tetrodotoxin and Glioma

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma x glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10(-7) M tetrodotoxin (TTX). (2) The only Na(V) mRNA with an increased expression level during neuronal differentiation was that for NaV1.7, as observed by real-time PCR analysis. (3) The increase in the level of NaV1.7 alpha subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of NaV1.7 alpha subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na+ channel, NaV1.7.

Topics: Cell Differentiation; Cell Line, Tumor; Glioma; Humans; Hybrid Cells; NAV1.7 Voltage-Gated Sodium Channel; Neuroblastoma; Neurons; Sodium; Sodium Channels; Tetrodotoxin

In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca2+ concentration ([Ca2+]i) occurred. Sodium nitroprusside (SNP), a nitric oxide donor and an iron-containing molecule, reduced chemical hypoxia-induced LDH release and cell death. These effects were counteracted by bepridil and by 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), two specific inhibitors of the Na+-Ca2+ exchanger. SNP also increased the activity of the Na+-Ca2+ exchanger as a Na+ efflux pathway, stimulated by Na+-free conditions and evaluated by monitoring [Ca2+]i in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca2+]i elevation, and this effect was blocked by bepridil. Chemical hypoxia-evoked cell death and LDH release were counteracted by the ferricyanide moiety of the SNP molecule, K3Fe(CN)6, and by ferric chloride (FeCl3), and this effect was counteracted by CB-DMB. In addition, the iron ion chelator deferoxamine reversed the protective effect exerted by SNP on cell injury. Collectively, these findings suggest that the protective effect of SNP on C6 glioma cells exposed to chemical hypoxia is due to the activation of the Na+-Ca2+ exchanger operating as a Na+ efflux-Ca2+ influx pathway induced by iron present in the SNP molecule.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amiloride; Animals; Bepridil; Calcium; Calcium Channel Blockers; Cell Hypoxia; Cell Survival; Chelating Agents; Chlorides; Deferoxamine; Enzyme Activation; Extracellular Space; Ferric Compounds; Ferricyanides; Fluoresceins; Glioma; L-Lactate Dehydrogenase; Molsidomine; Nitric Oxide; Nitroprusside; Sodium; Sodium Channels; Sodium-Calcium Exchanger; Staining and Labeling; Tetrodotoxin; Tumor Cells, Cultured; Vasodilator Agents

1. We report that NG108-15 (neuroblastoma x glioma) cells differentiated in defined serum-free media are capable of exhibiting stable automaticity (the spontaneous occurrence of regenerative action potentials) following exposure to extracellular perfusates containing NH4Cl. 2. Membrane depolarization (4-5 mV) concomitant with an increased pHi during NH4Cl exposure are followed by hyperpolarization (5-7 mV), sub-threshold oscillations, and spontaneous firing after the removal of NH4Cl. 3. Cells cultured in 10% serum did not exhibit automaticity. Cells cultured in serum-free media are twice as likely to show automaticity as those cultured in reduced (1.5%) serum media. 4. We have examined factors that contribute to the events following NH4Cl exposure, namely, membrane depolarization and hyperpolarization, subthreshold oscillations, and automaticity. The inward currents activated at more negative potentials and the ionic currents associated with pronounced afterhyperpolarization in NG108-15 cells cultured in serum-free media provide a basis for the repetitive activity in general and automaticity in particular.

Topics: Amiloride; Ammonium Chloride; Animals; Cell Differentiation; Cell Membrane; Glioma; Hybrid Cells; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Mice; Neuroblastoma; Ouabain; Rats; Tetrodotoxin; Time Factors; Vanadates; Verapamil

Astrocytes in vitro and in situ have been shown to express voltage-activated ion channels previously thought to be restricted to excitable cells, including voltage-activated Na+, Ca2+, and K+ channels. However, unlike neurons, astrocytes do not generate action potentials, and the functional role of voltage-activated channels in astrocytes has been an enigma. In order to study the function of Na+ channels in glial cells, we carried out ion flux measurements, patch-clamp recordings, and ratiometric imaging of [Na+]i during blockade of Na+ channels on rat spinal cord astrocytes cultured for 7-10 d. Acute blockade of astrocyte Na+ channels by TTX had multiple effects: (1) TTX reduced, in a dose-dependent manner, Na+/K(+)-ATPase activity measured as unidirectional influx of 86Rb+; (2) TTX depolarized astrocyte membrane potential at a rate of approximately 1 mV/min; (3) TTX (100 microM) reduced [Na+]i; and (4) prolonged exposure to micromolar TTX induced astrocyte death. All these effects of TTX could be mimicked by ouabain or strophanthidin, specific blockers of the Na+/K(+)-ATPase. The effects of TTX and ouabain (or strophanthidin) were not additive. These results suggest that TTX-blockable Na+ channels in glial cells serve functions that do not require their participation in action potential electrogenesis; in particular, we propose that glial Na+ channels constitute a "return" pathway for Na+/K(+)-ATPase function, which permits Na+ ions to enter the cells to maintain [Na+]i at concentrations necessary for activity of the Na+/K(+)-ATPase. Since astrocyte Na+/K(+)-ATPase is believed to participate in [K+]o homeostasis in the CNS, the coupling of Na+ flux through voltage-activated Na+ channels to ATPase activity may provide a feedback loop that participates in the regulation of K+ ion levels in the extracellular space.

Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Cell Line; Cells, Cultured; Electrophysiology; Ganglia, Spinal; Glioma; Membrane Potentials; Models, Biological; Ouabain; Rats; Rats, Sprague-Dawley; Rubidium; Sodium; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Tetrodotoxin; Time Factors; Tumor Cells, Cultured

In NG108-15 cells, bradykinin (BK) activates a potassium current (IK,BK) and inhibits the voltage-dependent calcium current (ICa,V). BK also stimulates a phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to IK,BK, through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by Gq and/or G11, two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of Gq/11 in the electrical responses to BK. GTP gamma S mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-Gq/11 alpha antibody into the pipette suppressed IK,BK, but not the inhibition of ICa,V by BK. Thus, BK couples to IK,BK via Gq/11, but coupling to ICa,V is most likely via a different, pertussis toxin-insensitive G protein.

Topics: Animals; Bradykinin; Calcium Channels; Electrophysiology; Enkephalin, Leucine; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Kinetics; Models, Biological; Neuroblastoma; Pertussis Toxin; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Potassium Channels; Tetrodotoxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Measurement of intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse NG108-15 neuroblastoma x glioma hybrid cells, using the fluorescent probe fura-2, revealed that 5-25 nM ciguatoxin (CTX) increased [Ca2+]i either in cells bathed in standard medium or after removal of external Ca2+ by a Ca(2+)-free medium supplemented with EGTA. Tetrodotoxin prevented the CTX increased [Ca2+]i suggesting that CTX-induced mobilization of intracellular Ca2+ depends on Na+ influx through voltage-gated Na channels. CTX-induced Ca2+ mobilization prevented subsequent action of bradykinin (1 microM) suggesting that CTX stimulates the inositol 1,4,5-trisphosphate-releasable Ca2+ store.

Topics: Animals; Bradykinin; Calcium; Ciguatoxins; Eels; Fluorometry; Fura-2; Glioma; Hybrid Cells; Mice; Neuroblastoma; Rats; Sodium; Tetrodotoxin; Tumor Cells, Cultured

Topics: Animals; Calcium; Ciguatoxins; Glioma; Hybrid Cells; Mice; Neuroblastoma; Neurotransmitter Agents; Rats; Synaptic Vesicles; Tetrodotoxin; Tumor Cells, Cultured

The actions of brevetoxin (PbTX-3) were studied on single, voltage-dependent sodium channels and whole-cell currents from the neuroblastoma x glioma cell line NG108-15. Purified PbTX-3 shifted the activation of sodium channels to membrane potentials negative to normal. PbTX-3 did not alter the single-channel mean open lifetime, suggesting that the toxin does not change the rate of sodium channel inactivation from the open state. There was also no change in single-channel conductance. These results indicate that brevetoxin increases sodium current at rest by shifting the voltage dependence of channel activation and that the resulting depolarization is limited by channel inactivation.

Topics: Animals; Dinoflagellida; Electric Conductivity; Glioma; Hybrid Cells; Marine Toxins; Membrane Potentials; Neuroblastoma; Neuromuscular Blocking Agents; Oxocins; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

Depolarization of differentiated neuroblastoma X glioma (NG108-15) cells with KCl (50 mM) or veratridine (50 microM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 +/- 7 to 595 +/- 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage-dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose-dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (-) stereoisomers of 202-791 showed agonist and antagonist properties, respectively. (+)-202-791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage-sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KCl treatment and also by divalent metal cations (micromolar range). High [K+]o-induced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 microM) and tetraethylammonium (10 microM). In contrast, veratridine-induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 microM). Veratridine-induced Ca2+ accumulation was slower (approximately 45 s), smaller in magnitude (approximately 30% of [K+]o-induced Ca2+ entry), and also enhanced by BAY K 8644 (approximately 50%). VSCC were identified in neuronal hybrid (NG108-15 and NCB-20) cells, but not in glial (C6BU-1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108-15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aminoquinolines; Animals; Bucladesine; Calcium; Cricetinae; Dihydropyridines; Fluorescent Dyes; Glioma; Humans; Hybrid Cells; Ion Channels; Membrane Potentials; Neuroblastoma; Potassium Chloride; Sodium; Tetrodotoxin; Veratridine

The influence of memantine on several properties of a neuronal cell line was tested. The aim was to get some insight into possible mechanisms of action of this drug which is therapeutically applicable in treatment of spasticity, Parkinson's disease, and cerebral coma. In neuroblastoma X glioma hybrid cells, memantine, at micromolar concentrations, blocked the depolarization induced by iontophoretically applied serotonin (5-hydroxytryptamine, 5-HT). In the hybrid cells, receptors of the 5-HT3 type mediated the depolarization, which was frequently accompanied by a series of action potentials. The inhibition by memantine of the serotonin response occurred fast and was completely reversible, irrespective of whether the cell showed a stable membrane potential or spontaneous action potentials. However, memantine did not alter spontaneous or electrically evoked action potential activity in the hybrid cells, and apparently did not block the underlying ionic conductances. Furthermore memantine did not affect either the cation permeability activated by substance P in the hybrid cells or the K+ channel triggered by bradykinin in a glioma cell line. Thus, memantine appears specifically to suppress the ion channel opened by serotonin in the hybrid cells. The interaction of memantine with serotonin receptors and the associated ion channels reported here, might give an important clue, as to a site of action of memantine in the nervous system.

Topics: Amantadine; Animals; Calcium; Cell Line; Glioma; Hybrid Cells; Memantine; Membrane Potentials; Neuroblastoma; Neurons; Serotonin; Serotonin Antagonists; Sodium; Tetrodotoxin

Veratridine induces membrane potential oscillations in non-excitable glioma cells, which are not affected by ouabain (2 mM) or by D600 (0.1 mM). In the presence of veratridine, scorpion toxin causes depolarization of the glioma cells to a positive value of the membrane potential. These effects of veratridine and of scorpion toxin are observed in Na+ but not in choline medium and are inhibited by tetrodotoxin. The response of the glioma cells to bradykinin has also been studied during these experiments. Previously bradykinin has been shown in these cells to induce a hyperpolarizing response caused by an increase in K+ conductance. This response to bradykinin can still be seen during the veratridine-induced oscillations of the membrane potential. In the glioma cells the uptake of guanidinium, a substitute for Na+, is enhanced by veratridine plus scorpion toxin. This stimulation is tetrodotoxin-sensitive. However, in the excitable neuroblastoma X glioma hybrid cells studied for comparison, veratridine causes membrane potential oscillations accompanied at the rising phase by one action potential or a train of action potentials. The results demonstrate that in non-excitable glioma cells tetrodotoxin-sensitive Na+ channels can be activated by veratridine and by scorpion toxin.

Topics: Animals; Biological Transport; Cell Line; Glioma; Guanidine; Guanidines; Hybrid Cells; Ion Channels; Membrane Potentials; Neoplasms, Experimental; Neuroblastoma; Rats; Scorpion Venoms; Sodium; Tetrodotoxin; Veratridine; Veratrine

Interaction of Li+ with the voltage-dependent Na+ channel has been analyzed in neuroblastoma X glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage-dependent Na+ channels. Veratridine and aconitine increased the uptake of Li+ to the same degree (EC50 30 microM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine-stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 micrograms/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage-dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.

Topics: Acetylcholine; Aconitine; Action Potentials; Animals; Clone Cells; Glioma; Hybrid Cells; Ion Channels; Lithium; Neuroblastoma; Scorpion Venoms; Sodium; Tetrodotoxin; Veratridine

The pathways of Li+ transport in neuroblastoma X glioma hybrid cells were studied at 2 mM external Li+. Five components of Li+ transport were identified. (1) A Na+-dependent Li+ countertransport system mediating Li+ transport in both directions across the plasma membrane. This transport pathway is insensitive to ouabain or external K+. It shows trans-stimulation (i.e. acceleration of Li+ extrusion by external Na+ and stimulation of Li+ uptake by internal Na+) and cis-inhibition (i.e. reduction of Li+ uptake by external Na+). (2) The Na+-K+ pump mediates Li+ uptake but not Li+ release in cells with physiological Na+ and K+ content. Li+ uptake by the pump in choline media is inhibited by both external Na+ and K+. In Na+ media, external K+ exhibits a biphasic effect: in concentrations up to about 1 mM, K+ accelerates, and at higher concentrations, K+ inhibits Li+ uptake by the pump. (3) Li+ can enter the voltage-dependent Na+ channel. Li+ uptake through this pathway is stimulated by veratridine and scorpion toxin, the stimulation being blocked by tetrodotoxin. Residual pathways comprise (4) a saturable component, which is comparable to basal Na+ uptake, and (5) a ouabain-resistant component promoting Li+ extrusion against an electrochemical gradient in choline media. The mechanisms for Li+ extrusion described here possibly explain how neuronal cells maintain the steady-state ratio of internal to external Li+ below 1 during chronic exposure to 1-2 mM external Li+.

Topics: Animals; Biological Transport; Cell Line; Glioma; Hybrid Cells; Kinetics; Lithium; Mice; Neuroblastoma; Ouabain; Potassium; Rats; Scorpion Venoms; Sodium; Tetrodotoxin; Veratridine

Electrical excitability is one of the various neuronal properties of neuroblastoma X glioma hybrid cells. At a Ca2+ concentration of 1.8 mM the action potential is inhibited by tetrodotoxin, suggesting that the inward current is carried by Na+ ions. In contrast, at a Ca2+ concentration of 20-36 mM and even in the absence of Na+, spikes (sometimes repetitive) with strong hyperpolarizing afterpotential occur, which are no longer affected by tetrodotoxin. They are, however, blocked by antagonists of Ca2+ like La3+, Co2+, Mn2+, and the synthetic compounds D-600 and BAY a-1040. This seems to indicate that at high concentrations of Ca2+, the inward current of the action potential is essentially carried by Ca2+. Sr2+, but not Mg2+ can effectively substitute for Ca2+. It slows down the time course of the action potential. Ba2+ depolarizes the membrane gradually. If Ca2+ is also present, Ba2+ causes a reduced depolarization and spontaneous action potentials with no hyperpolarizing after-potential are observed.

Topics: Action Potentials; Barium; Calcium; Cell Line; Cells, Cultured; Glioma; Humans; Hybrid Cells; Magnesium; Membrane Potentials; Neuroblastoma; Strontium; Tetrodotoxin

Topics: Amino Acids; Animals; Brain Neoplasms; Calcium; Cells, Cultured; Chloramphenicol; Cycloheximide; Female; Glioma; Membrane Potentials; Nerve Tissue Proteins; Neuroglia; Potassium; Rats; Sodium; Synaptic Membranes; Synaptosomes; Tetrodotoxin; Veratrine

Veratridine, an activator of action potential Na(+) ionophores, stimulated passive Na(+) uptake by electrically excitable neuroblastoma and muscle cells but had no effect on clonal cell lines defective in Na(+)-ionophore activity. Veratridine-dependent Na(+) uptake was completely inhibited by tetrodotoxin, a specific inhibitor of the action potential Na(+) ionophore. Half-maximal inhibition was obtained with 11 nM tetrodotoxin. Thus, veratridinedependent Na(+) uptake provides a specific and convenient means of assaying populations of cultured cells for action potential Na(+)-ionophore activity.

Topics: Action Potentials; Animals; Biological Transport; Cell Membrane Permeability; Clone Cells; Glioma; Hybrid Cells; L Cells; Mice; Muscles; Neuroblastoma; Neurons; Ouabain; Radioisotopes; Rats; Sodium; Sodium Isotopes; Stimulation, Chemical; Tetrodotoxin; Thymidine; Tritium