrix. These conditions promote a robust, radial, unfasciculated outgrowth of neurites that are tipped by elaborate growth cones. Routinely after 5 days, the neurite outgrowth intensity (NOI) was determined by measuring the optical density of the immunostained neurites (image analysis program OPTIMAS version 5.2) within defined areas, extending radially for up to 3 mm from the explant border. A dose-dependent inhibition of NOI was determined for the cytoskeleton-affecting drugs nocodazole (half maximal inhibition ([I50]), 0.02 microM), taxol ([I50], 0.016 microM), cytochalasin D ([I50], 0.006 microM), and tetramethyl lead ([I50], 0.05 microM). Likewise, NOI was decreased in a dose-dependent fashion by ML-9 and RO-31-8220, inhibitors of myosin-light chain kinase and protein kinase C (PKC), respectively. The addition of 1,2-dioctanoyl-s,n-glycerol, a potent activator of PKC, led, at 5 microM, to an increase and at 30 and 60 microM to a decrease in NOI. The described system provides a rapid, reproducible, and quantitative assay for the effects of exogenous factors on the mode and intensity of neurite outgrowth.

Topics: Actins; Animals; Aprotinin; Azepines; Chick Embryo; Cytochalasin D; Densitometry; Diglycerides; Enzyme Inhibitors; Fibrin; Gels; Indoles; Neurites; Neurotoxins; Nocodazole; Organ Culture Techniques; Paclitaxel; Protein Kinases; Spinal Cord; Tetraethyl Lead; Tubulin

Activation of protein kinase C (PKC) in quiescent Swiss 3T3 cells using either the tumor promoter phorbol 12,13-dibutyrate (PDB) or diacylglycerols increased the tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) by 3.8-fold. PDB stimulation of p125FAK tyrosine phosphorylation was detected within 1 min and reached a maximum within 5 min, considerably slower than PDB stimulation of 80K/MARCKS phosphorylation which was maximal within 1 min. In sharp contrast, bombesin-induced tyrosine phosphorylation of p125FAK reached a maximum (8-fold stimulation) within 1 min after addition of the peptide and occurred with a half-maximal effect of 0.08 nM, 6-fold lower than the half-maximal effect of bombesin on 80K/MARCKS phosphorylation. Down-regulation of PKC by prolonged treatment with PDB blocked the effect of PDB on p125FAK tyrosine phosphorylation but had no effect on the response to bombesin. A selective inhibitor of PKC, GF 109203X, markedly inhibited the stimulation of p125FAK tyrosine phosphorylation by PDB but had little effect on the response to bombesin, vasopressin, and endothelin. Bombesin stimulation of tyrosine phosphorylation could also be dissociated from mobilization of Ca2+ from intracellular stores. Depletion of the intracellular Ca2+ pool by treatment with the tumor promoter thapsigargin completely blocked the ability of bombesin to transiently increase the cytosolic Ca2+ concentration but had no effect on bombesin stimulation of p125FAK tyrosine phosphorylation. In contrast, cytochalasin D, an agent which selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced p125FAK tyrosine phosphorylation. Within the same concentration range (0.3-2 microM), the drug had no effect on other early events stimulated by bombesin, including Ca2+ mobilization and activation of PKC. These findings demonstrate that neither the PKC nor Ca2+ pathways are responsible for the rapid stimulation of p125FAK tyrosine phosphorylation by neuropeptide growth factors. Furthermore, the integrity of the actin cytoskeleton is essential for the effects of both PDB and bombesin.

Topics: 3T3 Cells; Actins; Animals; Bombesin; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Adhesion Molecules; Colchicine; Cytochalasin D; Cytoskeleton; Diglycerides; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Indoles; Intracellular Signaling Peptides and Proteins; Kinetics; Maleimides; Membrane Proteins; Mice; Myristoylated Alanine-Rich C Kinase Substrate; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Terpenes; Thapsigargin; Tyrosine

The cytochalasans, fungal metabolites that interact with actin, can affect lymphocyte proliferation; high concentrations inhibit lectin-induced proliferation and low concentrations augment it. The phorbol ester tumor promoter, PMA, alone is not mitogenic for primary lymphocytes but enhances the activity of mitogenic lectins. Because the cytochalasans have been reported to increase intracellular Ca2+ and because PMA activates protein kinase C, lymphocytes were treated with PMA and cytochalasin B (CyB) to determine if this combination would induce DNA synthesis. While this treatment by itself did not cause proliferation, lymphocytes cultured with PMA and CyB overnight, washed, and recultured with IL-2 proliferated to the same degree as lymphocytes stimulated with Con A. Three different cytochalasans, cytochalasin B, cytochalasin D, and chaetoglobosin C, all of which bind to cellular actin with different affinities and only one of which affects glucose transport, induced IL-2 receptors in combination with PMA. Flow cytometric analysis with an antibody to the IL-2 receptor alpha subunit confirmed the induction of receptors on CD8+ cells. However, no IL-2 was produced after the exposure of lymphocytes to the combination of cytochalasans and PMA. Therefore, there was sufficient signal to induce IL-2 receptor expression but not to induce IL-2.

Topics: Animals; Antibodies, Monoclonal; Cattle; CD8 Antigens; Cells, Cultured; Colchicine; Cycloheximide; Cytochalasin B; Cytochalasin D; Diglycerides; Flow Cytometry; Interleukin-2; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate