Effect of allopregnanolone on d-[3H]-aspartate release and [3H]-glutamate uptake in the hippocampus of kainate-treated mice.
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In order to determine whether the status epilepticus leads to alterations in the neurosteroid effect on excitatory amino acid transmission, we studied the influence of allopregnanolone on aspartate release and glutamate uptake in mouse hippocampus at various times after kainate administration. No significant differences in the K+-stimulated D-[3H]-aspartate release from the hippocampi of saline- and kainate-treated mice were observed; however, that parameter tended to fall in tissues collected I h after kainate administration. Allopregnanolone significantly attenuated the K+-stimulated D-[3H]-aspartate release from the hippocampi of control animals, as well at 24 h and 7 days after kainate injection; in contrast it did not affect amino acid release from the hippocampi collected 1 h after kainate administration. Kainate administration had no effect on [3H]-glutamate uptake after 1 and 24 h, but elevated that parameter on day 7. Allopregnanolone (10 and 100 microM) did not affect [3H]-glutamate uptake in control and kainate-treated mice. In conclusion, the present study indicates a loss of the inhibitory effect of allopregnanolone on the potasium-stimulated D-[3H]-aspartate release from mouse hippocampus during the kainate-induced status epilepticus; moreover, it excludes involvement of this neurosteroid in the regulation of hippocampal [3H]-glutamate uptake in both control and kainate-treated mice. (+info)
Activation of a glycine transporter on spinal cord neurons causes enhanced glutamate release in a mouse model of amyotrophic lateral sclerosis.
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The release of [(3)H]D-aspartate ([(3)H]D-ASP) or [(3)H]GABA evoked by glycine from spinal cord synaptosomes was compared in mice expressing mutant human SOD1 with a Gly(93) Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, and in control mice. Mice expressing mutated SOD1 were killed at the advanced phase of the pathology, when they showed signs of ingestion disability, because of paralysis of the posterior limbs. In control mice glycine concentration-dependently evoked [(3)H]D-ASP and [(3)H]GABA release. Potentiation of the spontaneous release of both amino acids is likely to be mediated by activation of a glycine transporter, since the effects of glycine were counteracted by the glycine transporter blocker glycyldodecylamide but not by the glycine receptor antagonists strychnine and 5,7-dichlorokynurenate. The glycine-evoked release of [(3)H]D-ASP, but not that of [(3)H]GABA, was significantly more pronounced in SOD1-G93A(+) than in control animals. (+info)
Immunological detection of D-beta-aspartate-containing protein in lens-derived cell lines.
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PURPOSE: Although the presence of biologically uncommon D-beta-aspartate (D-beta-Asp) in lens protein is thought to be related to aging, we recently found this isomer in lens alphaA-crystallin from human newborns. The objective of this study was to examine whether D-beta-Asp occurs in protein from lens-derived cell lines. METHODS: We examined the expression of D-beta-Asp-containing protein in the lens-derived cell lines alphaTN4-1 and N/N1003A, by western blot and immunoprecipitation analysis using a polyclonal antibody against Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr (peptide 3R), which corresponds to three repeats of positions 149-153 in human alphaA-crystallin. The anti-peptide 3R antibody, prepared in a previous study, is a useful tool for investigating D-beta-Asp-containing peptides. RESULTS: Western immunoblot and immunoprecipitation analysis showed that a 50 kDa protein in N/N1003A cells was strongly immunoreactive with the anti-peptide 3R antibody. Antibodies against alphaA- and alphaB-crystallin also stained this protein. On the other hand, the alphaTN4-1 cell line only expressed proteins of about 20 kDa, which also reacted to antibodies against alphaA-crystallin and alphaB-crystallin. CONCLUSIONS: The results indicate that the N/N1003A cell line expressed a 50 kDa D-beta-Asp-containing protein, which may share a common amino acid sequence with alphaA- and alphaB-crystallin. (+info)
Inhibitory effect of some neuroactive steroids on cocaine-induced kindling in mice.
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Some neuroactive steroids which positively modulate GABAA receptor activity suppress cocaine-induced kindling but a possible involvement of other neurochemical mechanism in their antiepileptogenic effect remains to be elucidated. To this end, in the present study, we evaluated effects of allopregnanolone, a positive modulator of the GABAA receptor; its isomer without GABAergic activity--isopregnanolone and a negative-modulator of GABAergic transmission--dehydroepiandrosterone sulfate on cocaine-induced kindling in mice. Animals were pretreated daily with either vehicle or neuroactive steroid and then given cocaine (45 mg/kg) for 12 days. After a 14-day washout period in which drugs were not administered, the mice were challenged with the same 45 mg/kg dose of cocaine. Isopregnanolone (5 mg/kg) and dehydroepiandrosterone sulfate (20 mg/kg) administered daily with cocaine decreased number of mice exhibiting seizures. Allopregnanolone (5 mg/kg) also showed strong tendency to suppress cocaine kindling, however, its effect did not reach statistical significance. None of the neuroactive steroids had effect on acute cocaine (75 mg/kg ip)-induced clonic seizures. Further biochemical study showed that the veratridine- but not K+ -stimulated release of D-[3H]-aspartate in hippocampal slices was higher in cocaine-kindled mice than in the control group. Isopregnanolone (100 microM) significantly attenuated the veratridine-induced D-[3H]-aspartate release in hippocampi of cocaine-kindled group. These data indicate that positive modulation of the GABAA receptors is not a critical feature of neuroactive steroids that would determine their ability to prevent the cocaine-induced kindling. (+info)
Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Muller cells.
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We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Muller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Muller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Muller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Muller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Muller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Muller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Muller cells by activation of glutamine synthetase. The Muller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Muller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina. (+info)
Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme.
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The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties. (+info)
Retinal bipolar cell input mechanisms in giant danio. II. Patch-clamp analysis of on bipolar cells.
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Glutamate receptors on giant danio retinal on bipolar cells were studied with whole cell patch clamping using a slice preparation. Cone-driven on bipolars (Cbs) and mixed-input on bipolars (Mbs) were identified morphologically. Most Cbs responded to the excitatory amino acid transporter (EAAT) substrate d-aspartate but not to the group III metabotropic glutamate receptor (mGluR) agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) or the AMPA/kainate receptor agonist kainate, suggesting EAATs are the primary glutamate receptors on Cbs. The EAAT inhibitor dl-threo-beta-benzyloxyasparate (TBOA) blocked all light-evoked responses of Cbs, suggesting these responses are mediated exclusively by EAATs. Conversely, all Mbs responded to d-aspartate and l-AP4 but not to kainate, indicating they have both EAATs and group III mGluRs (presumably mGluR6). The light responses of Mbs involve both receptors because they could be blocked by TBOA plus (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III mGluR antagonist) but not by either alone. Under dark-adapted conditions, the responses of Mbs to green (rod-selective) stimuli were reduced by CPPG but enhanced by TBOA. In contrast, both antagonists reduced the responses to red (cone-selective) stimuli, although TBOA was more effective. Furthermore, under photopic conditions, TBOA failed to eliminate light-evoked responses of Mbs. Thus on Mbs, rod inputs are mediated predominantly by mGluR6, whereas cone inputs are mediated mainly by EAATs but also by mGluR6 to some extent. Finally, we explored the interactions between EAATs and mGluR6 in Mbs. Responses to d-aspartate were reduced by l-AP4 and vice versa. Therefore mGluR6 and EAATs suppress each other, and this might underlie mutual suppression between rod and cone signals in Mbs. (+info)
Formation of D-tyrosyl-tRNATyr accounts for the toxicity of D-tyrosine toward Escherichia coli.
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D-Tyr-tRNATyr deacylase cleaves the ester bond between a tRNA molecule and a D-amino acid. In Escherichia coli, inactivation of the gene (dtd) encoding this deacylase increases the toxicity of several D-amino acids including D-tyrosine, D-tryptophan, and D-aspartic acid. Here, we demonstrate that, in a Deltadtd cell grown in the presence of 2.4 mm D-tyrosine, approximately 40% of the total tRNATyr pool is converted into D-Tyr-tRNATyr. No D-Tyr-tRNATyr is observed in dtd+ cells. In addition, we observe that overproduction of tRNATyr, tRNATrp, or tRNAAsp protects a Deltadtd mutant strain against the toxic effect of D-tyrosine, D-tryptophan, or D-aspartic acid, respectively. In the case of D-tyrosine, we show that the protection is accounted for by an increase in the concentration of L-Tyr-tRNATyr proportional to that of overproduced tRNATyr. Altogether, these results indicate that, by accumulating in vivo, high amounts of D-Tyr-tRNATyr cause a starvation for L-Tyr-tRNATyr. The deacylase prevents the starvation by hydrolyzing D-Tyr-tRNATyr. Overproduction of tRNATyr also relieves the starvation by increasing the amount of cellular L-Tyr-tRNATyr available for translation. (+info)