Kenneth R. Hightower

The role of the lens epithelium in development of UV cataract

In view of renewed interest in the lens epithelium as the initiation site for cataract development, it seemed timely to review recent studies which appear to establish UV damage in the lens epithelium as the cause of UV cataract. While UV photons can and do interact with lens proteins in the cortex and nucleus, experimental results from cultured lenses and tissue cultured epithelial cells also demonstrate both mutagenic and cytotoxic effects in the epithelium. This minireview examines UV-induced changes in lens physiology that appear to follow epithelial cell damage, including inactivation of critical enzymes of transport and metabolic processes. Changes in membrane function include altered cation transport, increased permeability, and altered biosynthesis. One potential scenario for the propagation of damage from the epithelium to the underlying fiber cells includes calcium elevation, an early event in cataract development and critical to many physiological processes.

Cytotoxic effects of internal calcium on lens physiology: A review

While calcium is possibly involved in cataractogenesis, it is unquestionably involved in normal lens physiology. Numerous reports have documented the many cellular processes in other tissues affected by alterations in cellular levels of calcium. The homeostasis of the lens is no less dependent on the critical balance of intracellular calcium. With advances being made in calcium-sensitive microelectrodes and pioneering studies progressing in ion channel electrophysiology, interest in calcium metabolism in the lens has been intensified. This report is an attempt to review recent findings that deal solely with biochemical changes resulting from calcium imbalances in the lens interior.

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.

Mechanisms involved in cataract development following near-ultraviolet radiation of cultured lenses

Cultured rabbit lenses were irradiated with UV (311 nm peak; 295-340 nm) for 30 to 60 min. The entire spectrum lies in the near-UV, the major component is UVB, with a minor portion (25%) of UVA, and is henceforth referred to as near-UV(B). Posterior irradiation caused no cataract and no significant ionic imbalances compared to anterior irradiation, which caused opacification and marked changes in sodium and calcium concentrations. Anterior irradiation also resulted in reduced Na/K-ATPase activity in the epithelium. ATPase activity was not immediately inhibited; rather, only after culture was enzyme activity reduced. The concentration of reduced glutathione (GSH) decreased rapidly in the epithelium and more slowly in the underlying lens fibers. Loss of GSH was more rapid and extensive when irradiation occurred in the presence of oxygen. Irradiation under anaerobic conditions resulted in opacification but was considerably less extensive than when irradiation of lenses occurred in the presence of 7% oxygen. Near-UV(B) damage following anaerobic irradiation and 20 hrs of culture resulted in an increase in sodium levels and loss of GSH; calcium levels were not significantly elevated. Since irradiation of tryptophan solutions produced small amounts of hydrogen peroxide, the possibility of hydrogen peroxide-mediated damage was investigated but no role could be substantiated. Peroxide detoxification by the epithelium of near-UV(B) cataracts was observed, as measured by its ability to eliminate hydrogen peroxide added as a bolus.

MEMBRANE DAMAGE IN UV‐IRRADIATED LENSES

Abstract The purpose of this study was to investigate three possible causes of membrane damage following UV irradiation: photooxidation of membrane thiol (SH) groups, peroxidation of membrane lipids and inhibited synthesis of membrane proteins. Thiol loss was not observed. Thin‐layer chromatography showed a four‐fold increase in several primary lipid peroxidation products such as hydroperoxyl lipids in the epithelial membrane preparations isolated from irradiated lenses. The formation of new hydroxyl lipid bands not seen in control preparations was also observed in isolated membranes from irradiated lenses. Irradiation in the presence or absence of oxygen produced lipid peroxidation products. Aerobic irradiation produced small, but statistically significant increases in lipid hydroxyls and hydroperoxyls relative to controls. Repair of initial damage might be compromised by the observed 60% reduction in rate of protein synthesis measured in lens membranes following irradiation. Synthesis was affected by means other than depleted potassium or elevated calcium levels.

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm2= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm2 of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm2 of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA‐irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz. 0.3 J/cm2. Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.

Regional distribution of calcium in alloxan diabetic rabbit lens

A diabetic rabbit model was developed for investigation of cataractogenesis and other changes in the anterior segment. Rabbits were fasted, injected with 0.7 mg/kg alloxan, fed 1% glucose solution for 24 hrs and returned to a normal diet. Animals showing and maintaining blood glucose of greater than 300 mg% within two days were used in this study. Concomitant with increase in blood glucose was a rise in aqueous humor glucose and osmolality, together with a decrease in ascorbate concentration. Vacuoles and small discrete opacities developed, and in some cases, at longer time periods complete opacity of anterior or posterior aspects was found. Total calcium content of the whole lens increased up to 2-fold, especially after 60 days, and was correlated with a decrease in lens transmittance of a He/Ne laser beam and also with high osmolality of the aqueous humor. Free calcium was six-fold higher in opaque areas than clear areas, and was 100-fold higher in vacuoles. It is suggested that, in addition to the recognized role in sugar cataractogenesis of osmotic stress due to sorbitol accumulation in the lens, changes of intracellular calcium in localized areas of the lens and stresses imposed by changes in aqueous humor osmolality may also be important.

A review of the evidence that ultraviolet irradiation is a risk factor in cataractogenesis

There are two approaches to the question of whether solar radiation contributes to human cataract. The first, epidemiological studies, investigates correlations between mans environmental UV dose and cataract frequency. The second, animal models, investigates the effects of varying UV strengths and spectra on lens opacificationin vivo orin vitro. While the latter approach typically provides for direct evidence, the data must still be extrapolated to human lenses. Results of physiological studies suggest that UV photons interact with proteins of the epithelial cell membranes, in particular tryptophan residues, transport ATPases and cytoskeletal proteins. One hypothesis is that damage to ion pumps and channels accumulates over the years as repair processes incompletely restore membrane function. Peroxidative damage is likely in view of the formation of UV-induced lipid peroxides in the lens epithelial membranes. Loss of homeostatic control of ions, particularly Ca+, leads to crystallin disorder in small regions of the underlying fiber cells. In our diabetic cataract studies, intracellular Ca++ electrodes detected large shifts in intracellular Ca++ before bulk-lens changes were apparent. Similar occurrences likely characterize UV cataract. Our lab is one of few studying lens physiology and how it is altered following transient exposures to UV-B and UV-A, both of which pass through the cornea. Some changes include: loss of epithelial cell GSH; elevated Ca++; loss of membrane voltage; impaired transport of Na+; increased permeability to ions and water; inhibition of critical enzymes; and a decrease in the rate of membrane synthesis.

Valinomycin cataract: the relative role of calcium and sodium accumulation.

Evidence was obtained which suggests that valinomycin-induced cataracts in cultured rabbit lenses are the result of calcium accumulation. Lenses exposed to 10−5m-valinomycin for 20 hr develop superficial cortical opacities accompanied by an increase in lens Na+, a decrease in lens K+, and an increase in lens Ca2+. Measurable hydration is not observed. Lenses exposed to valinomycin in the presence of low concentrations of Ca2+ in the media do not accumulate excessive calcium and do not become opaque in spite of significant increase in lens sodium. On the other hand, lenses cultured in the presence of ouabain accumulate excessive amounts of Na+, lose K+, maintain normal Ca2+ levels and remain transparent. Thus, Ca2+ accumulation, rather than an imbalance in Na+ and K+, appears to be responsible for lens opacification.

Metabolic studies on calcium transport in mammalian lens

Three important findings concerning the calcium pump in a mammalian lens are reported: 1) glycolysis is sufficient to support active transport of calcium in the young rabbit lens; 2) in addition to the epithelium posterior and anterior fibers are involved in calcium transport; 3) inhibition of glycolysis and the calcium pump result in calcium accumulation and subsequent opacification. That respiration does not contribute to the energy needs of the Ca++ pump is based on results which demonstrate that cyanide, dinitrophenol, and azide have no effect on 45Ca efflux when glucose is present. Moreover, incubation of lenses for 20 hrs. in the presence of cyanide fails to alter the internal concentration of calcium. The inhibition of glucose metabolism with iodoacetate (IAA) results in the accumulation of calcium to a level of 0.71mM and the formation of superficial subcapsular opacities. Evidence that fibers are also responsible for calcium extrusion consists of two findings: 1) removal of the epithelium from a lens results in calcium accumulation but the addition of IAA to such a lens results in a further gain in calcium; 2) the accumulation of 45Ca across the posterior surface of a lens partially immersed in medium is accelerated following inhibition of glycolysis.