Molecular characterization of the cystine/glutamate exchanger and the excitatory amino acid transporters in the rat lens.
OBJECTIVE
To determine whether the cyst(e)ine/glutamate exchanger (XC-) and the excitatory amino acid transporters (EAAT1 to -5) are expressed in the rat lens.


METHODS
A combination of molecular-based and immunocytochemical strategies was used to screen for the presence of the light-chain subunit of XC- (xCT) and the five known EAAT isoforms in the rat lens. An initial molecular profiling of xCT and EAAT1 to -5 expression was achieved by reverse transcription-polymerase chain reaction (RT-PCR). The presence of transporter proteins was verified by Western blot analysis and immunocytochemistry.


RESULTS
Transcripts for xCT and EAAT1 to -5 were detected by RT-PCR in lens fiber cells. Western blot analysis confirmed the expression of xCT and all five EAAT isoforms at the protein level. Immunocytochemistry revealed xCT expression to be present throughout the lens. Notably, changes in the subcellular distribution of xCT were shown to occur as a function of fiber cell differentiation. In the outer cortex, xCT labeling was predominantly cytoplasmic but progressively became more membranous with distance into the lens, due to xCT insertion into the broad sides of fiber cells. In the core, xCT labeling was localized around the entire membrane of inner fiber cells suggesting a redistribution of the exchanger. In contrast, EAAT expression was restricted to the outer cortex of the lens, with EAAT4/5 shown to be the predominant isoforms in cortical fiber cells. Western blot analysis of crude fiber membranes dissected from the outer cortex, inner cortex, and core region of the lens confirmed the presence of xCT in all three of these regions and demonstrated that EAATs were absent from the core region.


CONCLUSIONS
The molecular identification and localization of xCT and EAAT1 to -5 in the lens raises the possibility that in the outer cortex XC- and EAAT4/5 may work together to accumulate cysteine for GSH synthesis. The presence of xCT and the absence of the EAATs in the center of the lens suggest that XC- could operate with an alternative glutamate uptake pathway to accumulate cysteine where it can potentially act as a low-molecular-mass antioxidant.
Lim
Julie
J
Department of Physiology and the.
Lam
Yee Chai
YC
Kistler
Joerg
J
Donaldson
Paul J
PJ
D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D027182:Amino Acid Transport System y+ / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism*; D018846:Excitatory Amino Acids / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005973:Glutamine / Q000378:metabolism*; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D027322:Amino Acid Transport System X-AG; D027182:Amino Acid Transport System y+; D018846:Excitatory Amino Acids; D005973:Glutamine; D003545:Cysteine