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(Investigative Ophthalmology and Visual Science. 2000;41:2255-2261.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

A Human Lens Model of Cortical Cataract: Ca2+-Induced Protein Loss, Vimentin Cleavage and Opacification

Julie Sanderson, Julia M. Marcantonio and George Duncan

From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
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 µM ionomycin, or EMEM + 5 µM ionomycin + 5 mM EGTA (Ca2+ <1 µM). 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 (~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.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Reports from early this century described ionic disruption as a characteristic occurrence in most human cataractous lenses.1 It has since been shown that although pure nuclear cataracts, accounting for approximately 30% of cataracts extracted, have a normal internal ionic content, lenses with cortical cataract (pure or mixed) have increased lenticular Na+ and Ca2+ and decreased K+ content.2 The ionic alterations can occur throughout the whole lens, as is the case in mature cataracts, or they can occur in highly localized regions, as is the case with retrodots and focal cortical opacities.3 4 In the case of mature cortical cataract, there is a characteristic loss of dry weight that can be attributed to a decrease in the protein content of the lens. Furthermore, the degree of disruption in the ionic balance is correlated with the loss of protein.2 5 However, an analysis of human cortical cataracts does not provide information concerning a particular role for any individual ionic species, because all ionic levels are altered to some degree.2 The ionic changes have therefore been modeled in the organ-cultured bovine lens, where loss of soluble protein and lens opacification were found to be independent of Na+ and K+ disruption and lens hydration but were critically dependent on an increase in lens Ca2+.6 In vivo and in vitro animal models have implicated the Ca2+-activated protease calpain (EC.3.4.22.17) in the mechanism of cataractogenesis.7

It has recently become apparent that there are species and age differences in the response of lenses to increases in intracellular Ca2+.8 The most striking difference is that in young rodent lenses (in vitro and in vivo) nuclear opacities develop in response to treatments that increase lens Ca2+, whereas rabbit and bovine lenses, as well as older rodent lenses, undergo cortical opacification.6 7 8 Furthermore, human lenses are reported to contain only 3% of the calpain activity found in the rat lens, and no activity can be measured in human lens homogenates unless the endogenous inhibitor calpastatin is removed.9 Zigler et al.10 have also shown that the cultured primate lens is less sensitive to oxidative insult than the cultured rodent lens. If data from animal models are to be extrapolated to the process of cataract formation in man, information is needed from human experimental systems. Hightower and Farnum11 have reported that simply subjecting human lenses to elevated extracellular Ca2+ concentration (20 mM) for 48 hours results in the appearance of discrete cortical opacities. The present study was therefore undertaken to discover whether calcium has the same critical role to play in loss of protein from cortical cataracts that it has in animal lenses. Proteolysis and protein loss were studied by two techniques designed to increase greatly the sensitivity of the methods. Human lenses were first incubated in the presence of [3H]-amino acids to allow time for protein synthesis to occur and on exposure to conditions that would elevate internal calcium, the external medium was assayed for trichloroacetic acid (TCA)–precipitable radioactivity.6 Internal proteolysis was studied by Western blot methods probing for vimentin, because it is not only a critical cytoskeletal element in lens cell architecture, but it is also a recognized substrate for calcium-activated proteases such as calpain.12


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Lens Culture
Donor eyes were obtained from the East Anglian Eye Bank. The research followed the tenets of the Declaration of Helsinki regarding the use of human material. After removal of the cornea for transplantation, the eyes were placed in sterile containers and covered with Eagle’s minimum essential medium (EMEM) containing 200 U/ml penicillin and 200 µg/ml streptomycin. They were stored at 4°C before dissection. Lenses were placed in culture within 48 hours after the donor’s death. As far as possible, paired lenses were used for control and experimental protocols. Because of some experimental losses it was not always possible to report all data for all lenses.

Lenses were dissected by posterior approach and incubated for 30 minutes in bicarbonate-CO2–buffered EMEM (pH 7.4), containing 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin, and 50 µg/ml gentamicin. Thereafter, the lenses were maintained in EMEM with 50 µg/ml gentamicin at 35°C.

After a preculture period of 24 to 72 hours, lenses were allowed to incorporate [3H]-amino acids (Leu, Lys, Phe, Pro, and Tyr; 74 kBq/ml; Amersham, Little Chalfont, UK) into proteins during a 48-hour loading period. The end of this loading period equated to day 0 of the experimental period.

Experimental Protocol
During the experimental period, lens images were taken daily using a charge-coupled device (CCD) camera (UVP, Cambridge, UK) with Synoptics software (Synoptics, Cambridge, UK), and media were changed daily. After a 24-hour efflux period in control EMEM, the lenses were cultured under three experimental conditions: control EMEM (Ca2+ 1.8 mM); EMEM + 5 µM ionomycin; EMEM + 5 µM ionomycin + 5 mM EGTA (Ca2+ < 1 µM). Lenses in group 3 (ionomycin + EGTA) were cultured in the EGTA medium for 30 minutes before exposure to ionomycin to chelate external Ca2+ before introduction of the ionophore. The mean ages of lenses subjected to the various treatments did not differ significantly (Table 1) . At the end of the experiment, the lenses were removed from the medium and rolled on filter paper to remove medium, adhering nonlens tissue, and vitreous humor. Wet weight was determined before lenses were frozen in liquid nitrogen. Storage was at -70°C before analysis. All media were stored at -20°C before analysis of [3H]-amino acid and protein efflux.


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  		Table 1. Distribution of Donor Ages and Postexperimental Wet Weights of Human Lenses

 
Analysis of Amino Acid and Protein Efflux
The total [3H] activity of efflux medium was measured by counting a 1-ml aliquot in 10 ml of scintillator (OptiPhase "SuperMix"; Wallac Scintillation Products, Milton Keynes, UK), using a liquid scintillation counter (model 1409; Wallac). Ice-cold TCA was added to 1.5-ml aliquots of medium to a final concentration of 5%, and the samples were refrigerated to precipitate the protein. The samples were centrifuged at 12 000g for 30 minutes and the supernatants counted to determine the free [3H]-amino acid component of the efflux medium. The pellet was washed twice by resuspension in 5% TCA and centrifugation. Bovine serum albumin (0.2%) was added as a carrier at this stage. The pellet was then dried at 65°C, dissolved in 250 mM NaOH, and the sample counted to determine the content of [3H]-labeled protein in the efflux medium. Total protein content was measured in the efflux medium using a protein assay (Coomassie Plus; Pierce & Warriner, Chester, UK).

Analysis of Lens Proteins
Lenses were homogenized in 1 ml of extraction buffer composed of 6 mM phosphate buffer (pH 7.2) containing 100 mM KCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol plus 1 mM EGTA, 1 mM EDTA, 10 µM N-ethylmaleimide, 200 µM phenylmethylsulfonyl fluoride, and 5 µM E64 to prevent proteolysis during preparation. The homogenate was centrifuged at 12,000g for 30 minutes to separate the soluble from insoluble proteins. The pellet was washed three times by resuspension in 1 ml extraction buffer and centrifugation. An aliquot of the soluble protein fraction and each of the buffer washes were treated with TCA to a final concentration of 5% to separate the soluble proteins from the free amino acid pool. The protein pellets were washed three times by resuspension in 5% TCA and centrifugation. The washed pellets were dissolved in 250 mM NaOH for counting. The washed insoluble pellet from the lens homogenate was separated into the urea-soluble and urea-insoluble fraction by extraction in 8 M urea followed by centrifugation (12 000g for 30 minutes). The pellet was washed three times by resuspension in 4 M urea and centrifugation. The urea-insoluble pellet was dissolved in 250 mM NaOH for counting. The [3H] activity in each fraction, including all washes, was determined by scintillation counting, as described.

Vimentin Analysis
The urea-soluble fraction was separated by SDS-PAGE on 4% to 20% gradient gels (BioRad, Hemel Hempstead, UK). Proteins were either stained by a colloidal Coomassie blue G250 method13 or transferred to polyvinylidene fluoride membranes (Millipore, Watford, UK) and probed with monoclonal antibody to vimentin (clone V9; Sigma, Poole, UK). Detection was by enhanced chemiluminescence (ECL, Amersham).

Unless otherwise stated, all chemicals were obtained from Sigma. Statistical analysis was performed by paired Students t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Organ Culture of Human Lenses
In these experiments human lenses remained transparent throughout culture in control medium (EMEM). The lenses used were from donors in the age range 16 to 92 years (Table 1) . Older lenses from the beginning had increased cortical scatter and were more yellow, both characteristics of the aging lens in vivo.14 15 None of the lenses used had obvious focal cortical opacity. Figure 1 shows the appearance of lenses from 18- and 66-year-old donors cultured for 5 days.



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  		Figure 1. Dark field (forward scatter) and bright field (grid) digital images of human lenses from 18- (A) and 66- (B) year-old donors after culture for 5 days under control conditions.

 
The culture medium (EMEM) was serum free, and no supplements were found to be necessary for maintenance of transparency. However, a major criterion for successful culture was the length of the postmortem period, which had to be shorter than 48-hours for the lenses to remain viable over the 2-week culture period. All lenses showed increased light scatter in the region of the posterior suture when they were initially placed in the culture medium. However, these changes were reversible, with lenses of shorter postmortem time recovering more quickly.

A further biomarker of viability was the capacity of lenses to synthesize proteins in culture. All lenses incorporated [3H]-amino acids into protein with high consistency between paired lenses. The level of incorporation was dependent on donor age (Fig. 2) , with young lenses incorporating more [3H]-amino acids into protein than older lenses. This was not due to a decline in the transport of [3H]-amino acids into the lens, because the size of the [3H]-amino acid pool did not decrease with age (data not shown). Overall, the [3H]-amino acid incorporation into protein represented 16.1% ± 1.8% of the [3H]-amino acid pool at the end of loading, whereas the values from paired lenses of the youngest donor (16 years) were 25.1% and 23.1% and from the oldest donor (92 years) were 13.1% and 9.6%, respectively.



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  		Figure 2. Incorporation of [3H]-amino acid into the protein (TCA-precipitable) fraction of the lens over the 48-hour period in radioactive control medium before the start of the experimental protocols. Each point represents an individual lens. Incorporation was calculated by totalling the [3H] activity in the lens protein fractions at the end of the experimental period and the activity of [3H]-labeled protein in the efflux media sampled during the days after the initial 48-hour uptake period.

 
Lens Transparency
Raising the intracellular Ca2+ by exposure to ionomycin (5 µM) resulted in a loss of transparency that was initiated within the first 24 hours and progressed over the experimental period. Lenses from experimental day 1 (before addition of the ionomycin), days 3 and 7 are shown (Fig. 3) . Light scatter was located initially in the outermost cortical fiber cells and was most dense in the equatorial regions of the lens. The opacification progressed with time to the inner cortical fibers, but at no time was opacification of the nucleus observed. In lenses treated with ionomycin in medium in which the Ca2+ had been chelated with EGTA, the transparency changes were inhibited. Figure 3 shows paired lenses from a 30-year-old donor treated with ionomycin alone or ionomycin + EGTA. At day 3 the ionomycin-induced opacification was almost totally inhibited by Ca2+ chelation. By day 7 there was scatter in the equatorial region in ionomycin + EGTA–treated lenses, but it was restricted compared with scatter throughout the entire cortex in lenses treated with ionomycin alone.



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  		Figure 3. Ca2+-induced transparency changes in cultured human lenses. (A) Lenses from a 16-year-old donor cultured under control conditions ([Ca2+]out = 1.8 mM). (B,C) Paired lenses from a 30-year-old donor treated with 5 µM ionomycin in the presence of normal extracellular Ca2+ levels (B) and in medium in which external Ca2+ had been chelated to less than 1 µM with 5 mM EGTA (C). Digital images were taken on days 1, 3, and 7 of the experimental period. Within this relatively short period of exposure, only cortical changes were observed in the lenses. Note that the black dots in (B7) are artifacts and arise from bubbles of air trapped in the vitreous adhering to the lens.

 
Lens Weights
Lenses were weighed at the end of each experiment. There were variations due to the range of donor ages. Postexperimental lens weight increased with age within each of the treatment groups. In paired lenses, ionomycin increased lens wet weight compared with control, and Ca2+ chelation with EGTA reduced the ionomycin-induced increase (data not shown). However, because of the age-related variations, no statistically significant differences were observed when the data were pooled (Table 1) , although the means showed the same trends as the paired lenses.

Analysis of Efflux Medium
Exposure to ionomycin did not result in a marked perturbation of free [3H]-amino acid efflux, and in fact a slight decrease in loss was observed over time (Fig. 4) . This was in marked contrast to the efflux of [3H]-protein, which was very low in control lenses but greatly stimulated by exposure to ionomycin (Fig. 4B) . The highest rate of loss was observed in the 24-hour period after the initial exposure to the ionomycin. At the 24-hour time point, the [3H]-protein efflux was totally inhibited by chelation of the external Ca2+. By the end of the experiment, 41% of the labeled protein had been lost from the lenses, half of which was lost in the first 24 hours. This compares with a total loss of 12% in ionomycin + EGTA–treated lenses. When the medium was assayed directly for total protein, there was very little protein detectable in the medium from control lenses, whereas with ionomycin treatment, release of protein was observed (Fig. 4C) . The rate of loss was again greatest during the first 24 hours, slowing to a steady rate for the remainder of the experimental period. During the 6 days of exposure to ionomycin, lenses lost 2.4 ± 0.33 mg protein, compared with 0.07 ± 0.01 mg for control lenses and 0.22 ± 0.55 mg for ionomycin and EGTA lenses. These data clearly demonstrate that the loss of protein observed in ionomycin-treated lenses was dependent on the presence of free Ca2+ in the external medium.



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  		Figure 4. (A) Efflux of [3H]-labeled amino acids from control and experimental lenses. Note that the value at t = 0 represents the total activity in the free amino acid pool and that after a 7-day efflux period, approximately 20% of this activity still remains within each lens. (B) Efflux of [3H]-labeled proteins from the lens under the same experimental conditions as in (A). One hundred percent represents the total amount of radioactive amino acids incorporated into the protein fraction of the lens during the 48-hour pre-experimental period. (C) Efflux of total protein from the lens under same conditions as in (A). Each point represents mean ± SEM.

 
Lens Proteins
At the end of the efflux period, lens proteins were separated into water-soluble and -insoluble fractions, and the amount of [3H] label remaining was measured. Exposure of lenses to 5 µM ionomycin resulted in a loss of 44% of the [3H]-labeled soluble protein (Table 2) . This was inhibited by Ca2+ chelation, with no significant differences between the control and ionomycin + EGTA groups. The observed decrease in [3H]-labeled soluble protein can be accounted for by the efflux of the protein into the medium. Some redistribution of the water-insoluble fraction was observed, with a small decrease in the urea-soluble fraction (not significant) and a significant increase in the urea-insoluble fractions in ionomycin-treated lenses (P < 0.05; Table 2 ). These changes were not significantly inhibited in the ionomycin + EGTA–treated lenses.


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  		Table 2. Distribution of [3H]-Labeled Protein in Experimental Human Lenses

 
Calcium-Dependent Proteolysis
Cytoskeletal proteins are known substrates of calpain in the lens and other tissues.12 16 We have previously demonstrated that in whole rat and bovine lenses there is an almost total degradation of the cytoskeletal proteins spectrin, filensin and vimentin on incubation with Ca2+ ionophore.17 18 This is totally inhibited by Ca2+-free medium and reduced by calpain inhibitors. Cytoskeletal proteolysis was therefore investigated in greater detail in this series of experiments to compare the response of human and rat lenses to Ca2+ overload.

Figure 5 shows an SDS-PAGE gel of the urea-soluble fractions isolated from a pair of 16-year-old donor lenses cultured under control conditions (C) or supplemented with 5 µM ionomycin (I), and a pair of 66-year-old donor lenses cultured with ionomycin (I) or ionomycin with EGTA (IE). Comparison of the protein profiles of paired lenses does not show the major proteolytic changes observed in the rat.16 17 We therefore probed for cleavage products of vimentin, which is the most rapidly degraded lens cytoskeletal protein in in vitro experiments.18 The major vimentin band was seen at 56 kDa. In addition, a number of other vimentin-reactive bands were observed in lenses from both the 16- and 66-year-old donors. Three of these were dependent on the presence of ionomycin and external Ca2+: a cleavage product of approximately 48 kDa and two higher molecular weight products of approximately 115 kDa and 235 kDa. These data indicate that both Ca2+-dependent limited proteolysis and Ca2+-dependent cross-linking occurs in the human lens.



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  		Figure 5. (A) SDS-PAGE and (B) Western blot for vimentin of urea-soluble proteins from paired lenses. In both (A) and (B), lanes 1 and 2: urea-soluble proteins from control (C) and 5 µM ionomycin-treated (l) lenses from a 16-year-old donor; lanes 3 and 4: urea-soluble proteins from 5 µM ionomycin- (l) and 5 µM ionomycin + 5 mM EGTA (IE)-treated lenses from a 66-year-old donor. In (A) molecular weight markers are shown and the position of the cytoskeletal proteins spectrin, filensin and vimentin are indicated. In (B) native vimentin is marked on the left; Ca2+-dependent breakdown and cross-linked products of vimentin are marked by arrows on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The human lens is remarkably resilient when cultured in vitro. It can be maintained in a transparent state for prolonged periods in a relatively simple medium that contains neither serum nor growth factors. Furthermore, the age-related decline in lens growth19 is mirrored by the age-related decrease in protein-synthesis rates in cultured lenses (Fig. 2) .

Calcium overload occurs in a number of diseases, including cortical cataract.20 The present experiments are the first to characterize the response of the human lens to intracellular Ca2+ overload during long-term culture with extended protocols. Paired lenses tended to be equivalent in amino acid loading, incorporation into proteins, amino acid efflux, and transparency. Variations in these parameters between pairs of lenses were as would be anticipated because of the range of donor ages. This inherent variation, however, did not limit the power of the data in statistical analysis. Differences observed between treatment groups were highly significant, clearly demonstrating that cultured human lenses can be used successfully to investigate the mechanisms of cataractogenesis.

In the human lenses, Ca2+ overload resulted in opacification of the lens cortex. The Ca2+-induced loss of cortical transparency is consistent with data from other mammalian lenses, with the exception of the neonatal rodent lens, where a nuclear opacification is observed.7 The increased light scatter in the human lens was associated with an increased efflux of protein from the water-soluble protein fraction. This was shown to be a direct result of increased intracellular Ca2+, because chelation of the Ca2+ in the external medium was preventative. This human lens Ca2+-induced opacification (HLCO) model reflects observed changes in human cataractogenesis. Mature human cortical cataracts have increased Ca2+ content and decreased dry weight.2 5 The latter occurs because of a loss of crystallins from the soluble fraction, by insolubilization and efflux into the aqueous humor.21 22 These changes were paralleled in the HLCO model, in that ionomycin induced a loss of newly synthesized soluble protein and an increase in incorporation into the water-insoluble fraction (Table 2) . In addition, there was a mean increase in wet weight of approximately 20% in the lenses with Ca2+ overload. Because there is a concomitant loss of dry weight due to the efflux of protein from the lenses, the increase is due to increased lens hydration. Lens hydration and swelling have been observed both in animal models of cataract6 23 and in advanced human cortical cataract in vivo.5 The present model, involving an acute increase in internal calcium, produces cortical opacification within 1 week, whereas human cortical cataract may take years to develop in vivo. However, the features that the model and in vivo cataract have in common indicate that it can begin to bridge the gap between experimental animal models and human cataract.

There was an interesting absence of effect of Ca2+ overload on the overall rate of loss of amino acids from the lens. Certainly, if a general breakdown in structure was occurring throughout the lens over the exposure period, then the [3H]-amino acids, which are distributed throughout the lens,24 would be expected to be lost much more rapidly. They are, if anything, lost more slowly. It appears therefore that in these initial stages only the outer cortex is affected, wherein the newly synthesized (and therefore labeled) protein is located. It should be noted that less than 3% of the total protein is lost from the lens, but more than 40% of the newly synthesized protein is lost. The relatively low loss of total protein and free amino acids again indicates that internal structures are relatively intact. Indeed, we could observe no change in transparency in the nuclear regions. The small percentage change in total protein of the lens highlights the advantage of having the ability to observe changes in the newly synthesized proteins in the outer region that are first at risk. The data presented here are similar to those obtained previously from this laboratory in which an organ-cultured bovine lens model was investigated. Marcantonio et al.6 concluded that the lens behaved more as a stabilized gel system rather than simply as a collection of independent proteins encapsulated by membranes. They found that severe hydration of the lens with no increase in internal calcium, produced little protein loss, but a lesser extent of hydration accompanied by an increase in internal calcium produced a massive loss of protein, presumably by destabilizing the gel structure.

Increased intracellular Ca2+ results in the activation and modulation of a large number of enzymes. In relation to cataract, two families of enzymes have received particular attention: the calpains7 and the transglutaminases.25 In the human lens, calpain II activity has been investigated9 and found to be highest in the cortex of young donors and lowest in the nucleus of aged donors. Lenses also have been found to contain endogenous calpain inhibitor (calpastatin) in excess over calpain activity, and the level of calpastatin did not decrease with age.9 An excess of inhibitor over enzyme results in human lens homogenates demonstrating no calpain proteolytic activity, unless the calpastatin is removed. Using the cultured human lens, we have demonstrated the Ca2+-dependent limited proteolysis of vimentin, suggesting that in vivo the lens is able to overcome the inhibition within the cell. Furthermore, the proportion of native vimentin to the Ca2+-dependent vimentin breakdown product is comparable between the 16- and the 66-year-old lenses (Fig. 5B) . Regulation of calpain activity within the cell is known to be modulated by several factors in addition to Ca2+ and calpastatin, including autolytic cleavage and phospholipids (most potently phosphatidylinositol 4,5-biphosphate [PIP2]).26 Clearly, strict regulation of protease activity is necessary to prevent unscheduled proteolysis. Although proteolytic events can be identified in the human lens, the damage appears to be limited to a greater extent than in the rodent lens. This lessening of sensitivity to calpain activity in the human lens parallels the lesser sensitivity to H2O2 insult compared with the rodent.10

The activity of a second class of Ca2+-regulated enzymes, the transglutaminases, has also been observed in the human lens,27 although most research on this enzyme has been performed using freeze-thawed or homogenized animal lenses.25 27 Vimentin has recently been shown to be a substrate for lens transglutaminase.28 The Ca2+-dependent cross-linking of vimentin observed in the HLCO model suggests that the enzyme is activated in the human lens under conditions of intracellular Ca2+ overload and therefore supports a possible role for this enzyme, as well as calpain, in cataractogenesis. It will be interesting to investigate the interaction between the two Ca2+-activated systems in generating light scatter in the lens.

The major objective of these experiments was to determine whether Ca2+-induced opacification is observed in the human lens under physiologically relevant external Ca2+ concentrations. This has been clearly demonstrated. Parallels have been identified between the HLCO model and in vivo mechanisms of cataractogenesis in the human lens that may contribute to the ultimate goal of elucidation of the molecular mechanisms of human cortical cataract.


   Acknowledgements
 
The authors thank Peter Davies and Pam Keely at the East Anglian Eye Bank without whom this study would not have been possible.


   Footnotes
 
Supported by The Royal National Institute for the Blind (JS), and National Institutes of Health Grant EY10558 (JMM).

Submitted for publication August 31, 1999; revised January 24, 2000; accepted February 18, 2000.

Commercial relationships policy: N.

Corresponding author: Julie Sanderson, School of Biological Sciences, University of East Anglia, Norwich, UK. j.sanderson{at}uea.ac.uk


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Adams, DR (1929) The role of calcium in senile cataract Biochem J 23,902-912
  2. Duncan, G, Bushell, AR (1975) Ion analysis of human cataractous lenses Exp Eye Res 20,223-230[Medline][Order article via Infotrieve]
  3. Vrensen, GFJM, Willekens, B, De Jong, PTVM, et al (1994) Heterogeneity in ultrastructure and elemental composition of perinuclear lens retrodots Invest Ophthalmol Vis Sci 35,199-206[Abstract/Free Full Text]
  4. Duindam, JJ, Vrensen, GFJM, Otto, C, Greve, J. (1998) Cholesterol, phospholipid and protein changes in focal opacities in the human eye lens Invest Ophthalmol Vis Sci 39,94-103[Abstract/Free Full Text]
  5. van Heyningen, R. (1972) The human lens, 1: a comparison of cataracts extracted in Oxford (England) and Shikarrpur (W. Pakistan) Exp Eye Res 13,136-147[Medline][Order article via Infotrieve]
  6. Marcantonio, JM, Duncan, G, Rink, H. (1986) Calcium-induced opacification and loss of protein in the organ-cultured bovine lens Exp Eye Res 42,617-630[Medline][Order article via Infotrieve]
  7. Shearer, TR, David, LL (1991) Calpains in lens and cataract Mellgren, RL Murachi, T eds. Intracellular Calcium-Dependent Proteolysis ,265-274 CRC Press Boca Raton, FL.
  8. Fukiage, C, Azuma, M, Nakamura, Y, Tamada, Y, Shearer, T. (1998) Nuclear cataract and light scattering in cultured lenses from guinea pig and rabbit Curr Eye Res 17,623-635[Medline][Order article via Infotrieve]
  9. David, LL, Varnum, MD, Lampi, MD, Shearer, TR (1989) Calpain II in human lens Invest Ophthalmol Vis Sci 30,269-275[Abstract/Free Full Text]
  10. Zigler, JS, Lucas, VA, Du, X. (1989) Rhesus monkey lens as an in vitro model for studying oxidative stress Invest Ophthalmol Vis Sci 30,2195-2199[Abstract/Free Full Text]
  11. Hightower, KR, Farnum, R. (1985) Calcium induced opacities in cultured human lenses Exp Eye Res 41,565-568[Medline][Order article via Infotrieve]
  12. Shoeman, RL, Traub, P. (1991) Calpains and the cytoskeleton Mellgren, RL Murachi, T eds. Intracellular Calcium-dependent Proteolysis ,191-210 CRC Press Boca Raton, FL.
  13. Neuhoff, V, Arnold, N, Taube, D, Ehrhardt, W. (1988) Improved staining of proteins in PAGE including IEF with a clear background at nanogram sensitivity using Coomassie brilliant blue G250 and R250 Electrophoresis 9,255-262[Medline][Order article via Infotrieve]
  14. Weale, RA (1988) Age and transmittance of the human crystalline lens J Physiol 395,577-587[Abstract/Free Full Text]
  15. Bettelheim, FA, Ali, S. (1985) Light scatter of normal human lens, III: relationship between forward and back scatter of whole excised lenses Exp Eye Res 41,1-9[Medline][Order article via Infotrieve]
  16. Truscott, RJW, Marcantonio, JM, Tomlinson, J, Duncan, G. (1990) Calcium-induced opacification and proteolysis in the intact rat lens Invest Ophthalmol Vis Sci 31,2405-2411[Abstract/Free Full Text]
  17. Sanderson, J, Marcantonio, JM, Duncan, G. (1996) Calcium ionophore induced proteolysis and cataract: inhibition by cell permeable calpain antagonists Biochem Biophys Res Commun 218,893-901[Medline][Order article via Infotrieve]
  18. Marcantonio, JM, Duncan, G. (1991) Calcium-induced degradation of the lens cytoskeleton Biochem Soc Trans 19,1148-1150[Medline][Order article via Infotrieve]
  19. Harding, JJ, Crabbe, MJC (1984) The lens: development, proteins, metabolism and cataract Davson, H eds. The Eye Vol. 1B,207-492 Academic Press London.
  20. Duncan, G, Williams, MR, Riach, RA (1994) Calcium, cell signaling and cataract Prog Retinal Eye Res 13,623-652
  21. Spector, A. (1984) The search for a solution to senile cataracts. Proctor lecture Invest Ophthalmol Vis Sci 25,130-147[Free Full Text]
  22. Sandberg, HO, Closs, O. (1976) The alpha-crystallin content of aqueous humour in cortical, nuclear, and complicated cataract Exp Eye Res 22,75-84[Medline][Order article via Infotrieve]
  23. Harding, JJ (1991) Cataract. Biochemistry, Epidemiology and Pharmacology ,125-189 Chapman & Hall London.
  24. Kern, HL (1979) Transport of organic solutes in the lens Zadunaisky, JA Davson, H eds. Current Topics in Eye Research ,217-240 Academic Press New York.
  25. Lorand, L, Conrad, SM, Velasco, PT (1985) Formation of a 55 000-weight cross-linked ß crystallin dimer in the Ca2+-treated lens. a model for cataract Biochemistry 24,1525-1531[Medline][Order article via Infotrieve]
  26. Saido, TC, Sorimachi, H, Suzuki, K. (1994) Calpain: new perspectives in molecular diversity and physiological-pathological involvement FASEB J 8,814-822[Abstract]
  27. Hidasi, V, Muszbek, L. (1995) Transglutaminase activity in normal human lenses and in senile cataracts Ann Clin Lab Sci 25,236-240[Abstract]
  28. Clément, S, Velasco, PT, Murthy, SNP, et al (1998) The intermediate filament protein, vimentin, in the lens is a target for cross-linking by transglutaminase J Biol Chem 273,7604-7609[Abstract/Free Full Text]



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