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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The effects of N-(4,4-diphenyl-3-butenyl) derivatives of nipecotic acid (SKF-89976-A and SKF-100844-A) and guvacine (SKF-100330-A) on neuronal and astroglial γ-aminobutyric acid (GABA) uptake were investigated. In addition, the uptake of SKF-89976-A was studied using the tritiated compound. All of the compounds were found to be competitive inhibitors of GABA uptake irrespective of the cell type, with Ki values similar to or lower than those of the parent amino acids. Moreover, none of the compounds exhibited selectivity with regard to inhibition of neuronal and glial GABA uptake. In spite of the competitive nature of SKF-89976-A, the compound was not transported by the GABA carriers in the two cell types, because no saturable uptake could be demonstrated.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U-13C]glutamate (0.5 mM) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Local cerebral glucose metabolism (LCMRglc) and local cerebral blood flow (LCBF) were studied following implantation of a microdialysis fiber in rat dorsal hippocampus. Recovery time after implantation varied from 0 to 24 h. All rats showed pronounced disturbances in LCMRglc and LCBF during the first 2 h of implantation. The changes consisted of (a) a general decrease in blood flow and glucose phosphorylation and (b) small areas (spots) around the fiber with increased glucose phosphorylation and decreased blood flow. Animals allowed to recover for 24 h demonstrated a near normalization of LCMRglu and LCBF, and the focal disturbances (spots) of glucose phosphorylation and blood flow disappeared. The slight reduction in blood flow and glucose metabolism at this time must be accepted, because extension of the recovery period beyond 24 h may give interpretation problems due to the developing gliosis.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Using tritium-labelled 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) its tissue distribution and metabolism were investigated in adult mice and 4-day-old chicks after systemic administration of the drug. It was found not to be significantly metabolized in the brain since metabolites of THPO corresponding to only approximately 8% of the parent compound could be detected 30 min after administration of the drug intramuscularly in mice. In the liver, however, THPO was found to be metabolized to a considerable extent. In chicks THPO metabolites were found in the brain but they accounted for 〈 35% of the radioactivity. The brain concentration of THPO in mice and chicks corresponded to respectively 10 and 50% of the dose injected intramuscularly and the tissue level was essentially constant for at least 3 h after injection. Following systemic administration of THPO to mice and chicks the contents of aspartate, glutamate, glutamine, and γ-aminobutyric acid (GABA) in whole brain and in synaptosomes was determined. It was found that only GABA contents were affected being increased in synaptosomes from mice and decreased in whole brain in chicks. Doses of THPO. which in chicks but not in mice led to brain levels that were sufficient to inhibit glial GABA uptake, were found to protect chicks but not mice against isonicotinic acid hydrazide-induced seizures. The findings are compatible with the notion that THPO exerts its anticonvulsant activity by inhibition of astrocytic GABA uptake. Key Words: 4,5,6.7-Tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO)—Anticonvulsant—γ-Aminobutyric acid—Mice—Chicks—THPO metabolism. Schousboe A. et al. Tissue distribution, metabolism, anticonvulsant efficacy, and effect on brain amino acid levels of the glia-selective 7-aminobutyric acid transport inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol in mice and chicks. J. Neurochem. 47, 758–763 (1986).
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  • 5
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The transport kinetics of γ-aminobutyric acid (GABA), taurine, and β-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (KmGABA= 24.9 ± 1.7 μM; KmTau= 20.0 ± 3.3 μM; Kmβ-Ala= 73.0 ± 3.6 μM) and astrocytes (KmGABA= 31.4 ± 2.9 μM, KmTau= 24.7 ± 1.3 μM; Kmβ-Ala= 70.8 ± 3.6 μM). The maximal uptake rates (Vmax) determined were such that, in neurons, VmaxGABA〉 Vmaxβ-Ala=VmaxTau, whereas in astrocytes, Vmaxβ-Ala 〉 VmaxTau=VmaxGABA. Taurine was found to inhibit β-alanine uptake into neurons and astro cytes in a competitive manner, with Ki values of 217 μM in neurons and 24 μM in astrocytes. β-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 μM in neurons and 71 μM in astrocytes. However, β-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of β-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons. Taken together, the similarity between the results obtained for β-alanine and taurine uptake in addition to their competitive mode of mutual inhibition strongly suggest that these compounds share a common carrier in neurons and astrocytes and that neither amino acid in either cell type is transported appreciably, if at all, by the GABA carrier, and vice versa.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Metabolism of [U-13C5]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify 13C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-13C5]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-13C5]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-13C2]glutamate was observed in astrocytes incubated with [U-13C5]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using 13CO2 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 12 (1973), S. 2868-2873 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 36 (1981), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Glutaminc synthetase activity was investigated in developing primary astroglial cultures established from newborn mouse cerebral hemispheres. Between the 2nd and 4th week of culture there was little change in activity under our standard culturing conditions; however, when hydrocortiwne (10 μM) was added to the cultures for 48 h, the enzyme activity increased two- to fourfold, depending upon the age of the culture, with maximum response in 2-week-old cultures. The addition of dibutyryl cyclic AMP (dRcAMP) to the culture medium caused morphological differentiation of the astroglial cells but eliminated the response of the cells to hydrocortisone. Culturing in elevated serum levels, which delays morphological differentiation and inhibits astroglial cytodifferentiation after exposure to dBcAMP, shifted the time of maximal response to hydrocortisone from 2 to 3 weeks and prevented the abolishment of glutamine synthetase induction by dBcAMP. The induction of glutamine synthetase by hydrocortisone was prevented by actinomycin D (0.5 μg/ml), indicating its dependence upon RNA and protein synthesis. The present work thus confirms reports in the literature that hydrocortisone induces glutamine synthetase in neural tissues, but differs from the findings of Moscona and co-workers in the chick retina that intact tissues are required for the induction to occur.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 34 (1980), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a Km, of 28.8 ± 2.8 μM and a Vmax of 0.294 ± 0.022 nmol × min−1× [mg cell protein]−1. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC50) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: —The effects of a variety of acyclic or heterocyclic GABA analogues on GABA receptor binding and on high affinity transport of GABA in cultured astrocytes and mini-slices of brain cortex were studied. The receptor and transport sites were found to be stereospecific and they exhibited opposite stereoselectivity for (R)- and (S)-trans-4-amino-4-methylcrotonic acid and (R)- and (S)-β-proline. The most potent inhibitors of GABA binding were (RS)-4, 5-dihydromuscimol, muscimol, GABA, isoguvacine and isonipecotic acid with IC50values of, respectively, 0.009, 0.006, 0.033, 0.037 and 0.33 μM. Under the present experimental conditions the following compounds inhibited preferentially the glial transport system: (3RS, 4SR)-4-hydroxynipecotic acid, guvacine, (RS)-N-methylnipecotic acid, (RS)-β-proline and β-alanine (IC50 values 10, 25, 70, 320 and 1000 μM, respectively vs. 200, 100, 300, 1200 and 〉5000 for neuronal transport). On the other hand, (R)-trans-4-amino-4-methylcrotonic acid, (3RS, 4SR, 5SR)-4-hydroxy-5-methymipecotic acid and (RS)-3-hydroxy-5-aminovaleric acid preferentially inhibited neuronal transport as studied in mini-slices of brain cortex (IC50 values 160, 300 and 430 μM, respectively vs. 500, 〉 5000 and 1400 μM for glial transport).
    Type of Medium: Electronic Resource
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