Glutathione transport in immortalized HLE cells and expression of transport in HLE cell poly(A)+ RNA-injected Xenopus laevis oocytes.
OBJECTIVE
To determine reduced glutathione (GSH) transport in cultured human lens epithelial cells (HLE-B3) and plasma membrane vesicles and to study the expression of GSH transport in Xenopus laevis oocytes injected with poly(A)+ RNA from HLE-B3 cells.


METHODS
Confluent HLE-B3 cells pretreated with 10 mM DL-buthionine sulfoximine and 0.5 mM acivicin were used in GSH uptake studies. The uptake of 35S-GSH was performed for 30 minutes in either NaCl medium (Na+-containing) or choline chloride medium (Na+-free) at 37 degrees C and 4 degrees C. The molecular form of 35S uptake was determined by high-performance liquid chromatography. GSH uptake kinetics were studied in acivicin and buthionine sulfoximine-treated HLE-B3 cells in NaCl medium in the concentration range 0.01 microM to 50 mM. The transport of GSH and the effect of Na+ on uptake also were determined in mixed plasma membrane vesicles from HLE-B3 cells. In oocyte expression studies, HLE-B3 poly(A)+ RNA was injected into X. laevis oocytes and GSH uptake experiments were performed 3 days after injection. The uptake of 35S-GSH and GSH efflux rates were determined in HLE-B3 poly(A)+ RNA-injected oocytes.


RESULTS
No significant difference was found in the uptake of 1 mM GSH+/-acivicin (17.7+/-4.3 versus 15.7+/-1.4 picomoles/min(-1) per 10(6) cells). However, GSH uptake was significantly lower in Na+-free medium compared with Na+-containing medium (10.3+/-0.7 versus 16.8+/-0.9 picomoles/min(-1) per 10(6) cells; P < 0.01). GSH uptake in NaCl medium was carrier mediated. GSH uptake showed partial sodium dependency from 5 microM to 5 mM GSH in mixed plasma membrane vesicles from HLE-B3 cells. Oocytes injected with HLE-B3 poly(A) RNA expressed uptake and efflux of GSH. Uptake showed partial Na+ dependency at various GSH concentrations. The efflux rates were approximately 30-fold higher than those in water-injected oocytes (0.48+/-0.03 versus 0.016+/-0.005 (nanomoles per hour(-1) per oocyte, respectively). The molecular form of uptake in cultured cells and in oocyte studies was predominantly as intact GSH.


CONCLUSIONS
HLE-B3 cells and plasma membrane vesicles transported GSH by a carrier-mediated process. HLE-B3 poly(A)+ RNA injected X laevis oocytes expressed GSH transport. GSH uptake was partially Na+ dependent in all systems. HLE-B3 cells offer a useful model for characterizing GSH transport and for studying its regulatory role in the etiology of cataracts.
Kannan
R
R
Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, Los Angeles 90033, USA.
Bao
Y
Y
Mittur
A
A
Andley
U P
UP
Kaplowitz
N
N
D000818:Animals; D001692:Biological Transport; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005978:Glutathione / Q000493:pharmacokinetics*; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology; D014982:Xenopus laevis; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D007555:Isoxazoles; D012333:RNA, Messenger; D019328:Buthionine Sulfoximine; D012964:Sodium; D005723:gamma-Glutamyltransferase; D005978:Glutathione; C006303:acivicin