Characterization of glutamate uptake systems in astrocyte primary cultures from rat brain

Flott, Berenike E.; Seifert, W.

doi:10.1002/glia.440040307

Cited by 111 publications

(55 citation statements)

References 24 publications

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“…The Na+-dependent high-affinity uptake system exhibits a high specificity for glutamate and aspartate (25). Km values ranging from 14 to 220 ,uM have been reported (2,26), supporting the idea that this transport system may consist of several components.…”

Section: Resultsmentioning

confidence: 77%

“…For further characterization, uptake experiments were performed in the presence of DL-threo-3-hydroxyaspartate, the strongest known inhibitor of Na+-dependent glutamate transport assayed in brain slices (22) and astrocytes (2) (Fig. 3C).…”

Section: Resultsmentioning

confidence: 99%

“…Here the hybridization signal is restricted to the Purkinje cell layer. Control exper-DISCUSSION Current knowledge about the amino acid transporter, which limits the neurotransmitter action of L-glutamate released by glutamatergic synaptic terminals, has emerged from amino acid uptake studies on rat brain synaptosomes, brain slices, glia cell preparations, and primary cultures (2,23) as well as glioma and neuroblastoma lines (24). The Na+-dependent high-affinity uptake system exhibits a high specificity for glutamate and aspartate (25).…”

Section: Resultsmentioning

confidence: 99%

“…The excitatory signal is generally removed by reuptake of the amino acids into presynaptic terminals and surrounding glia cells by high-affinity transport systems, which appear to play an important role in the regulation of synaptic transmission (2). Conventional biochemical approaches have resulted in the partial purification of these proteins (3)(4)(5).…”

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confidence: 99%

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Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Storck

Schulte

Hofmann

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

1,072

707

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Transport systems specific for L-gutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/Laspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxyaspartate. In situ hybridization reveals a ikingly high density of GLAST mRNA in the Purkiqje cell layer qf cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.In the mammalian central nervous system L-glutamate is the main transmitter for most excitatory neurons, which are involved in complex physiological processes, such as learning and memory (1). The excitatory signal is generally removed by reuptake of the amino acids into presynaptic terminals and surrounding glia cells by high-affinity transport systems, which appear to play an important role in the regulation of synaptic transmission (2). Conventional biochemical approaches have resulted in the partial purification of these proteins (3)(4)(5). To date, three distinct systems have been described based on different ion requirements: a Na+-dependent system (3-6), a chloride-dependent system (7, 8), and a Na+-and Cl--independent system that is stimulated by Ca2+ (9, 10).Here we report the isolation of a eukaryotic glutamate/ aspartate transporter (GLAST) from rat brain and its characterization at the cDNAt and protein level. The deduced primary structure shows appreciable similarity to bacterial glutamate and dicarboxylate transporters. Expression of GLAST in Xenopus oocytes demonstrates that it is a highaffinity, Na+-dependent L-glutamate/L-aspartate transporter. GLAST mRNA is exclusively expressed in brain; it is primarily localized in the cerebellar Purkinje cell layer and is less dense throughout the cerebrum.MATERIALS AND METHODS supplier's instructions. Immunoprecipitation was performed using C-GLAST-GST, an affinity-purified antibody raised against a recombinant fusion protein consisting of the 49 C-terminal amino acids of GLAST and glutathione S-transferase (pGEX-lN vector; Amrad, Victoria, Australia).Injection of GLAST cRNA into Xenopus Oocytes and Analysis of the Expressed Amino Acid Transport. Oocytes were isolated from Xenopus laevis as described (13), separated by gentle agitation in collagenase type 11 (2...

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Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Storck

Schulte

Hofmann

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

1,072

707

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“…Glutamate uptake by control astrocytes was shown to be time-dependent, as had been reported previously (Flott and Seifert, 1991;Hara et aL, 1989;Piam' et aL, 1993). However, those astrocytes plated onto B25-35 for 7 days showed significantly lower glutamate uptakes regardless of the sampling times (1 min to 1 hr).…”

Section: Discussionsupporting

confidence: 83%

The role of astrocytes in Alzheimer's disease

Parpura-Gill

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This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer.The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction.In the unlikely event that the author did not send UME a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book.Photographs included in the original manuscript have been reproduced xerographically in this copy. Kgher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Copyright © Aleksandra Parpura-Gill, 1998. All rights reserved Signature was redacted for privacy. UMISignature was redacted for privacy.Signature was redacted for privacy.Signature was redacted for privacy. has not been confirmed, but is a possibility (Roses et al., 1990;Goate et aL, 1990).There are two subtypes of Alzheimer's disease determined by the age of onset (American Psychiatric Association, 1997). In Early Onset AD, symptoms of the disease begin before 65 years of age while in Late Onset AD they start after the age of 65. LateOnset AD is much more common than Early Onset. The course of Alzheimer's disease is characterized by an insidious beginning and progressive nature. Early deficits in memory are followed by other cognitive disturbances and personality changes, as well as loss of judgment and spatial orientation (American Psychiatric Association, 1997). Clinical Si^s of Alzheimer's DiseaseThe onset of dementia is an essential clinical feature of Alzheimer's disease and also a requirement for the diagnosis of the disease. Dementia is a syndrome characterized by progressive mental deterioration resulting from multiple cognitive deficits. The cognitive deficit must be severe enough to cause an inability of the patient to carry out occupational or social activities, and it has to represent a decline in the level of fiinctioning from a previous existing level (American Psychiatric Association, 1997;Terry and Katzman, 1983).Memory impairment as a component of dementia affects both short-term and long-term memory. Individuals suffering from dementia not only forget previously learned material, but also have significantly decreased ability to leam new material. Most often patients exhibit both for...

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Swelling of Müller cells induced by AP3 and glutamate transport substrates in rat retina

Izumi

Kirby-Sharkey

Benz

et al. 1996

Glia

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Characterization of glutamate uptake systems in astrocyte primary cultures from rat brain

Cited by 111 publications

References 24 publications

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

The role of astrocytes in Alzheimer's disease

Swelling of Müller cells induced by AP3 and glutamate transport substrates in rat retina

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