valinomycin and Neuroblastoma

Neuritogenesis is essential in establishing the neuronal circuitry. An important intracellular signal causing neuritogenesis is cAMP. In this report, we showed that an increase in intracellular cAMP stimulated neuritogenesis in neuroblastoma N2A cells via a PKA-dependent pathway. Two voltage-gated K(+) (Kv) channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA), inhibited cAMP-stimulated neuritogenesis in N2A cells in a concentration-dependent manner that remarkably matched their ability to inhibit Kv currents in these cells. Consistently, siRNA knock down of Kv1.1, Kv1.4, and Kv2.1 expression reduced Kv currents and inhibited cAMP-stimulated neuritogenesis. Kv1.1, Kv1.4, and Kv2.1 channels were expressed in the cell bodies and neurites as shown by immunohistochemistry. Microfluorimetric imaging of intracellular [K(+)] demonstrated that [K(+)] in neurites was lower than that in the cell body. We also showed that cAMP-stimulated neuritogenesis may not involve voltage-gated Ca(2+) or Na(+) channels. Taken together, the results suggest a role of Kv channels and enhanced K(+) efflux in cAMP/PKA-stimulated neuritogenesis in N2A cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium Channels; Cell Differentiation; Cell Line, Tumor; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Silencing; Intracellular Space; Ion Channel Gating; Mice; Neurites; Neuroblastoma; Neurogenesis; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Protein Kinase Inhibitors; Sodium Channels; Valinomycin

The effects of several K(+)-selective neutral ionophores on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells were examined. Specifically, alterations in membrane resting potential (V(m)), input resistance (R(in)) and electrically-induced action potential generation were determined upon bath application of enniatin (0.1-10 microg/ml), nonactin (0. 1-10 microM) and valinomycin (0.1-10 microM). Although some cells exhibited a slight hyperpolarization and/or reduced R(in), i.e. membrane electrical correlates of enhanced K(+) loss, neither V(m) nor R(in) were significantly altered by any of the ionophores. However, valinomycin and especially nonactin affected action potentials induced by electrical stimulation. This was apparent in the ablation of action potentials in some cells and in the occurrence of degenerative changes in action potential shape in others. The simultaneous administration of the neutral ionophores and the protonophore CCCP or the superfusion of enniatin, nonactin or valinomycin in high (50 mM) glucose-containing physiological solution did not yield more extensive alterations in V(m) or R(in). These data suggest that the neutral ionophores are unable to materially enhance K(+) flux above the relatively high resting level in NG108-15 cells. Thus, alterations in action potentials appear to be unrelated to K(+) transport activity.

Topics: Action Potentials; Analysis of Variance; Animals; Anti-Bacterial Agents; Cell Membrane; Depsipeptides; Dose-Response Relationship, Drug; Electric Stimulation; Glioma; Gramicidin; Hybrid Cells; Ion Transport; Ionophores; Macrolides; Membrane Potentials; Mice; Neuroblastoma; Neurons; Nigericin; Peptides; Potassium; Rats; Tumor Cells, Cultured; Valinomycin

Both lithium and valproate have been used in the treatment of manic-depressive illness with very limited understanding of their therapeutic mechanism of action. Recent literature suggests that blocking of potassium channels may be a common therapeutic mechanism of many antidepressant agents. To determine whether the commonly used antimanic agents could prevent potassium efflux-induced cell damage and apoptosis and the underlying mechanisms, we treated SH-SY5Y human neuroblastoma cells with the potassium ionophore, valinomycin (2-100 microM) and observed cell shrinkage, mitochondria damage, a significant increase in of lactate dehydrogenase (LDH) activity and caspase-3 protein expression. Cells treated with lithium (0.5-3 mM) or valproate (0.07-1.4 mM) alone produced no apoptotic morphological and biochemical changes while both mood stabilizers pretreatment reduced or prevented the apoptotic morphological changes. However, valinomycin-induced caspase-3 elevation was only prevented by lithium pretreatment while both lithium and valproate attenuated valinomycin-induced LDH release. Our results suggest that lithium and valproate share a common neuroprotective action against potassium efflux-induced cell apoptosis with different mechanisms.

Topics: Antimanic Agents; Apoptosis; Caspase 3; Caspases; Humans; Ionophores; L-Lactate Dehydrogenase; Lithium Chloride; Neuroblastoma; Tumor Cells, Cultured; Valinomycin; Valproic Acid

We have set up a model system for familial amyotrophic lateral sclerosis (FALS) by transfecting human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase (Cu,ZnSOD) or a mutant of this enzyme (G93A) associated with FALS. We have tested mitochondrial function and determined cytosolic Ca2+ concentration in control cells (untransfected) and in cells expressing either wild-type Cu,ZnSOD or G93A. We report that G93A induces a significant loss of mitochondrial membrane potential, an increased sensitivity toward valinomycin and a parallel increase in cytosolic Ca2+ concentration. The above phenomena are not related to total Cu,ZnSOD content and activity in the cell.

Topics: Amyotrophic Lateral Sclerosis; Calcium; Cloning, Molecular; Cytosol; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Neuroblastoma; Polymerase Chain Reaction; Recombinant Proteins; Superoxide Dismutase; Transfection; Tumor Cells, Cultured; Valinomycin

The effects of the flavonoid quercetin on cell proliferation and voltage-dependent K+ current were studied on mouse neuroblastoma x glioma hybrid cells. In the presence of 1% fetal calf serum, quercetin inhibited both cell proliferation and K+ current with effective doses inducing half-maximum effects of 10 microM and 70 microM respectively. Valinomycin (1 nM) antagonized 80% of the growth-inhibitory effects of 10 microM quercetin. The results suggest that at least a part of the anti-proliferative effect of quercetin is mediated by a K+ channel blockade. They further confirm a role for K+ channels in mitogenesis and cell proliferation.

Topics: Adenosine Triphosphate; Animals; Cell Division; DNA; Electric Conductivity; Fetal Blood; Glioma; Hybrid Cells; Mice; Neuroblastoma; Potassium Channels; Quercetin; Tumor Cells, Cultured; Valinomycin

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchicine, vinblastine) or cations (5-10 mM K+, Na+ or Mg2+) abolishes this increase of ornithine decarboxylase activity. (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212-219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six ionophores tested, valinomycin was the most potent one, with I50 value (concentration that gives 50% inhibition of ornithine decarboxylase activity) of 6.10(-9) M. Dibucaine and tetracaine were also effective inhibitors at 10(-4)-10(-5) M. The I50 values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1.10(-6) M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.

Topics: Anesthetics, Local; Animals; Blood; Carboxy-Lyases; Cell Line; Culture Media; Dibucaine; Dose-Response Relationship, Drug; Ionophores; Leukemia, Experimental; Mice; Neuroblastoma; Ornithine Decarboxylase Inhibitors; Tetracaine; Valinomycin

A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K+ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells.

Topics: Anilino Naphthalenesulfonates; Animals; Cell Differentiation; Cell Line; Cell Membrane; Energy Transfer; Membrane Potentials; Mice; Neuroblastoma; Spectrometry, Fluorescence; Valinomycin

Mouse neuroblastoma cells in stationary phase of growth display partially developed electrical properties. Addition of the K+ selective carrier valinomycin to these cells causes rapid enhancement of electrical excitability. We suggest that the appearance of molecules with properties similar to valinomycin is essential for the full expression of electrical excitability in differentiating neuroblastoma.

Topics: Action Potentials; Animals; Biological Transport, Active; Cell Line; Cell Membrane Permeability; Chlorides; Membrane Potentials; Mice; Neuroblastoma; Potassium; Sodium; Valinomycin