Molecular characterization of a reduced glutathione transporter in the lens.

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Abstract

PURPOSE: To characterize glutathione (GSH) transporter in the lens. METHODS: Poly (A) +RNA isolated from bovine lens was injected into Xenopus laevis oocytes. Oocytes were incubated for 1 hour in either NaCl or sucrose medium containing tracer GSH, and cell-associated radioactivity was determined. Glutathione efflux was determined in lens mRNA injected oocytes preloaded with GSH. Relationship of lens GSH transporter to the two recently cloned sodium-independent hepatic membrane GSH transporters was studied by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Bovine lens mRNA also was probed for gamma glutamyl transpeptidase (GGT) by RT-PCR. RESULTS: Uptake of tracer 35S-GSH could be demonstrated in X. laevis oocytes injected with poly (A) +RNA from bovine lens. Glutathione transport was carrier mediated (Km approximately 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-injected 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 was hybridized with the cDNA probe for the sodium-independent rat canalicular GSH transporter (RcGshT), the transcript for RcGshT was observed. RT-PCR confirmed the presence of RcGshT and showed the absence of rat sinusoidal GSH transporter (RsGshT) and GGT mRNA in rat lens. CONCLUSIONS: The authors have demonstrated for the first time that lens contains mRNA for RcGshT and expresses a low-affinity GSH transporter in oocytes. Glutathione efflux from the apical side of the anterior epithelium and progressive uptake, and inward efflux into cortical fibers, might be explained by expression of RcGshT alone or in combination with as yet unidentified GSH transporters.