Cultured Lens Epithelial Cells Research Articles

Carteolol Hydrochloride Suppresses the Generation of Reactive Oxygen Species and Rescues Cell Death After Ultraviolet Irradiation of Cultured Lens Epithelial Cells

Introduction: Anti-oxidant activities of adrenergic β-blockers are proposed in various organs. The aim of the present study was to investigate the effect of carteolol hydrochloride, an adrenergic β-blocker, on the production of reactive oxygen species (ROS) and the viable cell number after ultraviolet irradiation of cultured lens epithelial cells (LECs).Materials and Methodology:Cultured LECs were exposed to 0, 10–5, 10–4, and 10–3 M carteolol hydrochloride for 30 min followed by ultraviolet B (UVB) irradiation at intensity of 100, 200, or 400 mJ/cm2. The amount of ROS in the LECs was measured using dichlorodihydrofluorescein at 30 min after exposure to UVB. In addition, the number of living LECs was counted at 15 h after exposure to UVB.Results:Exposure to 10–3 M carteolol hydrochloride significantly decreased the amount of ROS after exposure to UVB at intensities of 100, 200, and 400 mJ/cm2. In addition, 10–3 M carteolol hydrochloride significantly increased the viable cell number after exposure to UVB at 400 mJ/cm2. However, 10–4 and 10–5M carteolol hydrochloride had no significant effect on ROS or the viable cell number in LECs.Discussions: Carteolol hydrochloride protects LECs against UVB irradiation by inhibiting the intracellular production of ROS.

Proliferation of cultured lens epithelial cells and association with possible risk factors for posterior capsular opacification

Abstract Purpose To determine the proliferative capacity of human lens epithelial cells (HLEC) in culture and investigate possible associations with clinical characteristics of the donors, such as age, gender, pseudoexfoliations, uveitis and diabetes. Methods Pieces of lens capsule and adhering lens epithelial cells were obtained through capsulorhexis at routine cataract surgery using phacoemulsification technique, with informed consent from the patients. These human lens epithelium specimens were cultured in a humidified CO2 incubator using standard culture medium and 5% fetal calf serum for two weeks. After one and two weeks respectively, cultured cells were stained with carboxy‐fluorescein diacetate succinimidyl ester (CFDA SE), after which image processing software was used to determine the area of the confluent epithelial cell layer in relation to the size of the original capsule specimen. Results The increase in the area of confluent HLEC showed a negative correlation with age at the second week of growth and with diabetes at the first week after surgery. Female gender was associated with higher rate of cell proliferation when considering the whole culture period. The presence of pseudoexfoliations in vivo or a history of uveitis did not significantly affect cell growth in culture postoperatively. Nor did installation of xylocain, trypan blue or phenylephrine in the anterior chamber during surgery affect cell proliferation. Conclusion High age, male gender and diabetes are associated with lower rate of proliferation of lens epithelial cells in culture and may hence be protective against posterior capsular opacification.

Sigma‐2 receptor agonists kill lens epithelial cells

Abstract Purpose The aim of the present study was to examine the effects of the sigma‐2 receptor agonist, siramesine, on morphology, growth, cell death, lysosomal function, and effects on extra‐lysosomal proteolytic systems in human lens epithelial cells. In addition we compared the effects to that obtained in other human cells in primary culture. Methods Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using Nomarski optics or calcein. Lysosomes were evaluated using acridine orange and Magic Red (RR‐cresyl violet). Nuclear morphology was studied using Hoechst 33342 and propidium iodide. Enzymatic activities in living cells or cell lysates were studied using fluorogenic substrates. Results Siramesine at low concentrations increased the cytoplasmic proteolytic activity of the proteasome and the calpain system. Effects were also observed with respect to lysosomal morphology, acidity and function. In addition, activation of caspase‐3 and the appearance of nuclei with an apoptotic morphology was found. Lens epithelial cells appeared to be more sensitive to siramesine than other cells. Conclusion Siramesine at low concentrations affects lens epithelial cells with perturbations of the major proteolytic systems and lysosomal morphology, resulting in caspase activation and cell death. Siramesine may be a possible substance for the treatment or prevention of posterior capsular opacification (PCO).

Altered Ferritin Subunit Composition: Change in Iron Metabolism in Lens Epithelial Cells and Downstream Effects on Glutathione Levels and VEGF Secretion

The iron storage protein ferritin is necessary for the safe storage of iron and for protection against the production of iron-catalyzed oxidative damage. Ferritin is composed of 24 subunits of two types: heavy (H) and light (L). The ratio of these subunits is tissue specific, and alteration of this ratio can have profound effects on iron storage and availability. In the present study, siRNA for each of the chains was used to alter the ferritin H:L chain ratio and to determine the effect of these changes on ferritin synthesis, iron metabolism, and downstream effects on iron-responsive pathways in canine lens epithelial cells. Primary cultures of canine lens epithelial cells were used. The cells were transfected with custom-made siRNA for canine ferritin H- and L-chains. De novo ferritin synthesis was determined by labeling newly synthesized ferritin chains with 35S-methionine, immunoprecipitation, and separation by SDS-PAGE. Iron uptake into cells and incorporation into ferritin was measured by incubating the cells with 59Fe-labeled transferrin. Western blot analysis was used to determine the presence of transferrin receptor, and ELISA was used to determine total ferritin concentration. Ferritin localization in the cells was determined by immunofluorescence labeling. VEGF, glutathione secretion levels, and cystine uptake were measured. FHsiRNA decreased ferritin H-chain synthesis, but doubled ferritin L-chain synthesis. FLsiRNA decreased both ferritin H- and L-chain synthesis. The degradation of ferritin H-chain was blocked by both siRNAs, whereas only FHsiRNA blocked the degradation of ferritin L-chain, which caused significant accumulation of ferritin L-chain in the cells. This excess ferritin L-chain was found in inclusion bodies, some of which were co-localized with lysosomes. Iron storage in ferritin was greatly reduced by FHsiRNA, resulting in increased iron availability, as noted by a decrease in transferrin receptor levels and iron uptake from transferrin. Increased iron availability also increased cystine uptake and glutathione concentration and decreased nuclear translocation of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium. Most of the effects of altering the ferritin H:L ratio with the specific siRNAs were due to changes in the availability of iron in a labile pool. They caused significant changes in iron uptake and storage, the rate of ferritin synthesis and degradation, the secretion of VEGF, and the levels of glutathione in cultured lens epithelial cells. These profound effects clearly demonstrate that maintenance of a specific H:L ratio is part of a basic cellular homeostatic mechanism.

Protective effects of metallothionein I and II against metal- and ultraviolet radiation-induced damage in cultured lens epithelial cells

Few quantitative analyses of the induction of metallothioneins (MTs) have been conducted, and there are no reports on the contribution of MTs to the protective mechanism against ultraviolet (UV) radiation in the lens. In this study, we quantitatively analyzed the induction of MTs and analyzed the resulting protective effects against both metal- and UV radiation-induced damage in the cultured lens epithelial cell line, alphaTN4-1. The induction profiles of MTs by ZnCl(2) treatment in alphaTN4-1 cells were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The cells in which MTs were induced were either treated with high concentrations of ZnCl(2) or CdCl(2), or irradiated with UV-C, UV-B, or UV-A radiation, followed by analysis of cell viability. The (3)H-thymidine incorporation rate was used as an indicator of cell viability. mRNA expression of MT-I and MT-II, the main MT isoform classes, was induced by ZnCl(2) treatment in a dose-dependent manner. MT induction increased the protective effects against both metaland UV-A radiation-induced cell damage. Our results suggest that MTs play an important role in the protection against damage induced by both toxic metals and UV-A radiation in lens epithelial cells.

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Gap junction-mediated intercellular communication (GJIC) is essential for the proper function of many organs, including the lens. GJIC in lens epithelial cells is increased by FGF in a concentration-dependent process that has been linked to the intralenticular gradient of GJIC required for lens transparency. Unlike FGF, elevated levels of TGF-beta are associated with lens dysfunction. We show that TGF-beta1 or -2 up-regulates dye coupling in serum-free primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) via a mechanism distinct from that utilized by other growth factors. Remarkably, the ability of TGF-beta and of FGF to up-regulate GJIC is abolished if DCDMLs are simultaneously exposed to both factors despite undiminished cell-cell contact. This reduction in dye coupling is attributable to an inhibition of gap junction assembly. Connexin 45.6, 43, and 56-containing gap junctions are restored, and intercellular dye coupling is increased, if the activity of p38 kinase is blocked. Our data reveal a new type of cross-talk between the FGF and TGF-beta pathways, as well as a novel role for TGF-beta and p38 kinase in the regulation of GJIC. They also provide an explanation for how pathologically increased TGF-beta signaling could contribute to cataract formation.

Molecular Cloning of Canine Excitatory Amino Acid Transporter 5 and Its Detection in Primary Lens Epithelial Cells

The full-length cDNA sequence of canine excitatory amino acid transporter (EAAT) 5 was determined in samples taken from the canine retina. The sequence was 2,467 bp long and was predicted to encode the 560 amino acid polypeptides. The deduced amino acid sequence of canine EAAT5 showed similarities of 91.8 and 92.7% to those of humans and rats, respectively. In canine, EAAT5 has a 49.4% identity with EAAT1, 43.7% with EAAT2, 46.4% with EAAT3, and 45.7% with EAAT4. RT-PCR analysis revealed EAAT5 expression in primary lens epithelial cell culture and the cerebellum, and Western blot analysis detected a single band of 60 kDa which confirmed EAAT5 protein expression in these cells. In addition, all subtypes of EAAT were detected in canine lens epithelial cells, indicating the pivotal role of EAATs in supplying glutamate, the precursor of antioxidant glutathione in the lens.

Cell death in lens epithelial cells after stimulation of the sigma‐2 receptor

Abstract Purpose The aim was to investigate the mechanisms of cell death in lens epithelial cells after administration of siramesine, a sigma‐2 receptor agonist. Methods Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using DIC or calcein as a cytoplasmic marker. Lysosomes were studied using acridine orange and MagicRed. Proteolytic activity of the proteasome, calpain, caspases and cathepsins in living cells or cell extracts were studied using different fluorogenic substrates. Results Siramesine at low concentrations increased the cytoplasmic proteolytic activity of the proteasome and the calpain system. Early effects was also observed with respect to lysosomal morphology, acidity and function. Activation of caspase‐3 and the appearance of nuclei with an apoptotic morphology were also found. Conclusion Siramesine at very low concentrations affects lens epithelial cells with perturbation of the major proteolytic systems, lysosomal morphology and results in caspase activation and cell death. Siramesine may be a promising substance for clinical studies concerning the treatment of PCO.

Oxidative effects on the glutathione enzyme systems

Abstract Purpose Lens ageing is associated with oxidative processes, with photo‐oxidation as one of the stressors. Cataract has traditionally been viewed as a one‐way progressive event, but in vivo and in vitro animal studies on ultraviolet radiation‐induced cataract during the last years show repair capacity in the lens. Little is known about what these repair systems are and how they might be influenced by pharmaceuticals. The glutathione redox enzymes have critical antioxidative functions and are a potential target for pharmaceutical intervention. Methods Primary cultures of lens epithelial cells were obtained from mouse and human pediatric lens capsules. The cells were stressed with varying concentrations of hydrogen peroxide. The short‐term activity pattern of primary glutathione‐reducing enzymes and other glutathione‐associated enzymes were determined. Results The main picture was a concentration‐dependent decrease in enzyme activity within 15 minutes, with subsequent recovery within one hour. Conclusion The time frame for enzyme inhibition is co‐inciding with that of maximally efficient exposure time in ultraviolet radiation‐induced cataract.

Effect of Culture Substrate and Culture Conditions on Lens Epithelial Cell Proliferation and α-smooth Muscle Actin Expression

The most common complication following cataract surgery is posterior capsule opacification. This results from migration, proliferation and transdifferentiation of residual lens epithelial cells (LECs). We studied the effect of several culture substrates and culture conditions on LEC proliferation and α-smooth muscle actin (α-SMA) expression. We used primary and secondary cultures of porcine LECs cultivated on collagen I, collagen IV, microscopic glass slides, and uncoated plastic dishes. We studied the cell proliferation and expression of α-SMA and α-, β-, and γ-crystallins. The effect of the medium exchange protocol was studied using the TOTL-86 rabbit epithelial lens cell line. There was no difference in growth characteristics of primary cultures on different substrates. In secondary cultures, LECs adhered better to collagen-coated surfaces. The culture substrate influenced LEC proliferation and α-SMA expression. The proliferation was greater when the medium was changed than when extra medium was added on the 4th day. The cells did not synthesize α-, βor γ-crystallin. The culture substrate influences the adhesion ability, proliferation and α-SMA expression in lens epithelial cells. In addition, it is necessary to consider the effects of the medium exchange protocol, serum supplementation, cell density and other cell culture conditions in lens epithelial cell experiments.

Involvement of integrins in mechanical stress-induced local Ca2+ influx, Ca2+ spots, in the presence of lysophosphatidic acid in cultured lens epithelial cells

Involvement of integrins in mechanical stress-induced local Ca2+ influx, Ca2+ spots, in the presence of lysophosphatidic acid in cultured lens epithelial cells

Upregulation and maintenance of gap junctional communication in lens cells

The cells of the lens are joined by an extensive network of gap junction intercellular channels consisting of connexins 43, 46, and 50. We have proposed, and experimentally supported, the hypothesis that fibroblast growth factor (FGF) signaling is required for upregulation of gap junction-mediated intercellular coupling (GJIC) at the lens equator. The ability of FGF to increase GJIC in cultured lens cells requires sustained activation of extracellular signal-regulated kinase (ERK). In other cell types, activation of ERK has been shown to block GJIC mediated by connexin43 (Cx43). Why ERK signaling does not block lens cell coupling is not known. Another unresolved issue in lens gap junction regulation is how connexins, synthesized before the loss of biosynthetic organelles in mature lens fiber cells, avoid degradation during formation of the organelle-free zone. We have addressed these questions using serum-free cultures (termed DCDMLs) of primary embryonic chick lens epithelial cells. We show that FGF stimulates ERK in DCDMLs via the canonical Ras/Raf1 pathway, and that the reason that neither basal nor growth factor-stimulated GJIC is blocked by activation of ERK is because it is not mediated by Cx43. In fibroblastic cells, the normally rapid rate of degradation of Cx43 after its transport to the plasma membrane is reduced by treatments that either directly (ALLN; epoxomicin) or indirectly (generation of oxidatively un/mis-folded proteins by arsenic compounds) prevent the ubiquitin/proteasome system (UPS) from acting on its normal substrates. We show here that Cx45.6 and Cx56, the chick orthologs of mammalian Cx50 and Cx46, behave similarly in DCDMLs. When organelles lyse during the maturation of fiber cells, they release into the cytosol a large amount of new proteins that have the potential to saturate the capacity, and/or compromise the function, of the UPS. This would serve to spare gap junctions from degradation during formation of the organelle-free zone, thereby preserving GJIC between mature fiber cells despite the lack of de novo connexin synthesis.

Hypoxia-Regulated Activity of PKCε in the Lens

To show that hypoxia is necessary to prevent opacification of the lens. Protein kinase C (PKC)-epsilon serves a role that is distinct from PKC-gamma when both PKC isoforms are expressed in the lens. PKCepsilon serves a very important role in hypoxic conditions, helping to prevent opacification of the lens. Digital image analysis, confocal microscopy, dye transfer assay, coimmunoprecipitation, Western blot analysis, and enzyme activity assays were used, respectively, to study opacification of the lens, intercellular communications, cellular localization of connexin-43 (Cx43), and the interactions between PKCepsilon, PKCgamma, and Cx43 in the lens epithelial cells. Hypoxic conditions (1%-5% of oxygen) were very important in maintaining clarity of the lenses of wild-type (WT) mice. Normoxic conditions induced opacification of the WT lens. Lenses from the PKCepsilon-knockout mice underwent rapid opacification, even in hypoxic conditions. Hypoxia did not induce apoptosis in the lens epithelial cells, judging by the absence of active caspase-3, and it did not change intercellular communication and did not affect the number and localization of junctional Cx43 plaques in the lens epithelial cell culture. Hypoxia activated PKCepsilon, whereas phorbol ester (TPA), oxidation (H(2)O(2)), and insulin-like growth factor-1 (IGF-1) activated PKCgamma and decreased the activity of PKCepsilon. Hypoxia did not induce the phosphorylation of the Cx43. Hypoxia-induced activation of PKCepsilon is very important in surviving hypoxia and maintaining the clarity of the lens. However, PKCgamma is utilized in the control of Cx43 gap junctions.

Acute effects of the sigma‐ receptor agonist on human lens epithelial cells

Abstract Purpose The aim of the present study was to examine the effect of the sigma‐2 receptor agonist, Siramesine, on morphology, growth, cell death, lysosomal function and effects on extra‐lysosomal proteolytic systems in human lens epithelial cells. Methods Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using Nomarski optics or calcein. Lysosomes were evaluated using acridine orange and MagicRed. Nuclear morphology was studied using Hoechst 33342 and propidium iodide. Enzymatic activities in living cells or cell lysates were studied using different fluorogenic substrates. Results Siramesine at low concentrations increased the cytoplasmic proteolytic activity of the proteasome and the calpain system. Effects was also observed with respect to lysosomal morphology, acidity and function. Activation of caspase‐3 and the appearance of nuclei with an apoptotic morphology were also found. Conclusion Siramesine at low concentrations affects lens epithelial cells with perturbation of the major proteolytic systems, lysosomal morphology and results in caspase activation and cell death. Siramesine may be a promising substance for clinical studies concerning the treatment of PCO.

Oxidative stress in lens epithelial cells after UVR exposure

Abstract Purpose The lens epithelium is the primary target in solar photooxidation. Thioltransferase is a repair enzyme capable of reactivating oxidized critical proteins and enzymes in the cells. Methods Primary lens epithelial cell cultures from wild type and thioltransferase knockout mice were exposed to various doses of UV radiation or hydrogen peroxide. The short‐term reaction pattern of several glutathione‐related enzymes were investigated, as well as long‐term growth rate and cell adhesion. Results UV radiation induces a reaction pattern comparable to that of hydrogen peroxide, with initial increase or decrease in activity of antioxidative enzymes, and a subsequent recovery within 1‐2 hours. Low dose UVR exposure induces cell stimulating effects similar to treatment with low peroxide concentration. Conclusion Oxidative stress from peroxide and UVR might be channeled through same oxidant cellular pathway.

Effect of Thioltransferase (Glutaredoxin) Deletion on Cellular Sensitivity to Oxidative Stress and Cell Proliferation in Lens Epithelial Cells of Thioltransferase Knockout Mouse

To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase(-/-)) lens epithelial cells (LECs) as a model. Primary LEC cultures were obtained from wild-type (TTase(+/+)) and TTase(-/-) mice. Characterization and validation of the cells were determined by immunoblotting for TTase and alpha-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H(2)O(2) stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase(-/-) cells was achieved with reagent. Primary LEC cultures obtained from wild-type (TTase(+/+)) and TTase(-/-) mice were characterized and found to contain lens-specific alpha-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase(-/-) cells and its presence in the wild-type cells. TTase(-/-) LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase(+/+) cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H(2)O(2) after challenge with low levels of H(2)O(2). Reloading of purified TTase into the TTase(-/-) cells restored the antioxidant function in TTase(-/-) cells to a near normal state. These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.

Cross-Talk between Fibroblast Growth Factor and Bone Morphogenetic Proteins Regulates Gap Junction-mediated Intercellular Communication in Lens Cells

Homeostasis in the lens is dependent on an extensive network of cell-to-cell gap junctional channels. Gap junction-mediated intercellular coupling (GJIC) is higher in the equatorial region of the lens than at either pole, an asymmetry believed essential for lens transparency. Primary cultures of embryonic chick lens epithelial cells up-regulate GJIC in response to purified fibroblast growth factor (FGF)1/2 or to medium conditioned by vitreous bodies, the major reservoir of factors (including FGF) for the lens equator. We show that purified bone morphogenetic protein (BMP)2, -4, and -7 also up-regulate GJIC in these cultures. BMP2, -4, or both are present in vitreous body conditioned medium, and BMP4 and -7 are endogenously expressed by lens cells. Remarkably, lens-derived BMP signaling is required for up-regulation of GJIC by purified FGF, and sufficient for up-regulation by vitreous humor. This is the first demonstration of an obligatory interaction between FGF and BMPs in postplacode lens cells, and of a role for FGF/BMP cross-talk in regulating GJIC in any cell type. Our results support a model in which the angular gradient in GJIC in the lens, and thus proper lens function, is dependent on signaling between the FGF and BMP pathways.

Effect of grape polyphenols on oxidative stress in canine lens epithelial cells

To evaluate whether the effects of oxidative stress could be attenuated in cultures of canine lens epithelial cells (LECs) by incubation with grape seed proanthocyanidin extract (GSE), resveratrol (RES), or a combination of both (GSE+RES). Primary cultures of canine LECs. LECs were exposed to 100MM tertiary butyl-hydroperoxide (TBHP) with or without GSE, RES, or GSE+RES. The dichlorofluorescein assay was used to detect production of reactive oxygen species (ROS), and immunoblot analysis was used to evaluate the expression of stress-induced cell-signaling markers (ie, the mitogen-activated protein kinase [MAPK] and phosphoinositide-3 kinase [PI3K] pathways). GSE and GSE+RES significantly reduced ROS production after a 30-minute exposure to TBHP. Only GSE significantly reduced ROS production after a 120-minute exposure to TBHP. Incubation with GSE reduced TBHP-induced activity of the MAPK and PI3K pathways. GSE inhibited key components associated with cataractogenesis, ROS production, and stress-induced cell signaling. On the basis of the data reported here, there is strong evidence that GSE could potentially protect LECs from the damaging effects of oxidative stress.

Intracellular Effects of NSAIDs/ASA in Oxidatively Stressed Human Lens Epithelial Cells in Culture

The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H₂O₂ in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H₂O₂-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H₂O₂-exposed HLECs in culture.

Iron Regulates L-Cystine Uptake and Glutathione Levels in Lens Epithelial and Retinal Pigment Epithelial Cells by Its Effect on Cytosolic Aconitase

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. 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. 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. 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.