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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 82 (1984), S. 259-268 
    ISSN: 1432-1424
    Keywords: ethylcholine mustard ; choline carrier ; affinity label ; translocation rates ; synaptosomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Ethylcholine mustard aziridinium (ECMA) inhibits choline transport in synaptosomes at a half-maximal concentration of about 20 μm. The rate of inhibition falls off rapidly after 10 min and the concentration dependency reaches a plateau at about 100 μm. The inhibition is not removed by washing the synaptosomes, and choline and hemicholinium-3 protect the carrier against attack by the mustard. Choline efflux, particularly that stimulated by choline in the medium (transactivation) is also inhibited by the aziridinium compound. Similarly choline influx activated by preloaded internal choline is inhibited by ECMA. The mustard can enter the synaptosomes in an active form but most of the carrier is alkylated when facing the outside. Prior depolarization of the synaptosomes causes an increase in the rate of inhibition by ECMA which is proportionally about the same as the increase in choline influx also caused by depolarization. At low ECMA concentrations the rate of inhibition is that of a first-order reaction with the carrier but at high ECMA concentrations the translocation of the carrier to the outward-facing conformation controls the rate of inhibition. Using a model of choline transport with some simplifying assumptions it is possible to estimate the amount of carrier; cholinergic synaptosomes carry about six times the concentration of carrier found in noncholinergic ones. In noncholinergic synaptosomes the carrier faces predominately out, the reverse in cholinergic ones. The rate constant of carrier translocation is increased by combination with choline some six- to sevenfold to about 3.5 min−1. The rate constant of ECMA attack on the carrier is about 440m −1 sec−1.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 48 (1987), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synapto-somes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 40 (1983), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Stores of methionine-enkephalin were labelled on the N-terminal by incubation of whole brain slices with [3H]tyrosine (10 °Ci/ml). The 3H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD2 and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μM cycloheximide. Isolated nerve terminals failed to incorporate any [3H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N-terminus and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [14C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N-terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 mM) elicited a Ca2+-dependent release of the synthesised [3H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [3H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 39 (1982), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 5
    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: Abstract: As in intact tissues, choline influx into synaptosomes is enhanced after a period of depolarization induced release of acetylcholine. The activation of uptake is dependent on the presence of Ca2+ and inhibited by high Mg2+ concentrations in the medium during depolarization. Choline transport in erythrocytes was not activated by prior treatment with potassium. The permeability constant of the synaptosome membrane to choline was found to be 2.7 × 10−8 cm·s−1 and to acetylcholine 1.8 ′ 10−8 cm·s−1. Choline influx has been studied after pre-loading synaptosomes with choline. Different radiolabels were used to measure efflux of preloaded choline and influx simultaneously. Isotopic dilution in flux studies was estimated and corrected for. Influx was stimulated by high internal concentrations of choline, and efflux similarly stimulated by high outside concentrations of choline. The maximal influx and efflux at saturating opposite concentrations of choline were equal with a value of about 500 pmol·min−1 per mg synaptosomal protein. A reciprocating carrier would explain the equality of the maximal influx and efflux. Acetylcholine competes with choline for binding to the carrier but is itself hardly transported. Increased acetylcholine concentrations were shown to inhibit both choline influx and efflux from the trans position. Raising intrasynaptosomal acetylcholine concentrations by pre-loading abolished the stimulation of influx by prior depolarization. It is proposed that high concentrations of acetylcholine immobilize the carrier on the inside of the synaptic membrane. The stimulation of choline influx consequent upon depolarization is caused by release of ACh which results in relief of this immobilisation. The enhanced supply of choline achieved by this mechanism is likely to be important in maintaining stores of the acetylcholine in vivo.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 57 (1991), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: : The objective of this study was to identify cellular proteins that are associated with foetal brain transplants effective in reinstating memory function in adult rats with brain lesions. Quantitative memory deficits can be created in rats by lesioning the cholinergic projection system, using ibotenic acid. Previous work suggested that injection of cell suspensions prepared from presumptive cholinergic cells of foetal basal forebrain into adult brain, after such lesions, are most effective in restoring cognitive function. It was not clear, however, whether it was the cholinergic nature of the transplants that was critical for their success or whether other factors were involved. In this study, the proteins present in transplanted tissues and control brains were analysed by two-dimensional polyacrylamide gel electrophoresis to identify markers for the cells that were specifically correlated with restoration of cognitive function. On each gel, the relative optical densities of the same 33 selected proteins were measured on an interactive computerised image analyser. The amount of each protein was compared between treatment groups and correlated with four behavioural measurements. Seven of the proteins analysed had levels of expression that were either related to transplantation or correlated with behavioural performance. The proteins of interest were divided into the following three groups: (1) transplant-related proteins, (2) cholinergic transplant-specific proteins, and (3) behaviour-related proteins. Notable among the proteins of interest was one of the cholinergic transplant-specific proteins that was positively correlated with three of the four behavioural measurements and was also the only protein among those analysed that was significantly correlated with choline acetyltransferase (ChAT) levels. This has been identified, by immunoblotting, as glial fibrillary acidic protein, an astrocytic cell marker. These results suggest, therefore, that at least two cell types, astrocytes and ChAT+-staining cells, play an important role in the successful recovery of cognitive function. This study also identified possible protein markers for cognitive performance. The level of expression of two of the proteins analysed was not affected by lesioning or transplantation, but was significantly correlated with behaviour. One of these proteins, whose amounts correlated negatively with behavioural measurements, has been identified as neurone-specific enolase, a brain-specific neuronal cell marker.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 42 (1984), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 41 (1983), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The release of lactate dehydrogenase and K+ when synaptosomes are exposed to resuspension in media of various osmolarity has been investigated in order to measure their disruption. Even when resuspended in distilled water a significant percentage (10–20%) of lactate dehydrogenase and K+ remains unreleased. The particles containing these substances sediment to the same density as synaptosomes. Synaptosomes retaining their internal organlles after hypoosmotic treatment can be seen in electron micrographs. Resealing of disrupted synaptosomes was measured by the inclusion of [14C]sucrose. The resealing is spontaneous, essentially complete (80–90%) within 20 min and not noticeably affected by temperature, pH, or the addition of fusogen. The synaptosome preparation after hypoosmotic disruption will therefore contain some undisrupted synaptosomes with some or all of their complement of cytoplasmic constituents, as well as resealed synaptosomes. The retention of the ability of the hypoosmotically treated preparation to convert [14C]choline to [14C]acetylcholine is demonstrated as an example of the disproportionate effect these undisrupted particles have on its properties.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 37 (1981), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The effect of ouabain and dihydroouabain on Na+-K+ ATPase, 86Rb uptake and the release of [14C]ACh (acetylcholine) from synaptosomal preparations of guinea pigs was compared. At low concentrations of glycoside (〈50 μm) there was a good correlation between the potency of ouabain and of dihydroouabain in inhibiting Na+-K+ ATPase and in causing the release of [l4C]ACh in a nondepolarising medium. Ouabain (200 μM) increased the release of [14C]ACh evoked by 25 mm-KCl, but not that evoked by 100μm-veratrine. The enhancement of release was independent of the presence of calcium. It was observed that in addition to [14C]ACh release, choline efflux was also stimulated by ouabain, independently of the presence of Ca2+. Experiments with hemicholinium-3 showed that the ouabain-induced increase in choline efflux was not due to an inhibition of reuptake. The effect of ouabain on intrasynaptosomal K+ concentration was measured in order to investigate the degree of depolarisation it caused. The decrease in K+ was found to be similar in magnitude and time course to that caused by veratrine. It was shown that ouabain-induced depolarisation caused an increased efflux of another positive ion (dibenzyldimethylammonium chloride) and retention of a negatively charged ion (chloride), as would be expected from the operation of the electrochemical potential gradient changing as a result of depolarisation. It is suggested that ouabain acts to stimulate ACh release from synaptosomes as follows: following blockage of the Na+-K+ ATPase there is rapid depolarisation which, if Ca2+ is present, provokes the normal Ca2+-dependent transmitter release process to occur. In addition, depolarisation accelerates the leakage of positive ions down their electrochemical potential gradient, but causes a retention of negative ions. Such an action does not depend on the presence of Ca2+, nor is it specific to transmitters.
    Type of Medium: Electronic Resource
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  • 10
    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: 3-Bromopyruvate inhibits pyruvate decarboxylase in brain homogenates and causes a 90% drop in acetylcholine tissue content at a concentration of 2 mM. Stereotaxic injection of 3-bromopyruvate into the basal forebrain causes after 7 days a 40% drop of acetylcholine concentration and pyruvate decarboxylase activity in the cortex and hippocampus, and greater decreases at the site of injection. However, values return to normal 18 days after injection. Choline ace-tyltransferase is partially inhibited only at the site of injection after 7 days. Choline transport and choline concentration are not affected at either 7 or 18 days after injection. Impairments in spontaneous alternation and in retention of passive avoidance were seen only 7 days after the injection. The results suggest that stereotaxic injection of bromopyruvate can induce discrete reversible cholinergic lesions on a time scale useful for behavioural experiments and for comparison with neurodegeneration.
    Type of Medium: Electronic Resource
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