V.N. Reddy

Metabolism of glutathione in the lens

The mechanisms by which high levels of reduced glutathione are maintained in the lens and the possible function of the tripeptide are discussed in this paper. It is clearly established that glutathione and its analogue, ophthalmic acid, are synthesized in situ by an analogous sequence of reactions involving at least one enzyme in common. The constituent amino acids required for their synthesis are derived to a large extent from the aqueous humor. Lens appears to be relatively impermeable to glutathione and contains a very low activity of the enzymes responsible for its breakdown. While the possibility that glutathione may be present in a bound form cannot be entirely excluded, the capsule and epithelium may serve as a barrier to the diffusion of this compound since the efflux of glutathione is much more rapid from decapsulated lens. It appears that in the lens, the level of glutathione is dependent upon glutathione reductase reaction in which oxidized glutathione is enzymatically reduced by NADPH. Glutathione may function as a protective agent for sulfhydryl enzymes and for maintaining protein sulfhydryls in the reduced state in addition to its participation as a coenzyme and in several oxidation-reduction reactions in the lens. The reduction in the level of glutathione that is observed during all types of cataract formation may be brought about by such factors as a decrease in the biosynthesis of this peptide, increase in permeability of lens membranes, and a relative deficiency in glutathione reductase as well as an increase in mixed disulfide formation with protein sulfhydryls. The diversion of NADPH from glutathione reductase to aldose reductase system may be an additional factor in the depletion of glutathione in cases of sugar cataracts.

Biochemical changes associated with the development and reversal of galactose cataracts

Abstract The rate of synthesis of glutathione (GSH) in lens extracts during cataract formation has been determined and found to be the same as that of extracts from normal lenses. It is concluded that unlike X-ray-induced cataracts the potential mechanism for GSH synthesis in galactose cataracts is unaffected. In addition, the levels of GSH, free amino acids, dulcitol, cations and the degree of hydration of the lens during the development and reversal of galactose cataract have been determined. Feeding of a normal diet even after the development of mature cataracts (20 days) leads to the disappearance of cortical opacities almost completely leaving an essentially clear lens with only a very fine pinhead nuclear opacity. The levels of GSH, taurine and other free amino acids, which fall rapidly dudring cataract formation, return to near normal values during the reversal phase even though the lenses are hydrated. In cnntrast to the near complete recovery of these constituents, sodium ion concentration remains four times higher than in the control lenses almost 4 weeks after diet reversal. The recovery of potassium ion during the same period is about 70% of controls. The continued hydration of the lenses during the reversal phase of cataract, despite the disappearance of dulcitol, is apparently related to the elevated sodium ion concentration. A possible explanation for the recovery of GSH and free amino acids in hydrated lenses has been suggested.

Calcium content and distribution in human cataract

Abstract The calcium content and distribution was measured in brunescent cataracts from India and cataracts from the United States classified according to guidelines of the Cooperative Cataract Research Group (CCRG). The severity and extent of opacification correlates well with the increase in Ca 2+ bound to membranes and insoluble proteins separated by differential centrifugation. Thus, bound Ca 2+ is approximately 30 ng/mg for the immature cataract. 100 ng/mg for the pale yellow. 200 ng/mg for the mature cataract and 300 ng/mg for the brown brunescent. These values can be compared to 16 ng/mg (0·2 m m ) for a freshly excised rabbit lens. To the extent that the mature cataract represents an advanced stage and the immature cataract an early stage of development, and likewise for the brown and pale yellow cataract, results of this study suggest that cataract development is not accompanied by, or the result of, a redistribution of ‘free’ and bound calcium. We find that the fraction of total calcium bound to membranes and insoluble proteins is the same in the early and advanced stages of U.S. and Indian cataracts. It appears that as calcium accumulates in the developing cataract, an increasing amount becomes membrane or protein bound while the level of diffusible Ca 2+ continues to increase. In the advanced stage as much as 85% of the calcium in the 10 000 g supernatant is dialyzable or diffusible. The remaining 15% is bound to watersoluble proteins and represents a sixfold to 15-fold increase in the mature and brown cataract, respectively. There is also a considerable increase in the amount of calcium found in the 1000 g pellet containing insoluble proteins and membrane fragments.

Ultrastructural changes during the development and reversal of galactose cataracts

Abstract Ultrastructural alterations in rat lens that accompany the induction of galactose cataract and the reinstatement of tissue transparency that occurs subsequent to removal of the animals from galactose diet were documented. Lenses examined at different times after the initiation of galactose diet exhibited edematous, liquefied fibers and cellular cysts. Electron dense aggregates were initially found in sections obtained from the pre-equatorial region and subsequently in the tissue from the anterior polar region. The location of the aggregates in the galactose-fed animals followed a spatio-temporal pattern which paralleled cataract maturation at the macroscopic level. The aggregates were not present in either the epithelium of the cataractous lenses or in the epithelium or fibers of lenses from animals that were fed glucose or laboratory chow alone. Mature cataracts were observed in rats fed galactose for 20 days. However, a reinstatement of fiber morphology and lens transparency was realized if these animals were subsequently placed on a galactose free diet. Normalization of fiber morphology and alignment was noted after the animals had been on a galactose free diet for 28 days. However, a nuclear opacity persisted throughout the 3 month observation period. The extent to which the repair of existing fibers and/or the formation of newly formed fibers contribute to the reversal of lens opacity remains to be ascertained.

High molecular weight protein aggregates in x-ray-induced cataract.

Abstract The nature and the possible mechanism of formation of high molecular weight (HMW) protein aggregates were studied in X-ray-induced cataract in rabbit. The HMW protein fraction (molecular weight >4×10 6 daltons) which constituted approximately 18% of the total soluble protein in both the cataractous cortex and nucleus was isolated by gel filtration chromatography. The concentration of sulfhydryl (-SH) groups per milligram of protein in the HMW fraction was three times higher than that of normal α-crystallin. In addition, 50% of the total concentration of sulfhydryl groups contained in the HMW protein was found to be present in the oxidized state; the increased oxidation of protein -SH groups in the X-irradiated lens being observed only during the final stage of cataract which was marked by complete opacity. Treatment of HMW protein with dithioerythritol and subsequent refractionation yielded two peaks; peak I which eluted as α-crystallin and the second fraction (peak II) which eluted in the position of low molecular weight β-crystallin. Peak I accounted for approximately 40% of the deaggregated protein and peak II 60%. There was little protein eluted in the position of γ-crystallin. Thus, it appears that the HMW protein of X-ray-induced cataract is comprised of both α- and β-crystallins joined by intermolecular disulfide bonds. SDS (sodium dodecyl sulfate) electrophoresis of HMW protein and peaks I and II showed that the major bands of both peaks I and II were represented in the bands exhibited by the HMW protein. The electrophoretic mobilities of the major bands of peaks I and II were similar to those of α-crystallin and β-crystallin, respectively. The amino acid composition of peak I was found to be comparable to α-crystallin while the composition of peak II was similar to β-crystallin with the major exception that the hydrolysates of proteins in both peaks I and II were nearly lacking in tyrosine.

Peroxide-Induced Damage in Lenses of Transgenic Mice with Deficient and Elevated Levels of Glutathione Peroxidase

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.

Crystallins and their synthesis in human lens epithelial cells in tissue culture.

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 Dulbeccos modified Eagles 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.

Disulfide cross-linking of urea-insoluble proteins in rabbit lenses treated with hyperbaric oxygen

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)

Blood-vitreous barrier to amino acids

Abstract The rate of loss of 14C-labeled cycloleucine (1-aminocyclopentane-1-carboxylic acid) from rabbit eye following intravitreal injection was found to follow a single exponential curve with a half life of 2·2 hr and was significantly reduced by the simultaneous introduction of 5 m m nonlabeled cycloleucine or methionine into the vitreous body. The transport rate was also found to decrease in the presence of 10−4 m ouabain. A similar inhibitory effect on the rate of loss of cycloleucine from the vitreous humor could be produced by a single systemic injection of sodium iodate. The steady state concentration of [14C]cycloleucine and various naturally occurring amino acids in the vitreous humor gradually increased and approached that in the plasma following systemic injection of sodium iodate. Treatment with iodate also resulted in an increased rate of accumulation of [14C]cycloleucine in the vitreous humor following its parenteral administration. The effect of iodate on amino acid transport was seen long before morphological changes in the pigment epithelium of the retina could be observed. It is concluded that the deficiency of amino acids in the vitreous humor, with respect to plasma, under steady state conditions is due to their constant removal from vitreous humor into blood by an active transport mechanism located in the pigment epithelium of the retina.

Transport of organic molecules in the lens

Abstract The ocular lens is capable of transporting amino acids, peptides and other organic molecules by carrier-mediated processes which are energy and temperature dependent. The energy for this active transport is derived chiefly from the anerobic metabolism of glucose. While the site of active transport is apparently located in the epithelial layer the question whether lens fiber membranes also have active transport mechanisms remains an unresolved problem. Although several amino acids exhibit overlapping affinities for various sites of transport, groups of compounds having structural similarities and certain stereochemical configurations show preference for a specific site of transport. The fundamental question of whether carrier-mediated system(s) for organic molecules in the lens is related directly to sodium-potassium activated ATPase also cannot be answered since the effect of ouabain on the transport of various nonelectrolytes is not always immediate as in the case of sodium and potassium ions. Similarly, further studies are needed to clarify the nature of the coupling mechanism between the transport of cations and organic molecules.