MOLECULAR CHARACTERIZATION OF A REDUCED GLUTATHIONE TRANSPORTER IN THE LENS

Purpose. To characterize glutathione (GSH) transporter in the lens. Me thods. Poly (A)(+)RNA isolated from bovine lens was injected into Xeno pus laevis oocytes. Oocytes were incubated for 1 hour in either NaCl o r sucrose medium containing tracer GSH, and cell-associated radioactiv ity was determined. Glutathione efflux was determined in lens mRNA inj ected oocytes preloaded with GSH. Relationship of lens GSH transporter to the two recently cloned sodium-independent hepatic membrane GSH tr ansporters was studied by Northern blot and reverse transcription-poly merase chain reaction (RT-PCR) analyses. Bovine lens mRNA also was pro bed for gamma glutamyl transpeptidase (GGT) by RT-PCR. Results. Uptake of tracer S-35-GSH could be demonstrated in X. laevis oocytes injecte d with poly (A)(+)RNA from bovine lens. Glutathione transport was carr ier mediated (K-m similar to 1.3 mM) and was sodium independent. High- performance liquid chromatography confirmed that the molecular form of uptake was predominantly (>98%) as it was for GSH. Poly (A)(+)RNA-inj ected oocytes preloaded with 16.5 nmol GSH-oocyte showed GSH efflux at a rate of 2.6 nmol/oocyte per hour. When bovine lens poly (A)(+)RNA w as hybridized with the cDNA probe for the sodium-independent rat canal icular GSH transporter (RcGshT), the transcript for RcGshT was observe d. RT-PCR confirmed the presence of RcGshT and showed the absence of r at sinusoidal GSH transporter (RcGshT) and GGT mRNA in rat lens. Concl usions. The authors have demonstrated for the first time that lens con tains mRNA for RcGshT and expresses a low-affinity GSH transporter in oocytes. Glutathione efflux from the apical side of the anterior epith elium and progressive uptake, and inward efflux into cortical fibers, might be explained by expression of RcGshT alone or in combination wit h as yet unidentified GSH transporters.