Identification of a novel, sodium-dependent, reduced glutathione transporter in the rat lens epithelium.
OBJECTIVE
To determine whether glutathione (GSH) transporter(s) other than the previously identified rat canalicular GSH transporter (RcGshT) is present in the lens.


METHODS
Poly (A) +RNA isolated from rat and guinea pig lens cortex and epithelium was injected into Xenopus laevis oocytes. The effect of sodium removal was determined by measuring cell-associated radioactivity in lenticular epithelium or cortex mRNA injected oocytes (pretreated with acivicin to inhibit gamma glutamyltranspeptidase) after 1 hour of incubation in NaCl medium or choline chloride (Na(+)-free) medium containing tracer GSH (plus unlabeled GSH). The effect of 2 mM bromosulfophthalein-GSH (BSP-GSH) on GSH uptake in the lens epithelium and cortex in NaCl medium at two GSH concentrations also was determined. The molecular form of uptake of GSH in lens epithelial mRNA-injected oocytes was examined by high-performance liquid chromatography. Western blot analysis was performed to study the presence of RcGshT in the cortex and epithelium.


RESULTS
Oocytes injected with mRNA from rat and guinea pig lens epithelium and cortex compartments expressed GSH transport. High-performance liquid chromatography confirmed that epithelial uptake was as intact GSH under conditions of inhibition of GSH synthesis with dl-buthionine sulfoximine. The mean GSH uptake (nmol/oocyte per hour) in epithelial mRNA-injected oocytes was significantly reduced (P < 0.01, n = 4 oocyte preparations) under Na(+)-free conditions compared to NaCl medium at 0.05 mM and 2 mM GSH in the medium. Uptake in cortical mRNA-injected oocytes was unaffected by Na+ removal. Lens epithelial uptake exhibited a strong inhibition by BSP-GSH at 0.05 mM (55%) and 2 mM (64%), whereas cortical uptake was unaffected by BSP-GSH. Western blot analysis identified RcGshT in the cortical and epithelial regions.


CONCLUSIONS
Results from the current study provide strong evidence for the presence of a hitherto unreported Na(+)-dependent, BSP-GSH inhibitable GSH transporter in the lens epithelium, which may mediate concentrative, basolateral uptake of aqueous GSH consistent with in situ eye perfusion studies. The Na(+)-independent, BSP-GSH insensitive RcGshT may function as an apical GSH effluxer in lens epithelium and in mediating concentration gradient driven inward GSH movement by uptake-efflux in the lens cortex.
Kannan
R
R
Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, Los Angeles 90033, USA.
Yi
J R
JR
Tang
D
D
Zlokovic
B V
BV
Kaplowitz
N
N
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004848:Epithelium / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006168:Guinea Pigs; D007904:Lens Cortex, Crystalline / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000494:pharmacology*; D013448:Sulfobromophthalein / Q000494:pharmacology; D014982:Xenopus laevis
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C086860:glutathione transporter; D013448:Sulfobromophthalein; D012964:Sodium; D005978:Glutathione