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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1α and -1β (MIP-1α and MIP-1β), which could be enhanced by interleukin (IL)-1β at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1β mRNA expression in NT2-N cells. Anti-IL-1β antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1β mRNA expression in NT2-N cells, suggesting that IL-1β in the MDM culture supernatants is a major factor in the induction of MIP-1β expression. Investigation of the mechanism(s) responsible for IL-1β-induced MIP-1α and -1β expression demonstrated that IL-1β activated nuclear factor kappa B (NF-κB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-κB, not only blocked IL-1β-induced activation of the NF-κB promoter but also decreased IL-1β-induced MIP-1α and -1β expression in NT2-N cells. These data suggest that NF-κB is at least partially involved in the IL-1β-mediated action on MIP-1α and -1β in NT2-N cells. IL-1β-mediated up-regulation of β-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LANS is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2′-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyrylcyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Cells dissociated from the forebrains of 21-day-old rats were enriched in oligodendroglia by Percoll gradient centrifugation, seeded on polylysine-coated surfaces, and cultured in a serum-containing medium. Incorporation by the cultures of tritium from d-[3H]galactose into the galactosyl residue of galactocerebroside (galC) increased in an almost linear fashion for 48 h with 1–8 μCi of d-[3H]galactose (30 mCi/μmol) per milliliter medium. Treatment for 2 days (day 1–3 after seeding) with 10−4M or 10−3M dibutyryl cyclic adenosine 3′,5′-monophosphate (db cyclic AMP) or 10−4M 8-bromo cyclic AMP stimulated galC radiolabelling. Incorporation of d-[3H]galactose into galC during a terminal 48-h radiolabelling period was not stimulated when the cells were continuously treated with these cyclic AMP analogues for 8 rather than 2 days.
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  • 5
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 ± 3.2 nmol/mg of protein/min and a Km of 449.2 ± 94.9 µM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 µM with a Vmax of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ? KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the “buffering” of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-d-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2–7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1–3, GluR5–7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 µM kainate or 5 mMl-glutamate, but not by a 24-h exposure to up to 10 mMα-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca2+-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Treatment of the human teratocarcinoma line NTera2/c1.D1 (NT2) with retinoic acid induces terminal neuronal differentiation. In a previous study, we found that the neurons obtained in this way express functional N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptor channels. We now show by reverse transcriptase-polymerase chain reaction and Southern blotting that these neurons transcribe each of the nine known non-NMDA glutamate receptor genes (GluR1-7, Ka-1, and Ka-2) and that four of these genes (GluR2, GluR6, GluR7, and Ka-1) are also transcribed by undifferentiated NT2 cells. Patch clamp studies demonstrate that individual non-NMDA glutamate receptor channels are readily isolated from NT2-derived neurons and that these channels are potently modulated by the desensitization blocker cyclothiazide. NT2-derived neurons are susceptible to kainate excitotoxicity but are not injured by prolonged exposure to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. We expect that the NT2-derived human neuronal culture system will facilitate studies of human neuronal non-NMDA glutamate receptor channels and of the pathophysiology of neuronal excitotoxicity.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 55 (1990), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20–40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 51 (1988), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The metabolism of glutamine was studied in cultured astrocytes by incubating these cells with [2-15N]-glutamine and using gas chromatography-mass spectrometry to quantitate the transfer of 15N to other amino acids. We found that astrocytes simultaneously synthesize and consume [2-15N]glutamine, with the respective synthetic and utilization rates being approximately equal (ca. 13.0 nmol min-1 mg protein-1). Considerable 15N was transferred to alanine and a significant amount to the essential amino acids leucine, tyrosine, and phenylalanine, the latter process denoting active reamination of cognate ketoacids. A net export of alanine into the medium was noted. Astrocyte glutamine utilization appeared to be mediated via both the phosphate-activated glutaminase (PAG) pathway and the glutamine aminotransferase pathway, the activity of which was about half that of PAG. The glutamine concentration in the incubation medium determined whether net synthesis or utilization of this amino acid occurred. When glutamine was omitted from the medium, net synthesis occurred. When it was present at a high (5 mM) level, net consumption was observed. At a physiologic (0.5 mM) concentration, neither net synthesis nor consumption was noted, although the 15N data indicated that glutamine was actively metabolized. An implication of this work is that astrocytes clearly are capable of both synthesizing and utilizing glutamine, and current concepts of a glutamate-glutamine cycle functioning stoichiometrically between astrocytes and neurons may be an oversimplification.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 50 (1988), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170k. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.
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