okadaic-acid and decamethrin

Voltage-dependent Kv2.1 K(+) channels, which mediate delayed rectifier Kv currents (I(K)), are expressed in large clusters on the somata and dendrites of principal pyramidal neurons, where they regulate neuronal excitability. Here we report activity-dependent changes in the localization and biophysical properties of Kv2.1. In the kainate model of continuous seizures in rat, we find a loss of Kv2.1 clustering in pyramidal neurons in vivo. Biochemical analysis of Kv2.1 in the brains of these rats shows a marked dephosphorylation of Kv2.1. In cultured rat hippocampal pyramidal neurons, glutamate stimulation rapidly causes dephosphorylation of Kv2.1, translocation of Kv2.1 from clusters to a more uniform localization, and a shift in the voltage-dependent activation of I(K). An influx of Ca(2+) leading to calcineurin activation is both necessary and sufficient for these effects. Our finding that neuronal activity modifies the phosphorylation state, localization and function of Kv2.1 suggests an important link between excitatory neurotransmission and the intrinsic excitability of pyramidal neurons.

Topics: Animals; Animals, Newborn; Blotting, Western; Cadmium Chloride; Calcimycin; Calcium Channel Blockers; Cell Count; Cells, Cultured; Cyclosporine; Delayed Rectifier Potassium Channels; Dendrites; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Ion Channel Gating; Ionophores; Kainic Acid; Membrane Potentials; Neuronal Plasticity; Nitrendipine; Nitriles; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Phosphorylation; Potassium; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Potassium Chloride; Pyramidal Cells; Pyrethrins; Rats; Seizures; Shab Potassium Channels; Time Factors; Translocation, Genetic

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses.

Topics: Animals; Benzylamines; Calcineurin; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cyclic AMP; Cyclic GMP; Cyclosporine; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Embryo, Mammalian; Embryo, Nonmammalian; Enzyme Inhibitors; Growth Cones; Humans; Models, Neurological; Nerve Growth Factors; Netrin-1; Neurons; Nitriles; Okadaic Acid; Phosphoprotein Phosphatases; Photolysis; Protein Phosphatase 1; Pyrans; Pyrethrins; Semaphorin-3A; Spinal Cord; Spiro Compounds; Sulfonamides; Time Factors; Tumor Suppressor Proteins; Xenopus

Disabled-2 (Dab2; also known as p96 and DOC-2) is a signal transduction protein that has been implicated in the control of cell growth. Dab2 is known to be a phosphoprotein, but little is known about the kinases that phosphorylate Dab2. We have found that Dab2 phosphorylation is markedly increased during the mitosis phase of the cell cycle. This phosphorylation is blocked by roscovitine, a selective inhibitor of cyclin-dependent kinases. Dab2 robustly coimmunoprecipitates from cells with the cyclin-dependent kinase cdc2, and purified cdc2 can phosphorylate purified Dab2 fusion proteins in vitro on multiple sites. Cellular phosphorylation of Dab2 by cdc2 promotes the association of Dab2 with Pin1, a peptidylprolyl isomerase that regulates the rate of Dab2 dephosphorylation. These findings reveal that Dab2 is differentially phosphorylated during the cell cycle by cdc2 and provide a potential feedback mechanism by which Dab2 inhibition of cell growth and proliferation may be regulated.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; Calcineurin; Calcineurin Inhibitors; CDC2 Protein Kinase; Cell Cycle; Cells, Cultured; Enzyme Inhibitors; Genes, Tumor Suppressor; HeLa Cells; Humans; Mitosis; NIMA-Interacting Peptidylprolyl Isomerase; Nitriles; Okadaic Acid; Peptidylprolyl Isomerase; Phosphoprotein Phosphatases; Phosphorylation; Proteins; Pyrethrins; Tumor Suppressor Proteins

Protein phosphatase 2A (PP2A) is a key signal transduction intermediate in the regulation of cellular proliferation and differentiation in vitro. However, the role of PP2A in the context of a developing organ is unknown. To explore the role of PP2A in the regulation of lung development, we studied the effect of PP2A inhibition on new airway branching, induction of apoptosis, DNA synthesis, and expression of epithelial marker genes in whole organ explant cultures of embryonic (E14) rat lung. Microdissected lung primordia were cultured in medium containing one of either two PP2A inhibitors, okadaic acid (OA, 0-9 nM) or cantharidin (Can, 0-3,600 nM), or with the PP2B inhibitor deltamethrin (Del, 0-10 microM) as a control for a PP2A-specific effect for 48 h. PP2A inhibition with OA and Can significantly inhibited airway branching and overall lung growth. PP2B inhibition with Del did not affect lung growth or new airway development. Histologically, both PP2A- and PP2B-inhibited explants were similar to controls. Increased apoptosis was not the mechanism of decreased lung growth and new airway branching inasmuch as OA-treated explant sections subjected to the terminal deoxynucleotidyltransferase dUTP nick end labeling reaction demonstrated a decrease in apoptosis. However, PP2A inhibition with OA increased DNA content and 5-bromo-2'-deoxyuridine uptake that correlated with a G(2)/M cell cycle arrest. PP2A inhibition also resulted in altered differentiation of the respiratory epithelium as evidenced by decreased mRNA levels of the early epithelial marker surfactant protein C. These findings suggest that inhibition of protein phosphatases with OA and Can halted mesenchymal cell cycle progression and reduced branching morphogenesis in fetal rat lung explant culture.

Topics: Animals; Antimetabolites; Apoptosis; Blotting, Northern; Bromodeoxyuridine; Calcineurin; Calcineurin Inhibitors; Cantharidin; Cell Differentiation; Cell Division; Cells, Cultured; Enzyme Inhibitors; Fetus; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Insecticides; Lung; Nitriles; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Proteolipids; Pulmonary Surfactants; Pyrethrins; Rats; Respiratory Mucosa; RNA, Messenger

Type II pyrethroid insecticides have been reported to be potent inhibitors of bovine brain calcineurin (EC 3.1.3.16, Enan E and Matsumara F, Biochem Pharmacol 43: 1777-1784, 1992). In concentrations up to 10(-5) M, none of the pyrethroid insecticides used in this study caused inhibition of the calcineurin-dependent dephosphorylation of the 19-amino acid phosphopeptide derived from the regulatory subunit R-II of the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase, which has been established as a good substrate for this enzyme. Neither did any of the compounds tested cause a shift in the inhibitory activity of okadaic acid (apparent Ki of 5 microM). The assumption that calcineurin is generally inhibited by pyrethroid insecticides is incorrect, and the interpretation of cellular experiments in which this assumption has been made must be revised.

Topics: Calcineurin; Calmodulin-Binding Proteins; Enzyme Inhibitors; Insecticides; Nitriles; Okadaic Acid; Phosphopeptides; Phosphoprotein Phosphatases; Pyrethrins