Lovastatin-induced cytoskeletal reorganization in lens epithelial cells: role of Rho GTPases.

PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells. METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis. RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin. CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.

The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper.

Oxidative effects on lens proteins have been linked with the formation of human age-related cataract, particularly nuclear cataract. This study investigated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on nuclear and cortical alpha-, beta- and gamma-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses were treated with 50 atm of either 100% N(2)(control) or 100% O(2)(experimental) for 3, 6, 16 and 48 hr. The levels of reduced glutathione (GSH) and water-soluble (WS) protein decreased more rapidly in the nucleus of the O(2)-treated lens than in the cortex. The first significant loss of WS protein in each of the two regions occurred when levels of GSH had decreased by at least 90% in either the nucleus (at 6 hr) or the cortex (at 16 hr). HPLC analysis of the nuclear WS proteins indicated that beta-crystallins were the first proteins affected by the oxidative stress. Soon after HBO-treatment was initiated (at 6 hr) and prior to insolubilization of protein, nuclear beta- and gamma-crystallins moved to the higher molecular weight alpha-crystallin fraction; 2-D gel electrophoresis and Western blotting indicated the presence of disulfide-crosslinked and non-crosslinked beta- and gamma-crystallins in this fraction. Significantly different HBO-induced effects were observed on lens cortical crystallins compared to those for the nucleus. For example, gamma-crystallins in the cortex shifted very soon after HBO-treatment (at 3 hr) to slightly higher molecular weights, possibly the result of protein/glutathione mixed disulfide formation; however, this phenomenon was not observed in the nucleus. Cortical beta- and gamma-crystallins remained in solution longer than nuclear proteins following HBO-treatment of the lenses, presumably the result of protection from the four-fold higher level of GSH (22 vs 6 m M) present in the lens periphery. Surprisingly, there was no movement of beta- and gamma-crystallins to alpha(H)- and alpha-crystallin fractions in the cortex of the O(2)-treated lens, in contrast to that observed for the nucleus. Cortical crystallins appeared to go directly from being soluble to being insoluble with no high molecular weight intermediate stage. The data suggested a possible chaperone-like function for alpha-crystallin in the nucleus of the stressed lenses, but not in the cortex. HBO-induced effects on lens nuclear supernatants, which mimicked those observed for intact lenses, could be nearly completely prevented by the copper-chelator bathocuproine, but not by the iron-chelator deferoxamine. Overall, the results provide additional evidence demonstrating an increased susceptibility of the lens nucleus to oxidative stress; the greater protective ability of the cortex may be linked to a higher capacity for beta- and gamma-crystallin/glutathione mixed disulfide formation, inhibiting disulfide-crosslinked insolubilization. The data also implicate copper as a catalyst for the autoxidation of -SH groups in the lens, and suggest that alpha-crystallin chaperone-like activity may play a greater role in the lens nucleus than in the cortex in preventing oxidative insolubilization of crystallins.

Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter.

PURPOSE: To measure lipid compositional and structural changes in lenses as a result of hyperbaric oxygen (HBO) treatment in vivo. HBO treatment in vivo has been shown to produce increased lens nuclear light scattering. METHODS: Guinea pigs, approximately 650 days old at death, were given 30 and 50 HBO treatments over 10- and 17-week periods, respectively, and the lenses were sectioned into equatorial, cortical, and nuclear regions. Lipid oxidation, composition, and structure were measured using infrared spectroscopy. Phospholipid composition was measured using (31)P-NMR spectroscopy. Data were compared with those obtained from lenses of 29- and 644-day-old untreated guinea pigs. RESULTS: The percentage of sphingolipid approximately doubled with increasing age (29-544 days old). Concomitant with an increase in sphingolipid was an increase in hydrocarbon chain saturation. The extent of normal lens lipid hydrocarbon chain order increased with age from the equatorial and cortical regions to the nucleus. These order data support the hypothesis that the degree of lipid hydrocarbon order is determined by the amount of lipid saturation, as regulated by the content of saturated sphingolipid. Products of lipid oxidation (including lipid hydroxyl, hydroperoxyl, and aldehydes) and lipid disorder increased only in the nuclear region of lenses after 30 HBO treatments, compared with control lenses. Enhanced oxidation correlated with the observed loss of transparency in the central region. HBO treatment in vivo appeared to accelerate age-related changes in lens lipid oxidation, particularly in the nucleus, which possesses less antioxidant capability. CONCLUSIONS: Oxidation could account for the lipid compositional changes that are observed to occur in the lens with age and cataract. Increased lipid oxidation and hydrocarbon chain disorder correlate with increased lens nuclear opacity in the in vivo HBO model.

Hyperbaric oxygen in vivo accelerates the loss of cytoskeletal proteins and MIP26 in guinea pig lens nucleus.

Previous studies have shown that treatment of guinea pigs with hyperbaric oxygen (HBO) produces certain changes in the lens nuclei of the animals which are typical of those occurring during aging. These include an increase in nuclear light scattering (NLS), elevation in levels of oxidized thiols, loss of water-soluble protein and damage to nuclear membranes. The present study investigated the effect of HBO-treatment in vivo on lens cytoskeletal proteins and MIP26 which are also known to undergo alteration with age. Young (2-month-old) and old (18-month-old) guinea pigs were treated 15 and 30 times with HBO (3 times per week with 2.5 atmospheres of 100% oxygen for 2.5 hr periods). SDS-PAGE and Western blotting showed that HBO-treatment of the older animals accelerated the age-related loss of five nuclear cytoskeletal proteins including actin, vimentin, ankyrin, alpha-actinin and tubulin, compared to levels present in age-matched controls (effects on spectrin and the beaded filaments were not investigated in this study). Treatment of the young animals with HBO produced losses which were primarily associated with concentrations of the nuclear alpha- and beta-tubulins; these cytoskeletal proteins were observed to be most sensitive to the induced oxidative stress, and were affected earliest in the study. Disulfide-crosslinking, rather than proteolysis, appeared to be the main cause of the HBO-induced cytoskeletal protein loss (elevated levels of calcium, which might have induced proteolysis, were not found in the experimental nuclei). Loss of MIP26 was observed only in the older guinea pigs treated 30 times with HBO; both disulfide-crosslinking and degradation to MIP22 were associated with the disappearance. Thus, nuclear MIP26 was susceptible to oxidative stress, but less so than the cytoskeletal proteins, particularly the tubulins. No cortical effects on either MIP26 or the cytoskeletal proteins were observed under any of the treatment protocols. No direct link was observed between an HBO-induced increase in NLS (observed in both the young and old animals using slit-lamp biomicroscopy) and losses of either MIP26 or the cytoskeletal proteins. The appearance of HBO-induced nuclear opacity without any change in the levels of nuclear sodium, potassium or calcium is similar to that observed previously for human senile pure nuclear cataracts. The results provide additional evidence that molecular oxygen can enter the nucleus of the lens and promote age-related events. The observed effects on MIP26 and the cytoskeletal proteins are indicative of an increased level of lens nuclear oxidative stress in the HBO model, possibly a precursor to nuclear cataract.

Nitric oxide synthesis in retinal photoreceptor cells.

Nitric oxide (NO) is known to be synthesized in several tissues and to increase the formation of cyclic GMP through the activation of soluble guanylate cyclases. Since cyclic GMP plays an important role in visual transduction, we investigated the presence of nitric oxide synthesizing activity in retinal rod outer segments. Bovine rod outer segments were isolated intact and separated into membrane and cytosolic fractions. Nitric oxide synthase activity was assayed by measuring the conversion of L-arginine to L-citrulline. Both membrane and cytosolic fractions were active in the presence of calcium and calmodulin. The activity in both fractions was stimulated by the nitric oxide synthase cofactors FAD, FMN, and tetrahydrobiopterin and inhibited by the L-arginine analog, L-monomethyl arginine. The Km for L-arginine was similar, about 5 microM for the enzyme in both fractions. However, the two fractions differed in their calcium/calmodulin dependence: the membrane fraction exhibited basal activity even in the absence of added calcium and calmodulin while the cytosolic fraction was inactive. But the activity increased in both fractions when supplemented with calcium/calmodulin: in membranes from about 40 to 110 fmol/min/mg of protein and in the cytosol from near zero to about 350 fmol/min/mg of protein in assays carried out at 0.3 microM L-arginine. The two enzymes also responded differently to detergent: the activity of the membrane enzyme was doubled by Triton X-100 while that of the cytosolic enzyme was unaffected. These results show that NO is produced by cytosolic and membrane-associated enzymes with distinguishable properties.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

The effect of hypertonicity on aldose reductase, alpha B-crystallin, and organic osmolytes in the retinal pigment epithelium.

PURPOSE: Aldose reductase (AR), an enzyme implicated in diabetic complications of ocular tissues, has been suggested to play a physiologic role in kidney and, possibly, other tissues by elevating the organic osmolytes in conditions of heightened extracellular tonicity. Hypertonicity has been shown to induce AR and alpha B-crystallin in some cells. To examine if similar mechanisms are operating in the retinal pigment epithelium (RPE), another target tissue of diabetic complications, we studied the effect of hypertonic media on the induction of AR, alpha B-crystallin, myoinositol, taurine, and other free amino acids. METHODS: Human RPE cells were cultured in normal and hypertonic media containing 150 mmol/l NaCl or 200 mmol/l cellobiose in combination with 30 mmol/l galactose from 0-8 days. Western blot analysis with antibodies were used to measure the expression of AR and alpha B-crystallin. Hybridization of northern blots using AR and alpha B-crystallin complementary DNA probes were employed for the measurement of the respective messenger RNA for these proteins. Changes in the levels of myoinositol, galactitol, taurine, and other free amino acids were determined biochemically. RESULTS: AR and alpha B-crystallin messenger RNA levels rose 16-fold and 4-fold, respectively, when human RPE cells were cultured for 3 days in media supplemented with either 150 mmol/l NaCl or 200 mmol/l cellobiose. AR and alpha B-crystallin protein levels also increased significantly, as seen by western blots. Consistent with the increased AR, galactitol accumulated to a greater extent when human RPE cells were grown in media containing 30 mmol/l galactose plus 150 mmol/l NaCl compared with cells grown in 30 mmol/l galactose alone. An 11-fold increase in cellular myoinositol and a 1.4-fold increase in taurine was observed in cells exposed to hypertonic media. CONCLUSIONS: These findings suggest that human RPE cells are responsive to hypertonic stress by elevating AR activity and use intracellular organic solutes in an interactive manner to help regulate intracellular tonicity.

Daptomycin compared with teicoplanin and vancomycin for therapy of experimental Staphylococcus aureus endocarditis.

The efficacies of daptomycin, teicoplanin, and vancomycin were compared in the therapy of experimental Staphylococcus aureus endocarditis. Rabbits infected with either of two methicillin-susceptible strains (SA-12871 or its moderately teicoplanin-resistant derivative SA-12873) or a methicillin-resistant S. aureus strain (MRSA-494) were treated with daptomycin, 8 mg/kg of body weight, every 8 h; teicoplanin, 12.5 mg/kg (low-dose teicoplanin [teicoplanin-LD], excluding MRSA-494) or 40 mg/kg (high-dose teicoplanin [teicoplanin-HD]) every 12 h; or vancomycin, 17.5 mg/kg every 6 h, for 4 days. Compared with no treatment daptomycin, teicoplamin-HD, and vancomycin significantly reduced bacterial counts of all test strains in vegetations and renal and splenic tissues (P less than 0.001). Teicoplanin-LD was equally effective against SA-12871 but failed against SA-12873, with three of six animals still being bacteremic at the end of therapy. For SA-12871, daptomycin was as effective as teicoplanin-HD and was superior to teicoplanin-LD and vancomycin (P = 0.02) in lowering vegetation bacterial counts. There were no differences between daptomycin, teicoplanin-HD, or vancomycin in the reduction of bacterial counts in tissues for any of the test strains. In rabbits infected with SA-12871, vegetations from 33% of teicoplanin-LD-treated, 6% of teicoplanin-HD-treated, and 13% of daptomycin-treated animals yielded organisms for which there were up to eightfold increases in the MICs. Resistance may have contributed to early death in one daptomycin-treated animal. No increases in the MICs for the test strain were detected in animals infected with SA-12873 or MRSA-494. We conclude that in this model and against these strains of S. aureus, daptomycin and teicoplanin-HD are as efficacious as vancomycin, but diminished susceptibility to both can develop during therapy.

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)

Exposure of rabbit lens to hyperbaric oxygen in vitro: regional effects on GSH level.

Previous studies have indicated that in vivo exposure to hyperbaric O2 may be associated with the development of nuclear cataract. In the present work, in vitro effects of hyperbaric O2 on rabbit lenses were investigated following culture of the lenses in an atmosphere of 99% O2 at pressures ranging between 1 and 100 atm. Treatment with O2 resulted in a significant decrease in the level of reduced glutathione (GSH) in the lenses even at the lower pressures studied (less than 8 atm). At 100 atm O2 the loss of GSH was 85% after a 3 hr exposure. At 8 atm O2 a significant drop in GSH concentration was shown to occur in the lens nucleus prior to loss of the tripeptide in the superficial cortex. O2-treated lenses became hazy in appearance, especially at the higher pressures, but did not become densely opaque. Pressures of N2 up to 100 atm had no effect on either lens transparency or on the concentration of GSH. Although oxidized glutathione (GSSG) was detected in the whole lens at pressures of O2 as low as 4 atm, no change in GSH level or evidence for GSSG accumulation was observed in the capsule-epithelium of the lens at pressures as high as 50 atm O2. Ninety percent of the GSSG present in lenses after exposure to 100 atm O2 could be reconverted to GSH by subsequent culture of the lenses under normal conditions. Exposure of lenses to 50 atm O2 produced a three-fold stimulation of hexose monophosphate shunt activity, equal to that which has been reported for treatment of lenses with 0.06 mM H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)