Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells

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.     

Blockade by Neurotransmitter Antagonists of Veratridine-Activated Ion Channels in Neuronal Cell Lines

Abstract: The voltage-dependent Na⁺ ionophore of various neuronal cells is permeable not only to Na⁺ ions but also to guanidinium ions. Therefore, the veratridine-(or aconitine-) stimulated influx of [¹⁴C]guanidinium in neuroblastoma × glioma hybrid cells was measured to characterize the Na⁺ ionophore of these cells. Half-maximal stimulation of guanidinium uptake was seen at 30 μ M veratridine. At 1 m M guanidinium, the veratridine-stimulated uptake of guanidinium was lowered to 50% by approximately 60 m M Li⁺, Na⁺, or K⁺ and by a few millimolar Mn²⁺, Co²⁺, or Ni²⁺. The basal, as well as the veratridine-stimulated, uptake of guanidinium was inhibited by the cholinergic antagonists (+)-tubocurarine ( K_i = 50 to 500 n M ) and atropine ( K_i = 5 to 30 μ M ) and the adrenergic antagonists phentolamine ( K_i = 5 μ M ) and propranolol ( K_i = 60 μ M ). The specificity of the inhibitory effects of these agents is stressed by the ineffectiveness of various other neurotransmitter antagonists. However, the corresponding ionophore in neuroblastoma cells (clone N1E-115) seems to be regulated differently. While phentolamine and propranolol inhibit the veratridine-activated uptake as in the hybrid cells, (+)-tubocurarine and atropine exert only a slight effect.     

Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.     

Membrane excitability expressed in human neuroblastoma cell hybrids

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.     

Reexamination of Opioid Stimulation of cGMP Formation in Cell Lines of Neuronal Origin

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings.2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma × glioma hybrid cells resembled the proportion of [Ca²⁺]_in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca²⁺ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines.3. The implication of the opioid-Ca²⁺-NO-cGMP cellular pathway for opioid activity in vivo is discussed.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

Effects of insulin on glucose uptake in cultured cells from the central nervous system of rodent.

1. Incubation of C6 glioma cultures with insulin resulted in a time and dose-dependent stimulation of 2-deoxy-D-glucose uptake. The maximal stimulation (160% of the control) was observed with 1 nM insulin and 0.05 nM caused half-maximum effect. 2. Incubation of NG 108-15 (neuroblastoma x glioma hybrid) and N2 neuroblastoma cells with 160 nM insulin did not result in a significant stimulation of this glucose uptake. 3. The basal level and stimulatory effect by insulin on this glucose uptake observed in C6 glioma cells were dependent on the presence of calcium in the medium. 4. Such an increase in glucose uptake in C6 glioma cells was also observed in the presence of diacylglycerol (DG) generating agents, such as carbachol (1 mM) and phospholipase C (0.05 unit/ml) or of DG analogs, such as sn-1,2-dioctanoyl glycerol (250 microM) and phorbol myristate acetate (1 microM). 5. Our results indicated that both calcium ion and DG levels play important roles in the regulation of glucose uptake in the glial cells, but not in neuronal cells from the brain.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

Mechanisms for activation and subsequent removal of cytosolic Ca2+ in bradykinin-stimulated neuronal and glial cell lines

Mechanisms for activation and for removal of cytosolic Ca2+ after stimulation with bradykinin were investigated in two neural cell lines by measuring cytosolic Ca2+ activity and 45Ca2+ fluxes. In the neuronal (neuroblastoma x glioma hybrid) and in the glial (rat glioma) cell lines, the transient, bradykinin-induced rise in cytosolic Ca2+ activity (determined by fura-2 or indo-1 fluorescence) was blocked by a bradykinin B2 receptor antagonist. Ca2+ ionophores (ionomycin and 4-Br-A23187) caused a comparable transient rise in cytosolic Ca2+ activity. After addition of ionophores, the Ca2+ response to bradykinin was reduced or completely blocked in both cell lines. At the concentrations used, the ionophores primarily depleted intracellular Ca2+ stores and prevented refilling of the stores. Thus, the bradykinin-induced rise of cytosolic Ca2+ activity seems to be mostly due to Ca2+ release from internal stores. In the neuronal but not in the glial cell line, a brief stimulation by bradykinin of 45Ca2+ uptake was followed by a long-lasting inhibition below control values. Thus, in the neuronal cells bradykinin presumably blocks Ca2+ channels by a readily reversible, pertussis toxin-insensitive mechanism. Excess cytosolic Ca2+ of the bradykinin-stimulated cells is mostly not resequestered into the internal Ca2+ pool accessible to bradykinin, but is mainly extruded through the plasma membrane, as indicated by (i) stimulation of 45Ca2+ release by bradykinin, (ii) quick reduction by bradykinin of cellular 45Ca2+ content of cells preequilibrated with 45Ca2+, and (iii) diminution of the ionophore-inducible Ca2+ response after the addition of bradykinin.     

Properties of the interaction of the sodium channel with permeant monovalent cations

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin-sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+-substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH = 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM). The experimental data are consistent with a model involving at least two allosteric cation-binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity. Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.     

Halothane inhibits the neurotoxin stimulated [14C]guanidinium influx through 'silent' sodium channels in rat glioma C6 cells

We have investigated the effect of pharmacological agents on [14C]guanidinium ion influx through sodium channels in C6 rat glioma and N18 mouse neuroblastoma cells. The sodium channels of the N18 cells can be activated by aconitine alone, indicating that they are voltage-dependent channels. In contrast, sodium channels in the C6 cells require the synergistic action of aconitine and scorpion toxin for activation and are therefore characterized as so-called silent channels. The general anesthetic halothane used at clinical concentrations, specifically inhibited the ion flux through the silent sodium channel of C6 rat glioma cells. The voltage-dependent channels of the N18 cells were insensitive to halothane at the concentrations tested.     

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.     

Modulation of sodium-sensitive GTPase by partial opiate agonists. An explanation for the dual requirement for Na+ and GTP in inhibitory regulation of adenylate cyclase

Opiates and opioid peptides inhibit adenylate cyclase and stimulate specific low Km GTPase activity in membranes from neuroblastoma x glioma NG108-15 hybrid cells. The effects of opiate agonists on both enzymes are mediated by high affinity stereospecific receptors and require Mg2+, GTP, and Na+. In the presence of Mg2+, Na+ inhibits basal GTPase activity; opiates stimulate GTP hydrolysis by antagonizing the Na+-induced inhibition. Activation of GTPase leads, in turn, to inactivation of GTP-stimulated adenylate cyclase activity. The intrinsic activities (or efficacies) of a series of opiates are identical for stimulation of GTPase and inhibition of adenylate cyclase. These results provide a mechanism for the dual requirement for Na+ and GTP in the inhibitory coupling of opiate receptors to the adenylate cyclase system in these cells and may be of general significance to the action of other inhibitory hormones.     

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.     

Phorbol esters and mitogenesis: comparison of the proliferative response of parental and Na+K+Cl- -cotransport-defective BALB/c 3T3 cells to 12-0-tetradecanoylphorbol-13-acetate

The ability of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl- cotransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 microM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30-35% increase in the saturation density of mutant cells compared to the 100-120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H-thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016-1.6 microM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl- cotransport activity decreases the response of these cells to the mitogenic action of TPA.     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

CALCIUM FLUXES IN CULTURED AND BULK ISOLATED NEURONAL AND GLIAL CELLS

Abstract— The influx and efflux of ⁴⁵Ca has been studied in cultured human glioma and mouse neuroblastoma cells and in isolated fractions enriched in synaptosomes, neuronal and astrocytic perikarya from rabbit brain.
The uptake of ⁴⁵Ca was somewhat more efficient in glioma compared to neuroblastoma cells, whereas there was little difference in the rate of ⁴⁵Ca uptake by isolated glial cells and neuronal perikarya. Isolated synaptosomes showed the highest rate of ⁴⁵Ca accumulation. An increase of K concentration to 50 m m in the medium, with a corresponding lowering of Na, stimulated both glioma and glial as well as synaptosomal ⁴⁵Ca uptake more markedly than the uptake by neuroblastoma cells and neuronal perikarya. Lowering the Na concentration and replacing it by choline had no effect on the cultured cells and astrocytes. Na-free media caused massive stimulation of ⁴⁵Ca influx in all fractions and cells tested.
The efflux of ⁴⁵Ca was studied after preloading of cells. Three phases could be resolved from the desaturation curves. All cells had nearly similar half-lives for ⁴⁵Ca efflux under standard conditions. Pulses of media containing 50 m m -K stimulated ⁴⁵Ca efflux from glioma cells and astrocytes more efficiently than from neuroblastoma cells, neuronal perikarya and synaptosomes. The stimulated release was exclusively seen in Ca-containing media in experiments with the cultured cells and in Ca-free media in experiments with cell perikarya. The effect of transmitter pulses on the release of ⁴⁵Ca was examined in a limited series. Acetylcholine and isoproterenol were found to stimulate ⁴⁵Ca release more actively from glia than from neurons.     

Comparison of ionic selectivity of batrachotoxin-activated channels with different tetrodotoxin dissociation constants

The purpose of these experiments is to test whether the differences between normal and tetrodotoxin-resistant Na+ channels reside in the selectivity filter. To do this, we have compared the selectivity of batrachotoxin-activated channels for alkali cations, organic cations, and nonelectrolytes in two neuroblastoma clonal cell lines: N18, which has normal tetrodotoxin (TTX) sensitivity, and C9, which is relatively TTX-resistant. We have also studied the effect of H+ on Na+ permeability and on the interaction between TTX and its receptor site in both cell lines. There is no qualitative difference between the two cell lines in any of these properties. In both cell lines the batrachotoxin-activated Na+ channels have a selectivity sequence of Tl+ greater than Na+ greater than K+, guanidinium greater than Rb+ greater than Cs+, methylamine. Also, in both cell lines H+ blocks Na+ channels with a pKa of 5.5 and inhibits the action of TTX with the same pKa. These observations indicate that the selectivity filters of the Na+ channels in C9 and N18 do not differ significantly despite the 100-fold difference in TTX-affinity. Our selectivity studies of batrachotoxin-activated Na+ channels for both cell lines suggest that these toxin-activated Na+ channels have a limiting pore size of 3.8 x 6.0 A, as compared to a pore size of 3.0 x 5.0 A for potential-activated Na+ channels.     

Inhibition of cell growth by K channel modulators is due to interference with agonist-induced Ca release

The effects of K⁺ channel modulators, tetraethylammonium, 4-aminopyridine and diazoxide, and high extracellular K⁺ on cell growth and agonist-induced intracellular Ca²⁺ mobilization were investigated. Two human brain tumour cell lines, U-373 MG astrocytoma and SK-N-MC neuroblastoma, were used as model cellular systems. K⁺ channel modulators and increased extracellular K⁺ concentration inhibited tumour cell growth in a dose-related fashion in both cell lines. In addition, agonist (carbachol or serum)-induced intracellular Ca²⁺ mobilization was also blocked by the pretreatment of growth-inhibitory concentrations of K⁺ channel modulators and high extracellular K⁺. Thus, these results suggest that K⁺ channel modulators are effective inhibitors of brain tumour cell growth and that their growth regulation may be due to the interference with the intracellular Ca²⁺ signalling mechanisms.     

Pregabalin and gabapentin inhibit substance P-induced NF-kappaB activation in neuroblastoma and glioma cells

Pregabalin and gabapentin are lipophilic amino acid derivatives of gamma-amino butyric acid that show anticonvulsant and analgesic activity against neuropathic pain. In this study, we investigated their actions on substance P-induced NF-kappaB activation in human neuroblastoma and rat glioma cells. Pregabalin and gabapentin decreased substance P-induced NF-kappaB activation in these cells. These drugs also inhibited NF-kappaB activation in rat spinal dorsal root ganglia cells pre-treated in vitro with substance P. These results suggest a previously undefined role of pregabalin and gabapentin in the regulation of inflammation-related intracellular signaling in both neuronal and glial cells.