High Affinity Glutamate Transport in Rat Cortical Neurons in Culture

Wang, Guang Jian; Chung, Hai Won; Schnuer, Jamie; Pratt, Kara G.; Zable, Anthony C.; Kavanaugh, Michael P.; Rosenberg, Paul A.

doi:10.1124/mol.53.1.88

Cited by 71 publications

(78 citation statements)

References 37 publications

(68 reference statements)

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“…The glutamate transporter EAAC1 was reported to be the major subtype found in hippocampal neurons [38]. In cultured cells, this transporter has an apparent K m of 17 µM for glutamate [39,40]. Again, this is a somewhat higher affinity than we measured, but within the same order of magnitude.…”

Section: Discussionsupporting

confidence: 51%

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer

Reis

Prado

Kalapothakis

et al. 1999

Biochemical Journal

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Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca2+-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca2+ entry via voltage-dependent Ca2+ channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca2+-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.

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Section: Discussionsupporting

confidence: 51%

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer

Reis

Prado

Kalapothakis

et al. 1999

Biochemical Journal

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“…Relative activity obtained was approximately 1:3:6 for EAAT1, EAAT2, and EAAT3. A possible explanation for the different percent inhibition of dihydrokainic acid versus kainic acid is the finding of partial inhibition of glutamate transport through a cortical neuronal transporter by dihydrokainic acid (38).…”

Section: Discussionmentioning

confidence: 99%

Na+-dependent Glutamate Transporters (EAAT1, EAAT2, and EAAT3) of the Blood-Brain Barrier

O’Kane¹,

Martı́nez-López²,

DeJoseph³

et al. 1999

Journal of Biological Chemistry

251

188

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Na؉ -dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na ؉ -dependent glutamate transport was described on the abluminal membrane of the bloodbrain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K ؉ -dependence, and an apparent K m of 14 M (aggregate of the three transporters) at a transmembrane potential of ؊61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.

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“…Forebrain neuronal cultures were prepared from 16-day Sprague-Dawley rat embryos (Charles River laboratories, Boston, MA) as previously described [68]. Cultures were initially plated on poly-L-lysine coated 24 well plastic plates using an 80/10/10 (v/v) mixture of Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY), Ham's F-12 (Sigma, St. Louis, MO), heat-inactivated iron supplemented calf serum (Hyclone, Logan, UT), containing 2 mM glutamine, 25 mM HEPES, 24 U/ml penicillin and 24 μg/ml streptomycin, incubated in a 5% CO 2 /95% air at 36°C.…”

Section: Experimental Procedures Neuronal Culturesmentioning

confidence: 99%

“…Medium was not subsequently changed. Using this technique, the cultures contained less than 1% astrocytes [68], typically about 0.2%. Neuronal cultures were used at 2-3 weeks after plating.…”

Section: Experimental Procedures Neuronal Culturesmentioning

confidence: 99%

NMDA receptor-mediated extracellular adenosine accumulation is blocked by phosphatase 1/2A inhibitors

Rosenberg

2007

Brain Research

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We have previously demonstrated that NMDA receptor-mediated extracellular adenosine accumulation in neuronal cultures is receptor-mediated and requires calcium influx. Because protein kinase C (PKC) is a calcium-dependent enzyme, we hypothesized that activation of PKC might be involved in NMDA-mediated adenosine accumulation. PKC inhibitors however, did not block NMDA-evoked adenosine accumulation, but rather, stimulated basal adenosine accumulation. These data suggested the possibility that NMDA receptor mediated adenosine accumulation involves net dephosphorylation rather than phosphorylation of one or more substrates. Thus, inhibition of kinases would be expected to increase adenosine accumulation and inhibition of phosphatases would be expected to block adenosine accumulation. To test this hypothesis, we used the phosphatase 1/2A inhibitors calyculin A and okadaic acid. Both inhibitors significantly reduced NMDA-evoked adenosine accumulation. In contrast phosphatase 2B inhibitors did not block NMDA-evoked adenosine accumulation. These data suggest that NMDAevoked adenosine accumulation is mediated by activation of phosphatase 1/2A. We have established previously that NMDA-mediated adenosine accumulation is associated with adenosine kinase inhibition. However, adenosine kinase is not a direct substrate for phosphatase 1/2A because inhibition of phosphatase 1/2A did not abolish NMDA-evoked adenosine kinase inhibition. Okadaic acid also had no effect on NO donor-evoked adenosine accumulation, which previously has been shown to be associated with adenosine kinase inhibition. Dephosphorylation of one or more proteins other than adenosine kinase as a consequence of NMDA receptor activation might play an important role in extracellular adenosine regulation, with important consequences for the regulation of excitatory synaptic transmission, plasticity, epileptogenesis, and excitotoxicity.

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High Affinity Glutamate Transport in Rat Cortical Neurons in Culture

Cited by 71 publications

References 37 publications

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer

Na+-dependent Glutamate Transporters (EAAT1, EAAT2, and EAAT3) of the Blood-Brain Barrier

NMDA receptor-mediated extracellular adenosine accumulation is blocked by phosphatase 1/2A inhibitors

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