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.     

Responses of neuroblastoma cells to iontophoretically applied acetylcholine

Dissociated mouse neuroblastoma cells were studied in vitro by using intracellular microelectrodes for electrical stimulation and recording. Some, but not all cells, which exhibited well developed action potentials to electrical stimulation also showed changes in membrane potential to iontophoretically applied acetylcholine (ACh). The types of responses to ACh varied. Short latency depolarizing responses to pulses of ACh (similar to those obtained with skeletal muscle) as well as sustained depolarization to steady ACh application (D response) occurred. A longer latency prolonged hyperpolarizing response (H response) and bi- and triphasic combinations of H and D responses were also seen. Pairs of cells showing morphologic contact were tested for the occurrence of effective synaptic coupling by placing intracellular microelectrodes in each cell. In none of 95 cases tested did spike activity produced by direct electrical stimulation of one cell elicit a synaptic potential of 200 μv or more in the other.     

Electrogenesis in mouse neuroblastoma cells in vitro

Intracellular microelectrode studies of passive membrane properties and action potential generation were carried out on cloned and uncloned mouse neuroblastoma cells in tissue culture. The cloned cells were studied between the eighth and tenth months and the uncloned cells between the third and fifth weeks after primary dissociation. Electrophysiologic measurements of cell membrane properties were made by passing stimulating current pulses across the cell membrane from an intracellular microelectrode and recording simultaneously from the same electrode, by means of a bridge circuit, the changes in membrane potential. The range of responses to electrical stimulation varied from passive increases in membrane potential to repetitive firing of action potentials. A 20 fold range in spike generating capability was found. Passive membrane properties (membrane potential, specific membrane resistivity, and specific membrane capacitance) were similar to those of sympathetic neurons in intact preparations. Seventy-nine percent of the cloned cell line compared to 94% of the uncloned line were capable of generating action potentials. Less than 2% of the cloned cells showed repetitive firing whereas 23% of the uncloned cells had this property. As in several types of normal neurons, the action potential mechanism was largely, although not completely, blocked by iontophoretic and bath applied tetrodotoxin.     

Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA.

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.     

Expression of monoamine oxidase A and B activities in mouse cybrids and hybrids

The inheritance of monoamine oxidase (MAO; EC1.4.3.4) was studied in cultured cells using techniques of somatic cell genetics. Cells of a mouse neuroblastoma variant line lacking MAO activity were fused to cytoplasts prepared from a mouse L cell line which expresses MAO activity and is resistant to chloramphenicol (a cytoplasmically inherited trait). The resulting cybrids were resistant to chloramphenicol, but did not recover MAO activity, indicating that the loss of activity in the neuroblastoma parent was not the result of an inherited lesion in a cytoplasmically transmitted gene. This cybrid cells were then fused to rat hepatoma cells expressing both A and B types of MAO activity. A resulting hybrid line, grown in medium containing hypoxanthine, aminopterin and thymidine (HAT) to select cells that had retained HPRT activity and hence the rat X chromosome, expressed both types of activity, but at a reduced level compared to the hepatoma parent. This finding indicates that the genetic lesion in the neuroblastoma cells resulting in loss of MAO activity is not phenotypically dominant, and that both A and B types of activity can be conferred together to neuroblastoma cells which normally express only the A type of MAO activity.     

MAH Cells and the Development of Rat Sympathetic Neurons

The generation of cell lines in the sympathoadrenal lineage has greatly facilitated our understanding of how precursor cells that do not respond to NGF give rise to mature NGF-dependent neurons. The neuronal developmental pathway in this lineage has been worked out by studying both primary precursor cells in culture and the v-myc-immortalized MAH cell line. MAH cells were established by retroviral infection of immunoisolated rat sympathoadrenal precursor cells. These cells have many of the characteristics of primary progenitor cells including neural precursor morphology, antigenic profile, and response to growth factors. MAH cells are able to recapitulate sympathetic development, giving rise to mature, postmitotic, NGF-dependent neurons. These cells have provided a model system for studying the factors, receptors, and modulating influences that play a role in the development of sympathetic neurons.     

Differential effects of various cAMP derivatives on the morphological and electrical maturation of neuroblastoma X glioma hybrid cells

The influence of adenosine 3′: 5′-cyclic monophosphate (cAMP) and some of its derivatives on the morphological differentiation and on the expression of electrical activity was investigated in neuroblastoma X glioma hybrid cells. This permanent cell line constitutes a well established culture system for studying neuronal properties in vitro. cAMP (1 mM) caused cell death. With 8-bromo-cAMP (0.1–1 mM) present giant multinuclear cells appeared, which were more obvious at 0.1 than at 1 mM 8-bromo-cAMP. 8-p-chlorophenylthio-cAMP (0.1–1 mM) induced an extension of neurites. These cellular processes were comparable to those elicited in the presence of db-cAMP (1 mM). However, only the cells treated with db-cAMP, but not those exposed to 8-p-chlorophenylthio-cAMP, were found to generate action potentials upon electrical stimulation. Neither dexamethasone nor carboxymethylcellulose, nor 8-bromo-cAMP could elicit the formation of processes in hybrid cells.     

Expression of human neuronal antigens in a mouse neuroblastoma x human dorsal root ganglion cell hybrid

Clonal mouse neuroblastoma cells were fused with cells from human foetal dorsal root ganglia and several continuously-growing hybrid clones isolated. One hybrid cell line (F2.1D1) containing a number of human chromosomes, was shown to retain the ability to extend neurites in response to dibutyryl cyclic AMP and to express various antigens characteristic of human foetal dorsal root ganglion neurons. The X-chromosome-controlled 12E7 antigen, human Thy-1 and the neuron-specific F12.A2B5 antigen were identified as surface components of the hybrid cells. None of these antigens were detected in the parental neuroblastoma cell line. In addition, using a species-specific monoclonal antibody, the hybrid cells were shown to synthesize human neurofilament protein. This is the first demonstration of the continued expression of a human species- and neuron-specific gene product in a human-mouse somatic cell hybrid.     

Expression of the neuron-specific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus-mediated cell fusion. The expression of 14-3-2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron-specific 14-3-2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno-types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial-specific protein S-100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.     

IKAP/hELP1 down-regulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor, and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP – down - regulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.     

DIFFERENTIATION OF NEUROBLASTOMA, GLIOMA, AND HYBRID CELLS IN CULTURE AS MEASURED BY THE SYNTHESIS OF SPECIFIC PROTEIN SPECIES: EVIDENCE FOR NEUROBLAST-GLIOBLAST RECIPROCAL GENETIC REGULATION

Abstract— Protein species from differentiating neuroblastoma, glioma, and hybrid neuroblastoma-glioma cell lines in cell culture were separated and identified initially in the first dimension by the use of isoelectric focusing gels and were further separated in the second dimension by SDS-acrylamide gels. There were two main classes of proteins identified: proteins which were dominantly expressed in neuroblastoma and also in hybrid cell cultures, and proteins which were expressed in glioma and also hybrid cell cultures. In general, proteins were identified which were significantly expressed in neuroblastoma cells and much reduced in glioma cultures, and also conversely so. The hybrid cell line expressed many of the neuroblastoma-type proteins and relatively fewer of the glioma type proteins. A specific protein species (2) was identified in hybrid cells and was not present in either parental neuroblastoma or glioma cultures. Protein z was expressed however by the co-culturing of neuroblastoma and glioma cells suggesting its induction is dependent on a soluble factor. Protein z in hybrid cells was demonstrated in both stained gels and by autoradiography. Chromosome analysis of hybrid cells confirmed the presence of both rat and mouse chromosomes. It is suggested that similar neuronal-glial interaction may be functional in the intact brain, and that similar reciprocal modulation between neurons and glia may be a central mechanism of differentiation in the nervous system.     

Studies on rat sympathetic neurons developing in cell culture. III. Cholinergic transmission.

Principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in culture with several types of non-neuronal cells. As described in the second paper of this series, the neurons in such mixed cultures formed two types of excitatory synapses with each other, electrical and chemical. Evidence is presented here that transmission at the chemical synapses was cholinergic. Four nicotinic ganglionic blocking agents (curare, hexamethonium, tetraethylammonium, and mecamylamine) strongly attenuated or eliminated the excitatory postsynaptic potentials (e.p.s.p.'s) at moderate concentrations; atropine at relatively high concentrations also blocked transmission. Iontophoretic application of acetylcholine (ACh) to the surface of the neurons gave rise to depolarizations that could be made to resemble the e.p.s.p.'s in size and time course; the ACh potentials and the e.p.s.p.'s were then similarly affected by nicotinic blocking agents. The sensitivity to ACh was often distributed nonuniformly on the neuronal surface; it was common to find small, sharply localized regions of high sensitivity. Catecholamines (norepinephrine, epinephrine, and dopamine) had only inhibitory actions; in a few experiments adrenergic blocking agents (phenoxybenzamine, propranolol) were found to have no effect on the e.p.s.p.'s. These observations leave no doubt that the neurons released ACh and had ganglionic, nicotinic ACh receptors on their surfaces. The significance of the fact that a high proportion of the sympathetic neurons in mixed cultures formed cholinergic synapses is discussed.     

Cholecystokinin-Induced Desensitization, Receptor Phosphorylation, and Internalization in the CHP212 Neuroblastoma Cell Line

Abstract: Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO-CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell-specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5-trisphosphate (IP₃) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP₃ responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist-bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO-CCKR cell line. This hybrid response supports the cell-specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.     

Differentiation of sympathetic and enteric neurons of the fowl embryo in grafts to the chorio-allantoic membrane

Summary Sympathetic cells (adrenergic neurons, SIF cells and chromaffin cells) and enteric neurons differentiate from migratory cells derived from the neural crest. The development of these cell types was studied in chorio-allantoic membrane (CAM) grafts, using combinations of tissues from domestic fowl embryos. Neural anlagen (neural tube and crest) of the vagal, cervico-thoracic and lumbo-sacral axial levels were equally capable of sympathetic differentiation, but this required somitic tissue for its significant expression. However, the vagal somites possessed only slight sympathogenic activity, thereby accounting for the negligible contribution of the vagal neural crest to the sympathetic nervous system.The same three levels of the neural anlage could furnish enteric neurons when combined directly with the aneuronal colo-rectum. However, the scale of this line of differentiation varied with the level of origin of the neural anlage, in contrast to the apparent equivalence in the ability to diffentiate as sympathetic cells. The density of enteric neurons in combinations with the vagal neural anlage was estimated as 60 times greater than the neuron density in combinations with the cervico-thoracic neural anlage. The lumbo-sacral neural anlage gave results similar to those of the cervico-thoracic level. Moreover, neural crest-derived pigment cells, positioned ectopically in the wall of the colo-rectum, were rare in combinations with the vagal neural anlage, but common in grafts with the other levels.When tested physiologically, the colo-rectum grown with the vagal neural anlage showed non-adrenergic, non-cholinergic inhibitory nervous activity in addition to the expected cholinergic excitatory responses. The neurons derived directly from vagal neural anlagen were similar to those that had reached the colo-rectum via their normal migratory pathways, when studied in terms of histological appearance, density of distribution and physiological responses.     

Cholinergic interneurons in the feeding system of the pond snail Lymnaea stagnalis. II. N1 interneurons make cholinergic synapses with feeding motoneurons.

The N1 neurons are a population of interneurons active during the protraction phase of the feeding rhythm. All the N1 neurons are coupled by electrical synapses which persist in a high Mg/low Ca saline which blocks chemical synapses. Individual N1 spikes produce discrete electrotonic postsynaptic potentials (PSPS) in other N1 cells, but the coupling is not strong enough to ensure 1:1 firing. Bursts of N1 spikes generate compound PSPS in the feeding motoneurons. The sign (excitation or inhibition) of the N1 input corresponds with the synaptic barrage recorded during the protraction phase. Discrete PSPS are only resolved in a Hi-Di saline. Their variation in latency and number can be explained by variation in electrotonic propagation within the electrically coupled network of N1 cells. The excitatory postsynaptic potentials (ESPS) in the 1 cell are reduced by 0.5 mM antagonists hexamethonium (HMT), atropine (ATR), curare (d-TC) and by methylxylocholine (MeXCh), all of which block the excitatory cholinergic receptor (Elliott et al. (Phil. Trans. R. Soc. Lond. 336, 157-166 (Preceding paper.) (1992)). The 1 cell EPSPS were transiently blocked by phenyltrimethylammonium (PTMA), which is both an agonist and antagonist at the 1 cell excitatory acetylcholine (ACh) receptor (Elliott et al. 1992). The inhibitory postsynaptic potential (IPSP) in the 3 cell is blocked by bath applications of MeXCh and PTMA, which both abolish the response of the 3 cell to ACh (Elliott et. al. 1992). The effects of the cholinergic antagonists on the response of 4 cluster and 5 cells to N1 stimulation matches their response to ACh (Elliott et al. 1992). It is concluded that the population of N1 cells are multiaction, premotor cholinergic interneurons.     

Human neuroblastoma cell lines having smooth muscle cell markers]

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural crest tumor) have at least two distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha -smooth muscle actin antibody. In addition, one of these cell lines (KP-N-SI) bound anti-desmin monoclonal antibodies as determined by indirect immunofluorescence. A total of eight cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to be the common neuroblastoma origin from each parent cell line by chromosomal analysis. Alpha-smooth muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by two-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3'UT) to human alpha-smooth muscle actin mRNA. This is the first report of the presence of alpha-smooth muscle actin and desmin in neuroblastoma cell lines. These data show that in addition to giving rise to cells with neural, Schwann cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth muscle cell markers.     

Conditions increasing the adrenergic properties of dissociated chick superior cervical ganglion neurons grown in long-term culture

Neurons dissociated from the embryonic chick superior cervical ganglion (SCG) were separated from ganglionic nonneuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 3-4 weeks in serum containing medium on a polyornithine substrate precoated with heart-conditioned medium. Both catecholamine (CA) and acetylcholine (ACh) are synthesized and accumulated by these neurons, but the amount of CA is higher and increases much more over time in culture than the amount of ACh. The cultures become therefore more adrenergic with time. We report here that the adrenergic properties of these cells can be enhanced. A 3-fold increase in CA synthesis, as expressed on a per neuron basis, is obtained by increasing neuron cell density 3- to 4-fold. ACh synthesis, however, is decreased at high neuronal density. Optimal CA production is obtained at densities of 120-150,000 neurons/cm2. This effect is due to direct cell contact since it cannot be transferred to low density cultures by medium conditioned by high density cultures. Nerve growth factor concentrations 5-10-fold higher than the amount necessary for optimal neuronal survival (1 microgram/ml 7S NGF) increases CA production but do not affect ACh synthesis. This effect is highest at low plating densities (20-30,000 neurons/cm2, 10-fold increase) and progressively decreases with increasing neuronal density. No increase is obtained in high density cultures where CA production is maximal. In addition, we made the novel observation that medium conditioned by chick liver cells in culture (LCM) increases CA production approximately 4-fold, whereas it does not increase ACh production by the SCG neurons. Work is in progress to biochemically characterize the active component(s) present in the LCM and to determine whether they favor the survival of a subpopulation of adrenergic neurons possible present in these ganglia. Alternatively, the adrenergic differentiation of neurons initially capable of synthesizing both CA and ACh could be selectively increased by LCM.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.