Kannan R, Yi J R, Zlokovic B V, Kaplowitz N
Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, Los Angeles 90033, USA.
Invest Ophthalmol Vis Sci. 1995 Aug;36(9):1785-92.
To characterize glutathione (GSH) transporter in the lens.
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.
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.
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.
对晶状体中的谷胱甘肽（GSH）转运体进行特性分析。
将从牛晶状体中分离得到的聚腺苷酸（Poly(A)+）RNA注射到非洲爪蟾卵母细胞中。卵母细胞在含有示踪剂GSH的NaCl或蔗糖培养基中孵育1小时，然后测定细胞相关放射性。在预先加载GSH的注射了晶状体mRNA的卵母细胞中测定谷胱甘肽外流。通过Northern印迹和逆转录-聚合酶链反应（RT-PCR）分析研究晶状体GSH转运体与最近克隆的两种非钠依赖性肝细胞膜GSH转运体之间的关系。还用RT-PCR检测牛晶状体mRNA中的γ-谷氨酰转肽酶（GGT）。
在注射了来自牛晶状体的聚腺苷酸（Poly(A)+）RNA的非洲爪蟾卵母细胞中可证明示踪剂35S-GSH的摄取。谷胱甘肽转运由载体介导（Km约为1.3 mM），且不依赖于钠。高效液相色谱证实摄取的分子形式主要是（>98%）GSH。预先加载16.5 nmol GSH-卵母细胞的注射了聚腺苷酸（Poly(A)+）RNA的卵母细胞显示谷胱甘肽外流速率为每小时2.6 nmol/卵母细胞。当牛晶状体聚腺苷酸（Poly(A)+）RNA与非钠依赖性大鼠胆小管GSH转运体（RcGshT）的cDNA探针杂交时，观察到RcGshT的转录本。RT-PCR证实了RcGshT的存在，并显示大鼠晶状体中不存在大鼠窦状GSH转运体（RsGshT）和GGT mRNA。
作者首次证明晶状体含有RcGshT的mRNA，并在卵母细胞中表达低亲和力的GSH转运体。前上皮顶端侧的谷胱甘肽外流以及逐渐摄取，以及向皮质纤维的内向外流，可能仅由RcGshT的表达或与尚未鉴定的GSH转运体共同表达来解释。
