PMID- 14994336
OWN - NLM
STAT- MEDLINE
DCOM- 20040503
LR  - 20131121
IS  - 0360-4012 (Print)
IS  - 0360-4012 (Linking)
VI  - 75
IP  - 6
DP  - 2004 Mar 15
TI  - Binding and transport of [3H](2S,4R)- 4-methylglutamate, a new ligand for 
      glutamate transporters, demonstrate labeling of EAAT1 in cultured murine 
      astrocytes.
PG  - 751-9
AB  - Transporters for L-glutamate (excitatory amino acid transporters; EAATs), 
      localized to astrocytes, are involved intimately in intermediary metabolism 
      within the brain. Because (2S,4R)-4-methylglutamate (4MG) has affinity for glial 
      EAATs, we employed [(3)H]4MG to define the characteristics of EAATs in cultured 
      murine astrocytes and describe new approaches to analyze EAAT function. Specific 
      binding of [(3)H]4MG in astrocytic membranes at 4 degrees C represented 90% of 
      total binding. Binding was rapid (apparent t(1/2) approximately 7 min) and 
      saturable. Saturation and Scatchard analyses indicated a single binding site 
      (n(H) = 0.8) with a K(d) of 6.0 +/- 1.5 microM and B(max) = 9.7 +/- 2.9 pmol/mg 
      protein. Binding of [(3)H]4MG to astrocytic homogenates was Na(+)-dependent and 
      inhibited by K(+). Compounds acting at EAATs, such as L-glutamate (Glu), 
      D-aspartate (D-Asp), L-(2S,3S,4R)-2-(carboxycyclopropyl)glycine and 
      L-trans-pyrrolidine-2,4-dicarboxylate displaced binding to nonspecific levels. 
      L-Serine-O-sulphate, an EAAT1-preferring ligand, fully displaced binding of 
      [(3)H]4MG. In contrast, inhibitors having preferential affinity for EAAT2, 
      L-threo-3-methylglutamate, dihydrokainate, and kainate, were relatively 
      ineffective binding displacers. Agonists and antagonists for Glu receptors failed 
      to significantly inhibit [(3)H]4MG binding. Studies with [(3)H]D-Asp reinforced 
      evidence that [(3)H]4MG was binding to EAATs. These data were consistent with 
      Western blot analyses, which indicated abundant expression of EAAT1 but not 
      EAAT2. [(3)H]4MG was also accumulated rapidly (apparent t(1/2) approximately 4 
      min) into whole astrocytes by a sodium- and temperature-sensitive process (K(m) 
      of 146 +/- 24 microM, V(max) = 336 +/- 27 nmol/mg protein/min), which possessed 
      an EAAT1-like pharmacologic profile. These findings confirm that 4MG is a 
      substrate for EAAT1 and that the binding assay developed using [(3)H]4MG can be 
      utilized in various preparations including cultured astrocytes.
CI  - Copyright 2004 Wiley-Liss, Inc.
FAU - Aprico, Karina
AU  - Aprico K
AD  - Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
FAU - Beart, Philip M
AU  - Beart PM
FAU - Crawford, Duncan
AU  - Crawford D
FAU - O'Shea, Ross D
AU  - O'Shea RD
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Neurosci Res
JT  - Journal of neuroscience research
JID - 7600111
RN  - 0 (Amino Acid Transport System X-AG)
RN  - 0 (Amino Acids, Dicarboxylic)
RN  - 0 (Excitatory Amino Acid Agonists)
RN  - 0 (Excitatory Amino Acid Antagonists)
RN  - 0 (Excitatory Amino Acid Transporter 1)
RN  - 0 (Glutamates)
RN  - 0 (Ligands)
RN  - 10028-17-8 (Tritium)
RN  - 14561-55-8 (4-methylglutamic acid)
RN  - 22255-17-0 ((alpha-carboxycyclopropyl)glycine)
RN  - 452VLY9402 (Serine)
RN  - 4SR0Q8YD1X (D-Aspartic Acid)
RN  - 626-69-7 (serine O-sulfate)
RN  - 6OTE87SCCW (6-Cyano-7-nitroquinoxaline-2,3-dione)
RN  - 9NEZ333N27 (Sodium)
RN  - RWP5GA015D (Potassium)
RN  - SIV03811UC (Kainic Acid)
SB  - IM
MH  - 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology
MH  - Amino Acid Transport System X-AG/*metabolism
MH  - Amino Acids, Dicarboxylic/pharmacology
MH  - Analysis of Variance
MH  - Animals
MH  - Animals, Newborn
MH  - Astrocytes/drug effects/*metabolism
MH  - Binding, Competitive
MH  - Blotting, Western/methods
MH  - Cell Membrane/drug effects
MH  - Cells, Cultured
MH  - D-Aspartic Acid/pharmacology
MH  - Dose-Response Relationship, Drug
MH  - Excitatory Amino Acid Agonists/pharmacology
MH  - Excitatory Amino Acid Antagonists/pharmacology
MH  - Excitatory Amino Acid Transporter 1/antagonists & inhibitors/*metabolism
MH  - Glutamates/*pharmacokinetics
MH  - Kainic Acid/pharmacology
MH  - Ligands
MH  - Mice
MH  - Potassium/metabolism
MH  - Serine/*analogs & derivatives/pharmacology
MH  - Sodium/metabolism
MH  - Temperature
MH  - Tritium/pharmacokinetics
EDAT- 2004/03/03 05:00
MHDA- 2004/05/05 05:00
CRDT- 2004/03/03 05:00
PHST- 2004/03/03 05:00 [pubmed]
PHST- 2004/05/05 05:00 [medline]
PHST- 2004/03/03 05:00 [entrez]
AID - 10.1002/jnr.20013 [doi]
PST - ppublish
SO  - J Neurosci Res. 2004 Mar 15;75(6):751-9. doi: 10.1002/jnr.20013.