NB2a Neuroblastoma Cells Research Articles

Unitary conductance of Na+ channel isoforms in cardiac and NB2a neuroblastoma cells.

Unitary conductances of native Na+ channel isoforms (gamma Na) have been determined under a variety of conditions, making comparisons of gamma Na difficult. To allow direct comparison, we measured gamma Na in cell-attached patches on NB2a neuroblastoma cells and rabbit ventricular myocytes under identical conditions [pipette solution (in mM): 280 Na+ and 2 Ca2+, pH 7.4; 10 degrees C]. gamma Na of NB2a channels, 22.9 +/- 0.9 pS, was 21% greater than that of cardiac channels, 18.9 +/- 0.9 pS. In contrast, respective extrapolated reversal potentials, +62.4 +/- 4.6 and +57.9 +/- 5.1 mV, were not significantly different. Several kinetic differences between the channel types were also noted. Negative to -20 mV, mean open time (MOT) of the NB2a isoform was significantly less than that of cardiac channels, and, near threshold, latency to channel opening decayed more rapidly in NB2a. On the basis of analysis of MOT between -60 and 0 mV, the rate constants at 0 mV for the open-to-closed (O-->C) and open-to-inactivated (O-->I) transitions were 0.42 +/- 0.11 and 0.47 +/- 0.11 ms-1 in NB2a and 0.10 +/- 0.06 and 1.19 +/- 0.07 ms-1 in myocytes. The slope factors were -38.9 +/- 8.7 and +10.7 +/- 6.1 mV in NB2a and -27.3 +/- 7.1 and +23.7 +/- 4.9 mV in myocytes. Transition rate constants were significantly different in NB2a and cardiac cells, but voltage dependence was not.

Transport of Carnitine in Neuroblastoma NB-2a Cells

Carnitine accumulation was measured in cultured neuroblastoma NB-2a cells. This process was found partially sodium dependent and its kinetics to be a sum of a saturable transport ( K m = 123 ± 13 μM) and diffusion ( D = 63 ± 7 pmol/mg protein/min/mM). On the contrary to previous reports on neural cells, the accumulation of carnitine was found insensitive to γ-aminobutyric acid (GABA). Measurements of carnitine accumulation in the presence of different compounds resulted in the conclusion that carnitine transport does not occur through the known systems specific toward choline and/or amino acids. For instance, an observed inhibition of carnitine transport by serine and cysteine, without any effect of alanine, excluded a possible role of ASC amino acid transport system. An involvement of a new transporter is thus postulated, specific toward compounds with a polar group in the β position with respect to the carboxylic group.

The toxicity of artemisinin and related compounds on neuronal and glial cells in culture

The antimalarial drug artemisinin and a number of its derivatives were tested for their effects on proliferation of undifferentiated neuroblastoma Nb2a cells and glioma C6 cells in culture as well as their ability to inhibit neurite outgrowth from Nb2a cells differentiated by removal of serum and addition of dibutyryl cyclic AMP. In the Nb2a and C6 cell cultures, all drugs except desoxyartemisinin significantly inhibited cell proliferation in a dose-related manner with the lowest effective concentration being that of artemisinin at 0.1 μM. Artemether, arteether, artemisinin and dihydroartemisinin also produced a dose-related decrease in the number of neurites/extensions formed by differentiating Nb2a cells, with an effect of dihydroartemisinin at a concentration as low as 1 nM. Desoxyartemisinin had no effect on extension/neurite formation. We propose a potential mechanism for neurotoxicity of artemisinin and its derivatives that involves the endoperoxide bridge which is also known to be necessary for their antimalarial action.

Exogenous thrombin inhibits neuritogenesis in cultured neuroblastoma cells but not in rat hippocampal neurons

Thrombin has been reported to inhibit neurite outgrowth from neuroblastoma cells grown in serum-containing medium after switching to serum-free medium. A test of the serum and substrate-dependence of this inhibition became possible with the development of Neurobasal/B27 serum-free medium. Inhibition of sprouting of Nb2a neuroblastoma cells by thrombin occurred from the substrate where it was bound to material adsorbed from serum. Neuritogenesis of primary hippocampal neurons was unaffected by exogenous thrombin on polylysine substrates with or without serum treatment. However, sprouting of hippocampal neurons was stimulated by treating the substrate with hirudin, a highly specific thrombin inhibitor. This suggests that hippocampal neurons are not directly responsive to added thrombin, perhaps because they produce their own thrombin.

Sodium channels in cultured neuroblastoma cells grown in high glucose or l-Fucose

Patch clamp techniques were used to record whole cell and single channel Na+ currents from NB41A3 neuroblastoma cells grown in culture. Cells were grown for two weeks in control medium or medium supplemented with 30 mM D-glucose of 30 mM L-fucose. Cells exposed to glucose or L-fucose had smaller whole cell Na+ currents than cells grown in unsupplemented medium, consistent with earlier studies (Yorek, Stefani & Wachtel, 1994). Whole cell macroscopic currents showed no change in activation or inactivation kinetics. Single channel current properties and opening probability were also unchanged. The number of [3H]saxitoxin binding sites, and therefore the total number of Na+ channels, was not reduced in cells grown in glucose or L-fucose (Yorek et al., 1994). Therefore, we conclude that some of the channels must have been rendered nonfunctional by the conditioning media. The finding that single channel properties are not altered suggests that channels become nonfunctional in an all-or-none manner.

Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element.

Effect of Golgi membrane phospholipid composition on the molecular species of GM3 gangliosides synthesized by rat liver sialyltransferase.

Previous studies have demonstrated that there is a change in the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase (LacCer alpha 2,3-ST) when the lipid composition of the Golgi membrane is altered (Kadowaki, Grant, and Williams, 1993. J. Lipid Res. 34: 905-914). To understand the basis of this phenomenon, the molecular species specificity of rat liver LacCer alpha 2,3-ST was determined under conditions in which the phospholipid class composition of the Golgi membrane was changed to resemble that of cultured mouse neuroblastoma NB2a cells, a cell line in which LacCer alpha 2,3-ST exhibits no molecular species specificity. The change in the lipid composition of the Golgi membrane was accomplished by incubating the Golgi membrane vesicles with nonspecific lipid transfer protein and a 10-fold excess of liposomes prepared with various proportions of purified rat liver lipids. The molecular species specificity of LacCer alpha 2,3-ST was also determined under conditions where the phospholipid molecular species composition but not the phospholipid class composition of the Golgi membrane was changed, and in which both the phospholipid class and molecular species compositions were changed by using liposomes prepared with lipids purified from a mouse brain tumor (ependymoblastoma). When using liposomes prepared with rat liver lipids, a change in the phospholipid class composition of the Golgi membrane to a composition similar to that of NB2a cells increased rather than decreased the molecular species specificity of rat liver LacCer alpha 2,3-ST.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of USF and c-Myc with a helix-loop-helix-consensus motif in the core promoter of the murine type II beta regulatory subunit gene of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase.

Previous studies showed that the core promoter of the mouse cAMP-dependent protein kinase regulatory subunit type II beta (RII beta) gene was composed of two functional elements. One element was GC rich and bound the Sp1 transcription factor. The second element contained a helix-loop-helix (HLH)-motif. Each element conferred transcriptional activity when inserted upstream of a reporter gene, chloramphenicol acetyltransferase and transfected into mouse NB2a neuroblastoma cells and Chinese hamster ovary (CHO) cells. The core promoter was further characterized by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factors and reduced the transcriptional activity of constructs containing the chloramphenicol acetyltransferase reporter when transfected into these cells. The results showed that the central nucleotides as well as the adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and interference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription factors were forming complexes with the HLH-consensus motif. The results suggest that RII beta transcription may be regulated, in part, by USF and c-Myc in NB2a and CHO cells.

Synergistic Effect of Choline and Carnitine on Acetylcholine Synthesis in Neuroblastoma NB-2a Cells

An influence of carnitine on acetylcholine synthesis from radiolabeled glucose was monitored in neuroblastoma NB-2a cells. Upon addition of carnitine the distribution of its derivatives was found significantly different than the values published for brain, the level of long-chain acyl derivatives being much higher and reaching 60%. Carnitine itself did not change acetylcholine level. Together with choline (20 μM), carnitine was observed to stimulate (by 36%) acetylcholine synthesis in a synergistic way, which indicated that both substrates could be limiting factors of this process in NB-2a cell line of neuroblastoma.

Phosphorylation States of Actin Filament Adenine Nucleotides in Detergent-Extracted Neuronal Cytoskeletal Fractions

High performance liquid chromatography of nucleotides from Triton X-100 cytoskeletal extracts has permitted analysis of the ATP and ADP content of actin filaments isolated intact from PC12 pheochromocytoma cells. We observed that the adenine nucleotide content matched the actin content of these cytoskeletal extracts, a finding consistent with the unit stoichiometry of nucleotide binding. Efficient assembly-linked ATP hydrolysis occurs in vivo, and based on a boundary hydrolysis model for nucleotide-promoted assembly, the observed ADP/ATP ratio indicates that the average microfilament in nonmuscle cells has 24 ATP-actin molecules at its growing end. Studies with NB41A3 neuroblastoma cells indicate that the ATP content of assembled actin filaments is about 3-4 times lower.

Glia‐derived nexin/protease nexin‐1 is expressed by a subset of neurons in the rat brain

Glia-derived nexin/protease nexin-1 (GDN/PN-1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN-1 in the developing and adult rat brain. In contrast to a transient widespread expression during pre- and postnatal development, some brain structures constitutively express GDN/PN-1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN-1. Furthermore GDN/PN-1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN-1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor.

Patterned neuronal attachment and outgrowth on surface modified, electrically charged fluoropolymer substrates.

Fluorinated ethylenepropylene copolymer (FEP) and polyvinylidene fluoride (PVDF) can generate static and transient electrical charges, respectively, after bulk molecular rearrangements induced by electrical charging techniques. Neurons cultured on electrically active FEP and PVDF show increased levels of nerve fiber outgrowth compared to electrically neutral material. The purpose of the present study was to determine if the addition of charged surface groups to the surfaces of FEP and PVDF would modify the influence of bulk electrical charges on cultured neurons. Mouse neuroblastoma (Nb2a) cells were cultured on electrically charged and uncharged FEP and PVDF substrates with covalently modified surfaces containing hydroxyl (OH) and amine (NH2) groups. Surface chemical modification was performed on the entire surface or in discrete striped regions. Nb2a cells cultured on electrically active FEP and PVDF showed greater levels of differentiation than cells on electrically neutral substrates. The presence of NH2 groups attenuated these responses in serum-containing media. Cells attached to NH2 rich surfaces generally displayed a flatter morphology and tended to remain attached for longer time periods. Cells cultured on stripe-modified substrates in serum-containing media showed a strong preferential attachment to modified regions, especially on NH2 stripes. In summary, bulk electrical charges are more important than surface charges in stimulating Nb2a cell differentiation. Surface groups serve to modulate neuronal morphology and confer specific attachment promoting properties in serum-containing media. The development of an optimal neuronal regeneration template may require the incorporation of specific bulk and surface properties.

Comparative toxicity of amyloid β-peptide in neuroblastoma cell lines: Effects of albumin and physalaemin

1. Synthetic amyloid beta-peptide was toxic to NB41A3 neuroblastoma cells in serum-free culture as judged by decreasing cell numbers and release of the cytosolic enzyme, lactic dehydrogenase. 2. Without amyloid beta-peptide, bovine serum albumin increased the number of cells surviving in culture. 3. In the presence of amyloid beta-peptide, BSA appeared to potentiate the amyloid beta-peptide toxicity. 4. The toxic dose response for amyloid beta-peptide varied between different cell lines (NB41A3, NB2a and IMR32), in a range of 100-1000 nM amyloid beta-peptide. 5. Amyloid beta-peptide toxicity was inhibited by the concurrent treatment of the cells with the tachykinin physalaemin with an ED50 of 10(-6) M.

Magnitude and polarity of a fluoroethylene propylene electret substrate charge influences neurite outgrowth in vitro.

Positively charged coating materials such as polylysine improve neuronal attachment in vitro. Due to the structural complexity of these charged molecules, it is unclear whether neuronal effects are due to charge or to physicochemical effects, or both. Polymeric materials with charge storage capabilities and defined surface properties may provide a model in which electrical charge and surface property effects can be separated. Fluorinated ethylenepropylene (FEP) films can store negative or positive charges injected through a corona charging process, thus generating a negative or positive external electrostatic field. In the present study, mouse neuroblastoma (Nb2a) cells were cultured on positive, negative, and uncharged FEP substrates, in both serum-containing and serum-free media. Cell attachment, differentiation, and neurite outgrowth were assessed 24, 48, 72 and 96 h after plating. Electron spectroscopy for chemical analysis (ESCA), contact angle analysis, and scanning electron microscopy (SEM) revealed no differences in surface chemistry and topography between positive, negative, and uncharged FEP. No significant differences in the levels of cell attachment on positive, negative, and uncharged substrates were observed. Significantly higher levels of neurite outgrowth, however, were observed with positive substrates as compared to negative and uncharged substrates, in both media conditions. Substrates charged to +1000 V showed greater levels of outgrowth compared to +500 and +3000 V, suggesting the presence of an optimal range of charge for neurite outgrowth. These results show that cell/charge interactions mediate cell effects on electrically charged substrates with identical surface chemistry, topography and adhesivity.

Effect of membrane lipids on the lactosylceramide molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide sialyltransferase

It has previously been shown that when the molecular species specificity of rat liver Golgi CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase was determined, using as the substrate lactosylceramide (LacCer) incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a pronounced variation in activity towards the various molecular species of LacCer (J. Lipid Res. 1989. 30: 1789-1797). In this paper, -the LacCer molecular species specificity of sialyltransferase from neuroblastoma NB2a cells was examined using five naturally occurring and three synthetic molecular species of LacCer. The enzyme activity was determined by following the formation of [14C]GM3 from CMP-[14C]neuraminic acid and individual molecular species of LacCer incorporated into liposomes. Nonspecific lipid transfer protein was included in the enzyme assay to facilitate the transfer of LacCer and other lipids between the liposomes and the membrane where sialyltransferase is located. In these enzyme assays the liposomes contained approximately 10 times more lipid phosphorus than either the microsomal fraction of NB2a cells or the Golgi fraction of rat liver. Thus, in the presence of nonspecific lipid transfer protein, the lipid composition of the membrane where sialyltransferase is located was modified to resemble the lipid composition of the liposomes. When the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the enzyme showed no specificity towards the various molecular species of LacCer. However, when the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a variation in activity towards the various LacCer molecular species similar to that observed with the liver Golgi enzyme using liposomes prepared with liver Golgi lipids. Likewise, when the molecular species specificity of rat liver Golgi sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the liver enzyme then showed no specificity towards the various molecular species of LacCer. These results indicate that the lipid environment of the membrane can alter the molecular species specificity of sialyltransferase towards its lipid substrate, LacCer.

Electrically charged polymeric substrates enhance nerve fibre outgrowth In vitro

The physical, chemical and electrical properties of synthetic guidance devices are known to influence nerve regeneration in vivo. In the present study, neurons were cultured directly on electrically charged polymer growth substrates to determine if local electrical charges enhance nerve fibre outgrowth in vitro. Piezoelectric polymers such as polyvinylidene fluoride (PVDF) generate transient surface charges under minute mechanical strain. Mouse neuroblastoma (Nb2a) cells were cultured directly on electrically poled (i.e. piezoelectric) and unpoled (i.e. nonpiezoelectric) PVDF substrates in serum-free and serum-containing media. Nerve fibre outgrowth was analysed 24, 48, 72 and 96 h after plating. Piezoelectric PVDF substrates generated 2–3 mV at 1200 Hz when placed on standard incubator shelves and unpoled PVDF substrates showed no output. Nb2a cells grown on piezoelectric substrates exhibited significantly greater levels of process outgrowth and neurite lengths at all time periods for both media conditions. Detailed surface characterization of PVDF substrates using electron spectroscopy for chemical analysis (ESCA) and a comprehensive wettability profile revealed that poled and unpoled PVDF was chemically indistinguishable and showed similar surface wettabilities and adhesive properties. Therefore, we conclude that enhanced process outgrowth was induced by the film's piezoelectric output, making poled PVDF a unique biomaterial for which cell/polymer interactions are mediated predominantly through bulk electrical properties rather than surface properties.

Modulation of neurite outgrowth in neuroblastoma cells by protein kinase C and platelet-activating factor.

Previous reports have shown that thrombin and activators of protein kinase C (PKC) inhibit neurite outgrowth (NOG) in neuroblastoma cells cultured in serum-free medium. Therefore, we tested the hypothesis that PKC activation mediates the effect of thrombin on NOG in murine neuroblastoma NB-2a cells. After 2 h in serum-free medium, 70% of the cells displayed neurites; addition of 300 ng/ml thrombin reduced NOG to 24% within 1 h. This inhibition was reduced after NB-2a cells were pretreated for 24 h with 200 nM phorbol dibutyrate down-regulate PKC. Thrombin and phorbol 12-myristate 13-acetate inhibited NOG in an additive way and the protein kinase inhibitors H-7, H-8, and HA1004 reversed the effect of thrombin on NOG with a rank order of activity consistent with PKC inhibition. Furthermore, PKC was translocated from the cytosol to a membrane-bound form 5 to 10 min after addition of thrombin. These findings indicate that thrombin inhibits NOG through a PKC-dependent pathway. Thrombin stimulates the synthesis of the phospholipid platelet-activating factor (PAF) in some cells. However, NOG was markedly stimulated when PAF or its analogue carbamyl-PAF were added to NB-2a cells in medium with serum. Furthermore, the PAF receptor antagonist SRI 63072 inhibited NOG in NB-2a cells in serum-free medium. These cells accumulated PAF with kinetics similar to that of NOG inducPAF was synthesized by the de novo pathway, as shown by the incorporation of [3H]choline. These findings suggest that PAF is a mediator of NOG in NB-2a cells. Thrombin neither stimulates nor inhibits PAF synthesis in these cells.

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RIIβ of type II cAMP-dependent protein kinase

The promoter and exon 1 of the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5′-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RIIβ 5′-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp −291 −121 . An enhancer element was located between bp −1426 −1018 .

Effect of Differentiation and Cell Density on Glycosphingolipid Class and Molecular Species Composition of Mouse Neuroblastoma NB2a Cells

The effects of cell density and retinoic acid-induced differentiation on the class and molecular species composition of mouse neuroblastoma NB2a cell glycosphingolipids were examined under conditions where the period of culture was controlled. The total amount of neutral glycosphingolipids per cell decreased both with differentiation and as the cells became confluent. The relative amount of the neutral glycosphingolipid classes was not affected by differentiation, whereas there were small but significant changes in the relative amount of the neutral glycosphingolipid classes as the cells became confluent. The total amount of the gangliosides was unaffected by either differentiation or cell density, but there were significant changes in the ganglioside class composition as a result of both cell density and differentiation, and the effects were additive. The molecular species of all the major neutral glycosphingolipid and ganglioside classes were essentially identical, and were altered only slightly by either differentiation or cell density.

Suramin, an anti-cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A

Protein kinase C purified from rat brain was found to be inhibited by suramin, a substance used originally in the therapy of antitrypanosomic infections and more recently proposed as antineoplastic agent. The inhibition of suramin was competitive with one of the substrates of the enzyme, ATP with a K i of 10 μM. At concentrations adequate to inhibit the isolated enzyme, suramin was shown to slow the rate of proliferation of neuroblastoma NB2A cells in vitro and to induce their differentiation as evidenced by typical morphological changes.