Differences between D- and L-aspartate binding to the Na+-dependent binding sites on glutamate transporters in frozen sections of rat brain

Takamoto, A. H. R.; Quiggin, L.B.; Lieb, I.; Shave, E.; Balcar, Vladimír J.; Yoneda, Yukio

doi:10.1016/s0024-3205(01)01478-3

Cited by 10 publications

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“…2). Interestingly, the distribution of [ 3 H]L-aspartate binding in rat brain closely resembles that of GLAST: it is significantly greater in the cerebellar cortex than in the forebrain regions [45,46]. As the binding to GLAST in frozen tissue is not likely to involve any active substrate transport, the most parsimonious conclusion from the observed correlation would seem to be that high affinity for the substrate binding site on GLAST is more important in the mechanism of the substrate-assisted redistribution of GLAST in astrocytes than the ability of the substrates to be actually transported.…”

Section: Discussionmentioning

confidence: 95%

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

et al. 2009

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Neurotransmitter L-glutamate released at central synapses is taken up and "recycled" by astrocytes using glutamate transporter molecules such as GLAST and GLT. Glutamate transport is essential for prevention of glutamate neurotoxicity, it is a key regulator of neurotransmitter metabolism and may contribute to mechanisms through which neurons and glia communicate with each other. Using immunocytochemistry and image analysis we have found that extracellular D-aspartate (a typical substrate for glutamate transport) can cause redistribution of GLAST from cytoplasm to the cell membrane. The process appears to involve phosphorylation/dephosphorylation and requires intact cytoskeleton. Glutamate transport ligands L-trans-pyrrolidine-2,4-dicarboxylate and DL-threo-3-benzyloxyaspartate but not anti,endo-3,4-methanopyrrolidine dicarboxylate have produced similar redistribution of GLAST. Several representative ligands for glutamate receptors whether of ionotropic or metabotropic type, were found to have no effect. In addition, extracellular ATP induced formation of GLAST clusters in the cell membranes by a process apparently mediated by P2 receptors. The present data suggest that GLAST can rapidly and specifically respond to changes in the cellular environment thus potentially helping to fine-tune the functions of astrocytes.

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Section: Discussionmentioning

confidence: 95%

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

et al. 2009

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“…55,56) The autoradiographic studies based on the labelling of brain sections in vitro will be discussed in more detail later because of their potential to show not only the regional distribution of the substrate-binding sites on GluT but, at the same time, also their neurochemical and pharmacological characteristics. The preferred radioligands in those experiments were either [ 3 H]Daspartate [57][58][59][60][61][62][63] or [ 3 H]L-aspartate. [62][63][64][65][66][67] Finally, in situ hybridisation (ISH) was employed to study regional and cellular distribution of GluT.…”

Section: Location Of Glutamate Transporters In the Cnsmentioning

confidence: 99%

“…The preferred radioligands in those experiments were either [ 3 H]Daspartate [57][58][59][60][61][62][63] or [ 3 H]L-aspartate. [62][63][64][65][66][67] Finally, in situ hybridisation (ISH) was employed to study regional and cellular distribution of GluT. 49,[68][69][70] In some cases, the results of ISH studies are not in complete agreement with the immunocytochemistry.…”

Section: Location Of Glutamate Transporters In the Cnsmentioning

confidence: 99%

“…The binding of radioligands was analysed autoradiographically (for details of the method see [63][64][65][66][67] . The distribution of the GLAST (EAAT1) and GLT (EAAT2) dioactive L-glutamate.…”

Section: H]d-aspartate Compared To the Distribution Of Glast (Eaat1) mentioning

confidence: 99%

“…non-fixed) tissue that behave, in terms of substrate selectivity and ionic dependence, as the substratebinding sites on GluT and does so in a quantitatively reproducible manner. [63][64][65][66][67] It is tempting, therefore, to suggest that the autoradiographical analysis of [ ture of GluT distribution in the tissue and it should be able to describe its characteristics in situ at the same time. But is it really so?…”

Section: Unresolved Problems and Future Direc-tionsmentioning

confidence: 99%

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Molecular Pharmacology of the Na+-Dependent Transport of Acidic Amino Acids in the Mammalian Central Nervous System.

Balcar

2002

Biological & Pharmaceutical Bulletin

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The crucial discovery of the excitatory properties of acidic amino acids [2][3][4][5] lead only gradually and with considerable delay, to the formulation of the hypothesis of the synaptic role of L-glutamate in the mammalian central nervous system (CNS) 6-9) (for reviews of early history of amino acid research, see [10][11][12][13] ). It may never be possible to identify any single experimental study after which L-glutamate became unequivocally accepted as the most important excitatory synaptic transmitter in the brain and spinal cord, but, it is certain that the discovery of the "high affinity uptake" accumulating L-glutamate into "synaptosomes" 14,15) (for reviews see 13,16,17) ) was an important step in the development of the concept of the glutamatergic neurotransmission in the CNS. As a putative mechanism for the necessary removal and inactivation of extracellular L-glutamate it provided a target for pharmacological studies and helped to establish a neurochemical link between L-glutamate and the mechanisms of synaptic transmission. 13,18,19) Most of the latter experiments were carried out in vitro using tissue "mini-slices" prepared from the rat cerebral cortex or cat spinal cord (for details of the methodology see 20,21) ). Rather than disrupting the nervous tissue by homogenization, the technique was using what might be called "neurochemical dissection". In more specific terms, it examined a large number of compounds, mostly glutamate and aspartate analogues but also a range of drugs, for possible effects on L-glutamate transport (GluT). Such approach had a potential to yield valuable information on the molecular properties of the substrate-recognition site on GluT,19) but, also, by testing neuroactive compounds with known sites of actions in brain, it helped to formulate hypotheses defining the most probable functions of the GluT in the mechanisms of the excitatory synaptic transmission.The pharmacology and biochemistry of GluT was later extensively investigated in cultured glial cells [22][23][24] (for a review see 25) ) but it was the data obtained in anaesthetised animals, using compounds (D-and L-threo-3-hydroxyaspartate, DL-t3OHA) identified earlier as powerful specific inhibitors of [ 3 H]L-glutamate uptake in brain slices, 26) that provided the first convincing evidence that GluT could influence the excitatory actions of L-glutamate in vivo, at least in the cat spinal cord.27) Furthermore, if the inhibition of GluT in vivo caused an increase in the extracellular concentrations of L-glutamate then, given the neurotoxic nature of excessive glutamatergic excitation, 28,29) similar inhibition of GluT applied to functioning synapses should, under suitable experimental conditions, result in the appearance of neurodegeneration. Such experiment was indeed performed and it was shown that an injection of DL-t3OHA into the rat striatum could cause the death of brain cells in vivo. 30) HETEROGENEITY OF GLUTAMATE TRANSPORTDuring the 1980's and early 1990's, it became apparent that, in order to account for th...

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d-Aspartate binding sites in rat Harderian gland

et al. 2009

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Radioligand binding of D-[(3)H]aspartic and L-[(3)H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for D-Asp and L-Glu was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (for D-Asp K(d) = 860 +/- 28 nM, B(max) = 27.2 +/- 0.5 pmol/mg protein; for L-Glu, K(d) = 580 +/- 15 nM and B(max) = 51.3 +/- 0.8 pmol/mg protein). L-[(3)H]glutamate had higher affinity and a greater percentage of specific binding than did D-[(3)H]aspartate. The pharmacological binding specificity of L-[(3)H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was L-Glu > D-Asp > NMDA > MK801 > D-AP5 > glycine. For D-[(3)H]aspartate, the data revealed an interaction of D: -Asp with either NMDA-type receptors or putative specific binding sites.

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Differences between D- and L-aspartate binding to the Na+-dependent binding sites on glutamate transporters in frozen sections of rat brain

Cited by 10 publications

References 0 publications

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

Molecular Pharmacology of the Na+-Dependent Transport of Acidic Amino Acids in the Mammalian Central Nervous System.

d-Aspartate binding sites in rat Harderian gland

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