Bradykinin induces hyperpolarizations in rat glioma cells and in neuroblastoma × glioma hybrid cells

The effect of the nonapeptide bradykinin on the membrane potential of permanent cell lines from neural origin was studied. A hyperpolarizing response of 10–30 s duration was produced when bradykinin was iontophoretically applied onto polyploid rat glioma cells (clone C6-4-2). Starting from the resting membra potential the peak value of the hyperpolarizing response was reached within 0.5–1.5 s. Then the potential returned more slowly to the origin value. The hyperpolarization was associated with an approximately 50% decrease in membrane resistance. Neither Na⁺ nor Cl⁻ seemed to be important for the hyperpolarizing response, since bradykinin elicited similar hyperpolarizations in cells exposed to media in which Na⁺ or Cl⁻ were replaced by choline or isethionate, respectively. Ca²⁺ fluxes are unlikely to be involved, since the addition of D600 did not affect the hyperpolarizations induced by bradykinin. However, a 10-fold increase in the concentration of K⁺ in the medium reduced the amplitude of the hyperpolarization by 40 mV. Thus, the hyperpolarization induced by bradykinin is associated with a decrease in membrane resistance which is likely to be caused by an increased K⁺-conductance. The glioma cells showed a desensitization upon repeated application of bradykinin. However, the sensitivity of the cells to bradykinin was restored after 3–8 min of incubation in the absence of bradykinin. Since an antagonist of bradykinin is not known, the specificity of the action of bradykinin is difficult to assess. Nevertheless, the hyperpolarizing response to bradykinin appears to be specific insofar as other peptides, i.e. lutoliberin, thyroliberin, neurotensin, substance P and apamin, exerted no effect on the membrane potential of the glioma cells. Bradikynin-elicited hyperpolarizations with characteristics similar to those described above could also be demonstrated in neuroblastoma × glioma hybrid cells, but not in multinucleated fibroblast cells.     

Characterization of angiotensin II binding sites on neuroblastoma × glioma hybrid cells

Mouse neuroblastoma X rat glioma hybrid cells, NG108-15, have recently been shown to contain immunoreactive angiotensin II (AII). In the present study, we have examined this hybrid cell line for the presence of specific AII binding sites using [125I] AII. Specific AII binding was saturable and reversible. Scatchard analysis revealed a linear plot with an affinity constant (Kd) of 0.323 nM and a binding capacity (Bmax) of 7.13 fmol/mg protein. Kinetic studies demonstrated an association rate constant (K+1) of 3.55 X 10(6) M(-1) sec-1 and a dissociation rate constant (K-1) of 4.18 X 10(-4) sec-1. Displacement curves, using concentrations of 10(-11) M to 10(-4) M of unlabeled AII, revealed high and low affinity components of the AII binding site with IC50's of 0.46 nM and 1.75 microM respectively. The AII antagonist, saralasin, had approximately equal potency with unlabeled AII at the high affinity site. Furthermore, structurally related and unrelated peptides had no significant inhibitory effect on AII binding. This study demonstrates that specific AII binding sites are present on NG108-15 cells, and that these binding sites are similar in kinetic character to the AII receptor that has been previously identified in membranes from mammalian brain. It is concluded that the NG108-15 hybrid cells may provide a useful continuous cell line model for investigating both the biochemical and molecular properties of the AII binding site.     

Acetylcholinesterase in neuroblastoma and neuroblastoma × glioma hybrid cells: Cellular localization and molecular forms

The cellular localization of acetylcholinesterase (ACHE) was investigated at the electron microscope (E.M.) in a neuroblastoma and neuroblastoma × glioma hybrid line, which differ for their ability to establish synaptic contacts. Only cells of the latter line show association of AChE to the plasmamembrane, while in the former the activity is mainly intracellular. Sucrose sedimentation analysis of AChE molecular forms has shown no significant differences in the distribution of the two forms, G₂ and G₄, between the two cell lines. On the contrary a marked difference is observed in the ability of the cell to release the enzyme in the culture medium. In fact the cells lacking AChE on their surface release in the medium a much higher proportion of their enzyme, than the cells showing AChE association to their plasmamembrane. The possible role of two alternative fates for AChE, secretion or membrane insertion, in determining the observed differences of enzyme localization is discussed.     

Proliferation and synapse formation of neuroblastoma × glioma hybrid cells: Effect of glia maturation factor

Glia maturation factor (GMF), extracted from bovine brain, stimulated DNA synthetis and proliferation of glioma cells and hybrid cells derived from glioma and neuroblastoma cells (NG108-15), but had no effect on neuroblastoma cells. The synapse formation of NG108-15 cells with rat striated myotubes was lower in the presence of GMF than the control and also lower after treatment with prostaglandin E₁ (PGE₁) plus theophylline, indicating that GMF did not induce functional differentiation of NG108-15 cells. The results show that expression of mitogenic action for GMF in the hybrid cells is a property derived from the glioma parent, and that NG108-15 is therefore an excellent model for studying glial-neuronal interactions.     

Binding assay for opioid peptides with neuroblastoma × glioma hybrid cells: Specificity of the receptor site

A sensitive and rapid radioreceptor assay has been developed to monitor opioid peptides in column effluents. It is based on competitive binding to NG 108-15 cells using [³H-Tyr]Leu-enkephalin as the displaced ligand. The specificity of binding to the cells of naturally occurring opioid peptides and synthetic analogs has been shown to be similar to that found with synaptic plasma membranes.     

A mouse neuroblastoma × rat glioma hybrid cell produces immunoreactive substance P-like material

A new hybrid cell line, designated NG115-401, produced a significant amount of immunoreactive (IR) substance P-like material. The production was measured by radioimmunoassay using specific antisera raised to substance P. The production rate was dependent upon cell growth, and maximum production occured at the same stage (late logarithmic stage) as the time of appearance of choline acetyltransferase activity, one of the characteristic neuronal properties of the cells. On the other hand, no IR-substance P was found in hybrid cells other than NG115-401, i.e. NG115-301 and NG115-303 cells, which were derived from the same parent mouse neuroblastoma N115TG-2 cells. Furthermore, in an attempt to survey IR-substance P production in several clonal cells, consisting of some mouse neuroblastoma cells and a rat glioma cell, and several of their hybrid cells, we could find no cell line producing a significant amount of IR-substance P except for NG115-401 hybrid cells. The cellular production of IR-substance P in NG115-401 hybrid cells was also confirmed by detecting the material in all 14 subpopulations of this hybrid cells. Partial characterization of IR-substance P-like material produced by NG115-401 hybrid cells was carried out by its application to gel-filtration column chromatography. Only high molecular weight material (approximately 26,000) appeared in the column eluates.     

Bradykinin induces rise of free calcium in nuclei of neuroblastoma × glioma hybrid NG 108-15 cells

Confocal fluorescence microscopy was used to study the bradykinin-induced calcium signals in the neuro-blastoma × glioma cell line NG 108–15. We found that bradykinin induced a rise in free calcium, not only in the cytoplasm but also in the nucleus. The nuclear and cytosolic calcium concentrations were not significantly different and rose to about 1.2 :μM. The signal was mediated by the B₂-receptor subtype as confirmed using the specific antagonist Hoe 140. Both the onset and the intensity of the calcium signals were concentration-dependent. The rise of nuclear calcium level was independent of extracellular calcium and suppressed by thapsigargin which is known to deplete inositol 1,4,5-trisphosphate-sensitive calcium stores. Bradykinin-induced calcium increase desensitizes rapidly. This desensitization was shown not to involve activation of protein kinase C. © 1995 Wiley-Liss, Inc.     

Transport pathways for lithium ions in neuroblastoma × glioma hybrid cells at ‘therapeutic’ concentrations of Li

The pathways of Li⁺ transport in neuroblastoma × 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⁺.     

Translocation of phosducin in living neuroblastoma × glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein

Activation of G protein-coupled receptors triggers translocation of certain proteins from cytoplasm to cell membrane located targets. One of these cytosolic proteins is phosducin (Phd) which has been described to compete with G protein-coupled receptor kinases for Gβγ dimers attached to the cell membrane, thereby attenuating desensitization of activated receptors. These features of protein redistribution prompted us to examine whether stimulation of membrane associated E-prostaglandin receptors coupled to Gs causes Phd to migrate towards the plasma membrane. We made use of enhanced green fluorescence protein (EGFP), a reporter protein, to follow redistribution of Phd both by means of confocal microscopy and biochemical techniques in living neuronal NG 108-15 hybrid cells challenged with prostaglandin E1 (PGE1). The cells were transiently transfected to express Phd fused to the C-terminus of EGFP, or to express EGFP only. Overexpression of the proteins is implied by FACS analysis as well as by western blot technique, and the functional integrity of EGFP-tagged Phd was confirmed by its ability to elevate cAMP accumulation. Time-lapse imaging of single living cells by means of confocal microscopy revealed that exposure to prostaglandin causes EGFP/Phd, which is evenly spread throughout the cell, to relocate towards the membrane within few minutes. Fluorescence associated with the cell nucleus displayed little rearrangement. The principle finding that prostaglandin triggers translocation of Phd from cytosol to the cell periphery was verified with membranes prepared from EGFP/Phd expressing cells. We found maximal concentrations of membrane associated fluorescent material 5 to 7 min upon prostaglandin exposure. The present study reports for living NG 108-15 hybrid cells that PGE1 stimulation causes cytosolic Phd to translocate towards the membrane, where it is believed to bind to G protein subunits such as Gβγ and Gs.     

beta-Endorphin in neuroblastoma x glioma hybrid cells

Acid extracts from mouse Neuroblastoma x rat Glioma hybrid cells have been purified by means of Sep-Pak C-18 and fractionated by high performance liquid chromatography. Each fraction has been submitted to a sensitive beta-endorphin radioimmunoassay and an immunoreactivity peak at camel beta-endorphin retention time was found.     

Acetylcholinesterase in neuroblastoma and neuroblastoma x glioma hybrid cells: cellular localization and molecular forms

The cellular localization of acetylcholinesterase (AChE) was investigated at the electron microscope (E.M.) in a neuroblastoma and neuroblastoma x glioma hybrid line, which differ for their ability to establish synaptic contacts. Only cells of the latter line show association of AChE to the plasmamembrane, while in the former the activity is mainly intracellular. Sucrose sedimentation analysis of AChE molecular forms has shown no significant differences in the distribution of the two forms, G2 and G4, between the two cell lines. On the contrary a marked difference is observed in the ability of the cell to release the enzyme in the culture medium. In fact the cells lacking AChE on their surface release in the medium a much higher proportion of their enzyme, than the cells showing AChE association to their plamamembrane. The possible role of two alternative fates for AChE, secretion or membrane insertion, in determining the observed differences of enzyme localization is discussed.     

Localization of [3H]serotonin in neuroblastoma x glioma hybrid cells

Synthesis, localization and release of serotonin (5-HT) were studied in cholinergic neuroblastoma X glioma NG108-15 cells. The content of 5-HT and tryptophan hydroxylase activity rose substantially when hybrid cells were differentiated by prostaglandin E1 plus theophylline, or dibutyryl cAMP. Localization of [3H]5-HT taken up into differentiated NG108-15 cells was examined by electron microscopic radioautography. Silver grains were observed mostly in neurites, indicating that neurites of differentiated NG108-15 cells are the preferential uptake site of [3H]5-HT. Statistical analysis of the results of electron microscopic radioautographs revealed that silver grains had a high affinity for dense core vesicles of 60-170 nm diameter, though grains were also located over endoplasmic reticulum, mitochondria and cytosol. Dense core vesicles were abundant in neurites, and less numerous in cell bodies of the hybrid cells. [3H]5-HT taken up into NG108-15 cells was released by potassium stimulation in the presence of Ca2+. The results indicate that NG108-15 hybrid cells manifest many properties comparable to those of serotonergic neurons.     

Developmental time courses of Na and Ca spikes in neuroblastoma X glioma hybrid cells

The developmental time course of membrane excitability in neuroblastoma X glioma hybrid cells (NG108-15) was analyzed by measuring the electrical potential at each culture day after dBcAMP treatment. The maximum rate of rises of Na and Ca spikes which reflect their inward currents were measured in appropriate medium respectively. The Ca spike began to appear at 2-3 days in culture and reached its maximum on the fifth day. The Na spike began to appear at 3-4 days in culture and was completed on the seventh day. After then Ca and Na spikes remained constant and did not decrease even on the eighteenth day. Electrical excitability of the hybrid cells, as a whole, developed stepwise and was completed on the seventh day of culture.     

Pharmacologic responses of cells of a neuroblastoma x glioma hybrid clone and modulation of synapses between hybrid cells and mouse myotubes

Cells of the hybrid clone NG108-15 responded to 5-hydroxytryptamine (5-HT), dopamine or acetylcholine with graded depolarizations involving membrane conductance increases. Responses desensitized during continuous application of the neurotransmitters, and responses to 5-HT and dopamine cross-desensitized: a desensitizing application of one neurotransmitter also desensitized the hybrd cell to the other neurotransmitter. 5-HT and acetylcholine did not cross-desensitize. The hybrid cell 5-HT response was not attenuated by D-LSD, and was blocked by 10(-5) M morphine, although not via binding to naloxone-sensitive opiate receptors. 5-HT or the prostaglandin PGF2alpha caused the release of acetylcholine at the synapses of hybrid cells with mouse myotubes. Application of 5-HT or PGF2alpha also facilitated the synaptic release elicited by hybrid cell action potentials. Following treatment with the antimitotic agent cytosine arabinoside, co-cultures of hybrid cells and mouse myotubes exhibited plentiful synaptic connections only if maintained in medium containing 1 mM dibutyryl cAMP (dBcAMP). After X-irradiation, co-cultures were synaptically active even in the absence of dBcAMP. Thus, methods have been found to regulate both the short-term and long-term synaptic activity of NG108-15 hybrid cells.     

Bradykinin evoked depolarization of a novel neuroblastoma × DRG neurone hybrid cell line (ND723)

Application of bradykinin (Bk) to neuroblastoma × dorsal root ganglion (DRG) neurone hybrid cells (ND723) evoked an inward (depolarizing) current associated with an increase in membrane conductance. This response was antagonized byd-Arg⁰, Hyp³, Thi^5,8,d-Phe⁷-Bk, but was not mimicked by des-Arg⁹-Bk, indicating the involvement of B₂-receptors. The response was unaltered by replacement of extracellular Na⁺ byN-methylglucamine. Replacement of extracellular Cl⁻ by gluconate⁻ shifted the estimate reversal potential to a more positive value, while the use of potassium acetate filled recording electrodes shifted the reversal potential to a more negative value, and reduced the response amplitude, indicating the importance of Cl⁻ in the response. This response to Bk was mimicked by the calcium ionophore ionomycin. Bk stimulated the formation of inositol 1,4,5-trisphosphate (IP₃), and increased the release of arachidonic acid. In addition, Bk produced an increase in [Ca²⁺]_i, as determined by microspectrofluorimetry. This was due to the release of Ca²⁺ from intracellular stores, since the response was unaltered when the cells were bathed in Ca²⁺-free solution. In summary, Bk depolarizesND723 cells, probably through the activation of a chloride conductance. It seems likely that this is secondary to the rise in cytosolic Ca²⁺ concentration, due to the release of Ca²⁺ from internal stores by IP₃. This Ca²⁺-activated chloride response is present in some sensory neurones, although its role in the activation of sensory neurones by Bk is at present unclear.     

Formation of synapses between cells of a neuroblastoma X glioma hybrid clone and mouse myotubes

Synapses form between cells of a neuroblastoma X glioma hybrid clone and cultured mouse skeletal myotubes. The synapses are cholinergic, and the acetylcholine release mechanism is dependent on calcium ions. The transmitter output of the synapses is low, with considerable variability in the latency and amplitude of the postsynaptic responses to presynaptic action potentials. The fine structure of physiologically identified functional junctions was examined electron microscopically. Small (50 nm) clear vesicles were seen presynaptically and there were areas with a wide (approx. 50 nm) gap containing basement membrane-like material between the pre- and postsynaptic cells. In addition, in some regions there was a densely staining material lining the muscle membrane and some suggestion of infolding of the muscle membrane. In none of the cases, however, have areas been found where small, clear vesicles cluster around pre- and postsynaptic membrane densities. Thus, functional synapses can occur in the absence of the highly organized synaptic structure seen at mature synapses.     

Glutamate receptor activation can trigger electrical activity in human glioma cells

Cells from major types of gliomas, i.e. oligodendrogliomas and glioblastomas, are able to generate action potentials upon a current injection similar to neurons ( Patt et al. (1996) Neuroscience, 71 , 601–611; Labrakakis et al. (1997b ) J. Neuropath. Exp. Neurol., 56, 243–254. Here, we report that activation of ionotropic glutamate receptors by the selective agonist, kainate, or by glutamate itself, depolarized the tumour cells in culture and living slices from tumour tissue, and can elicit volleys of action potentials, as recorded with the patch-clamp technique. Sixty-six percent of the glioblastoma cells, 44% of the astocytoma and 86% of the oligodendroglioma cells responded to glutamate and the specific agonist of AMPA/kainate receptors, kainate. The involvement of non-NMDA (N-methyl-d -aspartate) receptors is further supported by the observation that both kainate and glutamate currents were blocked by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). The receptor activation was accompanied by an increase in cytosolic Ca²⁺, as recorded with a fura-2 microfluorometric system. The Ca²⁺ elevation was mediated by the activation of Ca²⁺ channels due to membrane depolarization. The presence of voltage-gated Ca²⁺ channels was confirmed by patch-clamp experiments. Taken together, these findings imply that the electrophysiological properties of glioma cells are more reminiscent of those of neurons than of glial cells.     

F11 neuroblastoma × DRG neuron hybrid cells express inhibitory μ- and δ-opioid receptors which increase voltage-dependent K currents upon activation

The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express μ- and δ-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of μ and δ, but not κ, receptor expression in F11 cells. Radioligand binding assaysshow that F11 cell membranes bind the μ- and δ-opioid receptor agonists, DAGO and DPDPE with K_d = 4.5 and 4.9 nM and B_max = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K⁺ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the κ-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, ß-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K⁺ current. The opioid-induced increase in voltage-dependent membrane K⁺ current in F11 cells resembles the inhibitory effect elicited by μ- and δ-opioid agonists in primary cultures of mouse DRG neurons.     

Effects of methylmercury on electrical responses of neuroblastoma cells

The effects of methylmercury on a variety of electrophysiological properties of the N1E-115 neuroblastoma cells were studied using microelectrode and voltage clamp techniques. The action potential was reduced in amplitude with an apparent dissociation constant of the order of 20 microM in the face of relatively small membrane depolarization. Voltage clamp experiments revealed that both peak sodium current and steady-state potassium current were suppressed by 20-60 microM methylmercury, with a stronger effect on the sodium current than on the potassium current. The protein reagents dithiodipyridine and N-ethylmaleimide suppressed both currents. Acetylcholine receptor/channel complexes are vulnerable to the action of methylmercury; the nicotinic fast depolarizing response, the muscarinic hyperpolarizing response, and the muscarinic slow depolarizing response, were all suppressed by 10-30 microM methylmercury. In contrast, the dopamine induced response was not affected by methylmercury at 30 microM. It was concluded that methylmercury impairs both sodium and potassium channel gating mechanisms and suppresses acetylcholine receptor/channel complexes. It remains to be seen whether the effect of chronic exposure is similar to that seen after acute and high level exposure in the present study.     

Transmitter responsiveness in two newly isolated clones of neuroblastoma X glioma hybrid

Mouse neuroblastoma clone N1E-115 cells and rat glioma clone C6 cells were hybridized and two new clones were isolated. One clone, designated NG115-301, possessed weak electric excitability to an applied current pulse, while another clone, NG115-401, generated an action potential in response to the pulse. The former clone responded to serotonin and catecholamines with slow hyperpolarizations, while the latter clone responded to catecholamines with transient depolarizations. Both clones did not respond to acetylcholine. These types of responses have not been reported in any available clones. These clones may enrich the repertoire of cell clones useful for the characterization of transmitter reception mechanisms in the nervous system.