Inhibitory effects of chlorogenic acid on aldose reductase activity in vitro and cataractogenesis in galactose-fed rats.

Chlorogenic acid (5-O-caffeoylquinic acid, CA), a phenolic compound found ubiquitously in plants, has antidiabetic effect in diabetic animal models. In this study, we investigated the inhibitory effect of CA on diabetic cataractogenesis. We evaluated the aldose reductase (AR) activity during cataract development in 50% galactose-fed rats, an animal model of sugar cataract. Galactose-fed rats were treated orally with CA (10 and 50 mg/kg body weight) once a day for 2 weeks. In vehicle-treated galactose-fed rats, lens opacity was increased, and lens fiber swelling and membrane rupture were observed. In addition, AR protein was highly expressed in lens epithelial cells and lens cortical fibers of galactose-fed rats. However, CA inhibited the rat AR activity in vitro, and the administration of CA prevented the development of sugar cataract through the inhibition of AR activity. These observations suggest that CA is useful for the treatment of sugar cataract.

Exogenous oestrogen exposure, female reproductive factors and the long-term incidence of cataract: the Blue Mountains Eye Study.

PURPOSE: To assess the association between exogenous oestrogen exposure, female reproductive factors and the long-term incidence of cataract. METHODS: People aged 49+ years living in a defined geographic region were invited to participate in this prospective population-based survey. Of 2072 women (83% of those eligible) who were examined during 1992-1994 at baseline, around 75% of survivors were re-examined at 5- and 10-year follow-up visits. Lens photographs were assessed using the Wisconsin Cataract Grading System by masked graders. An interviewer-administered questionnaire was used at each visit to collect information on female reproductive factors and the use of the oral contraceptive pill and postmenopausal hormone replacement therapy. RESULTS: After adjusting for age, smoking, socioeconomic status, steroid use, hypertension and diabetes, ever having used the oral contraceptive pill had a mild protective effect against incident cortical cataract (odds ratio 0.70, 95% confidence interval 0.50-0.98). However, hormone replacement therapy, age at menarche, age at menopause, duration of exposure to endogenous oestrogen, parity and type of menopause were not found to have significant associations with the incidence of any type of cataract or cataract surgery. CONCLUSION: Apart from a weak protective association of oral contraceptive pill use and the development of cortical cataract, we found no significant longitudinal associations between exogenous oestrogen exposure, female reproductive factors and the long-term incidence of cataract in this older population-based cohort.

Whole-cell patch clamping of isolated fiber cells confirms that spatially distinct Cl- influx and efflux pathways exist in the cortex of the rat lens.

PURPOSE: To test the hypothesis that lens fiber cells use different combinations of transport proteins to mediate Cl influx and efflux in order to regulate their steady state volume. METHODS: Cells were isolated from rat lenses by enzymatic dissociation in the presence of Gd(3+), and short and long fiber cells were assigned to peripheral efflux and deeper influx zones, respectively. Electrical properties were of isolated cells, and whole lenses were analyzed by using whole-cell patch clamping and intracellular microelectrodes, respectively, before and after exposure to hyposmotic challenge and/or the addition of [(dihydronindenyl)oxy] alkanoic acid (DIOA). RESULTS: Cells from the influx zone were dominated by an outwardly rectifying Cl(-) conductance, and exposure to hyposmotic challenge increased this conductance. Cells isolated from the efflux zone were dominated by K(+) conductance(s) with only a minimal contribution from the Cl(-) conductance. Exposure of cells that exhibited a minimal baseline Cl(-) conductance to hyposmotic challenge caused swelling and a transient increase in Cl(-) current. In other cells that initially lacked a Cl(-) conductance, hyposmotic challenge caused swelling, but no increase in outward current. However, the subsequent addition of DIOA exacerbated swelling and activated a Cl(-) current. Under isosmotic conditions, addition of DIOA also induced cell swelling and the transient activation of a Cl(-) current. In whole lenses, exposure to hyposmotic challenge increased the contribution of an anion conductance to the membrane potential. CONCLUSIONS: In peripheral cells, Cl(-) efflux is primarily mediated by potassium chloride cotransporters (KCCs) and its activity can be upregulated by hyposmotic challenge. In addition, these cells also contain a Cl(-) channel that exhibits a variable baseline activity level and that can be recruited to effect regulatory volume decrease if the KCC transporters are inhibited.

Gap junctions contain different amounts of cholesterol which undergo unique sequestering processes during fiber cell differentiation in the embryonic chicken lens.

PURPOSE: To determine the possible changes in the distribution of cholesterol in gap junction plaques during fiber cell differentiation and maturation in the embryonic chicken lens. The possible mechanism by which cholesterol is removed from gap junction plaques is also investigated. METHODS: Filipin cytochemistry in conjunction with freeze-fracture TEM was used to visualize cholesterol, as represented by filipin-cholesterol complexes (FCCs) in gap junction plaques. Quantitative analysis on the heterogeneous distribution of cholesterol in gap junction plaques was conducted from outer and inner cortical regions. A novel technique combining filipin cytochemistry with freeze-fracture replica immunogold labeling (FRIL) was used to label Cx45.6 and Cx56 antibodies in cholesterol-containing gap junctions. Filipin cytochemistry and freeze-fracture TEM and thin-section TEM were used to examine the appearance and nature of the cholesterol-containing vesicular structures associated with gap junction plaques. RESULTS: Chicken lens fibers contain cholesterol-rich, cholesterol-intermediate and cholesterol-free gap junction populations in both outer and inner cortical regions. Filipin cytochemistry and FRIL studies confirmed that cholesterol-containing junctions were gap junctions. Quantitative analysis showed that approximately 86% of gap junctions in the outer cortical zone were cholesterol-rich gap junctions, whereas approximately 81% of gap junctions in the inner cortical zone were cholesterol-free gap junctions. A number of pleiomorphic cholesterol-rich vesicles of varying sizes were often observed in the gap junction plaques. They appear to be involved in the removal of cholesterol from gap junction plaques through endocytosis. CONCLUSIONS: Gap junctions in the young fibers are enriched with cholesterol because they are assembled in the unique cholesterol-rich cell membranes in the lens. A majority of cholesterol-rich gap junctions in the outer young fibers are transformed into cholesterol-free ones in the inner mature fibers during fiber cell maturation. A distinct endocytotic process appears to be involved in removing cholesterol from the cholesterol-containing gap junctions, and it may play a major role in the transformation of cholesterol-rich gap junctions into cholesterol-free ones during fiber cell maturation.

Cl- influx into rat cortical lens fiber cells is mediated by a Cl- conductance that is not ClC-2 or -3.

PURPOSE: Exposure of organ-cultured lenses to Cl(-) channel blockers under isotonic conditions induces a localized cortical zone of extracellular space dilations. The purpose of this study was to investigate whether elongated lens fiber cells from this zone contain an anion conductance that mediates Cl(-) influx and whether two chloride channel isoforms known to be expressed in the lens (ClC-2 and -3) are responsible. METHODS: Fiber cells were isolated by enzymatic dissociation in the presence of Gd(3+) and Co(2+) and their electrical properties analyzed by whole-cell patch clamping. Cells from the zone of extracellular space dilations were selected for analysis on the basis of cell length. RT-PCR and immunocytochemistry were used to determine whether ClC-2 or -3 channel isoforms are expressed in fiber cells located in the zone of extracellular space dilations. RESULTS: Cells from the zone of extracellular space dilations were typically >120 microm in length and exhibited an outwardly rectifying Cl(-) conductance that was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and displayed an anion selectivity sequence of I(-) > Cl(-) >> gluconate. ClC-2 and -3 were found to be expressed at the transcript and protein level in lens fiber cells, but subsequent immunocytochemical studies indicated that expressed proteins did not colocalize with cell membranes in the zone of extracellular space dilations, being predominately cytoplasmic in nature. CONCLUSIONS: Taken together, the data indicate that extracellular space dilations are due to the inhibition of a Cl(-) channel(s) that normally mediates Cl(-) influx into cortical lens fiber cells under isotonic conditions. The molecular identity of this channel remains to be determined.

Induction of cortical cataracts in cultured mouse lenses with H-89, an inhibitor of protein kinase A.

PURPOSE: To compare the effects of two serine-threonine protein kinase inhibitors in a mouse lens culture system previously designed to investigate cortical cataracts caused by L-buthionine sulfoximine (BSO), inhibitor of GSH biosynthesis. METHODS: Cataract development in HL-1 medium was evaluated visually or by measurement of lens Na+/K+ ratio through atomic absorption. Protein changes were evaluated by 32P-labeling, 2D-gel electrophoresis, phosphorimaging and mass spectrometry. Results. H-7 (50 microM), inhibitor of protein kinase A (PKA) and protein kinase C (PKC), did not cause cataracts, but inhibited BSO cataract development. By contrast, 25 microM H-89, selective inhibitor of PKA, caused large annular cortical cataracts and 100-fold elevation of Na+/K+ within 30 hr in day 10 lenses, in either the presence or absence of BSO. H-89 cataracts were also seen in day 12 and day 21 lenses. 32P-labeling of day 12 lenses pretreated with H-89 displayed more than 80% decrease in phosphorylation of alphaA crystallin, a known substrate of PKA, in the insoluble protein fraction. 2D-gel electrophoresis of day 12 H-89 cataract lens fractions revealed limited degradation of alpha and beta crystallins, degradation of cytoskeletal proteins, and elevated lens Ca2+ (>4 nmol/mg wet wt.), suggesting Ca2+-activated proteolysis. Conclusions. High Na+/K+ cataracts are induced by H-89, selective inhibitor of PKA, but not by H-7, an inhibitor of both PKA and PKC that impeded BSO-induced Na+/K+ elevation and cataract. These results suggest contrasting effects of PKA and PKC on lens cation transport and cortical cataract development.

Inhibition of fiber cell globulization and hyperglycemia-induced lens opacification by aminopeptidase inhibitor bestatin.

PURPOSE: To examine the role of calcium-dependent and -independent proteolytic activity in the globulization of isolated fiber cells and glucose-induced lens opacification. METHODS: Fiber cells from rat lens cortex were isolated, and the [Ca(2+)](i) and protease activity in the isolated fibers were determined by using a calcium binding dye and the protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (BOC-Leu-Met-CMAC). The activity of calpain in the lens cortex homogenate was determined with fluorescein-casein in the presence of Ca(2+) and that of fiber cell globulizing aminopeptidase (FCGAP) with BOC-Leu-Met-CMAC and reduced glutathione (GSH) in the absence of Ca(2+). The lens proteases-calpain and the novel aminopeptidase FCGAP were partially purified by diethylaminoethyl (DEAE) gel column chromatography. Single fiber cells were isolated from rat lens, plated on coverslips, and placed in a temperature-controlled chamber. Their globulization time was determined by the appearance of light-scattering globules in the absence and the presence of protease inhibitors including the aminopeptidase inhibitor bestatin. To investigate the effect of the protease inhibitors E-64 and bestatin on the prevention of hyperglycemic cataract, the rat lenses were cultured in medium 199 in the presence of 5.5 and 50 mM glucose and in the absence and the presence of protease inhibitors. Changes in light transmission by the lenses were determined by digital image analysis. RESULTS: Normal levels of lens fiber cell [Ca(2+)](i), determined by using a cell-permeable dye were approximately 100 nM, and the protease activity determined with BOC-Leu-Met-CMAC was maximum at [Ca(2+)](i) of approximately 500 nM. A large fraction of the FCGAP that cleaves BOC-Leu-Met-CMAC was separated from calpain, which cleaves fluorescein-casein, by diethylaminoethyl (DEAE) gel column chromatography. The FCGAP did not bind to the column, whereas calpain bound to the column and was eluted by approximately 180 mM NaCl. Unlike calpain, the FCGAP did not require calcium for activation and did not cleave fluorescein-casein. However, the Ca(2+)-dependent calpain activated FCGAP, indicating that the latter may exist in pro-protease form. The FCGAP was selectively inhibited by the specific aminopeptidase inhibitor bestatin, indicating that FCGAP could be an aminopeptidase. However, the FCGAP was found to be immunologically distinct from leucine aminopeptidase and calpain. Perfusion of the isolated rat lens fiber cells with Ringer's solution led to their globulization in 30 +/- 3 minutes. Addition of 0.5 mM of the protease inhibitors E-64 and leupeptin increased the globulization time to 60 and 100 minutes, respectively, whereas no globulization of the fiber cells was observed for 4 hours in the presence of 0.05 mM bestatin. In rat lens cultured in medium containing 50 mM glucose, both E-64 and bestatin (0.05 mM each) significantly reduced the extent of opacification, indicating that an aminopeptidase, downstream to a Ca(2+)-dependent protease, may be involved in mediating cataractogenic changes. CONCLUSIONS: In addition to calpain, a Ca(2+)-independent novel protease, FCGAP, a novel aminopeptidase, represents a significant fraction of the total proteolytic activity in the lens. Inhibition of FCGAP by bestatin attenuates Ca(2+)-induced globulization of the isolated fiber cells in vitro and hyperglycemia-induced opacification of cultured rat lens.

The role of Src family kinases in cortical cataract formation.

PURPOSE: The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. METHODS: A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. RESULTS: Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. CONCLUSIONS: The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression.

PKC-gamma phosphorylation of connexin 46 in the lens cortex.

PURPOSE: To identify the role of PKC-gamma in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex. METHODS: The association between PKC-gamma and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors. RESULTS: Co-localization of PKC-gamma and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-gamma was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-alpha was not found in lens cortex or nuclear regions. PKC-gamma could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 microM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-alpha pseudosubstrate inhibitor was not observed. CONCLUSIONS: PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is suggested.

A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification.

PURPOSE: Cortical cataract in humans is associated with Ca2+ overload and protein loss, and although animal models of cataract have implicated Ca2+-activated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purpose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS: Donor human lenses were cultured in Eagle's minimum essential medium (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induce a Ca2+ overload. Lenses were loaded with [3H]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 microM ionomycin, or EMEM + 5 microM ionomycin + 5 mM EGTA (Ca2+ < 1 microM). Efflux of proteins and transparency were monitored daily. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were probed with anti-vimentin antibody (clone V9) and detected by enhanced chemiluminescence. RESULTS: Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental period. Ionomycin treatment resulted in cortical opacification, which was inhibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin also led to an efflux of [3H]-labeled protein, amounting to 41% of the labeled protein over the 7-day experimental period, compared with 12% in ionomycin + EGTA-treated lenses. Efflux was accounted for by loss from the lens soluble protein (crystallin) fraction. Western blot analysis of the cytoskeletal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 kDa in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (approximately 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS: Human lenses can be successfully maintained in vitro, remaining transparent for extended periods. Increased intracellular Ca2+ induces cortical opacification in the human lens. Ca2+-dependent cleavage and cross-linking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacification (HLCO) model enables investigation of the molecular mechanisms of opacification, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.

Acute effect of ethanol on lens cation homeostasis.

This study examined the effect of ethanol on the calcium homeostasis of the bovine lens. After acute exposure of the whole lens to physiologically related ethanol concentration, the calcium content of the lens cortex increased from 0.345 +/- 0.075 to 0.476 +/- 0.047 micromol/g (p < 0.05). In contrast, other cation levels such as sodium, potassium, and magnesium did not change. In the study of the lens calcium transport, ethanol caused an increase in the calcium permeability of the lens lipid membrane by about 12% at 30 mM ethanol. Ethanol did not alter the calcium pump activity at ethanol concentration up to 400 mM. Above 600 mM ethanol, the calcium pump was almost completely inhibited. It has been suggested that moderate to heavy alcohol consumption is a risk factor for cataracts. This study indicates that acute ethanol exposure can cause a loss in the lens calcium homeostasis, which maybe one of the cellular mechanisms to contribute to the cataract development in the alcoholic individual.

Calcium homeostasis of isolated single cortical fibers of rat lens.

PURPOSE: To investigate the calcium homeostasis in single fiber cells isolated from rat ocular lens cortex and to quantify the changes in the concentration of free intracellular calcium [Ca2+]i during the process of disintegrative globulization. METHODS: Individual fiber cells from the cortex of the adult rat lens were isolated by treatment with trypsin in ion-free buffered sucrose. The isolated fiber cells were loaded with the acetoxymethyl esters of Fluo-3 or Calcium Green-2, or with Fluo-3 and Fura Red, and changes in [Ca2+]i of single cortical fibers were measured using a microfluorometer. The time course of increase of [Ca2+]i in fiber cells exposed to Ringer's solution was measured, and the effects on the increase of [Ca2+]i of calcium channel blocker, verapamil, Na-Ca exchange inhibitors Ni2+ and Zn2+, and protease inhibitor, leupeptin, Na+-free and K+-free media and Ca2+-containing isotonic sucrose solution, were investigated. RESULTS: In Hepes sucrose solution (containing approximately 1.5 microM Ca2+), the isolated fiber cells maintained stable values of [Ca2+]i at 99.6+/-10 nM (n = 32). Exposure of the isolated fibers to Ringer's solution (containing 2 mM Ca2+) led to a monoexponential increase of [Ca2+]i at a rate of 0.12 min(-1). This increase in [Ca2+]i was accompanied by disintegration of the isolated fibers into discrete but resealed globules. Changes in [Ca2+]i, monitored by using a two-dye ratiometric method using Fura Red and fluo-3, showed a progressive increase in [Ca2+]i in fibers exposed to Ringer's solution, preceding globulization. The [Ca2+]i in the globules in Ringer's solution, determined using Calcium Green-2, was 3.6+/-0.7 microM (n = 23). Compared with that in fibers in Ringer's solution, the rate of increase of [Ca2+]i in fibers was much slower in the presence of 50 microM verapamil (0.047 min[-1]), in Na+-free (0.086 min[-1]) and in K+-free (0.062 min[-1]) Ringer's solution, or when the fibers were suspended in Hepes-sucrose solution, containing 2 mM Ca2+ (0.046 min[-1]). After 30 minutes, the [Ca2+]i of fiber cells exposed to Ringer's solution, containing 2 mM Ni2+ (574.7+/-29 nM; n = 7) or Zn2+ (402.6+/-77 nM; n = 7) was significantly lower (P < 0.001) compared with that in fiber cells exposed to Ringer's solution alone (1995+/-461 nM, n = 10). In Ringer's solution, leupeptin delayed globulization without significantly affecting the increase in [Ca2+]i. The [Ca2+]i of fiber cells isolated from outer and inner cortex and suspended in Hepes-sucrose was comparable; however, after 15 minutes of exposure to Ringer's solution, [Ca2+]i in fibers from the outer cortex was approximately three times higher than [Ca2+]i in those from the inner cortex. CONCLUSIONS: Exposure to high (millimolar) concentrations of calcium in the external medium leads to an increase in [Ca2+]i of isolated individual fiber cells, which precedes disintegrative globulization. The protective effects of Na+-free and K+-free solutions on globulization appear to be due to a lower rate of increase of [Ca2+]i. Part of the calcium influx may be mediated by L-type calcium channels and by Na-Ca exchange, operating in reverse. Proteolytic inhibitors do not affect the increase in [Ca2+]i but delay globulization by inhibiting calcium-mediated proteolysis. The isolated fiber cells and the disintegrated globules maintain a 100- to 300-fold gradient of calcium across their plasma membranes.

Contribution of osmotic changes to disintegrative globulization of single cortical fibers isolated from rat lens.

In this study the contribution of osmotic changes to disintegrative globulization of lens cortical fibers was examined. Single fiber cells were isolated by trypsinization of adult rat lens cortex, and morphological changes elicited by exposure to different external solutions were monitored optically. The survival of the fiber-shaped cells was analysed in accordance with the Weibull distribution. Changes in [Ca2+]i were measured using the fluorescent calcium-sensitive dye-Fluo-3. Exposure of isolated fiber cells to Ringer's solution (containing 2 mm Ca2+) led to an exponential increase in [Ca2+]i with a time constant of 10.2+/-0.8 min, and caused disintegrative globulization in 25+/-4 min (=Tg). The process of globulization as well as the rate of increase in [Ca2+]i was delayed by removing Cl- ions from the external media. Globulization was also delayed by adding 20% bovine serum albumin (Tg=107+/-3 min) or chloride channel inhibitors 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, and tamoxifen. When the fiber cells were suspended in isotonic (280 mm sucrose) HEPES-sucrose (HS) or HEPES-EDTA-sucrose (HES) solution, no globulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mm) HES solution led to disintegration of fiber cells in 75+/-7 min. Disintegration of the fiber induced by hypotonic HES solution could be delayed by either 0. 05 mm leupeptin (Tg=97+/-6 min) or by pre-loading the fibers with BAPTA (Tg=100+/-4 min). Inhibition of membrane calcium transport by 0.5 mm La3+ had no effect on Tg in hypotonic HES. Addition of 2 mm Ca2+ to HES solution accelerated globulization, and Tg was 57+/-4, 69+/-5 and 102+/-6 min for hypo-, iso- and hyper- tonic solutions, respectively. Transient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results suggest that in ionic media, part of the calcium influx in isolated fiber cells is mediated by the influx of chloride ions. In the absence of other ions, the fiber cells still accumulate calcium, although this calcium influx was independent of medium tonicity. Globulization-induced by hypotonic sucrose solution appears to be mediated by the activation of intracellular proteases and by cell swelling-induced release of calcium from internal stores. Such swelling-mediated disintegrative globulization of fiber cells may be of significance in understanding the cellular basis of diabetic cataracts.

On the use of Zn2+ to discriminate endonucleases activated during apoptosis.

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.

Loss of cytoskeletal proteins and lens cell opacification in the selenite cataract model.

This study of lens protein composition found that some cytoskeletal proteins were degraded during the earliest stages of cataract formation. Cataract was induced in 13-14 day old rats by a single subcutaneous injection of sodium selenite (19 mumol kg-1). By 24 hr after the injection of selenite, the ratio of insoluble to soluble protein increased as lens opacification began. The increase in insoluble protein aggregates was correlated with an accelerated loss of proteins having molecular weights of 42, 55/57 and 235 kDa which reacted with antibodies to the cytoskeletal proteins actin, tubulin/vimentin and spectrin, respectively. We observed the loss of 49, 60 and 90 kDa proteins which were not identified. In the lenses of animals protected from protein aggregation and opacification by administration of 1.5 mmol kg-1 pantethine, the pattern of proteins in SDS-PAGE gels resembled the pattern for proteins from transparent lenses of normal untreated animals and loss of cytoskeletal proteins was prevented.

Age-related decline in ubiquitin conjugation in response to oxidative stress in the lens.

Accumulation of damaged proteins is a major age-related change in lenses of virtually all species and is associated with lens opacification. Proteolytic removal of the damaged proteins may play an important role in maintaining the transparency of the lens. In many tissues, selective removal of abnormal or damaged proteins occurs via a ubiquitin-dependent proteolytic pathway. Ubiquitin, an 8.5 kDa polypeptide, selectively binds to proteins to form ubiquitin-protein conjugates. This ubiquitin-protein conjugate is, in most cases, a signal for protein degradation. In this work, age-related changes in rat lens in the following aspects were detected: (a) levels of the ubiquitin-protein conjugates, (b) some of the enzymes involved in ubiquitin conjugation in rat lenses, and (c) ability to respond to oxidative damage. Endogenous ubiquitin-protein conjugates were detected in epithelium, cortex and nucleus of lenses from young and old rats. The levels of endogenous high molecular weight (HMW) ubiquitin-protein conjugates in each developmental zone of the lenses from young rats were higher than that in the counterparts of lenses from old animals. Peroxide-treatment generally resulted in elevated levels of endogenous HMW ubiquitin-protein conjugates although masses of bulk proteins remain unchanged. The increases in ubiquitin-protein conjugates in the epithelial sections of young and old lenses upon oxidative stress were comparable. In the cortex of young lenses, there was a significant oxidation-related increase in ubiquitin-protein conjugates. There was a similar trend but diminished response in the cortex of old lenses. Nuclear fibers from young lenses also showed an oxidation-induced increase in the level of ubiquitin-protein conjugates. This response was not observed in nuclear fibers of old lenses. The ability to form HMW-ubiquitin conjugates with exogenous 125I-labeled ubiquitin in the lens also increased upon oxidative stress. The extent of the increase in the de-novo ubiquitin conjugating activity upon exposure to oxidation in old lens was much smaller than in young lens. Ubiquitin-activating enzyme (E1), and ubiquitin conjugating enzymes (E2(17k), E2(20k) and E2(25k) were detected by thiol ester assays or Western blot analysis. No significant age-related changes in the levels of E1, E2(17k), E2(20k) and E2(25k) were detected. The activity of E1 and E2(17k) increased upon exposure to H2O2. These data indicate that lens has the ability to increase ubiquitin conjugation activity in response to oxidative stress and this ability is attenuated upon aging. The age-related decrease in the ability to mount a ubiquitin-dependent response upon oxidation may contribute to the accumulation of damaged proteins in the old lenses.

Identification of a novel, sodium-dependent, reduced glutathione transporter in the rat lens epithelium.

PURPOSE: To determine whether glutathione (GSH) transporter(s) other than the previously identified rat canalicular GSH transporter (RcGshT) is present in the lens. METHODS: Poly (A) +RNA isolated from rat and guinea pig lens cortex and epithelium was injected into Xenopus laevis oocytes. The effect of sodium removal was determined by measuring cell-associated radioactivity in lenticular epithelium or cortex mRNA injected oocytes (pretreated with acivicin to inhibit gamma glutamyltranspeptidase) after 1 hour of incubation in NaCl medium or choline chloride (Na(+)-free) medium containing tracer GSH (plus unlabeled GSH). The effect of 2 mM bromosulfophthalein-GSH (BSP-GSH) on GSH uptake in the lens epithelium and cortex in NaCl medium at two GSH concentrations also was determined. The molecular form of uptake of GSH in lens epithelial mRNA-injected oocytes was examined by high-performance liquid chromatography. Western blot analysis was performed to study the presence of RcGshT in the cortex and epithelium. RESULTS: Oocytes injected with mRNA from rat and guinea pig lens epithelium and cortex compartments expressed GSH transport. High-performance liquid chromatography confirmed that epithelial uptake was as intact GSH under conditions of inhibition of GSH synthesis with dl-buthionine sulfoximine. The mean GSH uptake (nmol/oocyte per hour) in epithelial mRNA-injected oocytes was significantly reduced (P < 0.01, n = 4 oocyte preparations) under Na(+)-free conditions compared to NaCl medium at 0.05 mM and 2 mM GSH in the medium. Uptake in cortical mRNA-injected oocytes was unaffected by Na+ removal. Lens epithelial uptake exhibited a strong inhibition by BSP-GSH at 0.05 mM (55%) and 2 mM (64%), whereas cortical uptake was unaffected by BSP-GSH. Western blot analysis identified RcGshT in the cortical and epithelial regions. CONCLUSIONS: Results from the current study provide strong evidence for the presence of a hitherto unreported Na(+)-dependent, BSP-GSH inhibitable GSH transporter in the lens epithelium, which may mediate concentrative, basolateral uptake of aqueous GSH consistent with in situ eye perfusion studies. The Na(+)-independent, BSP-GSH insensitive RcGshT may function as an apical GSH effluxer in lens epithelium and in mediating concentration gradient driven inward GSH movement by uptake-efflux in the lens cortex.

Modelling cortical cataractogenesis. XVIII. In vitro diabetic cataract reduction by venoruton. A flavonoid which prevents lens opacification.

The effect of a novel flavonoid, venoruton (a mixture of mono-, di-, tri- and tetrahydroxyethylrutosides) has been investigated in healthy rat lenses and compared with diabetic cataract modelled in vitro. One mM venoruton was added to medium simulating healthy and diabetic conditions for the incubated lenses; damage was followed by either stereoscopic photography of the lenses under a Cooperative Cataract Research Group operating microscope or with our recently developed method: the leakage of lactate dehydrogenase (LDH) into the lens culture media. The increased LDH activity in the medium and observable development of the opacity were correlated with cell damage, which has been found to be associated with globular degeneration and cataract formation. The extent of opacification and LDH release is reduced if 1 mM venoruton is included in the medium. The protective effect may be related to antioxidant activity against reactive oxygen species: decreased luminol luminescence was shown after venoruton addition to either superoxide-generating hypoxanthine plus xanthine oxidase, or hydrogen peroxide.

The role of prostaglandins E2 and F2 alpha in ultraviolet radiation-induced cortical cataracts in vivo.

PURPOSE: Previous work has shown that exposure of lens epithelial cells or rabbit eyes in vivo to ultraviolet B (UVB) radiation enhanced prostaglandin (PG)E2 synthesis. Such enhanced PGE2 synthesis was related to the increased DNA synthesis that followed UVB exposure. The current study examined the relationship between enhanced prostaglandin synthesis and UVB-induced cataract formation. METHODS: Seventy albino (New Zealand white) rabbit eyes were exposed to UVB radiation in vivo. Fluence of radiation at the cornea was 2.8 J/cm2, 5.6 J/cm2, or 11.2 J/cm2. Eyes were examined 24 hours after UVB exposure and for as long as 10 days by slit lamp biomicroscopy. Mass spectrometry was used to measure PGE2, PGF2 alpha, and 6-keto-PGF1 alpha content of the lens and iris-ciliary body using authentic standards. To determine the effect of inhibition of prostaglandin synthesis on UVB-induced cataract formation, animals were given indomethacin intraperitoneally. Other pharmacologic agents, such as PGE2, PGF2 alpha, and misoprostol, were applied topically to the eye. The effect of UVB on K+ pump was determined by incubating isolated lenses with [86Rb+]. RESULTS: Twenty-four hours after UVB exposure, PGE2 and PGF2 alpha concentrations in aqueous humor were increased by 100- and 30-fold, respectively. Lens PGE2 and PGF2 alpha increased by 6- and 4-fold, respectively, after UVB radiation exposure. Pretreatment of animals with indomethacin prevented the rise in lens and aqueous humor PGE2 and PGF2 alpha levels. Furthermore, indomethacin was partially protective against UVB cataract formation and lowered cataract severity from stage 3 to stage 1, but it did not prevent UVB-induced lens changes completely. Topical application of PGE2 before UVB exposure completely prevented cataract formation in the UVB-exposed eye. In contrast, topical administration of PGF2 alpha increased cataract severity. UVB-induced cataract formation preceded changes in [86Rb]+ uptake in lenses subsequently incubated in K(+)-free Tyrode's. CONCLUSIONS: Enhanced synthesis of cyclooxygenase products of arachidonic acid metabolism in the lens is associated with UVB-induced cataract formation in albino rabbit eyes, and inhibition of cyclooxygenase by indomethacin decreased the severity of cataracts. PGE2, the principal arachidonic acid metabolite, appears to have a protective role because pretreatment of the eye with topical PGE2 completely prevented UVB-induced cataract formation, whereas PGF2 alpha increased the severity of the cataract. The evidence presented for a role of PGF2 alpha in the development of cataract suggests that caution be exercised in the use of PGF2 alpha derivatives in the therapy of glaucoma.

Mechanism of calcium-induced disintegrative globulization of rat lens fiber cells.

PURPOSE: To study the role of calcium and calcium-dependent processes in the disintegrative globulization of isolated single rat lens cortical fibers. METHODS: The authors isolated viable and morphologically intact single fiber cells from rat lens cortex and studied the effect of 1 mM [Ca2+]o on the globulization of fiber cells from the outer and inner cortex. They investigated the effects of the calcium-channel blocker, verapamil; an inhibitor of calcium transport, lanthanum; various protease inhibitors; Na+ -free and K+ -free media; calcium ionophore, A23187; and calcium chelator, BAPTA, on the globulization of fiber cells exposed to 1 mM [Ca2+]o. RESULTS: Perfusion with Ringer's solution containing 1 mM [Ca2+]o, caused disintegration and globulization of the isolated fibers in 32.3 +/- 1 minute, and the addition of 10 microM A23187 to the superfusing solution reduced the time to complete globulization (tg) to 19.4 +/- 0.3 minutes. However, the addition of protease inhibitors, leupeptin, calpain inhibitor I, E-64, or pepstatin (0.5 mM each) to the superfusing solution, increased tg to 105 +/- 3.5, 84.2 +/- 7.8, 60.7 +/- 3.5, and 48.3 +/- 3.1 minutes, respectively. The tg also increased (96.4 +/- 3.5 minutes) when the fibers were preincubated with BAPTA-AM or when they were exposed to 1 mM [Ca2+]o in Na+ - or K+ -free Ringer's solution (tg = 66.7 +/- 5.3 and 58.9 +/- 3.9 minutes, respectively) or in Ringer's solution containing 1 mM [Ca2+]o + 50 microM verapamil (tg = 49.3 +/- 3.8 minutes). Single fibers isolated from the outer cortex of the lens were less susceptible to extracellular calcium than those isolated from the inner cortex. CONCLUSIONS: Increased calcium influx and the attendant elevation of [Ca2+]i are necessary for disintegrative globulizaiton of lens fiber cells. Calcium influx appears to be mediated partially by the L-type calcium channels and the background calcium leak. Protection by protease inhibitors suggests that membrane fragmentation, caused by elevated [Ca2+]i, results from proteolytic damage to the fiber cytoskeleton. Besides underscoring the central role of calcium homeostasis in preserving the morphologic integrity of the cortical fibers, this study suggests a possible cellular mechanism for the formation of supranuclear cataract.