Structural Selectivity and Molecular Nature ofl-Glutamate Transport in Cultured Human Fibroblasts

Cooper, Benedict; Chebib, Mary; Shen, Jie; King, Nicholas J. C.; Darvey, Ivan G.; Kuchel, Philip W.; Rothstein, Jeffrey D.; Balcar, Vladimír J.

doi:10.1006/abbi.1998.0626

Cited by 42 publications

(29 citation statements)

References 36 publications

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“…For example, in cultured fibroblasts, the regulation of Na ϩ -dependent Laspartate uptake parallels the activation and translocation of PKC⑀, suggesting a specific role of PKC⑀ in the regulation of transport activity in these cells (Franchi-Gazzola et al, 1994). However, it is not known which Glu transporters are regulated under these conditions (Cooper et al, 1998). In NIH 3T3 cells, the Pit-2 subtype of phosphate transporter is specifically regulated by PKC⑀, but it is not clear whether this effect is related to redistribution of the transporter or a change in intrinsic activity (Jobbagy et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Regulation of the Neuronal Glutamate Transporter Excitatory Amino Acid Carrier-1 (EAAC1) by Different Protein Kinase C Subtypes

2002

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In previous studies, we have shown that activation of protein kinase C (PKC) rapidly (within minutes) increases the activity and cell surface expression of the glutamate transporter EAAC1 in two systems that endogenously express this transporter (C6 glioma cells and cocultures of neurons and astrocytes). However, the magnitude of the increase in activity is greater than the increase in cell surface expression. In addition, certain compounds completely block the increase in cell surface expression but only partially attenuate the increase in activity. We hypothesized that PKC increases EAAC1 activity by increasing cell surface expression and catalytic efficiency and that two different subtypes of PKC mediate these effects. To address these hypotheses, the PKC subtypes expressed by C6 glioma cells were identified. Of the PKC subtypes that are activated by phorbol esters, only PKC␣, PKC␦, and PKC⑀ were observed. Gö 6976, a compound that blocks PKC␣ at concentrations that do not inhibit PKC␦ or PKC⑀, partially inhibited the increase in uptake but completely abolished the increase in EAAC1 cell surface expression. The 'Gö 6976-insensitive' increase in activity was not associated with a change in total transporter expression but was associated with an increase in the V max . Na ϩ -dependent glycine transport was not increased, providing indirect evidence that the Gö 6976-insensitive increase in activity was not caused by a change in the Na ϩ electrochemical gradient required for activity. Finally, by down-regulating different subtypes of PKC, we found evidence that PKC⑀ mediates the increase in EAAC1 activity that is independent of changes in cell surface expression and found further evidence that PKC␣ mediates the increase in cell surface expression. The potential relationship of the present work with a previously identified role for PKC␣ in certain forms of synaptic plasticity is discussed.

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Section: Discussionmentioning

confidence: 99%

Regulation of the Neuronal Glutamate Transporter Excitatory Amino Acid Carrier-1 (EAAC1) by Different Protein Kinase C Subtypes

2002

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“…Moreover, reduced expression of GLT1 (EAAT2) in global transient ischemia animal model [56] and in ALS patients [30,47] seems responsible for the elevation of extracellular glutamate concentration and neuronal death. Molecular and/or functional characterizations of glutamate transporters were performed in different cell culture models from CNS and peripheral tissues, in order to investigate the regulation of their expression, translocation and activity by various endogenous and exogenous factors [11][12][13]21,35,38,49].…”

Section: Introductionmentioning

confidence: 99%

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer’s disease

Zoia

Cogliati

Tagliabue

et al. 2004

Neurobiology of Aging

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Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na + -dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V max , without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

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“…91) Several explanations for this particular structural requirement have been put forward. 67,92) Among other main features of the substrate-selectivity was an aspartate analogue with a substitution on C2 (a carbon atom, 2-methyl-DL-aspartate) having a lower affinity for uptake than either the aspartate enantiomers or L-glutamate, while, in contrast, substitutions on C3 were well tolerated at least in the case of aspartate analogues (DL-t3OHA). Large difference between threo and erythro isomers of DL-3OHA indicated that the optimum bound conformation was narrowly defined and any structural alterations that would prevent the substrate molecule from attaining it could result in a major loss of the ability to interact with the substrate-recognition site.…”

Section: Substrate Specificity and Structural Re-quirements Of Glutmentioning

confidence: 99%

“…In a study done more than 25 years later, using a very different experimental model, many of the initial results were reproduced with only minor differences that could be explained by a greater precision of the more recent technique. The experimental model used in those studies was the uptake of [ 3 H]L-glutamate by cultured human fibroblasts that were shown to express at least four glutamate transporters 92) and the most significant departure from the data in brain slices was that of b-glutamate appearing as a "weak" rather than "strong" inhibitor.…”

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

confidence: 99%

“…As pointed out earlier, there has been a wealth of information on the substrate specificity and structural requirements of GluT 19,26,34,[92][93][94][95][96][97][98][99][100][101]103,105,[107][108][109] even in relation to individual EAAT's 110,112,114,[179][180][181][182][183] yet this structure-activity data has not so far been satisfactorily matched with the protein architecture of the putative substrate-binding regions. Moreover, some of the EAAT's may form multimeric complexes 184) and most of them also act as chloride channels 185) ; for a review see ref.…”

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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Structural Selectivity and Molecular Nature ofl-Glutamate Transport in Cultured Human Fibroblasts

Cited by 42 publications

References 36 publications

Regulation of the Neuronal Glutamate Transporter Excitatory Amino Acid Carrier-1 (EAAC1) by Different Protein Kinase C Subtypes

Regulation of the Neuronal Glutamate Transporter Excitatory Amino Acid Carrier-1 (EAAC1) by Different Protein Kinase C Subtypes

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer’s disease

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

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