Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

Abstract

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.

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

“…In the present studies okadaic acid did inhibit the effect of D-asp on the distribution of GLAST and this is consistent with phosphorylation/ dephosphorylation of GLAST being involved in the mechanism of the rapid regulation of GluT. This is in accordance with the findings obtained using glial plasmalemmal vesicles [35] or astrocytes where GLAST is known to be the principal glutamate transporter [8,36]. The present observations are also consistent with the effect of the PKC-delta inhibitor rottlerin [37].…”

Characterization of the Tritium-Labeled Analog of L-threo-β-Benzyloxyaspartate Binding to Glutamate Transporters

“…In contrast, the Hill coefficients Table 2. for substrate transport (L-glutamate or D-aspartate), measured by uptake assays or electrophysiological studies, were reported to be 1.9 to 3.3, which most likely reflects the requirement for two to three Na ϩ ions to complete the uptake process (Klöckner et al, 1993;Sugawara et al, 1998;Kimmich et al, 2001). These results suggest that Na ϩ binding in the ligand binding step is different from Na ϩ binding in the substrate-uptake process.…”

Glutamate, Excitatory Amino Acid Transporters, Xc−Antiporter, Glutamine Synthetase, and γ-Glutamyltranspeptidase in Human Corneal Epithelium

“…The detection of multiple EAATs in the cornea is consistent with the reports of multiple EAATs in retina and lens, [14][15][16][17][18]22,25 as well as cultured brain astrocytes, retinal pigmented epithelial cells, and fibroblasts. [41][42][43] The detection of immunoreactive glutamate in the flat superficial epithelial cell layer positive for multiple EAATs, Xc − antiporter, and GGT suggests that multiple transport and enzyme systems actively regulate glutamate in flat superficial epithelial cells at the tear fluid interface where glutamate, aspartate, and taurine (synthesized from cystine) are elevated above plasma levels. 9,36,44 [Cumulative research results suggest that taurine plays an important role in calcium modulation, the development of the central nervous system, and the process of cell migration.…”