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  • 1
    ISSN: 1432-1106
    Keywords: l-Glutamate and l-aspartate ; Neurotransmission ; High affinity uptake ; Na+-dependent binding ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Binding of [3H]l-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [3H]l-aspartate binding was strongly inhibited by l-threo-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of l-glutamate and l-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-d-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 μM weakly inhibited) of the Na+-dependent [3H]l-aspartate binding to suggest that [3H]l-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na+ in the incubation medium. It is concluded that [3H]l-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na+-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [3H]l-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Neurochemical correlates of GABAergic synaptic transmission [binding, uptake, metabolism, and tissue content of γ-aminobutyric acid (GABA)] were investigated in the cortex of rats that had been given 27 mM bromide in drinking water for periods of time ranging from 1 day to 1 month. No effect of bromide on any of the parameters was found and it is concluded that chronic administration of bromide has no profound effect on GABAergic inhibitory system in the rat cortex.
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Metabotropic glutamate receptors (mGluR) modulate neuronal function. Here, we tested the effect on metabolism of a range of Group I and II mGluR ligands in Guinea pig brain cortical tissue slices, applying 13C NMR spectroscopy and metabolomic analysis using multivariate statistics. The effects of Group I agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) depended upon concentration and were mostly stimulatory, increasing both net metabolic flux through the Krebs cycle and glutamate/glutamine cycle activity. Only the higher (50 µm) concentrations of CHPG had the opposite effect. The Group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), consistent with its neuroprotective role, caused significant decreases in metabolism. With principal components analysis of the metabolic profiles generated by these ligands, the effects could be separated by two principal components. Agonists at Group II mGluR [(2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG IV) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC)] generally stimulated metabolism, including glutamate/glutamine cycling, although this varied with concentration. The antagonist (2S)-α-ethylglutamic acid (EGLU) stimulated astrocyte metabolism with minimal impact on glutamate/glutamine cycling. (RS)-1-Aminophosphoindan-1-carboxylic acid (APICA) decreased metabolism at 5 µm but had a stimulatory effect at 50 µm. All ligand effects were separated from control and from each other using two principal components. The ramifications of these findings are discussed.
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  • 4
    ISSN: 1440-1681
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: 1. It has been suggested that Na+/K+-ATPase and Na+-dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na+/K+-ATPase activity using Rb+ uptake by ‘minislices’ (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb+ was determined by estimating Rb+ in aqueous extracts of the minislices, using atomic absorption spectroscopy.2. We determined the potencies of several known substrates/inhibitors of GluT, such as l-trans-pyrrolidine-2,4-dicarboxylate (LtPDC), dl-threo-3-benzyloxyaspartic acid, (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and l-anti,endo-3,4-methanopyrrolidine dicarboxylic acid, as inhibitors of [3H]-l-glutamate uptake by cortical prisms. In addition, we established the susceptibility of GluT, measured as [3H]-l-glutamate uptake in brain cortical prisms, to the inhibition of Na+/K+-ATPase by ouabain. Then, we tested the hypothesis that the Na+/K+-ATPase (measured as Rb+ uptake) can respond to changes in the activity of GluT produced by using GluT substrates as GluT-specific pharmacological tools.3. The Na+/K+-ATPase inhibitor ouabain completely blocked Rb+ uptake (IC50 = 17 µmol/L), but it also potently inhibited a fraction of GluT (approximately 50% of [3H]-l-glutamate uptake was eliminated; IC50 〈 1 µmol/L).4. None of the most commonly used GluT substrates and inhibitors, such as l-aspartate, d-aspartate, L-CCG III and LtPDC (all at 500 µmol/L), produced any significant changes in Rb+ uptake.5. The N-methyl-d-aspartate (NMDA) receptor agonists (R,S)-(tetrazol-5-yl)-glycine and NMDA decreased Rb+ uptake in a manner compatible with their known neurotoxic actions.6. None of the agonists or antagonists for any of the other major classes of glutamate receptors caused significant changes in Rb+ uptake.7. We conclude that, even if a subpopulation of glutamate transporters in the rat cerebral cortex may be intimately linked to a fraction of Na+/K+-ATPase, it is not possible, under the present experimental conditions, to detect regulation of Na+/K+-ATPase by GluT.
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  • 5
    ISSN: 1573-7381
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The distribution of purinergic (P2x1) receptors on smooth muscle cells in relation to autonomic nerve varicosities in the rat urinary bladder has been determined using immunofluorescence and confocal microscopy. P2x1 receptors were visualized using rabbit polyclonal antibodies against the extracellular domain of the P2x1 receptor, and varicosities were visualized using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2x1 receptor clusters were observed on the smooth muscle cells of the detrusor, namely, a large ellipse of mean long axis 1.23 ± 0.21 μm and short axis 0.92 ± 0.17 μm and a smaller spherical cluster with a mean diameter of 0.40 ± 0.04 μm. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per μm2 or two orders of magnitude more than the larger-sized clusters. The large clusters are in general located beneath varicosities, with only 4.5% of P2x1 clusters not possessing an overlying varicosity. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2x1 and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2x1 receptor clusters. On occasions, two or more small SV2-labelled varicosities about 0.7 μm in diameter each with a receptor patch were found juxtaposed to each other; these might represent the splitting up of a single large varicosity. These observations are discussed in relation to the identity of the autonomic neuromuscular junction.
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Cyclic AMP response element binding protein (CREB) is a constitutive transcription factor that activates transcription following stimulus-dependent phosphorylation at Ser133, implicated in synaptic plasticity and neuronal survival pathways. The prevailing view that CREB is exclusively nuclear has been questioned by several studies, and, for example, mitochondrial localization has been reported. Using subcellular fractionation of rat brain cortex coupled with western immunoblotting with Ser133-phospho-CREB (pCREB) antibodies, we found a robust pCREB immunoreactivity (IR) in mitochondria-enriched fractions. The pCREB antibodies also stained the mitochondria, in addition to nuclei, of glial cells in primary cortical cultures. However, two CREB antibodies against different epitopes and gel shift assay detected the CREB protein mainly in the nuclear fraction. The two-dimensional electrophoretic mobility of mitochondrial pCREB IR differed markedly from the nuclear CREB/pCREB IR, indicating that the pCREB antibody cross-reacts with another mitochondrial protein. Immunoprecipitation of the mitochondrial pCREB IR produced three bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as E2, E1 α-subunit, and E1 β-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the α-subunit. In conclusion, this study confirms the presence of pCREB-like IR in brain mitochondria that, after careful scrutiny, turned out to be pyruvate dehydrogenase rather than authentic CREB.
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The effects of the enantiomers of (±)-CAMP and(±)-TAMP [(±)-cis- and(±)-trans-2-aminomethylcyclopropanecarboxylic acids,respectively], which are cyclopropane analogues of GABA, were tested onGABAA and GABAC receptors expressed in Xenopuslaevis oocytes using two-electrode voltage clamp methods. (+)-CAMP wasfound to be a potent and full agonist at homooligomeric GABACreceptors (KD∼40 μM andImax∼100% at ρ1;KD∼17 μM and Imax∼100% at ρ2) but a very weak antagonist atα1β2γ2L GABAAreceptors. In contrast, (-)-CAMP was a very weak antagonist at bothα1β2γ2L GABAAreceptors and homooligomeric GABAC receptors (IC50∼900 μM at ρ1 and ∼400 μM atρ2). Furthermore, (+)-CAMP appears to be a superior agonist tothe widely used GABAC receptor partial agonistcis-4-aminocrotonic acid (KD∼74μM and Imax∼78% at ρ1;KD∼70 μM and Imax∼82% at ρ2). (-)-TAMP was the most potent of thecyclopropane analogues on GABAC receptors (KD∼9 μM and Imax∼40% atρ1; KD∼3 μM andImax∼50-60% at ρ2), but it was also amoderately potent GABAA receptor partial agonist(KD∼50-60 μM and Imax∼50% at α1β2γ2LGABAA receptors). (+)-TAMP was a less potent partial agonist atGABAC receptors (KD∼60 μM andImax∼40% at ρ1; KD∼30 μM and Imax∼60% atρ2) and a weak partial agonist atα1β2γ2L GABAAreceptors (KD∼500 μM andImax∼50%). None of the isomers of (±)-CAMP and(±)-TAMP displayed any interaction with GABA transport at theconcentrations tested. Molecular modeling based on the present resultsprovided new insights into the chiral preferences for either agonism orantagonism at GABAC receptors.
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  • 9
    ISSN: 0173-0835
    Keywords: Cardiomyocytes ; P2X receptors ; Autonomic control of the heart ; Heart failure ; Dilated cardiomyopathy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This is the first report of the analysis of the ATP-specific P2X1 receptor subunit in human hearts. We have examined homogenate samples of human left atria for the presence of P2X1 receptors using Western blots. Anti-P2X1 immunoreactivity was detected in populations of nondiseased atria as well as in atria from explanted hearts from patients with terminally failing heart conditions such as dilated cardiomyopathy. At least three groups of P2X1 immunoreactive proteins were detected in the Western blots with approximate molecular mass values of 50, 70, and 160 kDa. We report changes in expression of their 50 and 70 kDa components. These changes may be related to the type of deficit in these hearts since the changes have been observed in hearts with decreased ejection fractions characteristic of dilated cardiomyopathy.
    Additional Material: 2 Ill.
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  • 10
    ISSN: 1573-6903
    Keywords: GABA ; cell cultures ; ontogenetic development ; cerebral cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract High-affinity uptake of [3H]γ-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V max) and, to a smaller extent, theK m of [3H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [3H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [3H]GABA, [3H]ACHC, or [3H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.
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