The effect of hydrogen peroxide on sarco/endoplasmic and plasma membrane calcium ATPase gene expression in cultured human lens epithelial cells.

The loss of calcium homeostasis in the lens of the eye appears to be a factor contributing to lens opacity. In the human lens, calcium homeostasis depends on the Ca²âº-ATPase pumps found only in the epithelium. A plasma membrane calcium pump, PMCA2 is upregulated in human cataractous lenses. To determine if oxidation caused the plasma membrane Ca²âº-ATPases (PMCA) or sarcoplasmic/endoplasmic Ca²âº-ATPases (SERCA) to become upregulated, we cultured a human lens epithelial cell line, in the presence of hydrogen peroxide. We observed an increase in PMCA1, PMCA2 SERCA2b and SERCA3 mRNA levels and protein expression with increasing hydrogen peroxide concentrations and treatment times. Hydrogen peroxide caused a rise in the intracellular calcium which could be an initiating factor in the concerted upregulation of PMCA1 and SERCA3. Our data support the idea that oxidative stress could contribute to a selective rise in PMCA/SERCA expression in human cataractous lenses.

Plasma membrane Ca2+-ATPase expression in the human lens.

The focus of the study was to characterize plasma membrane calcium-ATPase pump (PMCA) isoform expression in the human lens and cultured lens epithelial cells as a basis for future studies of calcium homeostasis in the lens. Proteins and mRNA expression were analysed using Western Immunoblotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Clear human lenses from the Kentucky Lions Eye Bank and an immortalized human lens epithelial cell line (HLE B-3) were used. RT-PCR products of PMCA1, PMCA2, and PMCA4 primers were detected at 429, 557, and 849bp, respectively. All these products were identified as PMCA isoforms by sequence analysis. Protein bands at approximately 130, 115, and 135kDa were detected by Western blot analysis for PMCA1, PMCA2 and PMCA4, respectively. PMCA3 was not detected at protein or mRNA level in any human lens sample or cell culture, but was detected in the rat brain cortex used as a control. Several bands with lower molecular weights, especially for PMCA2, were detected in the epithelial samples and probably represent break down products of PMCA2. No PMCA proteins or breakdown products were detected in the nuclear or cortical fractions from human lenses. PMCA1, 2, and 4 proteins and mRNAs are expressed in human lens epithelium and cultured epithelial cells; PMCA3 is not. PMCA was not detected at all in the lens fibre cells. The calcium pump must be selectively processed, independent of other membrane proteins such as the Na-K-ATPase pumps, because the distribution of the Na-K-ATPase pump is asymmetrical in the epithelium and present throughout the lens whereas the calcium pumps are not. The findings of this study provide a basis for further studies to examine the role and modulation of PMCA isoforms in calcium homeostasis and in the development of cataract.

Regulation of sarco/endoplasmic Ca2+ -ATPase expression by calcium in human lens cells.

Maintenance of cellular calcium levels is critical to cell function. Loss of calcium homeostasis might be a contributing factor to the development of cataract in the lens. In lens epithelium, calcium is involved in cell signaling and its precise regulation is vital. In this study, we investigated the regulation of sarco/endoplasmic reticulum Ca(2+)-ATPase2b (SERCA2b) and SERCA3 isoform expression in cultured epithelial B-3 cells from human lenses. Both mRNA and membrane proteins samples were collected for semi quantitative RT-PCR using GAPDH as a control. Western blot analyses were performed on membrane samples.Thapsigargin, a SERCA isoform inhibitor which causes increased cytosolic levels elicited dose-and time-dependent up-regulation of SERCA3 at both mRNA and protein levels; SERCA2b expression was unaffected. Both EGTA and actinomycin partially inhibited the thapsigargin-induced SERCA3 up-regulation. These results indicate that the up-regulation of SERCA3 by thapsigargin is dependent on a calcium-mediated pathway that is likely to occur at the transcription level.

Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin.

The conformational features of dihydrosphingomyelin (DHSM), the major phospholipid of human lens membranes, were investigated by 1H and 31P nuclear magnetic resonance spectroscopy. Several postulates emerge from the observed trends: (a) in partially hydrated samples of DHSM in CDCl3 above 13 mM, at which lipid-lipid interactions prevail, the amide proton is mostly involved in intermolecular H-bonds that link neighboring phospholipids through bridging water molecules. In the absence of water, the NH group is involved in an intramolecular H-bond that restricts the mobility of the phosphate group. (b) In the monomeric form of the lipid molecule, the amide proton of the major conformer is bound intramolecularly with one of the anionic and/or ester oxygens of the phosphate group. A minor conformer may also be present in which the NH proton participates in an intramolecular H-bond linking to the OH group of the sphingoid base. (c) Complete hydration leads to an extension of the head group as water molecules bind to the phosphate and NH groups via H-bonds, thus disrupting the intramolecular H-bonds prevalent at low concentrations.

Conformational studies of sphingolipids by NMR spectroscopy. II. Sphingomyelin.

Sphingomyelin (SM) is the most prevalent sphingolipid in the majority of mammalian membranes. Proton and 31P nuclear magnetic resonance spectral data were acquired to establish the nature of intra- and intermolecular H-bonds in the monomeric and aggregated forms of SM and to assess possible differences between this lipid and dihydrosphingomyelin (DHSM), which lacks the double bond between carbons 4 and 5 of the sphingoid base. The spectral trends suggest the formation of an intramolecular H-bond between the OH group of the sphingosine moiety and the phosphate ester oxygen of the head group. The narrower linewidth and the downfield shift of the resonance corresponding to OH proton in SM suggest that this H-bond is stronger in SM than in DHSM. The NH group appears to be involved predominantly in intramolecular H-bonding in the monomer. As the concentration of SM increases and the molecules come in closer proximity, these intramolecular bonds are partially disrupted and the NH group becomes involved in lipid-water interactions. The difference between the SM and DHSM appears to be not in the nature of these interactions but rather in the degree to which these intermolecular interactions prevail. As SM molecules cannot come as close together as DHSM molecules can, both the NH and OH moieties remain, on average, more intramolecularly bonded as compared to DHSM.

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.

Plasma membrane calcium ATPase gene expression in bovine lens epithelium.

Plasma membrane calcium adenosine triphosphatase (Ca(2+)-ATPase) is an energy-dependent protein responsible for transporting cytosolic calcium across the plasma membrane. Multiple plasma membrane Ca(2+)-ATPase isoforms are expressed from four genes (PMCA1-4) and alternative mRNA splicing. We have studied PMCA gene expression in bovine lens epithelium tissues by reverse transcription-polymerase chain reaction, Southern blot, and Northern blot hybridization. All four PMCA genes are expressed in the lens epithelium, the PMCA3 transcript being the most abundant. The transcripts for PMCA1, PMCA2, and PMCA4 exist in decreasing order of abundance. There is no evidence for the expression of any novel PMCA genes in bovine lens epithelium.

Expression of sarco/endoplasmic reticular Ca(2+)-ATPase in human lens epithelial cells and cultured human lens epithelial B-3 cells.

PURPOSE: Sarco/Endoplasmic Reticulum Ca(2+ )-ATPase (SER-CAs) isoforms, SERCA2b (100Kd) and SERCA3 (97Kd), are co-expressed in some non-muscle tissues. Both types of SERCAs play important roles in Ca(2+) regulation in non-muscle tissues such as brain and platelets. We tested whether these two SERCAs are present in human lens epithelial cells, human lens epithelial B-3 cells (HLE B-3 cells) and rat lens epithelial cells providing a basis for further studies the role of SERCAs in cataract formation. METHODS: Lens epithelial cells from rat lens, human lens and cultured human lens epithelial B-3 cell line (HLE B-3) were used to extract mRNA and membrane proteins. Reverse transcription polymerase chain reaction (RT-PCR) was employed to detect SERCA3 and SERCA2b mRNA using SERCA2b and SERCA3 primers. Western blotting was used to detect SERCA3 and SERCA2b proteins using anti-SERCA2b and 3 antibodies. RESULTS: A 270 bp fragment was amplified with SERCA2b primers and a 186 bp fragment amplified with SERCA3 primer in human lens epithelial cells, human lens epithelial B-3 cells and rat lens epithelial cells. Both fragments are the expected SERCA2b and SERCA3 products. The sequences of these two fragments in human lens epithelial cells are 100% and 98% homologous to the published partial SERCA2b and SERCA3 mRNA sequence, respectively. The amino acid sequences translated from both PCR products are 100% homologous to the published SERCA2b and SERCA3 sequences in GeneBank. Western blotting confirmed that the expected 100 KDa and a 97 KDa proteins are recognized by anti-SERCA2b and anti-SERCA3 antibodies. CONCLUSION: Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCAs) isoforms 2b and 3 mRNAs are expressed in human lens epithelial cells, the cultured human lens epithelial B-3 cell line (HLE B-3) and rat lens epithelial cells. SERCA2b and SERCA3 may play an important role in the regulation of calcium homeostasis in lens epithelial cells.

Alpha-crystallin/lens lipid interactions using resonance energy transfer.

Resonance energy transfer was used to study the interaction of alpha-crystallin with lens cortex lipid vesicles. The binding of alpha-crystallin to cortex lipid vesicles and the preincubation temperature dependence of the binding were confirmed. In this study, the tryptophan of alpha-crystallin was used as the energy donor, and the fluorescence probe N-(5-dimethylaminonaphthalene-1-sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (dansyl DHPE) was chosen as the energy acceptor. Lens cortex lipid vesicles were preincorporated with dansyl DHPE. Energy transfer from the tryptophan of alpha-crystallin to dansyl DHPE was found and the energy transfer efficiency was calculated. There was a higher energy transfer efficiency between alpha-crystallin and dansyl DHPE when alpha-crystallin was preincubated at 65 degrees C compared to 22 degrees C. Data confirmed the binding of alpha-crystallin to lens cortex lipid and showed that alpha-crystallin bound more closely to the surface of cortex vesicles when it was preincubated at a higher temperature. This is probably due to the exposure of hydrophobic surfaces when alpha-crystallin is preincubated at a higher temperature.

Ca(2+)-ATPase activity and lens lipid composition in reconstituted systems.

Lens lipid composition and lipid hydrocarbon chain structure change with age, region and cataract. Since the lens Ca(2+)-ATPase pump is important to the maintenance of calcium homeostasis and lens clarity, muscle sarcoplasmic reticulum Ca(2+)-ATPase was reconstituted with bovine lens lipids and dihydrosphingomyelin, the rare and major phospholipid of the human lens. Ca(2+)-ATPase activity was found to be about 5 times lower when the pump was reconstituted into dihydrosphingomyelin or lens lipids compared to native sarcoplasmic reticulum lipids. The addition of cholesterol to levels ranging from 13-53 mole%, had no affect on reconstituted Ca(2+)-ATPase activity. Ca(2+)-ATPase activity correlated with the degree of hydrocarbon chain saturation. The greater levels of saturation are a consequence of the high sphingolipid content in the reconstituted systems. These data support the hypothesis that changes in lens lipid composition or structure could affect Ca(2+)-ATPase activity in human lenses. Because the mechanisms governing Ca(2+)-ATPase activity in vivo are much more complex than in these simple reconstituted systems, this study represents an initial step in the elucidation of the relationships of endogenous membrane lipid composition-structure and function.

Effect of oxidation on Ca2+ -ATPase activity and membrane lipids in lens epithelial microsomes.

Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. The level of Ca2+ -ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.

Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes.

Membrane lipid composition varies in different tissues and species. Since a defined lipid composition is essential to the function of many membranes, the relationship between membrane lipid composition and structure was determined using infrared and Raman spectroscopy in four membranes containing a calcium pump: rabbit fast and slow twitch muscle sarcoplasmic reticulum and human and bovine lens fiber cell membranes. We found that membrane sphingolipid and phosphatidylcholine content were correlated to a decrease and increase, respectively, in the infrared lipid CH2 symmetric stretching band frequency. We interpret the change in frequency as a change in lipid hydrocarbon chain structural order. This was confirmed by Raman order parameters. The high degree of hydrocarbon chain saturation found in the variable amide chains of sphingolipids is likely to account for this correlation. Lipid phase transition temperature and cooperativity also correlated to sphingolipid and phosphatidylcholine content, and are the forces defining the order in at physiological temperature in the samples studied. Ca(2+)-ATPase caused an increase in the CH2 symmetric stretching frequency in fast twitch muscle sarcoplasmic reticulum (interpreted as an increase in hydrocarbon chain disorder), but had no effect on slow twitch muscle sarcoplasmic reticulum lipid hydrocarbon chain structure. In the natural systems studied, we find that it is the lipid hydrocarbon chain saturation that defines lipid hydrocarbon chain order.

Membrane lipid alpha-crystallin interaction and membrane Ca2+ -ATPase activities.

PURPOSE: To determine the effect of alpha-crystallin binding on lens membrane lipid characteristics and the stability of Ca2+ -ATPase activity when challenged with H2O2 or elevated temperatures. METHODS: Alpha-Crystallin binding to muscle sarcoplasmic reticulum membranes was quantified using a centrifugation protocol. Alpha-Crystallin binding to lens epithelial lipids was measured by a fluorescence energy transfer technique. Lipid phase transition temperature and lipid order was measured using fluorescence spectroscopy. Ca2+ -ATPase activity was measured using classical biochemical assays. RESULTS: The main phase transition temperatures of multilamellar vesicles composed of sphingomyelin or lipids extracted from bovine lens were 40 degrees C and 20 degrees C, respectively. In the presence of saturating amounts of alpha-crystallin, the phase transition temperature and lipid order of both sphingomyelin and lens lipid membranes remained almost the same as that without alpha-crystallin. The interaction of alpha-crystallin and lipid is likely to be restricted to the membrane surface. The binding of alpha-crystallin did not influence the oxidative or thermal inactivation of the Ca2+ -ATPase pump. CONCLUSION: Alpha-Crystallin-lens membrane binding does not protect the Ca2+ -ATPase pump from thermal derangement or oxidation by H2O2.

Acute effect of ethanol on lens cation homeostasis.

This study examined the effect of ethanol on the calcium homeostasis of the bovine lens. After acute exposure of the whole lens to physiologically related ethanol concentration, the calcium content of the lens cortex increased from 0.345 +/- 0.075 to 0.476 +/- 0.047 micromol/g (p < 0.05). In contrast, other cation levels such as sodium, potassium, and magnesium did not change. In the study of the lens calcium transport, ethanol caused an increase in the calcium permeability of the lens lipid membrane by about 12% at 30 mM ethanol. Ethanol did not alter the calcium pump activity at ethanol concentration up to 400 mM. Above 600 mM ethanol, the calcium pump was almost completely inhibited. It has been suggested that moderate to heavy alcohol consumption is a risk factor for cataracts. This study indicates that acute ethanol exposure can cause a loss in the lens calcium homeostasis, which maybe one of the cellular mechanisms to contribute to the cataract development in the alcoholic individual.

Temperature induced structural changes of beta-crystallin and sphingomyelin binding.

The study of the binding of alpha-crystallin to membranes is potentially important for understanding the function of alpha-crystallin in the ocular lens and the formation of cataracts. Using fluorescence probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3 -phosphoethanolamine, triethylammonium salt (NBD-PE) and (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid, dipotassium salt (bis-ANS), the temperature dependence of the binding of alpha-crystallin to sphingomyelin liposomes, and the structural changes of alpha-crystallin and sphingomyelin induced by temperature were studied. The influence of the binding of alpha-crystallin on the mobility of the head group region of liposomes of sphingomyelin was dependent on the thermal history of alpha-crystallin. Binding of alpha-crystallin to sphingomyelin caused a decrease in the anisotropy of the fluorophore NBD-PE at or below 37 degrees C. However, when alpha-crystallin or the mixture of alpha-crystallin/sphingomyelin were preincubated near the secondary structure phase transition temperature of 60 degrees C, an increase of the anisotropy of NBD-PE (decrease of lipid head group mobility) was observed when measured at 22 degrees C or 37 degrees C. An inflection near 47 degrees C in the curve of fluorescence anisotropy of bis-ANS pre-incorporated into the alpha-crystallin corresponded to a 3 degrees or 4 degrees structural change of alpha-crystallin. alpha-Crystallin either increases or decreases the flexibility of the head group of sphingomyelin liposomes depending on its structure.

Lipid interactions with human antiphospholipid antibody, beta 2-glycoprotein 1, and normal human IgG using the fluorescent probes NBD-PE and DPH.

Recurrent venous thrombosis, arterial thrombosis and pregnancy losses are clinical manifestation associated with antiphospholipid antibody (aPL) that recognizes negatively charged phospholipid antigens. Enzyme-linked immunosorbent assays (ELISA) are generally used to determine the presence and specificity of aPL. In this paper, a fluorescence spectroscopy method has been applied, through monitoring the alteration of fluorescence intensity and anisotropy of a fluorophore that was incorporated in liposomes to explore the changes of molecular structure or configuration elicited by the binding aPL with phospholipid antigens. The bilayer surface was markedly ordered by aPL binding as indicated by the surface-sensitive probe NBD-PE. The binding of aPL on the bilayer surface is saturable. The saturation concentration of aPL is 40% (w/w, aPL/lipid) for cardiolipin membranes. The binding of aPL on cardiolipin took place in the absence of beta 2-GP1. The addition of beta 2-GP1 further increased the anisotropy and decreased the intensity of fluorescence. The binding of aPL is predominantly attributed to electrostatic interaction, but the configuration of the acyl chains of phospholipid also plays a role. It is found that the thermal history is important for aPL binding. The incubation at 37 degrees C is more favorable for aPL binding than ambient temperature. Normal human serine (IgG-NHS) did not elicit any distinct change of NBD-PE fluorescence, which indicates it does not interact with the lipid.

Influence of cholesterol on the interaction of alpha-crystallin with phospholipids.

The influence of cholesterol on the binding of alpha-crystallin to pure phospholipid membranes was studied. The rationale of this investigation stems from two unique aspects of human lens cells: an unusually high level of cholesterol in the membranes and the specific binding of alpha-crystallin to membranes. In the absence of cholesterol, binding of alpha-crystallin liposomes composed of either sphingomyelin, disteroyl-phosphatidylcholine or egg-phosphatidylcholine caused a decrease in the fluorescence intensity and anisotropy of the fluorophore NBD-PE. Since this fluorescence probe resides in the polar headgroup region of the membrane, the observed changes indicated that the binding of alpha-crystallin affected the structure of these membrane regions. The ability of alpha-crystallin to modulate membrane structure suggests yet another potential role for this lens protein. Addition of cholesterol markedly decreased the binding of alpha-crystallin to liposomes composed of either sphingomyelin or disteroylphosphatidylcholine and antagonized the capacity of bound alpha-crystallin to decrease membrane surface order. This antagonism could be explained by the ability of cholesterol to directly decrease the anisotropy of the fluorophore in sphingomyelin membranes unexposed to alpha-crystallin. Thus, with cholesterol present, a further decrease in membrane order upon subsequent binding of alpha-crystallin was less likely. The results obtained with the sphingomyelin liposomes are considered most meaningful, since sphingomyelins are the principal phospholipids in the human lens nuclear membrane and cholesterol preferentially interacts with sphingomyelin. We conclude that cholesterol in lipid membranes can antagonize the binding of alpha-crystallin and thus interfere with the capacity of bound alpha-crystallin to alter membrane order. We suggest that such actions of cholesterol might serve to preserve lens membrane structure in the physiological state where the concentration of soluble alpha-crystallin is great.

Age-related lipid oxidation in human lenses.

PURPOSE: To quantify age-related changes in products of lipid oxidation in human lenses and to relate these changes to membrane hydrocarbon chain structure. Deviation from a well-defined membrane-lipid composition and structure could result in alterations in membrane function and disruption of the homeostasis of the cell. METHODS: Infrared spectroscopy was used to detect lipid compositional and structural changes in human lens membranes associated with age and cataracts. RESULTS: Lipid oxidation increased linearly threefold relative to total phospholipids in subjects ranging in age between 1 and 85 years, as was evident by increases in trans double bonds, lipid carbonyls, and secondary products. There was no statistical difference between the levels of lipid oxidation in the cortex or nucleus. Lipid hydrocarbon chain order (rigidity) increased from approximately 40% at birth to 70% at 80 years of age. Changes in lipid order correlated with changes in the relative content of membrane phosphatidylcholine and sphingomyelin, and with the level of lipid oxidation. CONCLUSIONS: Lipid oxidation increased linearly and uniformly throughout the human lens with age. The change in lipid oxidation with age correlated to a change in lipid order.

Calcium ATPase activity and membrane structure in clear and cataractous human lenses.

PURPOSE: Maintenance of calcium homeostasis is imperative for the clarity of the lens. Ca(2+)-ATPase is essential for the removal of cytosolic calcium, either across the plasma membrane or through intracellular organelles such as the endoplasmic reticulum. In this study, membranes prepared from clear lens epithelium were compared to membranes prepared from cataractous lens epithelium. METHODS: Human lens membranes were prepared by a protocol utilizing homogenization and centrifugation. Ca(2+)-ATPase activity was measured biochemically using Gamma-32P labeled ATP. Lipid order was measured using infrared and Raman spectroscopy. RESULTS: Ca(2+)-ATPase activity was similar in membranes prepared from cataractous lenses that were classified as nuclear subcapsular, nuclear and brunescent cataracts. Ca(2+)-ATPase activity was approximately 50% less in membranes prepared from cataractous lenses in comparison to clear lenses. Because clear lenses from Indian donors was unavailable, clear human lenses were used as a qualitative control for the measurement for Ca(2+)-ATPase activity. Lipid order was measured in lens fibers from cataractous and clear lenses from the United States donors. Lipid order increased from 55% in the hydrocarbon chains from clear lens fibers to 84% in cataractous lens fibers. CONCLUSIONS: These findings support the hypothesis that membranes are deranged in cataractous tissue, which should lead to altered levels of calcium.

Calcium permeability in large unilamellar vesicles prepared from bovine lens cortical lipids.

The loss of calcium homeostasis in the lens is thought to play an important role in cataract formation. Although both lens Ca(2+)-ATPase and membrane lipid permeability are essential to calcium homeostasis, membrane permeability has been studied less extensively. In the present study, the calcium permeability of large unilamellar vesicles (LUVs) prepared from bovine lens cortical lipids has been characterized. When a calcium gradient had been established by the addition of CaCl2 to the external medium, calcium influx through the membrane was monitored by calcium concentration sensitive changes in Fura-2 fluorescence as a function of time. The calcium permeability coefficient, P, was 4.46 x 10(-13) cm s-1 at 37 degrees C. This value was about 4 fold higher than that for LUVs prepared from egg phosphatidylcholine and many times higher than that for LUVs prepared from sphingomyelin. These results provide a basis for future studies of factors that influence the permeability of lens cell membranes to calcium.