Iron regulates L-cystine uptake and glutathione levels in lens epithelial and retinal pigment epithelial cells by its effect on cytosolic aconitase.
OBJECTIVE
The authors previously published the novel finding that iron regulates L-glutamate synthesis and accumulation in the cell-conditioned medium (CCM) by increasing cytosolic aconitase activity in cultured lens epithelial cells (LECs), retinal pigment epithelial (RPE) cells, and neurons. The present study was designed to determine whether iron-induced L-glutamate accumulation in the CCM regulates L-cystine uptake and glutathione (GSH) levels through the aconitase pathway in LECs and RPE cells.


METHODS
The presence of xCT, the light chain of X(c)(-), a glutamate/cystine antiporter, was analyzed by RT-PCR, immunoblotting, and immunocytochemistry. Uptake of L-[(35)S]cystine and L-[(3)H]glutamate was measured in the presence or absence of transporter inhibitors. L-cystine uptake and intracellular GSH concentration were measured in the presence or absence of iron-saturated transferrin, the iron chelator dipyridyl (DP), or oxalomalic acid (OMA), an aconitase inhibitor.


RESULTS
LECs and RPE cells express xCT, as evidenced by RT-PCR analysis and immunoblotting. xCT was localized by immunocytochemistry. The authors found that the iron-induced increase in L-glutamate availability increased L-cystine uptake, with subsequent increases in GSH levels. In addition, L-glutamate production, L-cystine uptake, and GSH concentration were inhibited by OMA and DP, indicating a central role for iron-regulated aconitase activity in GSH synthesis in LECs and RPE cells.


CONCLUSIONS
These results demonstrate for the first time that iron regulates L-cystine uptake and the downstream production of GSH in two mammalian cell types. It is possible that the increase in intracellular antioxidant concentration induced by iron serves as a protective mechanism against the well-established capacity of iron to induce oxidative damage.
Lall
Marilyn M
MM
Department of Molecular Biomedical Sciences, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
Ferrell
Jenny
J
Nagar
Steve
S
Fleisher
Lloyd N
LN
McGahan
M Christine
MC
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D027182:Amino Acid Transport System y+ / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003553:Cystine / Q000378:metabolism*; D003600:Cytosol; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism*; D015151:Immunoblotting; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism; D010070:Oxalates / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014168:Transferrin / Q000494:pharmacology*
D027182:Amino Acid Transport System y+; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D010070:Oxalates; D012333:RNA, Messenger; D014168:Transferrin; C110714:holotransferrin; C015407:oxalomalic acid; D003553:Cystine; D015082:2,2'-Dipyridyl; D000154:Aconitate Hydratase; D005978:Glutathione