Na(+)-dependent glutamate transporter in human retinal pigment epithelial cells.
OBJECTIVE
To characterize glutamate uptake in the human retinal pigment epithelial (HRPE) cell line 165 and to determine the feasibility of using this cell line as an experimental model for glutamate transport studies.


METHODS
Confluent monolayers of the HRPE cells cultured in 35-mm petri dishes were used to study glutamate uptake with regard to its ion dependence, substrate specificity, and kinetics. The radioactivity associated with the cells was measured by a liquid scintillation counter.


RESULTS
Glutamate uptake was noticeably stimulated by the presence of Na+ in uptake buffer. Acidic amino acids (100 microM; L-glutamate, L-aspartate, D-aspartate, and L-cysteate) and glutamate transporter inhibitors (100 microM; dihydrokainic acid, DL-threo-beta- hydroxyaspartic acid, and L-trans-pyrrolidine-2,4-dicarboxlic acid) interacted with uptake of radiolabeled glutamate. The activity of glutamate uptake depends on Na+ concentration. Glutamate uptake in the presence of Na+ does not have anion dependence. The uptake of glutamate was enhanced by external acidic environment and inhibited by 0.5 mM DIDS: Glutamate receptor antagonists (100 microM; [+/-]-2-amino-4-phosphonobutyric acid and 6-cyano-7- nitroquinoxaline-2,3-dione) did not inhibit glutamate uptake. Kinetic analysis shows that this transporter consists of at least two saturable systems.


CONCLUSIONS
The results of the present study demonstrate that a glutamate transporter is expressed in the HRPE cells. Its characteristics are similar to those of glutamate transporters observed in the RPE of laboratory animals. The human cell line 165 will be a useful tool for characterization of glutamate transport in the RPE. This study also provides clear evidence for the presence of a glutamate transporter in the human RPE.
Miyamoto
Y
Y
School of Optometry, University of California-Berkeley 94720.
Del Monte
M A
MA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D027322:Amino Acid Transport System X-AG; D000596:Amino Acids / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002412:Cations / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity
D027322:Amino Acid Transport System X-AG; D000596:Amino Acids; D002412:Cations; D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates; D006023:Glycoproteins; D018698:Glutamic Acid; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid