Characterization of Na+-coupled glutamate/aspartate transport by a rat brain astrocyte line expressing GLAST and EAAC1.
D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.
Kimmich
G A
GA
Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY 14642, USA.
Roussie
J
J
Manglapus
M
M
Randles
J
J
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism* / Q000502:physiology*; D000409:Alanine / Q000494:pharmacology; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D027341:Excitatory Amino Acid Transporter 1; D050580:Excitatory Amino Acid Transporter 3; D005731:Ganglionic Stimulants / Q000494:pharmacology; D019833:Genistein / Q000031:analogs & derivatives / Q000494:pharmacology; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D020033:Protein Isoforms / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D013687:Telencephalon / Q000166:cytology*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D027341:Excitatory Amino Acid Transporter 1; D050580:Excitatory Amino Acid Transporter 3; D005731:Ganglionic Stimulants; D050491:Glutamate Plasma Membrane Transport Proteins; D007476:Ionophores; D020033:Protein Isoforms; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; C493398:Slc1a1 protein, rat; C493392:Slc1a3 protein, rat; D027981:Symporters; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; C036040:serine O-sulfate; D012964:Sodium; D019833:Genistein; D011505:Protein-Tyrosine Kinases; C027917:tetramethylammonium; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine; D007608:Kainic Acid