Glucose-dependent glycosylation of secretory glycoprotein in mouse myeloma cells.

The mouse myeloma tumor, MOPC-46, produces a kappa-type immunoglobulin light chain that may be isolated from the urine of tumor-bearing animals. This protein possesses a single carbohydrate side chain, attached by glycosylamine linkage to asparagine residue 28. When viable single cell suspensions of the tumor are incubated in vitro in minimum essential medium containing sodium pyruvate as a source of carbon and energy, the major protein synthesized and secreted corresponds to a nonglycosylated form of the kappa light chain. However, when glucose or mannose are substituted for sodium pyruvate as a source of carbon, the immunoglobulin light chain is synthesized and secreted in the fully glycosylated, native form. The dependence of normal glycosylation of the protein on the presence of either glucose or mannose in the medium is relatively specific for these compounds since substitution with either fructose, galactose, glycerol, ribose, or N-acetylglucosamine was ineffective. The nonglycosylated protein produced in the presence of sodium pyruvate was characterized as nonglycosylated MOPC-46 light chain by immunoprecipitation and gel electrophoresis. An identical nonglycosylated protein was produced by tumor cells in the presence of glucose when the incubation mixtures contained tunicamycin.     

Oleic acid derived metabolites in mouse neuroblastoma N18TG2 cells

Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.     

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.     

Interaction of neuroblastoma C 1300 N18 cells with liposomes and intermembrane metabolism of lipids

The methods of isotopic and fluorescent labels have shown that interaction of cells of neuroblastoma S 1300 N 18 with small one-layer neutral liposomes prepared of the egg phosphatidyl choline with the addition of different amounts of cholesterol is realized by two mechanisms: the transmembrane transfer of cholesterol by the concentration gradient and membrane lipid metabolism proper, the ratios of cholesterol phospholipids in the biological and artificial membranes being equal. A dependence is established of the neuroblastoma cell viability on the activity of the cholesterol membrane metabolism. A problem on the mechanism which causes the death of cells during the interaction with phosphatidyl-cholesterol liposomes is under discussion.     

Transmembrane lipid transport between lecithin liposomes and neuroblastoma C1300 N18 cells

The method of double isotopic labels was used to study dynamics of lipid metabolism between neuroblastoma C 1300 N 18 A 1 cells and lecithin liposomes which contained 4.5-5 mumol of lecithin in 1 ml of the suspension. The cell lipids were labelled by radioactive carbon and cultivated on the medium with [1-14C] sodium acetate, phosphatidylcholine of liposomes was labelled by tritium. It is shown that 15-30 min long incubation with liposomes causes a sharp decrease of the cholesterol esters amount with a simultaneous fall of the free cholesterol level. The total content of phospholipids in this case remains unchanged though there occurs the noticeable exchange of labelled phospholipids between cells and liposomes. The cholesterol content in the plasma membranes of cells lowers sharply. The neuroblastoma cells are able to compensate arising changes in the cholesterol level for 45-60 min after which they progressively die. 90 min later only an insignificant part of the population (about 10% of cells) is retained.     

Study of the intermembrane lipid metabolism between neuroblastoma C 1300 N18 cells and liposomes using the double isotope label technic

Methodological approaches are developed for simultaneous study of the intermembrane transfer of lipids by the concentration gradient, equilibrium metabolism of lipids between neuroblastoma and liposome cells as well as of translocation and metabolism of exogenous and endogenous lipids in cells. Methods to calculate the content of endogenous and exogenous lipids in cells and liposomes taking into account their possible metabolism are suggested.     

Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells

The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG.     

Abnormal glycosylation of myelin-associated glycoprotein in quaking mouse brain

Brain slices from actively myelinating (26–28 days) quaking and normal littermates were dual-labeled with radioactive mannose and fucose for 2 h. Following the incubation myelin was isolated by sucrose density gradient centrifugation and the incorporation of sugars into the major myelinassociated glycoprotein (MAG) determined. The incorporation of mannose (an internal monosaccharide) and fucose (a terminal monosaccharide) was impaired in quaking by approximately 70 and 83% respectively as compared to control. The mannose/fucose ratio in quaking myelin was approximately 70% higher than in control. The results indicate an abnormal processing of the N-linked oligosaccharide moiety of MAG in quaking oligodendrocytes.     

Interaction between sodium channels in mouse neuroblastoma cells

Single sodium channels in mouse neuroblastoma cells (N1E 115) were studied in cell-attached patches. During a series of consecutive responses to depolarizing pulses, records with and without channel opening were seen to form clusters rather than appearing randomly. The probability of finding open channels on a record seemed to increase with increasing number of channel openings. The open times of channels became shorter with increasing closed time interval measured between consecutive channel openings. Overlapping openings showed a voltage-dependent open time, in contrast to single openings which had voltage-independent open time. On the basis of these observations interaction between neighbouring sodium channels is suggested.Abbreviations RP resting potential - OT channel open time     

Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells

Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth.     

The ultrastructure of mouse neuroblastoma cells in tissue culture.

Neuroblastoma cells grown in suspension culture are round and have no distinctive structural characteristics. However, cells transferred to substrates flatten, develop long neurites, and assume the morphology of normal neurons. The resemblance of monolayered neuroblastoma cells to normal neurons is amplified by treatment with hypertonic medium; under these conditions, cell division is inhibited and the neurites become long and differentiated. The treated cells contain clusters of clear vesicles, 400-600 A in diameter, which are morphologically indistinguishable from the synaptic vesicles of normal neurons. Specialized cell contacts are observed between the treated cells as well as between confluent cells grown in normal medium.     

Neurotransmitter metabolism in murine neuroblastoma cells.

Murine neuroblastoma cells in culture are able to synthesize the putative neurotransmitters--acetylcholine, dopamine, norepinephrine, tyramine, octopamine, histamine, serotonin and γ-aminobutyric acid (GABA). They possess not only synthetic, but also degradative enzymes involved in metabolism of these transmitters, and many of these enzymes increase in activity as the cells “differentiate”. Catecholamines, and perhaps other transmitters, appears to be stored within membrane-limited vesicles which accumulate within the process endings of these cells. Uptake of some transmitters, GABA, glycine, dopamine and norepinephrine, shows characteristics of the high affinity transport systems observed in other neuronal populations; uptake of choline and other amino acids is similar to that in non-neuronal populations. Cells show receptor sensitivities to acetyl-choline, dopamine, norepinephrine, prostaglandins E₁ and morphine, as demonstrated by electrophysiologic, toxin binding and cyclic nucleotide studies.     

Glia maturation factor beta regulates the growth of N18 neuroblastoma cells

Glia maturation factor beta (GMF-beta) is a 17-kDa growth regulating protein isolated from the brain. The effect of bovine GMF-beta on neurons was tested on the neuroblastoma line N18 and the pheochromocytoma line PC12. GMF-beta inhibited the proliferation of N18 cells and promoted their neurite outgrowth, with an increase in neurofilament protein, but had no effect on PC12 cells. This was in contrast to nerve growth factor (NGF) which regulated PC12 but not N18. Acidic fibroblast growth factor (FGF), on the other hand, had a weak effect on PC12 but none on N18. Antisera against GMF-beta and NGF neutralized the biological activity of the corresponding growth factors but showed no cross-neutralization. Fluorescence visualization revealed the binding of GMF-beta to N18 cells but not to PC12 cells; the opposite was true with NGF.     

Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.     

Insulin-like growth factor I receptors on mouse neuroblastoma cells. Two beta subunits are derived from differences in glycosylation

We have characterized receptors for the insulin-like growth factor (IGF-I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell-free system, IGF-I and insulin stimulated the phosphorylation of 95-kDa and 105-kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF-I receptor beta subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor beta subunit is a separate single 95-kDa protein. Enzymatic digestion of IGF-I receptor beta subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS-PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N-linked complex-type carbohydrate chains on the extracellular domain of beta subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion. Analysis of IGF-I receptor beta subunit subtypes in these cells may provide insights into the mechanism of action of IGF-I on neural tissues.     

Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.     

Rapid simultaneous isolation of microsomes and plasma membranes from neuroblastoma C 1300 N 18 cells

The method is suggested to isolate simultaneously microsomes and plasma membranes of neuroblastoma S 1300 N 18 cells by means of differential centrifugation in the step density gradient of Percoll/Ficoll with a high degree of purification determined from the activity of marker enzymes (acetyl cholinesterase Na+,K+-ATPase, alkali phosphatase, glucose-6-phosphatase, succinate-dehydrogenase, acid phosphatase) as well as from the content of DNA and RNA and with a sufficiently high protein yield. The purified fractions of microsomes and plasma membranes are established to contain no phosphatidyl glycerol and cardiolipin--safety markers of mitochondrial membrane purification. A degree of separation of microsomes, plasma membranes and proteins dissolved in cytosol may be estimated by the activity of the cholesterol-synthesizing system of enzymes with the use of sterol-transferring protein.     

Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells.

The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors.     

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 2 · 10}{}^{\mathrm{?7}}\text{M}$). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.     

Muscarinic pharmacology of the inhibition of adenylate cyclase in N18TG2 neuroblastoma cells

This study characterizes the muscarinic cholinergic receptors associated with the inhibition of adenylate cyclase on N18TG2 neuroblastoma cell membranes. Agonists could be divided into two classes: oxotremorine, acetylcholine, carbachol and arecoline exerted the most efficacious and potent inhibition, while McN-A343, bethanechol and AHR-602 were partial agonists. Both quinuclidinyl benzilate and atropine maximally antagonized the inhibitory effect of McN-A343, carbachol and oxotremorine. Pirenzepine was almost as potent as atropine in reversing the inhibitory effect of McN-A343, but was 300 times less potent than atropine or quinuclidinyl benzilate in antagonizing the effects of either carbachol or oxotremorine. Gallamine was ineffective as an antagonist at concentrations up to 1 mM. These results suggest that the receptors that modulate this inhibition are of the M₂ type, since they were activated by carbachol, acetylcholine and oxotremorine, but much less by McN-A343 and AHR-602 (both M₁ selective agonists). The full agonists were blocked by atropine and quinuclidinyl benzilate but not by low concentrations of pirenzepine (M₁ selective antagonist).