Oxidative modification of lens crystallins by H2O2 and chelated iron

Crystallins are the soluble structural proteins that constitute approximately 90% of the dry mass of the eye lens. The present study attempts to elucidate possible mechanisms whereby the H2O2 present in the eye could contribute to the oxidative modification of lens crystallins. The data indicate that exposure of solutions of crystallins to H2O2 and EDTA-chelated iron leads to covalent crosslinking of polypeptides, loss of intrinsic protein fluorescence, and the generation of a novel fluorophor emitting in the 420 nm range. These changes closely mimic oxidative modifications that occur in lens proteins in vivo. Exposure of the proteins to H2O2 in the absence of chelated iron failed to generate detectable levels of these modifications. These findings are contrasted with earlier studies of lenses in organ culture where H2O2 alone produced marked damage while the further addition of chelated iron protected the lenses from oxidation.     

Crystalline lens induction of lipid peroxidation

The level of lipid peroxidation products (LPP) was determined in the aqueous humor from the anterior chamber of patients with cataract and donor eyes. The content of LPP in senile cataract aqueous humor was shown to be significantly increased. To determine the possible mechanism of LPP increase in aqueous humor, human lenses at different stages of cataract as well as transparent human and rabbit lenses were incubated for 3 hours in 3.0 ml medium containing liposomes (0.5 mg/ml) prepared from phospholipids from the egg yolk and 0.14 M NaCl + 0.01 M TRIS-HCl buffer, pH 7.4). Corrections were made for phospholipid autooxidation. The level of LPP accumulation in the medium was determined by MDA assay. The rate of LPP production increased significantly in transparent lenses and in early senile cataract, as compared to controls and advanced (mature) cataracts. EDTA (1 mM), superoxide dismutase (114 u/sample), catalase (900 u/sample), chelated iron (III): Fe3+-ADP addition to the incubation medium depressed the level of LPP accumulation. This suggests the participation of Fe2+, O2-., H2O2 in the mechanism of LPP production in the lens. The induction of lipid peroxidation in the lens can be significant for leukotriene and prostaglandin synthesis in the eye.     

Volume regulation in lens epithelial cells and differentiating lens fiber cells

Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cells triggers a regulatory volume decrease (RVD) that is usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H2O water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 mM sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypo- or hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.     

Formation of hydrogen peroxide by lens proteins: protein-derived hydrogen peroxide as a potential mechanism of oxidative insult to the lens.

The exposure of dialyzed preparations of lens crystallins to copper (II) ions causes a decrease in protein surface thiol and the production of hydrogen peroxide (H2O2). H2O2 production by gamma and beta crystallin subfractions (which contain the greatest level of thiol) is the predominant source of this H2O2. Protein surface thiols are probable sources of H2O2 formation since N-ethyl maleimide treatment of lens proteins and zinc ions inhibit H2O2 production. These data are consistent with a hypothesis that transition metal-catalyzed oxidation of protein contributes to cataractogenic lens protein oxidations.     

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.     

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.     

Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells.

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.     

Protective effects of selenium, vitamin C and vitamin E against oxidative stress of cigarette smoke in rats

Cataractous lenses have been found to have an altered distribution of the intracellular ionic environment: the concentrations of potassium and magnesium being decreased and the concentrations of sodium and calcium increased. These changes arise as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. In this study flame atomic absorption spectroscopy (AAS) was used for calcium, magnesium, iron and zinc determination, and flame atomic emission spectroscopy (AES) was used for sodium and potassium contents in normal and cigarette smoke-exposed rat lenses. The methods are sensitive enough to detect quantitatively all six cations in a single rat lenses. In this work, six elements, including Ca2+, K+, Na+, Zn2+, Fe2+ and Mg2+ in experimental rat eye lenses and normal transparent lenses were determined. It was found that the concentrations of Ca2+, Na+, Zn2+, and Fe2+ were increased dramatically while K+ and Mg2+ decreased in smoke-exposed rat lenses when compared to the control rat lenses. There were no significant changes between 'smoked' rats supplied with vitamin C and control groups. A positive correlation was found also in the other two groups of 'cigarette smoked' animals supplemented with selenium plus vitamin E and selenium when compared with 'cigarette smoked' without any supplements. These data provide support for the hypothesis that cigarette smoking increases the risk of cataract formation. We investigated whether vitamin C is the most important antioxidant in the body. The roles of diet with optimum amounts of antioxidant vitamins C and vitamin E and the antioxidant mineral selenium are discussed.     

Visualizing ocular lens fluid dynamics using MRI: manipulation of steady state water content and water fluxes

Studies using various MRI techniques have shown that a water-protein concentration gradient exists in the ocular lens. Because this concentration is higher in the core relative to the lens periphery, a gradient in refractive index is established in the lens. To investigate how the water-protein concentration profile is maintained, bovine lenses were incubated in different solutions, and changes in water-protein concentration ratio monitored using proton density weighted (PD-weighted) imaging in the absence and presence of heavy water (D(2)O). Lenses incubated in artificial aqueous humor (AAH) maintained the steady state water-protein concentration gradient, but incubating lenses in high extracellular potassium (KCl-AAH) or low temperature (Low T-AAH) caused a collapse of the gradient due to a rise in water content in the core of the lens. To visualize water fluxes, lenses were incubated in D(2)O, which acts as a contrast agent. Incubation in KCl-AAH and low T-AAH dramatically slowed the movement of D(2)O into the core but did not affect the movement of D(2)O into the outer cortex. D(2)O seemed to preferentially enter the lens cortex at the anterior and posterior poles before moving circumferentially toward the equatorial regions. This directionality of D(2)O influx into the lens cortex was abolished by incubating lenses in high KCl-AAH or low T-AAH, and resulted in homogenous influx of D(2)O into the outer cortex. Taken together, our results show that the water-protein concentration ratio is actively maintained in the core of the lens and that water fluxes preferentially enter the lens at the poles.     

Heparin-binding properties of lactoferrin and lysozyme.

1. Binding of biotin-heparin to immobilized lactoferrin and lysozyme was optimum at pH 6.0, 100 mM NaCl. Complex interactions between NaCl and CaCl2 concentrations were observed for heparin binding to both proteins. 2. The metal ions Cu2+, Zn2+, Fe2+ and Fe3+ inhibited heparin binding, with half-maximal inhibition of binding to lactoferrin occurring between 600 microM and 1 mM and for lysozyme between 500 and 800 microM. 3. Binding of biotin-heparin to both proteins was inhibited to varying degrees by heparin, heparan sulfate, chondroitin sulfate A, dextran sulfate and DNA.     

Effect of saline acclimation on body water and sodium compartmentalization in Pekin ducks (Anas platyrhynchos)

The compartmentalization of body fluids was measured in individual Pekin ducks ( Anas platyrhynchos) drinking freshwater and after sequential acclimation to 300 mM NaCl and 400 mM NaCl. Total body water, extracellular fluid volume, plasma volume and exchangeable sodium pool were measured using (3)H(2)O, [(14)C]-polyethylene glycol, Evans Blue dye, and (22)Na dilution, respectively. Following acclimation to 300 mM NaCl, body mass decreased, but total body water and total exchangeable sodium pool were unaltered. Na and water were redistributed from the extracellular fluid (interstitial fluid) compartment into the intracellular fluid compartment. Following further acclimation to 400 mM NaCl, body mass, total body water and intracellular fluid volume decreased, but exchangeable sodium pool and extracellular fluid volume were unchanged. Our results suggested that, when Pekin ducks drink high but tolerable salinities, they maintain total body water, but redistribute Na(+) and water from interstitial fluid to the intracellular fluid compartment. When stressed beyond their ability to maintain total body water, they lose water from the intracellular fluid.     

Interactions of iodide ions with isolated photosystem 2 particles

The effects of I- ions on O2 evolution by photosystem 2 particles, which were depleted of the 18-kDa and the 23-kDa extrinsic proteins of the O2 evolution complex by NaCl washing (dPS2 particles) were examined. In the absence of Cl- (incompetent dPS2) I- stimulated O2 evolution up to 3-6 mM, depending on the associated cation, and inhibited it at higher concentrations. In the presence of Cl- (competent dPS2), I- was inhibitory at all concentrations. The inhibition was reversible, it occurred at a site preceding Tyrz (Tyr residue mediating electron transfer from H2O to photosystem 2), and it interfered noncompetitively with the reactivation of incompetent dPS2 with Cl-. Furthermore, the organic salts tetrabutyl ammonium iodide and tetraphenyl phosphonium iodide proved to be stronger inhibitors than the inorganic NaI. This is interpreted as an indication of a negatively charged surface, situated behind a hydrophobic permeability barrier. Permeant organic cations, being better compensators of the inner surface charge than Na+, are also more apt in facilitating access of the I- ions to the inhibitory site in the vicinity of Tyrz.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Ionic composition of the rectal contents and excreta of the reduviid bug Triatoma infestans

We have investigated the chemical composition of the rectal contents, faeces and urine of the blood-sucking bug Triatoma infestans. This is the environment in which the important disease-causing organism, Trypanosoma cruzi, lives. Directly after feeding of Triatoma infestans, the pH of the excreta switched from an acidic to an alkaline pH and, 1 day later, back to a slightly acidic pH. The osmolality varied in the initial excreta and in the rectal contents on the day following the meal between 300 and 460 mosmol/kg H(2)O, but after an additional day it increased to 350-970 mosmol/kg H(2)O. Determinations by ion capillary electrophoresis showed that sulphate and phosphate dominated the rectal contents in unfed bugs. After feeding, the first four drops of fluid excreta were mainly a sodium chloride solution (>150 mM for each). One to 10 days after feeding strong individual variations in the concentrations of individual ions were evident, especially for potassium and sodium. Mean concentrations of chloride remained at about 70 mM; sulphate and phosphate showed an increase within the first 1 or 2 days and then reached a level of about 160 and 210 mM, respectively. The rectal contents of long-term starved bugs contained high concentrations of phosphate and potassium; sulphate and sodium were slightly lower.     

Effects of deionized water on viability of the zebra mussel,Dreissena polymorpha

1. Zebra mussels exposed to deionized water (DW, changed daily) begin to die within several days. More than half the animals die in DW within a week.2. Animals allowed to reattach themselves prior to exposure to DW live somewhat longer than detached animals exposed to DW but are still sensitive to the toxic effects of DW.3. Small animals die in response to DW faster than large animals.4. The toxic effect of DW can be suppressed by addition of 0.5 mM NaCl, 0.25 mM MgSO₄, or small amounts of aquarium water.5. KCl is about 10 times more toxic to zebra mussels when applied in DW than when applied in aquarium water.6. The toxic effects of 37 mg KCl/1 DW are not prevented by the addition of 0.5 mM NaCl or 0.25 mM MgSO₄.7. Stimulation of potassium transport mechanisms by DW may account for the increased toxicity of KCl in DW. Toxic effects of DW and increased toxicity of potassium in DW may have applications in controlling zebra mussel infestations.     

Protein-bound kynurenine is a photosensitizer of oxidative damage

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.     

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment.

Single and multiple quantum nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the motional dynamics of sodium and potassium ions in concentrated protein solution, represented in this study by cortical and nuclear bovine lens tissue homogenates. Both ions displayed homogeneous biexponential magnetic relaxation behavior. Furthermore, the NMR relaxation behavior of these ions in lens homogenates was consistent either with a model that assumed the occurrence of two predominant ionic populations, "free" and "bound," in fast exchange with each other or with a model that assumed an asymmetric Gaussian distribution of correlation times. Regardless of the model employed, both ions were found to occur in a predominantly "free" or "unbound" rapidly reorienting state. The fraction of "bound" 23Na+, assuming a discrete two-site model, was approximately 0.006 and 0.017 for cortical and nuclear homogenates, respectively. Corresponding values for 39K+ were 0.003 and 0.007, respectively. Estimated values for the fraction of "bound" 23Na+ or 39K+ obtained from the distribution model (tau C greater than omega L-1) were less than or equal to 0.05 for all cases examined. The correlation times of the "bound" ions, derived using either a two-site or distribution model, yielded values that were at least one order of magnitude smaller than the reorientational motion of the constituent lens proteins. This observation implies that the apparent correlation time for ion binding is dominated by processes other than protein reorientational motion, most likely fast exchange between "free" and "bound" environments. The results of NMR visibility studies were consistent with the above findings, in agreement with other studies performed by non-NMR methods. These studies, in combination with those presented in the literature, suggest that the most likely role for sodium and potassium ions in the lens appears to be the regulation of cell volume by affecting the intralenticular water chemical potential.     

Divalent metal cation binding properties of human prothymosin alpha.

The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.     

Determination of calcium, sodium, potassium and magnesium concentrations in human senile cataractous lenses

Cataractous lenses have been found to have a distribution of the intracellular ionic environment, the concentrations of potassium and magnesium decreasing and the concentrations of sodium and calcium increasing relative to the cytosol of most cells. This arises as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. These changes have been found to be initiated as a result of normal ageing of the human lens. In this study, total Ca2+, K+, Na+ and Mg2+ contents have been determined in human normal and cataractous lenses using atomic absorption and flame emission spectroscopy. The normal human lens Ca2+ is between 0.15 and 0.5 miromol g(-1) fresh lens weight; in senile cataracts the value increased up to 9.31 micromol g(-1) ( p < 0.0001). The normal levels of Na+, Mg2+ and K+ are 20, 5.5 and 60 micromol g(-1) respectively; these changed to 136.10, 3.60 and 9.33 micro mol g(-1), respectively in cataractous senile human lenses ( p < 0.002, p < 0.002 and p < 0.01). The remarkable differences in these elements may play some role in cataractogenesis.