Marjorie F. Lou

Redox regulation in the lens

The high content of glutathione (GSH) in the lens is believed to protect thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size. However, ageing lenses or lenses under oxidative stress show an extensively diminished size of GSH pool with some protein thiols being S-thiolated by oxidized non-protein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC) or protein-S-S-gamma-glutamylcysteine. It was shown in an H(2)O(2)-induced cataract model that PSSG formation precedes a cascade of events before cataract formation, starting with protein disulfide crosslinks, protein solubility loss and high molecular weight aggregation. Furthermore, this early oxidative damage in protein thiols can be spontaneously reversed in H(2)O(2) pretreated lenses if the oxidant is removed in time. This dethiolation process appears to have mediated through a redox-regulating enzyme, thioltransferase (TTase), which is ubiquitously present in microbial, plant and animal tissues, including the lens. The GSH-dependent, low molecular weight (11.8 kDa) cytosolic enzyme plays an important role in oxidative defense and can modulate key metabolic enzymes in the glycolytic pathway. The enzyme repairs oxidatively damaged proteins/enzymes through its unique catalytic site with a vicinal cysteine moiety, which can specifically dethiolate protein-S-S-glutathione and restore protein free SH groups for proper enzymatic or protein functions. Most importantly, it has been demonstrated that thioltransferase has a remarkable resistance to oxidation (H(2)O(2)) in cultured human and rabbit lens epithelial cells under oxidative stress conditions when other oxidation defense systems of GSH peroxidase and GSH reductase are severely inactivated. A second repair enzyme, thioredoxin (TRx), which is NADPH-dependent, is widely found in many lower and higher life forms of life. It can dethiolate protein disulfides and thus is an extremely important regulator for redox homeostasis in the cells. Thioredoxin has been recently found in the lens and has been shown to participate in the repair process of oxidatively damaged lens proteins/enzymes. These two enzymes may work synergistically to regulate and repair thiols in lens proteins and enzymes, keeping a balanced redox potential to maintain the function of the lens.

Glutathione depletion in the lens of galactosemic and diabetic rats

Depletion of lens glutathione (GSH) occurs quickly and drastically following induction of diabetes or galactosemia in rats as well as in lens culture. The explanation for this dramatic loss of GSH has been investigated by many laboratories but the solution has been elusive. There are several possible causes for the change in the reducing power of the lens under hyperglycemia. (a) The enzyme glutathione reductase which reduces oxidized glutathione to GSH is inhibited. (b) The cofactor NADPH which both the aldose reductase of polyol pathway and glutathione reductase require becomes depleted under hyperglycemia to the point that there is an insufficient amount for glutathione reduction. (c) Membrane permeability is increased, due to osmotic-induced lens hydration. We explored all the above possibilities in the mechanism of GSH depletion and studied the effect of aldose reductase inhibitor (ARI) on osmotic change. We found that under hyperglycemic condition, there was no change in the enzyme glutathione reductase activity. There was an initial drop in NADPH level but there was sufficient remaining for glutathione reductase use. Both NADPH and glutathione depletion could be prevented completely by ARI. In addition, ARI could also prevent any hyperglycemic-induced abnormal transport and leakage of amino acids. We have therefore concluded that only the decreased membrane transport of amino acids which are needed for glutathione biosynthesis and the simultaneous loss of GSH through leaky membrane as initiated by the polyol pathway can be responsible for the drastic GSH depletion.

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein-thiol mixed disulfides in human lens☆

Protein-thiol mixed disulfide formation has been implicated as a possible mechanism for the protein-protein aggregation in cataractogenesis. Previously we have found that two species of thiols are bound to proteins: GSH (PSSG) and cysteine (PSSC). In this study we found these molecules are ubiquitous in animal lenses with the highest levels in human, dog and rat, and lowest in monkey. However, the relative amount of PSSG to PSSC is quite different in each animal species. The ratio of PSSG/PSSC was 1/10 in rat lens, 4/1 in human and dog lenses and 2/1 in monkey lens. We also studied the effect of aging on the protein-thiol mixed disulfide levels in human donor lenses between 3 months and 88 years. Lens GSH levels were inversely related to age, similar to earlier reports, but PSSC levels increased linearly with age. PSSG levels showed a triphasic pattern with an initial sharp and linear increase from a low content in infants to a highest level at age 20; fell back about 50% to a new steady state level that was maintained for four more decades; finally, above 60 years, the levels in some lenses were two to three-fold higher while some lenses remained at the same low value. PSSC in human lens appeared to concentrate in the nuclear region and in the water insoluble proteins while PSSG was more evenly distributed. Besides the aging effect on the protein-thiol mixed disulfides, oxidative stress also potentiated protein modification in the human lens.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing.

EGF is an essential growth factor needed for epithelial cell proliferation and wound healing of the cornea, but the molecular mechanism is not understood. Although studies have shown that EGF in some non-phagocytic cells induces ROS generation, little is known about the role of ROS in corneal epithelial cells. Therefore, we examined the potential physiological role of ROS in corneal cell proliferation, adhesion and wound healing using rabbit or human corneal epithelial cells, and pig whole cornea organ culture as models. EGF (5 ng/ml)-induced ROS in serum-starved RCE or HCE cells were captured as DCFH fluorescence and detected by confocal microscopy. The elevation of ROS was eradicated when the cells were pretreated with an antioxidant N-acetylcysteine (NAC) or mannitol, or with inhibitor to NADPH oxidase (DPI), or to lipoxygenase (NDGA). EGF-induced ROS generation correlated with cell growth and activation of Akt and MAPK signaling pathways, while NAC eliminated all these effects. EGF-stimulated cell adhesion or migration in cell culture was greatly suppressed in the presence of NAC while EGF-facilitated epithelial cell wound healing in corneal organ culture was also blocked by NAC. This is the first demonstration of a novel ROS physiological function in corneal wound healing.

The role of protein-thiol mixed disulfides in cataractogenesis

Protein-thiol mixed disulfides in lenses have been implicated in the mechanism of protein-protein disulfide and other cross-linking leading to protein aggregation. The methodology for the detection and quantitation of protein-thiol mixed disulfides has been successfully established in our laboratory. Examination of mixed disulfides at different stages during development of a cataract may give relevant information on the mechanism of cataractogenesis, and whether oxidation is a part of that mechanism. In this study we investigated the involvement of mixed disulfides in cataract formation by using the H2O2-exposed lens as a model. Rat lenses, after being exposed to 0.5 mM H2O2 in culture showed an inverse relationship between the GSH loss and the protein-GSH formation with no effect on the protein-cysteine level. The H2O2-induced protein modification was also demonstrated indirectly by isoelectric focusing. The rate of protein-GSH production is dependent on the time of exposure and the concentration of H2O2. Age also plays some role as the lens GSH level decreases and the protein-thiol mixed disulfides increase as the animal becomes older. Lenses of older rats did not display more susceptibility to H2O2-induced mixed disulfide formation. The two protein-thiol mixed disulfides have a well-defined pattern of distribution in the rat lens. Most of the protein-GSH was found in the cortex and the water-soluble protein fraction whereas more protein-cysteine was found in the nucleus and water-insoluble protein fraction. Lens of older rat has more protein-cysteine as well as more water-insoluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Up-regulation of K+ channels in diabetic rat ventricular myocytes by insulin and glutathione

OBJECTIVE The cardiac pathogenesis of diabetes mellitus involves oxidative stress that elicits profound changes in myocardial glutathione, an endogenous regulator of cell function. This study examined the role of glutathione in regulating K(+) channel activity in isolated ventricular myocytes from diabetic rats and its relationship to insulin signaling. METHODS AND RESULTS Colorimetric analysis of extracts of ventricular tissue from Sprague-Dawley rats showed that the basal level of reduced glutathione (GSH) was significantly less in rats with experimental diabetes compared with sham controls, consistent with oxidative stress conditions. This change in GSH status paralleled a significant decrease in the activity of gamma-glutamylcysteine synthetase, a major pathway involved in GSH homeostasis. Voltage-clamp studies confirmed that, compared with control myocytes, K(+) channels carrying the transient outward current (I(to)) are down-regulated in the diabetic state and that this electrophysiological change is reversed by in vitro treatment with insulin for 2-3 h. Incubation of diabetic rat myocytes with GSH also normalized I(to) density compared with untreated myocytes, but with a longer time course than insulin. To determine if up-regulation of I(to) by insulin was mediated by alterations in myocyte GSH, insulin-responsiveness of diabetic rat myocytes was tested in the presence of 1,3-bis-chloroethyl-nitrosourea, an inhibitor of glutathione reductase, or buthionine sulfoximine, a blocker of gamma-glutamylcysteine synthetase. Neither blocker alone altered I(to) density in diabetic rat myocytes when compared with untreated cells, but each blocked the effect of insulin to up-regulate I(to). CONCLUSIONS These data suggest that oxidative stress-induced alteration in GSH redox state plays an important role in regulating I(to) channel function and that GSH homeostasis in ventricular myocytes is functionally coupled to insulin signaling.

miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H2O2) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H2O2. These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.

Roles of the 15-kDa selenoprotein (Sep15) in redox homeostasis and cataract development revealed by the analysis of Sep 15 knockout mice.

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.

The dual effect of oxidation on lipid bilayer structure.

Sphingomyelin membranes were prepared with different levels of oxidative damage caused bytert-butyl hydroperoxide (TBH). Temperature-induced changes in membrane hydrocarbon chain packing (phase transitions) were monitored using infrared spectroscopy. Lipid phase transition characteristics were evaluated from thermodynamic parameters fitted to the experimental transition curve data. At temperatures below the lipid phase transition Tc, hydrocarbon chains pack in an ordered state whereas above the Tc the hydrocarbonchains pack in a disordered state. Compared to the non-oxidized control, the packing of the hydrocarbon chains of mildly oxidized sphingomyelin (<10 nmol TBH/mg lipid) was no different at all temperatures below the Tc, and was more ordered above the Tc. The hydrocarbon chains of strongly oxidized sphingomyelin (>10nmol TBH/mg lipid) were more disordered at temperatures above and below the Tc compared to the control samples. These results suggest that lipid oxidation has a dual effect on lipid order. A more ordered or disordered state may result depending on the degree of oxidation and the state of lipid order prior to oxidation. These results could be important for explaining the structural changes in oxidized membranes high in sphingomyelin such as those found in the ocular lens and liver plasma membranes.