共查询到20条相似文献,搜索用时 15 毫秒
1.
Tatsuro Koike 《生物化学与生物物理学报:生物膜》1978,509(3):429-439
A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K+ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells. 相似文献
2.
Opioid activities of beta-casomorphins 总被引:1,自引:0,他引:1
Victor Brantl Hansjörg Teschemacher Julia Bläsig Agnes Henschen Friedrich Lottspeich 《Life sciences》1981,28(17):1903-1909
β-Casomorphin-7 (H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH) and its analogues: β-casomorphin-6, (-5) and (-4) (derived by sequential removal of respectively one, two or three amino acid residues from the C-terminus), were tested for their opioid activities in a variety of assay systems. Each of the four peptides displayed opioid activity in an opiate receptor binding assay, the isolated mouse vas deferens (MVD), the guinea-pig ileum longitudinal muscle myenteric plexus preparation (GPI) and produced naloxone-reversible analgesia after intracerebroventricular injection into rats. In contrast, none of the peptides displayed opioid activity in the isolated rat vas deferens preparation (RVD). β-Casomorphin-5 was the most potent compound in all the assays employed. Each β-casomorphin was more potent on the GPI than on the MVD. In view of the fact that the GPI, MVD and RVD are populated predominantly by μ-, δ- and ε-receptors, respectively, the β-casomorphins probably represent μ-type opiate receptor agonists. 相似文献
3.
Gangliosides administered exogenously are well-known effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro-2A cells with bovine brain ganglioside mixture (BBG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BBG concentration of 25 microM. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on ganglioside-stimulated neurite outgrowth. A similar result was obtained with psi-tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro-2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded. 相似文献
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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. 相似文献
6.
Han NL Wen J Lin Q Tan PL Liou YC Sheu FS 《International journal of biological sciences》2007,3(5):263-273
Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways. 相似文献
7.
Neurite outgrowth of neuroblastoma cells: dependence on adhesion surface--cell surface interactions 总被引:1,自引:2,他引:1
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《The Journal of cell biology》1984,98(3):1010-1016
Neurite outgrowth of C 1300 neuroblastoma cells, which were dispersed from adherent cultures or grown in suspension, was studied on different protein-coated surfaces. Of 29 different surface structures studied, including surfaces treated with various fibronectins, lectins, glycosidases, or glycosyltransferases capable of stimulating fibroblast spreading, only the surfaces coated with plasma fibronectin or with a protein mixture secreted by C6 glioma cells displayed an extensive activity in the sprouting assay. Neurite outgrowth was inhibited by brain gangliosides and by colominic acid (a sialic acid polymer). A 50% inhibition of neurite outgrowth of N18 neuroblasts induced by the glioma cell proteins was observed at the following approximate concentration: 100 microM (0.2 mg/ml) GD1A ganglioside, 20 microM (0.04 mg/ml) GT1B ganglioside, and 5 mg/ml colominic acid. Specificity of inhibition was suggested by the finding that a few polyanionic substances tested were not inhibitory in the sprouting assay, and that the type of gangliosides inhibiting sprouting were found to be major sialoglycolipids of the neuroblasts. A hypothesis is discussed, according to which neurite outgrowth of neuroblasts is stimulated by adhesion involving interactions of the adhesion-mediating protein with cell surface carbohydrates characteristic of brain gangliosides. 相似文献
8.
Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells
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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. 相似文献
9.
Mouse neuroblastoma cells have been tested for their responsiveness to iontophoretically applied acetylcholine, norepinephrine, dopamine, and serotonin using intracellularly recorded membrane potential and resis tance as indicators of response. Acetylcholine can elicit either a membrane depolarization (D-response) or hyperpolarization (H-response) or a compound D-H response from different neuroblastoma cells of a given culture. Some 60 to 70% of tested cells gave no measurable response. A change of culture medium (from DMEM to F-12) increased the incidence of H-responses 2-fold. Dopamine elicited H-responses in about one-quarter of cells tested, while no responses were elicited by norepinephrine or serotonin in a small sample of cells. 相似文献
10.
Neurite outgrowth of neuroblastoma cells induced by proteins covalently coupled to glass coverslips 总被引:4,自引:0,他引:4
A method using proteins covalently coupled to glass surfaces has been applied to studies on the differentiation of neuroblasts. Neuroblastoma cells from clones N 18 and NIE 115 adhere to surfaces coated with fibronectin or laminin and extend rapidly growth cone-containing neurites. Some lectin-coated surfaces are also able to support neurite outgrowth, although the activities are lower than those of fibronectin and laminin. We discuss the biochemical requirements of the surface structures capable of inducing a differentiated neuronal morphology to neuroblastoma cells, and also consider the possible relationship of the results to physiological differentiation phenomena. 相似文献
11.
Electrogenesis in mouse neuroblastoma cells in vitro 总被引:12,自引:0,他引:12
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. 相似文献
12.
Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition 总被引:4,自引:0,他引:4
Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites. 相似文献
13.
Arachidonic acid (AA) incorporation into phospholipids and cyclooxygenase and lipoxygenase mediated metabolism of arachidonic acid were studied in homogenized and intact Neuro-2A cells. When 3H8-AA was added to homogenized cells and incubated 20 minutes, 39% of the label was converted to prostaglandins (PGs), 10% to hydroxy-eicosatetraenoic acid (HETE) and 26% was incorporated into phospholipids. PGE2 and PGF2a were the major PGs produced. Synthesis of PGs was blocked by 10 microM indomethacin and synthesis of PGs and HETE was blocked by 10 microM eicosatetraynoic acid (ETYA). The cell homogenate produced the 13,14-dihydro-15-keto metabolites of PGE2 and PGF2a from 3H8-AA and also converted exogenous 3H7-PGE2 and 3H8-PGF2a to metabolites. When intact cells were labeled for 24 hours with 14C1-AA and the cells and media then analyzed, 75% of the radioactivity was incorporated into cellular phospholipids, 0.8% was converted to PGs and metabolites and 0.7% converted to HETE. Cells prelabeled for 24 hours were washed and incubated for 30 minutes in fatty acid free media. There was a 23% release of AA from phospholipids. One-fifth of the released AA was converted to HETE. PG synthesis in the intact resting cells was low. In summary, the Neuro-2A cell provides a good model system for studying arachidonic acid metabolism and incorporation into phospholipids in cells of neuronal origin. 相似文献
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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. 相似文献
17.
Veith NM Plattner H Stuermer CA Schulz-Schaeffer WJ Bürkle A 《European journal of cell biology》2009,88(1):45-63
The causative agent of transmissible spongiform encephalopathies (TSE) is PrPSc, an infectious, misfolded isoform of the cellular prion protein (PrPC). The localisation and trafficking of PrPSc and sites of conversion from PrPC to PrPSc are under debate, particularly since most published work did not discriminate between PrPC and PrPSc. Here we describe the localisation of PrPC and PrPSc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrPC with proteinase K, PrPSc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrPSc was detected in late endosomes/lysosomes. As we detected PrPSc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrPC is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrPC/PrPSc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions. 相似文献
18.
Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). (3)H-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction. (3)H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function. 相似文献
19.
Javaneh Tamiji Dorota A. Crawford 《Biochemical and biophysical research communications》2010,398(3):450-173
Prostaglandin E2 (PGE2) is a key lipid-derived compound which mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). Recent studies have shown that altered PGE2 signalling due to abnormal lipid peroxidation and oxidative stress may underlie some pathologies of the nervous system. The prenatal exposure to the drug misoprostol, a prostaglandin type E analogue, has also been linked to a number of neurodevelopmental defects. In the present study, we use ratiometric calcium imaging with fura-2AM as a calcium indicator to determine the effects of PGE2 and misoprostol on calcium homeostasis in growth cones of mouse neuroblastoma (Neuro-2a) cells. Our results show that both drugs increase the amplitude of calcium transients in growth cones of Neuro-2a cells and induce neurite retraction. Moreover, quantitative real-time PCR also revealed that the mRNA expression level of the four EP receptors was significantly higher during the neurogenesis period in mouse indicating the importance of PGE2 signalling in the nervous system. 相似文献
20.
Interaction between sodium channels in mouse neuroblastoma cells 总被引:2,自引:0,他引:2
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 相似文献