Glutathione transport in immortalized HLE cells and expression of transport in HLE cell poly(A)+ RNA-injected Xenopus laevis oocytes.

摘要

PURPOSE: 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.