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Most proliferating cells follow a series of orderly transitions from one phase to another. These transitions are usually controlled by timed degradation of cell cycle regulators by the ubiquitin-proteasome pathway (UPP). There are no published reports regarding the timing of phases of the human lens cell cycle or regarding cell cycle-related changes in UPP components. Objectives of this study were to characterize the timing of the phases of the human lens epithelial cell cycle and to explore potential functions of critical components of the UPP in controlling lens cell cycle. Human lens epithelial cells were synchronized at G0/G1 phase by contact inhibition. Cell cycle progression upon subculturing was monitored by FACS analysis. It took approximately 40 hr for HLEC to complete one cell cycle, approximately 20 hr for G1 phase, approximately 8-10 hr for S phase and approximately 10 hr for the combination of G2 and M phases. Proteasome-dependent degradation of p21WAF and p27Kip, the dominant Cdk inhibitors, was associated with the G1/S phase transition in these cells. Proteasome inhibition experiments indicate that proteolysis is the predominant process which is responsible for the variations in these regulators during the cell cycle. Levels of specific ubiquitin conjugating enzymes, Ubc7 and Ubc10, increased 6 and 2-fold at the G2/M phase and S/G2/M phases, respectively. Levels of these E2s decreased precipitously upon completion of the M phase. In contrast, levels of ubiquitin activating enzyme (E1) and Ubc3 remained constant during the cell cycle. Cul1, a component of the SCF (an E3), remained relatively constant during cell cycle. The up-regulation of Ubc7 and Ubc10 during the G2/M and S/G2/M phases suggests that these enzymes may be involved in controlling the cell cycle progression at this phase. Taken together, the data indicate that expression of key components of the UPP in the human lens epithelial cells is regulated in a cell cycle-dependent manner. Some of the variations in levels of ubiquitin conjugating enzymes are suggestive of previously undescribed functions.

Purpose: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells., Methods: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis., Results: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin., Conclusions: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.

LEP503 is a novel gene product isolated from lens epithelial cells by a subtractive cDNA cloning strategy. It is highly conserved in different vertebrate species and developmentally regulated in postnatal rat lens, suggesting that LEP503 may be an important lens epithelium gene involved in the processes of lens epithelial cell differentiation. The expression of LEP503 is highly restricted to lens epithelial cells in vivo. To investigate the molecular mechanisms regulating the promoter of the human LEP503, we cloned and characterized the promoter of the human LEP503 gene. The transcription start site was localized to a nucleotide C 22 base pairs (bp) 5' of the initiation methionine codon. By reporter gene transfection experiments, we found that approximately 2.5-kb of LEP503 5'-flanking sequence directed high level luciferase activity in human lens epithelial cells; further deletion analysis revealed positive regulatory element between bp -401 and +22. Mutation analysis in each of the seven potential binding sites for transcription factors within the region between -401 and +22 showed that the AP-1 element at -131 and the Sp1 element at -48 are the most important sites for the tissue-specific expression of LEP503. Consistent with lens epithelial cell-restricted expression of LEP503 mRNA, we found that the approximately 2.5-kb 5'-flanking sequence directed high-level promoter activity in lens epithelial cells but not in other cell types. Understanding the LEP503 promoter will allow us to investigate lens epithelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to lens epithelial cells. The LEP503 gene is mapped to human chromosome 1q22, the same location to which zonular pulverulent cataract was previously mapped.

We isolated a clone encoding a protein from a human lens epithelial cell (LEC) cDNA library with antibody (Ab) from a cataract patient and named it "lens epithelium-derived growth factor" (LEDGF). LEDGF is found to be identical to p75, a coactivator of both transcription (1) and pre-mRNA splicing (2). In serum-free medium LEDGF stimulated growth of LECs, cos7 cells, skin fibroblasts, and keratinocytes, and prolonged cell survival. Without LEDGF, the aforementioned cells did not survive. Also in serum-free medium, Ab to LEDGF neutralizing LEDGF blocked cell growth and caused cell death. Thus, LEDGF, a regulatory factor, may play an important role for growth and survival of a wide range of cell types., (Copyright 2000 Academic Press.)

Lens capsule consists of several kinds of extracellular matrix (ECM) which may play an important role in cell attachment, migration and proliferation of lens epithelial cells as a basement membrane. We have investigated the effects of ECM on cell attachment, proliferation and migration in a human lens epithelial (HLE) cell line. The HLE cell line, SRA 01/04, which was transfected with large T-antigen of SV40 was cultured in the absence of serum. Culture plates were coated with human type IV collagen, laminin or fibronectin. The number of cells were counted at 30-180 min and 3, 5 and 7 days of culture. The rate of BrdU incorporation was measured to study the cell proliferation. The cell migration was measured 1, 3, 5 and 7 days after seeding cells. Integrins, the receptors of ECM, were also detected using antibodies for the cell membrane antigens (CD49b, CD49c, CD49e) by an immunohistochemical method. Although less than 10% of cells attached to the non-coated plate and 50-60% of cells attached to the ECM-coated plates, there was no difference of cell attachment among each ECM used. The cell attachment was almost complete during the first 30 min of culture. Cell proliferation was not enhanced, but cell survival was aided by culture on the ECM components for up to 7 days. The area of cell attachment enlarged on the ECM-coated plates, whereas no migration was observed on the non-coated plate. These data indicate that ECM is the essential factor for cell attachment and increases migration of HLE cells., (Copyright 1999 Academic Press.)

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation., (Copyright 1999 Academic Press.)

Purpose: To identify differentially expressed genes in a human lens epithelial cell line exposed to oxidative stress., Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) differential display was used to evaluate differential gene expression in a human lens epithelial cell line (SRA 01-04) when cells were exposed for 3 hours to a single bolus of 200 microM hydrogen peroxide. Differentially expressed genes were identified through DNA sequencing and a nucleotide database search. Differential expression was confirmed by northern blot and RT-PCR analyses., Results: Using 18 primer sets, 28 RT-PCR products were differentially expressed between control and hydrogen peroxide-treated cells. In stressed cells, mitochondrial transcripts nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 and cytochrome b were downregulated 4-fold. Of the cytoplasmic mRNAs, glutamine cyclotransferase decreased 10-fold, whereas cytokine-inducible nuclear protein, alternative splicing factor 2, and beta-hydroxyisobutyryl-coenzyme A hydrolase increased 2-, 4-, and 10-fold, respectively. Analysis of mitochondrial transcripts in a 24-hour time course showed that NADH dehydrogenase subunit 4 mRNA decreased by 2-fold as early as 1 hour after oxidative stress, whereas the rate of decrease was slower for cytochrome b, cytochrome oxidase III, and 16S rRNA., Conclusions: Oxidative stress induced specific expressed gene changes in hydrogen peroxide-treated lens cells, including genes involved in cellular respiration and mRNA and peptide processing. These early changes may reflect pathways involved in the defense, pathology, or both of the lens epithelium, which is exposed to oxidative stress throughout life.

Although primary cultures of human lens epithelial (HLE) cells provide important information concerning the role of epithelium in normal lens and cataract formation, the lack of a cell line precludes a broad range of studies on the metabolism and molecular biology of these cells. We have, therefore developed an HLE cell line. Primary cultures of HLE cells were transfected with plasmid vector DNA containing a large T antigen of SV40. The immortalized cells were characterized with regard to morphology, growth rate, karyotype, and expression of crystallins, aldose reductase and other enzymes. A single clone of the immortalized cells, SRA 01/04, formed a monolayer and grew constantly over 130 passages. Isozyme phenotype showed that SRA 01/04 was of human origin, and the chromosome counts were in the hypotetraploid range. Western blot analysis showed that the cells expressed a very low level of crystallins (alphaA and betaB2) and aldose reductase. Messenger RNA (mRNA) for both alpha and beta crystallins was detected by reverse transcription polymerase chain reaction (RT-PCR) in both early and late passages. Sequence analysis of the PCR products, corresponding to alphaA and betaB2 crystallins in the cell line and in primary cultures of HLE, revealed a 100% match with published human alphaA and betaB2 sequences. These characteristics were unchanged in the cell line in early and late passages. This is the first report of the presence of alphaA and transcripts of mRNA for both alphaA and betaB2 in an established human cell line. This new HLE cell line makes it possible to undertake many future studies on the role of epithelium in lens and cataract formation., (Copyright 1998 Academic Press.)

Purpose: To investigate the ability of the nitroxide free radical and superoxide dismutase mimic 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL) to protect against x-ray-induced lens DNA damage and cataract formation in the rabbit., Methods: Eleven gray (Gy) x-rays was delivered twice, with a 48-hour interval, to the same eye of 5-week-old rabbits. Fifteen minutes before each x-ray, 150 microliters aqueous humor was removed from the anterior chamber and replaced with 150 microliters citrate buffer containing 0 mM or 100 mM TEMPOL. The development of cataract was classified into seven stages by slit-lamp examination. DNA strand breaks were measured in the lens epithelium of x-rayed rabbits using a single-cell gel electrophoresis method., Results: The level of total TEMPOL in the aqueous humor of rabbits at 15 minutes after intracameral injection of the compound was 21 mM with 84% present in the oxidized form (determined by electron paramagnetic resonance spectroscopy). At 19 weeks after x-ray, rabbits irradiated without TEMPOL showed either stage 5 (complete posterior subcapsular opacity) or stage 6 (mature) cataracts, whereas the animals that had first been injected with TEMPOL developed only stage 2 to stage 4 cataracts (the difference between the two groups was significant at P < 0.01). Intracameral injection of TEMPOL resulted in a decrease of nearly 70% in the level of DNA strand breaks produced by a single 11-Gy dose of x-ray. In vitro studies showed that TEMPOL was reduced rapidly by ascorbic acid but not by reduced glutathione. Oxidized but not reduced TEMPOL (TEMPOL-H) was an effective radioprotector in cultured rabbit lenses, and TEMPOL was nearly completely bioreduced in the plasma and aqueous humor of animals that were fed the compound in drinking water., Conclusions: TEMPOL was effective in protecting against lens epithelial DNA damage and cataract formation in x-rayed rabbits. Although a number of mechanisms are possible, the protective effect may be associated with the ability of TEMPOL to protect against radiation-produced peroxides by acting as a superoxide dismutase mimic and to oxidize Fe2+ bound to DNA, thus preventing formation of the hydroxyl radical and subsequent DNA damage through the Haber-Weiss mechanism.

Purpose: High levels of ascorbic acid are known to be present in the aqueous humor of many diurnal species, whereas nocturnal animals have low concentrations of the compound. The purpose of this study was to test the hypothesis that the high concentration of aqueous ascorbate in diurnal animals protects the lens against ultraviolet (UV)-induced damage to the eye. This study compares the effect of UV-B-induced DNA strand breaks on the lens epithelia of guinea pigs and rats after depletion or elevation of aqueous humor ascorbate, respectively., Methods: Eyes of guinea pigs and rats were exposed to UV-B radiation (0.25-0.75 J/cm2 on the cornea) for 10 minutes, and DNA strand breaks in lens epithelium were measured by single-cell gel electrophoresis. Ascorbic acid concentration in the aqueous humor, lens, and lens-capsule epithelium were assayed by spectrophotometric and electrochemical methods. For depletion of aqueous humor and lens ascorbate in guinea pigs, the animals were maintained on an ascorbate-deficient diet. Aqueous ascorbic acid was elevated in the rat by intraperitoneal injections of sodium ascorbate (1 g/kg)., Results: The ascorbate concentration in the aqueous humor of the normal rat was approximately 3% that of the guinea pig, whereas the concentration of the compound in the lens of the normal rat was 10% that of the guinea pig. Guinea pigs fed an ascorbate-deficient diet showed a dramatic drop of more than 80% in aqueous humor ascorbate in the first week, whereas lens ascorbate decreased by approximately 25% during this time period. After a single intraperitoneal injection of sodium ascorbate in the rat, aqueous humor ascorbic acid increased nearly 30 times that in the control, whereas lens ascorbate increased by approximately 30%. The extent of DNA damage in the lens epithelium of a normal rat exposed to UV-B was significantly greater than that occurring in lenses of normal guinea pigs after exposure to the same dose of radiation. Lenses from ascorbate-deficient guinea pigs showed 50% more DNA damage than those from normal guinea pigs after UV exposure, whereas the lenses in ascorbate-injected rats exhibited significant protection against UV-induced DNA strand breaks., Conclusions: High levels of ascorbic acid in the aqueous humor had a protective effect against UV-induced DNA damage to lens epithelium. The results were consistent with the hypothesis that high ascorbic acid in diurnal animals protects the lens against the cataractogenic effect of UV radiation in sunlight.

Purpose: To investigate the biochemical mechanisms involved in the cataract induced by lovastatin, a commonly used cholesterol-lowering agent., Methods: The effects of lovastatin on lens transparency and on lens epithelial cell proliferation and structure have been investigated using organ-cultured rat lenses and cultured epithelial cells from human and rabbit lenses, respectively. Lens histologic and morphologic changes were recorded microscopically. Small GTP-binding protein profiles were determined by [alpha-32P] GTP overlay assays., Results: Rat lenses organ cultured for 7 days with lovastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, developed frank subcapsular opacity. Lens epithelial cells (both human and rabbit) demonstrated extensive morphologic changes and inhibition of proliferation when treated with lovastatin. Histologic sections of lovastatin-treated lenses showed partial to complete degeneration of the central epithelium, distortion of elongating epithelial cells, and extensive vacuole formation in the equatorial regions of the cortex. Supplementation of the medium with DL-mevalonic acid (a precursor of isoprenoids whose synthesis is inhibited by lovastatin) prevented the lovastatin-induced changes in whole lenses or in lens epithelial cell cultures, whereas supplementation with cholesterol had no such effect. GTP-binding proteins accumulated in the soluble fractions of lovastatin-treated lens epithelial cells. This was consistent with a blockade in isoprenylation preventing normal association with membranes., Conclusions: The findings suggest that impairment of the function of small GTP-binding proteins, due to a lovastatin-induced blockade in their isoprenylation, affects lens cell structure and proliferation in tissue culture and induces lens opacity in organ culture. These findings are consistent with the proposed roles of small GTP-binding proteins as molecular switches that regulate fundamental cellular processes, including growth, differentiation, and maintenance of cell structure.

Circulating autoantibodies against lens antigens are prevalent in patients with age-related cataract (ARC), but their pathogenic significance is unknown. We hypothesized that these autoantibodies are cytotoxic for lens epithelial cells (LECs). To test this hypothesis. We incubated LECs with mouse polyclonal or monoclonal antibodies against beta-crystallin (anti-beta) in the presence or absence of guinea pig complement. We found that anti-beta in the presence of the complement bound to and killed mouse LECs (MLECs) and human LECs (HLECs). Sera obtained from patients with ARC also were cytotoxic to both HLECs and MLECs in culture. Heat-inactivated human sera were not cytotoxic to LECs in the absence of the complement, but were cytotoxic to both HLECs and MLECs in the presence of additional complement. These results support the hypothesis that autoantibodies against lens antigens are cytotoxic to LECs, and that cell death may involve complement-mediated pathways.

Transgenic mice with elevated glutathione peroxidase (GSHPx) activity and gene knockout animals with a deficiency of the enzyme were used to investigate the role of GSHPx in defending the lens against H2O2-induced damage. The effects of peroxide on cultured lenses were determined by using light and transmission electron microscopy to evaluate morphological changes occurring in the epithelium and superficial cortex of the central and equatorial regions of the lens. DNA single-strand breaks in the epithelium were also examined. Following a 30-min exposure to 25 microM H2O2, lenses from normal animals showed distinct changes in the morphology of both the epithelium and superficial cortex. The damage to these cells was extensive in lenses of gene knockout mice in which activity of GSHPx was undetectable. In marked contrast, lenses of transgenic mice, which had 5-fold higher activities of GSHPx, were able to resist the cytotoxic effects. Similar to damage to cell morphology, the extent of DNA strand breaks was significantly lower (40% of control) in H2O2-exposed lenses as compared to normal lenses while DNA damage in gene knockout lenses was 5 times greater than that of GSHPx-rich transgenic lenses. The present studies extend our previous findings on the role of the glutathione redox cycle in the detoxification of peroxide and demonstrate that an increase in GSHPx activity protects the lens against peroxide-induced changes in cell morphology and DNA strand breaks.

Recent studies have demonstrated that several growth factors enhance fiber differentiation in cultured human lens epithelial (HLE) cells in early passages. However, these effects gradually decrease in cells of later passages. The purpose of this investigation is to test the hypothesis that the decreasing effect of growth factors on fiber differentiation in later passages may be due to a decrease or the inactivation of growth factor receptors as a function of serial subcultures. Specimens of HLE cells were obtained from infants. First through to fourth passage cells were treated with 10 ng ml-1 of epidermal growth factor, basic fibroblast growth factor or insulin-like growth factor-I. Fiber differentiation was determined from spontaneous lentoid formation by phase-contrast and transmission electron microscopy. Growth factor binding to the receptor on the cell surface was determined by transmission electron microscopy using the conjugates of colloidal gold and growth factors, and the number of receptors on the cell surface were also quantified by immunocytochemistry. Spontaneous lentoid formation was enhanced by all of the growth factors studied in the first passage. However, in the second and third passage only double layering of cells without characteristic fiber differentiation was observed while in the fourth passage, growth factors had no effect on differentiation. The number of growth factor bindings as well as the number of growth factor receptors gradually decreased with the number of passages. The loss of effect of growth factors on fiber differentiation with increasing number of passages correlated with the decrease in receptor number.

Purpose: A successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance. This study examines the effects of four different growth factors on proliferation and differentiation in HLE cells in early subcultures., Methods: Specimens of HLE cells were obtained from infants. First- or second-passage cells were cultured in the presence of 10(-2) to 10(2) ng/ml acidic and basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF-I). Cell proliferation was determined from cell number, and fiber differentiation was assessed from the time of appearance, the number of lentoids formed, and the expression of gamma-crystallin., Results: Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater than 10(-1) ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on proliferation appeared only at a concentration of 10(2) ng/ml. EGF, bFGF, or IGF-I at 10 ng/ml affected the time of appearance and the number of lentoids formed within 5 to 7 days. In contrast, lentoids were observed after 42 days without the addition of growth factors. Lentoid formation was accompanied by the expression of gamma-crystallin., Conclusions: EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation in early subcultures.

A sensitive, electrochemical method was employed for the simultaneous measurement of reduced and oxidized glutathione in lens cortex, nucleus and capsule epithelia of rabbit lenses, normal human lenses and human cataracts. In addition, aqueous humor from cataract patients was also analyzed. The level of GSSG in the nucleus of human cataracts was significantly higher than that in the nucleus of normal eye bank lenses. The capsule epithelium of intracapsular extracted cataracts possessed high levels of reduced glutathione, despite the fact that much of the glutathione in the cortex and nucleus of the lenses was depleted. Levels of GSH in the aqueous humor of cataract patients were several times higher than those reported for normal aqueous humor. Electrochemical detection proved to be a useful technique for analysis of reduced and oxidized glutathione in lens and aqueous humor, especially when sample size is small, such as for capsule epithelium.

Several recent reports have indicated that ganglioside treatment both in vivo and in vitro has a protective effect on the loss of membrane permeability resulting from inhibition of transport enzyme(s) in different experimental models. In this study we have investigated the effect of monosialoganglioside on oxidation-induced changes in organ-cultured rabbit lenses and in cultured dog lens epithelium and human retinal pigment epithelial cells. Exposure of organ-cultured lenses to 0.5 mM hydrogen peroxide for 1 hr increased the efflux of 86Rb from intact lenses and loss of myoinositol from the capsule epithelium. Pretreatment of the lenses with monosialoganglioside significantly reduced the efflux rate of 86Rb and loss of myoinositol. Monosialoganglioside also prevented morphological changes induced by 0.1 mM hydrogen peroxide in dog lens epithelium and loss of cell viability caused by docosahexaenoic acid in dog lens epithelium and in human retinal pigment epithelial cells. In contrast to the protective effect of monosialoganglioside on permeability and morphological changes in cultured cells, it had no effect against single-strand breaks of DNA in dog lens epithelium resulting from exposure to hydrogen peroxide, X-ray and UV-B radiation. Although the molecular mechanisms by which monosialoganglioside prevents permeability and morphological changes induced by hydrogen peroxide and docosahexaenoic acid are not known, it appears that this ganglioside serves as a membrane stabilizer rather than as a free-radical scavenger.

Purpose: Recent evidence suggests that taurine and myoinositol may serve as organic osmolytes in a number of cells, including lens and retinal pigment epithelia, but the mechanism for their increased accumulation in response to hypertonic stress is not known. To assess whether NaK ATPase contributed to the elevated levels of taurine and myoinositol in cells exposed to hypertonic media, we measured the activity of NaK ATPase, which is known to be implicated in the transport of these substances, in human lens and retinal pigment epithelia cultured in isotonic and hypertonic media., Methods: Primary cultures of human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells were maintained in isotonic and hypertonic media for varying periods of time, and the activity of NaK ATPase and the levels of taurine and myoinositol were measured in cells cultured under two different conditions. The possible involvement of the transport enzyme in the accumulation of the two osmolytes was also investigated by inhibiting the enzyme with ouabain., Results: When primary cultures of HLE and HRPE were exposed to hypertonic medium containing NaCl (600 mOsm) or cellobiose (500m Osm) for 72 hours, the concentration of taurine and myoinositol in HLE cells increased by 218% and 558% of control, respectively, in NaCl medium, whereas the corresponding increases in cellobiose medium were 147% and 439%. In HRPE cells, the increase in myoinositol levels in the two hypertonic media was more dramatic than that in taurine. Concomitant with the increase in the concentration of the osmolytes, there was an increase in NaK ATPase activity in both cell types. Although the accumulation of taurine in HLE cells in hypertonic media in a 6-hour culture was essentially prevented by 10(-8) mmol/l ouabain, myoinositol levels were affected to a lesser, but still significant, extent. In HRPE cells, which were cultured for 24 hrs in the presence of 10(-6) mmol/l ouabain, there was a more direct correlation between the inhibition of NaK ATPase and the decreased accumulation of taurine and myoinositol in the hypertonic media., Conclusion: Although the exact mechanism by which NaK ATPase activity increases in response to hypertonic stress remains to be established, the increased activity of the enzyme is related to the enhanced accumulation of the organic osmolytes, taurine, and myoinositol, in HLE and HRPE cells cultured in hypertonic medium.

Purpose: This study examined the extent to which human lens epithelial (HLE) cells in tissue culture retain the potential for differentiation, expression of lens-specific marker proteins, and the synthesis of lens capsule, the major characteristics of lens epithelium in vivo., Methods: Primary cultures of HLE cells were maintained for up to 450 days. Transmission and immunoelectron microscopy were used to study the thickness of the synthesized capsule and the formation of type IV collagen and laminin, two major protein components of the basement membrane of lens capsule in vivo., Results: In a long-term HLE culture system, without subcloning, lens fiber differentiation and capsular synthesis were maintained over a period of 450 days. In these cultures, the cell sheet showed three distinct zones: (1) a central zone with tight monolayer; (2) a mild peripheral zone with irregularly aggregated multilayer; and (3) a peripheral zone with loose monolayer. The basement membrane-like material was synthesized in the central zone and lentoids, which serve as a model for fiber differentiation, developed primarily in the mid peripheral zone. No capsular material or lentoids were observed in the peripheral zone. The capsule-like material was 2 to 2.5 microns thick and showed the presence of type IV collagen and laminin, as detected by antibody reaction., Conclusion: This study demonstrates for the first time that HLE cells in long-term cultures synthesize a continuous sheet of capsule-like material. The findings also suggest that reformation of a tight cell-to-cell relationship or generation of high cell density similar to that found in vivo may be an important factor for the synthesis of lens capsule.

The localization of Na,K-ATPase in the lens is quite controversial. We explored this problem through immunoelectron microscopic examination of rat and human lens. Unlike previously reported results, we have found that Na,K-ATPase is localized in the basal plasma membrane, but not in the lateral or apical plasma membrane of both rat and human lens epithelium. The lens fiber lacked immunoreaction. Localization of Na,K-ATPase was also investigated in the cultured human lens epithelium and in lentoid. Immunoreaction was detected in the apical (facing the media) plasma membrane of the lens epithelium cultured on the lens capsule, whereas the reaction was observable in both apical and basal plasma membrane of the lens epithelium cultured on the biopore membrane filters. Immunoreaction in lentoid was observed in the surface plasma membrane. These data indicate that the polarized distribution seen in the in situ lens epithelium changes when these cells are cultured, and that Na,K-ATPase in the cultured lens cells including lentoid is located in the plasma membrane which is in contact with the growth media. This change in polarity of Na,K-ATPase distribution in cultured epithelial cells may be dictated by the need to maintain ion homeostasis by extrusion of sodium ions across the cell membrane facing the media.

The polyol pathway was investigated in primary cultures of human retinal pigment epithelial (HRPE) cells and the results were compared with those in human lens epithelial (HLE) cells cultured under similar conditions. Significant levels of galactitol were formed in HRPE cells cultured for 72 hr in a medium containing 30 mmol/l D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy (TEM). Biochemical analysis revealed a significant depletion of cellular myoinositol, taurine, and a number of other free amino acids similar to those in HLE cells. These morphologic and biochemical changes observed in HRPE cells cultured in high galactose medium were inhibited or prevented by the inclusion of an aldose reductase inhibitor in the medium, further supporting the view that vacuole formation is due to the osmotic effect of polyol formation mediated by aldose reductase. The similarity of intracellular vacuole formation resulting from polyol accumulation and the biochemical changes in HRPE and HLE cells strongly suggests that a common mechanism is involved.

The formation of excess sugar alcohol mediated by aldose reductase (AR) and its intracellular accumulation in lens with resultant hydration is thought to be the initiating mechanism in the pathogenesis of diabetic and galactosemic cataracts. AR is also involved in other diabetic complications including retinopathy and neuropathy. Therefore, there is heightened interest in developing effective AR inhibitors (ARIs) for possible clinical use in human diabetes. However, the evaluation of these drugs for potential clinical use requires that the compounds be evaluated in appropriate target tissues since AR from different tissues is known to exhibit differential susceptibility to ARIs. The relative efficacy of ARIs in human lens epithelium (HLE) and human retinal pigment epithelium (HRPE) was studied by measuring the degree of inhibition of galactitol formation at various concentrations of ARI following incubation of cells in high galactose media for 72 hrs. Regardless of the structural characteristics of the ARIs investigated, higher doses were required to inhibit polyol synthesis in HRPE as compared to HLE cells. Based on ED50 values, dose required for 50% inhibition, the order of potencies against both HLE and HRPE enzymes was AL-4114 greater than AL-3152 greater than AL-1576 greater than tolrestat greater than statil greater than sorbinil. Since some ARIs are known to be bound to plasma proteins, it is conceivable that the observed differences in ED50 values could be due to differential binding to serum proteins in the culture medium. This possibility was examined by employing cultures of dog lens epithelium (DLE). These cells, which synthesize much higher levels of galactitol than HLE and HRPE, could be maintained in serum-free media for short periods (4 hrs) of time. The results, which demonstrate that the extent of polyol inhibition was the same in the presence or absence of serum, suggest that the differences in the potency of the inhibitors may reflect their inherent activity against AR in HLE and HRPE cells.

Crystallin expression in human lens epithelial cells in culture and a number of non-lenticular tissues was studied by the technique of immunoblotting using monoclonal antibodies. The expression of alpha A, beta 5 and beta 6 crystallins per unit number of cells increased with passage number while alpha B appeared to be constant Lentoid bodies derived from cultured human lens epithelial cells not only expressed gamma-crystallin and MP26 as previously demonstrated, but also produced alpha A, alpha B, beta 5 and beta 6 crystallins. In human non-lenticular tissues including ciliary body, vitreous body, neural retina, cultured retinal pigment epithelial cells and scleral fibroblasts, alpha B-crystallin was detected, but was undetectable in cornea and iris. Alpha A was present only in the lens. These studies demonstrate that HLE cells maintain the ability to synthesize crystallins through several passages. Following differentiation, they not only synthesize gamma-crystallin and MP26 but continue to express alpha- and beta-crystallins similar to differentiated lens fiber cells in vivo. Consistent with previous observations, the expression of alpha B-crystallin does not appear to be specific for the lens.

The congenital hereditary cataracts and microphthalmia in the miniature schnauzer dog are inherited by an autosomal recessive mode. To understand the genetic basis of these diseases, the authors purified and analyzed leukocyte deoxyribonucleic acid (DNA) from affected and normal animals using a candidate gene approach. Because the genes that encode the lens-specific proteins, specifically, alpha, beta, and gamma crystallins and the membrane protein (MP26), are known to maintain the structure and function of the lens, the authors used complimentary DNA (cDNA) fragments that corresponded to the above genes to search for the mutations at their loci in the affected animals. They found no evidence of the gene deletion and rearrangement in any of the five loci. In addition, the hybridizable sequences of the dog DNA to the specific probes for the human chromosome 4 and 18 loci, which are reported to be involved in the abnormality of the human eye, seem to be unaffected. These data support the notion that the hereditary cataracts and microphthalmia in the dog may be associated with genes other than those reported for several animal systems.

'Reactive oxygen species', generated within the lens, are implicated in the deepening of nuclear pigmentation leading to browning of the human cataractous lens. The present study, which was carried out in senile brunescent cataracts, deals with changes in the antioxidant glutathione redox cycle defense system during the progression of browning (yellow to brown) in different regions of the human lens. A significant reduction in the levels of reduced glutathione was noted in the cortical and nuclear regions of the brown lens as compared to yellow lens, despite normal glutathione reductase activity. The rate limiting enzyme of the hexosemonophosphate shunt pathway, glucose 6-phosphate dehydrogenase, and the important oxidant scavenger enzyme, glutathione peroxidase, showed significant changes in opposite directions (glucose 6-phosphate dehydrogenase increased and glutathione peroxidase decreased), only in the cortical region of the brown lens as compared to yellow lens. The significance of the present findings in relation to the overall biochemical mechanisms underlying browning of the lens tissue in human cataracts is discussed.

Human lens epithelial (HLE) cells in tissue culture accumulated significant levels of galactitol when they were cultured for 72 hr in medium containing 30 mM D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy. The number and size of intracellular vacuoles increased when the culture period was extended to 7 days. In addition, polyol accumulation was accompanied by loss of myoinositol. None of these changes occurred in cells exposed to 30 M L-galactose which is not a substrate for aldose reductase. The accumulation of galactitol, intracellular vacuole formation and loss of myoinositol observed in D-galactose-exposed cells were prevented by the inclusion of the aldose reductase inhibitor, sorbinil, in the culture medium. Comparison of the relative efficacies of two aldose reductase inhibitors indicate that AL 1576 is nearly 20 times more potent than sorbinil in inhibiting the human lens enzyme. It is concluded that vacuole formation in HLE cells is due to the osmotic effect of polyol formation brought about by the action of aldose reductase and that the etiology of human diabetic cataract may also involve the polyol pathway.

Differentiation of human lens epithelial (HLE) cells cultured in vitro was drastically accelerated when the cells were cultured on cell-substrate adhesion-free surfaces. Spontaneous differentiation of the lens epithelial cells in monolayer cultures could be recognized with the appearance of lentoid bodies after 40-50 days if maintained without further passage. Although dissociated HLE cells reconstituted into monolayers consistently on the haptotactic substrates (either gold-coated biopore membrane or regular plastic dishes), the cells from the same batches exclusively formed cell aggregates when cultured on either biopore membrane or agarose-coated plastic dishes (nonhaptotactic). The cells on nonadhesion substrate first aggregate, then synchronously develop into lentoids by the 10th day of culture. The differentiation of HLE cells into lentoid structures with lens-fiber characteristics was documented by both ultrastructural and biochemical markers, such as loss of cytoplasmic organelles, formation of gap junctions, and the expression of gamma-crystallin and MP26. The system, in which differentiation of epithelial cells can be induced predictably in a short period of time, provides an excellent model for the study of differentiation and gene expression in human lens cells cultured in vitro.

The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 were investigated in cultured rabbit lens epithelial cells. Exposure of cells to H2O2 was carried out following inhibition of either of the two antioxidant systems. Two different procedures were used to expose the cells to extracellular H2O2, one in which a low, steady state level of 0.025 mM H2O2 was maintained in the culture medium with the use of glucose oxidase and the other in which H2O2 was added to the medium as a single pulse at levels ranging from 0.03 to 0.5 mM. When lens cells were treated with a low, steady state level of H2O2, the glutathione redox cycle was the primary means of defense against oxidative damage. Cells with fully active catalase but with inhibited glutathione reductase were not able to resist the cytotoxic effects of a 0.025 mM level of extracellular H2O2. Under these conditions the cells were nearly completely depleted of reduced glutathione within 15 min. The cellular damage observed after 1.5 hr of culture included loss of cell-to-cell contact, rounding up of the cells and formation of numerous blebs. In contrast, cells with completely inhibited catalase but with an unimpaired glutathione redox cycle suffered few damaging effects from a 3-hr exposure to 0.025 mM H2O2. When lens cells were pulsed with a single challenge of 0.5 mM H2O2, both the glutathione redox cycle and catalase were found to be essential for survival of the cells. While control cells were able to withstand the pulse of H2O2, cells with impaired activities of either the glutathione redox cycle or catalase were killed. Control cells treated with 0.5 mM H2O2 may have been protected from damage by the fact that the cellular level of GSH never dropped below 35% of normal. The cause of cell death following inhibition of catalase appeared to be related to an inability of the cells to remove peroxide from the culture medium, at a rapid rate, following the H2O2-pulse. Although cells with impaired glutathione reductase activity removed H2O2 from the medium at a rate comparable to that of control cells (due to uninhibited catalase activity), they did not survive the challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper presents an overview of the current state of our knowledge concerning the metabolism and function of glutathione (GSH) in the lens, with particular reference to the contributions of Dr Jin H. Kinoshita to this field. Glutathione in the lens is synthesized from its constituent amino acids and degraded by mechanisms involving transpeptidation and hydrolysis. The turnover of GSH in the lens is due to its catabolism rather than transport of GSSG as is the case in red blood cells and some other tissues. Three aspects of the functional role of GSH in cataract formation are considered. First, GSH may be important in maintaining protein thiols in the reduced state, thus preventing the formation of high molecular weight protein aggregates which are the basis for light scattering and lens opacification. A second function may be to protect membrane -SH groups that are important in cation transport and permeability. A third functional role is to detoxify hydrogen peroxide and other organoperoxides. The glutathione redox cycle is intimately involved in the detoxification of H2O2 which is normally present in the aqueous humor.

The role of reduced glutathione (GSH) in lens membrane function was studied by depleting GSH with 1-chloro-2,4-dinitrobenzene (CDNB), a reaction catalyzed by GSH-S-transferase. Depletion of GSH in the lens epithelium by 70-90% led to a decrease in uptake and increase in efflux of 86Rb. ATP levels and Na+/K+-ATPase activity were normal while there was a slight decrease in lactate production. The results provide the first direct evidence that depletion of endogenous GSH per se does not lead to inactivation of Na+/K+-ATPase. However, lenses deficient in GSH when challenged with a normally tolerated level of H2O2 showed significant inactivation of membrane ATPase without a further increase in membrane permeability. Pretreatment with CDNB resulted in a 3-fold stimulation of the hexose monophosphate shunt activity which is attributed to the unexpected finding of a significant increase in the level of oxidized glutathione in the lens. It is concluded that deficiency of GSH causes a marked increase in membrane permeability and such lenses are susceptible to oxidative damage resulting in inactivation of the Na+/K+ pump, thus leading to ionic changes and cataract development.

A method has been developed to isolate and characterize beta-crystallins of rabbit lens cortex. Chromatographic separation of water-soluble structure proteins of rabbit lens cortex on a Sephacryl S-200 gel column yielded four beta-crystallin peaks (beta1, beta2, beta3 and beta4), all eluting between alpha and gamma-crystallins. Their molecular weights were estimated to be 250,000, 130,000, 60,000, and 37,000 daltons, respectively. SDS-gradient gel electrophoresis of these beta-crystallins gave rise to characteristic polypeptides; beta1, two polypeptides of 30,000 and 23,000 daltons; beta2, one major polypeptide of 33,000; beta3; two polypeptides of 28,000 and 26,000; and beta4, two polypeptides of 22,500 and 11,200 daltons. From a knowledge of the molecular weights and the ratio of the polypeptides in each crystallin, their oligomeric structure was calculated to be 5:5, 4, 1:1, and 1:1. The relative abundance of these four beta-crystallins was found to be 25.6%, 7.2%, 27.2%, and 2.8% of the total water-soluble proteins of the lens cortex.

Using the techniques of quasielastic light scattering spectroscopy we have studied in vivo the effect of X-irradiation on the diffusivity of lens proteins as a function of position in the lens and time after irradiation. The findings were compared with those for normal lenses in vivo. The experimentally observed correlation functions show marked changes in the protein diffusivity well before substantial biochemical changes take place.

Rat lenses in organ culture were exposed to activated species of oxygen generated in the culture medium either by xanthine oxidase and hypoxanthine or by riboflavin and visible light, two systems which have been shown to produce superoxide and H2O2. In each case there was marked damage to carrier-mediated transport systems of the lens. Under standard culture conditions this damage was strongly inhibited by catalase, but not by superoxide dismutase (SOD). By the addition to the medium of chelated iron, hydroxyl radicals were produced in a Fenton reaction with a concomitant decrease in H2O2 levels. With both oxygen radical-generating systems, the addition of chelated iron strongly inhibited lens damage. This inhibitory effect could be reversed by the addition of SOD with the chelated iron. Under such conditions SOD converts superoxide anion to H2O2, thereby preventing reduction of the chelated iron and thus stopping the generation of hydroxyl radicals. Increased lens damage following addition of SOD to the iron-containing systems correlated with higher H2O2 concentrations, and was inhibited by catalase. These findings suggest that, when generated in the fluids surrounding the lens, H2O2 poses a much greater oxidative stress for the lens than do the superoxide or hydroxyl free radicals.

Our previous studies on cultured rabbit lens epithelial cells from 4-day-old rabbits showed that the glutathione redox cycle plays an important role in detoxifying H2O2, a potentially damaging oxidant present in the aqueous humor. Here we report the effect of donor age and cell density on the ability of cultured rabbit lens epithelial cells to detoxify H2O2. Lens epithelial cells (8 x 10(5] from a 4-day-old and an 8-year-old rabbit were cultured for 3 hr in minimal essential medium (MEM) or in MEM containing 0.01-0.1 mM H2O2 maintained with glucose oxidase. We determined the effect of H2O2 on the level of reduced glutathione (GSH), hexose monophosphate shunt activity, cell growth, and morphology. For growth studies, cells were exposed to the desired concentration of H2O2 for 3 hr and then cultured in MEM plus 10% rabbit serum for 7 days and counted. Young and old untreated cells contained high levels (30-40 nmol/8 x 10(5) cells) of GSH. Cells from 4-day-old rabbits tolerated 0.03 mM H2O2 with no effect on GSH and a minimal decrease in subsequent cell growth. However, in the older cells, GSH and growth were substantially diminished following treatment with 0.03 mM H2O2. Cells plated out at high density (8 x 10(5] were more tolerant of 0.03 mM H2O2 than cells plated out at low density (5 x 10(4]. Maximum shunt activity in the younger cells exposed to H2O2 was twice that of the older cells and occurred at a higher level of H2O2 (0.04 compared with 0.03 mM). Enzyme activities in untreated young and old cells were comparable for hexokinase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase. However, glutathione reductase activity was 50% lower in the cells from the 8-year-old rabbit. The toxicity of H2O2 to cultured lens epithelial cells was directly related to donor age and inversely related to cell density. The damage in the older lens epithelial cells at 0.03 mM H2O2 was apparently due, in part, to a diminished response of the glutathione redox cycle to oxidative challenge.

In vivo exposure of human patients and experimental animals to hyperbaric O2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100% O2 for 24 hr. intermolecular disulfide-linked proteins formed in both the cortical and nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by greater than 95% in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal electrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were beta- and gamma-crystallins. Also involved in the cross-linking was the enzyme G-3PD but not the main intrinsic membrane protein. MIP26 kDa. Treatment of the nuclear urea-insoluble fraction of O2-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. It was determined that an increase of approximately one disulfide group per 10(5) Da molecular weight corresponded to cross-linking of nearly 20% of the urea-insoluble protein present in the O2-treated lenses. Experiments carried out at 8 atmospheres O2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O2. After 8 hr of treatment of lenses with 8 atmospheres O2 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62% in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres O2 produced a further increase in protein disulfide in the nuclear region. The formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric O2 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies have been made of the effects of X-ray on various lens reducing systems, including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS) and of certain enzymes, including GSH reductase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PG). It was found that during several weeks following X-irradiation but prior to cataract formation, there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the X-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the X-rayed lens was found to decrease significantly relative to controls 1 week prior to cataract formation, and the ratio of NADPH to NADP+ in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the X-rayed lens. A corresponding decrease occurred in the activity of the HMS in X-rayed lenses as measured by culture in the presence of 1-14C-labeled glucose, G-6-PD was partially inactivated in the X-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to X-irradiation. The data indicate that X-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high-molecular-weight protein aggregates may be initiated.

Explants of epithelial cells from young human lenses of 5-12 months of age, obtained from patients who underwent surgery for retinopathy of prematurity, were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. Without exception, every piece of the anterior capsule explant showed cell outgrowth within 48-72 h and resulted in confluent monolayer culture within 2 weeks. From these monolayer cultures, two to three passages of subcultures were obtained by routinely seeding cells in a ratio of 1:4. The doubling times for these human lens epithelium (HLE) cultures during the first 4 weeks of two passages were found to be 24-36 h. In a majority of cultures through the first three passages, more than 12 population doublings were attained. However, no lentoid bodies were formed during this period. These cells were studied for the presence of crystallins and their synthesis. Using SDS-polyacrylamide gel electrophoresis, the presence of alpha- and beta-crystallins was demonstrated in HLE cells through three passages. The amount of alpha-crystallin in the first two passages amounted to nearly 13% of the total protein, but decreased significantly in the third passage. The presence of crystallins was corroborated by antibody reaction to the specific crystallins. Indirect immunofluorescence revealed the presence of actin and vimentin in these cell cultures. The synthesis of crystallins in HLE cultures was shown by the incorporation of [35S]methionine which was time dependent. The crystallin synthesis was found to decrease in third passage when the cell growth slowed down without consistent formation of confluent monolayer. These studies have demonstrated that primary cultures of HLE cells can be successfully grown from young lenses through several passages which continue to express the characteristic crystallins of the epithelial cells.

The present study was designed to examine the possible role of calcium in the opacification of x-ray-induced cataract in rabbit. The results demonstrate that the concentration of calcium in x-rayed lenses, just prior to lens hydration (7.5 weeks postirradiation), was twice that present in contralateral control lenses. At this stage of immature cataract, the lens nucleus remained transparent and maintained a normal level of calcium, but the lens cortex, containing regions of subcapsular opacification, accumulated a level of calcium that was twice that of the control. In the completely opaque mature cataract, (8-9 weeks postx-ray), both the cortex and nucleus had gained significant amounts of calcium. As the concentration of total calcium increased in the immature x-ray cataract, the amount of the cation bound to membranes and insoluble proteins of the cytosol also increased comparably. However, the relative proportion of calcium in the various fractions remained unaltered in the immature cataract; in both control lenses and immature cataracts, 20% of the total calcium remained in the membrane pellet and 70% was located in the soluble protein fraction. Only in the mature stage of cataract was a shift in the distribution of calcium apparent, as the proportion of calcium in the soluble protein fraction increased to 90%. Although only 7% of the total calcium in a mature cataract was bound to membrane, the amount represented a fivefold increase over the control. The results of this study demonstrate that an elevation in lens calcium accompanies the opacification process in x-ray cataract. The work also suggests that changes in calcium levels are not likely to result from inactivation of Ca-ATPase.

The effect of some protective agents on cataract development is briefly reviewed and new evidence is presented on the efficacy of a phosphorothioate compound (Amifostine) in inhibiting the development of X-ray-induced cataract. Morphological studies showed that at the end of 4 months, lenses from X-irradiated rats which had not received any drugs showed liquefaction in the equatorial region and at the posterior pole, as well as a marked swelling of the fibers in the anterior cortex. Animals which received 1.16g/kg of WR77913 showed considerable protection against the development of radiation induced cataracts with morphological changes in the lens being less severe than in animals receiving no drugs. When animals were treated with 0.5g/kg of Amifostine (WR2721) the lenses showed much greater protection against cataract development than with WR77913. Amifostine appears to be more effective than WR77913 in inhibiting X-ray-induced cataract development.

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4-6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 10(5) cells)-1 3 hr-1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0.01 mM. Participation of the glutathione redox cycle in the H2O2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1-14C]-labeled glucose. The oxidation of [1-14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0.04-0.05 mM H2O2, while the corresponding increase in oxidation of [6-14C]-labeled glucose was only 1.6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0.03-0.04 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 10(5) epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

The Na-K ATPase activity of rabbit lens was measured at various times after exposure to a single dose of 2000 rads of X-ray and was compared with that in contralateral control eyes. A decrease in enzyme activity in both whole lens and in isolated capsule-epithelium was first observed 3 to 4 weeks after irradiation and became increasingly marked at 7.5 weeks after X-ray. These findings are consistent with our earlier observations that active transport of cations is reduced in these lenses and support the view that loss of membrane ATPase is responsible for the impairment of the cation pump in X-irradiated lenses. Despite a significant loss of the enzyme, X-irradiated lenses were able to maintain near normal levels of total cations (Na+ + K+), thus accounting for their normal hydration. The results of the changes in lens Na+ and K+ levels revealed that between 4 and 7.5 weeks after X-ray, the gain in Na+ was compensated by an equivalent loss of K+. A breakdown of this relationship of 1:1 exchange of Na+ for K+ is accompanied by a disproportionate increase in Na+ and water.

Relative amounts of two water-soluble beta-crystallin polypeptides with molecular weights of 26,000 (26K) and 28,000 daltons (28K) were measured in a number of species and also in different age groups of rats. The data indicate an age-dependent increase in the quantity of the 26K which is concomitant with a decrease in the 28K polypeptide. The ratio of these two polypeptides in the total water-soluble fraction is similar to that observed in the purified beta-crystallins. Purified beta 3-crystallin of the rabbit and its equivalent "beta L" of the bovine lens display charge heterogeneity upon DEAE chromatography, suggesting subtle qualitative changes in these crystallins. The presence of these two polypeptides in the urea-soluble fraction of lens preparations is also an indication of qualitative changes. The 26K polypeptide of beta-crystallins studied here is different from the 26K main intrinsic membrane protein in that, unlike the latter, it does not aggregate upon heating in SDS-buffer.