calpain and Cataract

Topics: Alzheimer Disease; Calpain; Cataract; Diabetes Mellitus, Type 2; Female; Humans; Isoenzymes; Male; Muscle Proteins; Muscular Dystrophies, Limb-Girdle; Mutation

Globally, cataract accounts for the majority of cases of treatable blindness and the lens opacification associated with cataract is primarily due to the insolubilisation of crystallins, proteins essential for the transparency of the lens. Recent studies have suggested that a major cause of this insolubilisation may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of Ca2+ and elevated levels of lens Ca2+ is a condition strongly associated with cataract. Calpain 2 appears to be the major calpain involved in animal cataractogenesis and the strongest candidate of the calpains for a role in human cataractogenesis but despite intensive study, the mechanism(s) underlying activation of the enzyme both in cataractogenesis and normal lens function are unclear. Recently, the high-resolution structure of calpain 2 was recently solved and a structural basis for the Ca2+-dependence of the enzyme's activity has been putatively established. Other recent studies have suggested that membrane interaction(s) may play a role in lowering the Ca2+-requirements of calpain 2 activation and most recently, strongly supporting this suggestion, several lipid interactive regions in the enzyme have been identified. Here, we review progress in understanding of the role played by calpain 2 in cataractogenesis and the possible use of inhibitors of the enzyme as anti-cataract agents.

Topics: Calcium; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Lens, Crystalline; Models, Biological; Models, Molecular; Solubility; Vision, Ocular

There is emerging evidence to suggest that the unregulated Ca(2+)-mediated proteolysis of essential lens proteins by calpains might be a major contributor to some forms of cataract in both animals and humans. Moreover, recently solved calpain structures have revealed molecular-level details of the activation mechanism used by these proteases, enabling the structure-based design of potent calpain inhibitors with the potential to act as anti-cataract agents. These agents offer the first real hope of an urgently needed alternative to the surgical treatment of at least some forms of cataract and relief from a life-depreciating condition on a global scale.

Topics: Animals; Calcium; Calpain; Cataract; Cattle; Crystallins; Cysteine Proteinase Inhibitors; Guinea Pigs; Haplorhini; Humans; Isoenzymes; Lens, Crystalline; Mice; Rabbits; Rats

Premature visual impairment due to lens opacification is a debilitating characteristic of untreated diabetes. Lens opacification is primarily due to the insolubilization of crystallins, proteins essential for lens optical properties, and recent studies have suggested that a major cause of this insolubilization may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of calcium (Ca2+) and elevated levels of lens Ca2+ is a condition associated with both diabetic cataractogenesis and other forms of the disorder. A number of calpains have been identified in the lens, including calpain 2, calpain 10 and two isozymes of calpain 3: Lp82 and Lp85. The use of animal hereditary cataract models have suggested that calpain 2 and/or Lp82 may be the major calpains involved in murine cataractogenesis with contributions from calpain 10 and Lp85. However, calpain 2 appears to be the major calpain involved in murine diabetic cataractogenesis and the strongest candidate of the calpains for a role in human types of cataractogenesis. Here, we present an overview of recent evidence on which these observations are based with an emphasis on the ability of calpains to proteolyse lens crystallins and calpain structural features, which appear to be involved in the Ca2+-mediated activation of these enzymes.

Topics: Calpain; Cataract; Diabetes Complications; Humans; Lens, Crystalline

The number of mammalian calpain protease family members has grown to 14 on last count. Overactivation of calpain 1 and calpain 2 (and their small subunit) has long been tied to acute neurological disorders (e.g. stroke and traumatic brain injury) and recently to Alzheimer's disease. Loss-of-function mutations of the calpain 3 gene have now been identified as the cause of limb-girdle muscular dystrophy 2A. Calpain 10 was recently identified as a susceptibility gene for type 2 diabetes, whereas calpain 9 appears to be a gastric cancer suppressor. This review describes our current understanding of the calpain family members and their mechanistic linkages to the aforementioned diseases as well as other emerging pathological conditions.

Topics: Alzheimer Disease; Animals; Calpain; Cataract; Diabetes Mellitus, Type 2; Disease; EF Hand Motifs; Humans; Multigene Family; Muscular Dystrophies; Nervous System Diseases; Stomach Neoplasms

Topics: Alzheimer Disease; Amino Acid Sequence; Animals; Apoptosis; Binding Sites; Calcium-Binding Proteins; Calpain; Cataract; Cell Cycle; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Long-Term Potentiation; Muscular Dystrophies; Parkinson Disease; Substrate Specificity

Topics: Animals; Calcium; Calpain; Cataract; Central Nervous System Diseases; Humans; Muscular Dystrophies; Myocardial Ischemia; Proteins

Selenite overdose cataract, an experimental model of nuclear cataract produced in young rats is reviewed. Topics include procedures for cataract production and assessment, metabolic and molecular changes in the epithelium of the lens, calcium accumulation, activation of calcium-activated protease system, mechanisms for crystallin precipitation, anti-cataract drug testing and relevance to human cataract.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cataract; Chemical Precipitation; Crystallins; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Disease Models, Animal; Drug Evaluation, Preclinical; Epithelium; Forecasting; Lens, Crystalline; Rats; RNA, Messenger; Sodium Selenite

Recent advances in understanding the mechanism of selenite cataract have resulted from locating the cleavage sites on proteolyzed beta-crystallins from the cataract, mimicking the insolubilization of crystallins found in the cataract in an in vitro system, studying cataract produced in lenses cultured in selenite, and permanently or temporarily reducing the rate formation of selenite cataract by use of various inhibitors. The present review discusses the selenite cataract as a useful model for understanding the role calcium-induced proteolysis in cataract formation.

Topics: Amino Acid Sequence; Animals; Calcium; Calpain; Cataract; Crystallins; Disease Models, Animal; Lens, Crystalline; Molecular Sequence Data; Selenium

It has been suggested that proteases are involved in removal of damaged or obsolete proteins and/or that the activation of proteases could contribute to cataract formation. This review summarizes the properties of several recently studied lens endopeptidases including: trypsin-like protease, multicatalytic endopeptidase complex, membrane bound proteases, and calpain. Properties discussed include composition, substrate specificity, distribution, changes in activity during aging, and regulation. Additionally, properties of the lens ubiquitin conjugation system are reviewed. When possible, an attempt was made to relate these findings to whether the lens proteolytic activity was involved in clearing damaged proteins, or whether it could contribute to cataract formation. Clearing of damaged or obsolete lens proteins may involve the participation of several protease activities. Findings suggest that lens protease activities are lost at variable rates during aging, and differ in concentration between species. It was concluded that the consequence of proteolytic activity in the lens may depend closely on the compliment of proteolytic activities found. For instance, proteases causing only partial degradation of lens proteins may predominate in lenses undergoing cataract formation, while proteases assisting in the removal of partially degraded proteins are lost. The partially degraded lens proteins, as well as other denatured lens proteins, may then accumulate and lead to cataract formation.

Topics: Aging; Animals; Aquaporins; Calpain; Carrier Proteins; Cataract; Cell Membrane; Cysteine Endopeptidases; Endopeptidases; Eye Proteins; Humans; Lens, Crystalline; Ligases; Membrane Glycoproteins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Trypsin; Ubiquitin-Conjugating Enzymes

Selenite cataract is a fairly recently described, experimental animal model for cataract (1). Selenite cataract has been extensively characterized histologically (2) and biochemically (3,4). The model has been particularly useful for studies on the roles of calcium accumulation and lens proteolysis in cataract formation (4). This review describes current knowledge of the biochemical mechanism for selenite cataract and indicates how the model may be used for further understanding of cataractogenesis in general.

Topics: Animals; Biomechanical Phenomena; Calcium; Calpain; Cataract; Cold Temperature; Crystallins; Lens, Crystalline; Oxidation-Reduction; Peptide Hydrolases; Rats; Rats, Inbred Strains; Selenious Acid; Selenium; Solubility

βA3/A1-crystallin is a lens structural protein that plays an important role in maintaining lens transparency via interactions with other crystallins. While the function of βA3/A1-crystallin in the retina is well studied, its functions in the lens, other than as a structural protein, remain unclear. In the current study, we generated the lens-specific βA3/A1-crystallin conditional knockout mouse (named βA3/A1ckO) and explored phenotypic changes and the function of the crystallin in the lens. The βA3/A1ckO mice showed congenital cataract at birth and exhibited truncation of lens proteins. Several truncated protein fragments were recovered as a pellet during a low-speed centrifugation (800 rpm, 70 x g) followed by a relatively higher speed centrifugation (5000 rpm, 2744 x g). Mass spectrometric analysis of pellets recovered following the two centrifugations showed that among the fragments with Mr < 20 kDa, the majority of these were from β-tubulin, and some from phakinin, αA-crystallin, and calpain-3. Further, we observed that in vitro activation of calpain-3 by calcium treatment of the wild-type-lens homogenate resulted in the degradation of calpain-3, αA-crystallin and β-tubulin and insolubilization of these proteins. Based on these results, it was concluded that the activation of calpain 3 resulted in proteolysis of β-tubulin, which disrupted cellular microtubular structure, and caused proteolysis of other lens proteins (αA-crystallin and phakinin). These proteolyzed protein fragments become insoluble, and together with the disruption of microtubular structure, and could be the causative factors in the development of congenital nuclear cataract in βA3/A1cKO mice.

Topics: Animals; Calpain; Cataract; Crystallins; Lens, Crystalline; Mice; Mice, Knockout; Proteolysis; Tubulin

Topics: Actins; Animals; Calcinosis; Calcium; Calpain; Cataract; Collagen; Fibrosis; Heterozygote; Lens, Crystalline; Macrophages; Mice; Mice, Inbred C57BL; TRPM Cation Channels

As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of. Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 μmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30.. Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, β-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, β-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting.. FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.

Topics: alpha-Crystallins; Animals; Animals, Newborn; Antioxidants; beta-Crystallins; Blueberry Plants; Calpain; Cataract; Eye Proteins; gamma-Crystallins; Gene Expression Regulation; Glutathione; Glutathione Peroxidase; Heme Oxygenase-1; Lens, Crystalline; Malondialdehyde; NF-E2-Related Factor 2; Oxidative Stress; Poly(ADP-ribose) Polymerases; Polyphenols; Rats; Rats, Sprague-Dawley; Sodium Selenite; Superoxide Dismutase

Cataract, a leading cause of blindness, is of special concern in diabetics as it occurs at earlier onset. Polyol accumulation and increased oxidative-nitrosative stress in cataractogenesis are associated with NFκB activation, iNOS expression, ATP depletion, loss of ATPase functions, calpain activation and proteolysis of soluble to insoluble proteins. Tocotrienol was previously shown to reduce lens oxidative stress and inhibit cataractogenesis in galactose-fed rats. In current study, we investigated anticataract effects of topical tocotrienol and possible mechanisms involved in streptozotocin-induced diabetic rats. Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Diabetic rats were treated with vehicle (DV) or tocotrienol (DT). A third group consists of normal, non-diabetic rats were treated with vehicle (NV). All treatments were given topically, bilaterally, twice daily for 8 weeks with weekly slit lamp monitoring. Subsequently, rats were euthanized and lenses were subjected to estimation of polyol accumulation, oxidative-nitrosative stress, NFκB activation, iNOS expression, ATP levels, ATPase activities, calpain activity and total protein levels. Cataract progression was delayed from the fifth week onwards in DT with lower mean of cataract stages compared to DV group (p<0.01) despite persistent hyperglycemia. Reduced cataractogenesis in DT group was accompanied with lower aldose reductase activity and sorbitol level compared to DV group (p<0.01). DT group also showed reduced NFκB activation, lower iNOS expression and reduced oxidative-nitrosative stress compared to DV group. Lenticular ATP and ATPase and calpain 2 activities in DT group were restored to normal. Consequently, soluble to insoluble protein ratio in DT group was higher compared to DV (p<0.05). In conclusion, preventive effect of topical tocotrienol on development of cataract in STZ-induced diabetic rats could be attributed to reduced lens aldose reductase activity, polyol levels and oxidative-nitrosative stress. These effects of tocotrienol invlove reduced NFκB activation, lower iNOS expression, restoration of ATP level, ATPase activities, calpain activity and lens protein levels.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Administration, Topical; Animals; Antioxidants; Blood Glucose; Body Weight; Calpain; Catalase; Cataract; Diabetes Mellitus, Experimental; Gene Expression; Glutathione; Lens, Crystalline; Male; NF-kappa B; Nitric Oxide Synthase Type II; Nitrosation; Oxidative Stress; Pilot Projects; Rats, Sprague-Dawley; Superoxide Dismutase; Time Factors; Tocotrienols

The present study investigates whether highbush blueberry leaf polyphenols prevent cataractogenesis and the underlying mechanisms. Chlorogenic acid, quercetin, rutin, isoquercetin and hyperoside were quantified in Vaccinium corymbosum leaf decoction (BBL) using HPLC-DAD. Wistar rats were injected subcutaneously with 20 μmol selenite (Na2SeO3)/kg body weight on postnatal (PN) day 10 (Se, n = 8-10/group) only or also intraperitoneally with 100 mg dry BBL/kg body weight on PN days 11 and 12 (SeBBL group, n = 10). Control group received only normal saline (C). Cataract evaluation revealed that BBL significantly prevented lens opacification. It, also, protected lens from selenite oxidative attack and prevented calpain activation, as well as protein loss and aggregation. In vitro studies showed that quercetin attenuated porcine lens turbidity caused by [Formula: see text] or Ca(2+) and interacted efficiently with those ions according to UV-Vis titration experiments. Finally, rutin, isoquercetin and hyperoside moderately inhibited pure human μ-calpain. Conclusively, blueberry leaf extract, a rich source of bioactive polyphenols, prevents cataractogenesis by their strong antioxidant, chelating properties and through direct/indirect inhibition of lens calpains.

Topics: Analysis of Variance; Animals; Antioxidants; Blueberry Plants; Calcium; Calpain; Cataract; Chelating Agents; Chlorogenic Acid; Disease Models, Animal; Eye Proteins; Lens, Crystalline; Lipids; Plant Extracts; Quercetin; Rats; Rats, Wistar; Selenious Acid; Swine

βA3/A1-crystallin is an abundant structural protein of the lens that is very critical for lens function. Many different genetic mutations have been shown to associate with different types of cataracts in humans and in animal models. βA3/A1-crystallin has four Greek key-motifs that organize into two crystallin domains. It shown to bind calcium with moderate affinity and has putative calcium-binding site. Other than in the lens, βA3/A1 is also expressed in retinal astrocytes, retinal pigment epithelial (RPE) cells, and retinal ganglion cells. The function of βA3/A1-crystallin in the retinal cell types is well studied; however, a clear understanding of the function of this protein in the lens has not yet been established. In the current study, we generated the βA3/A1-crystallin knockout (KO) mouse and explored the function of βA3/A1-crystallin in lens development. Our results showed that βA3-KO mice develop congenital nuclear cataract and exhibit persistent fetal vasculature condition. At the cellular level KO lenses show defective lysosomal clearance and accumulation of nuclei, mitochondria, and autophagic cargo in the outer cortical region of the lens. In addition, the calcium level and the expression and activity of calpain-3 were increased in KO lenses. Taken together, these results suggest the lack of βA3-crystallin function in lenses, alters calcium homeostasis which in turn causes lysosomal defects and calpain activation. These defects are responsible for the development of nuclear cataract in KO lenses.

Topics: Alleles; Amino Acid Motifs; Animals; Autophagy; beta-Crystallin A Chain; Binding Sites; Calcium; Calpain; Cataract; Cell Nucleus; Crystallins; Cytoplasm; Female; Gene Expression Regulation; Heterozygote; Homeostasis; Immunohistochemistry; Lens, Crystalline; Lysosomes; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy; Mitochondria; Polymerase Chain Reaction; RNA, Messenger

To compare the expression of calpain and caspase-12 in human lens epithelial cells (LECs) of cataract patients with or without diabetic retinopathy (DR).. Prospective, comparative case series.. Patients were divided into 4 groups: patients without diabetes mellitus (DM) (Group 1), patients with DM and without diabetic retinopathy (DR) (Group 2), diabetic patients with nonprolifeative DR (NPDR) (Group 3), and diabetic patients with proliferative DR (PDR) (Group 4). Data on DM duration and glycated hemoglobin (HbA1c) level were collected. The LECs were obtained during cataract surgery and immunohistochemical staining was performed for calpain and caspase-12. The reactivity score of each specimen was determined using confocal microscopy.. A total of 40 patients (40 eyes) were evaluated, and each group was composed of 10 patients. The expression of calpain and caspase-12 was highest in Group 4, followed by Group 3, Group 2, and Group 1 (P < .001). The expressions were significantly higher with a longer duration of DM, increased HbA1c level, or advanced DR.. Endoplasmic reticulum stress may be associated with apoptosis of LECs, resulting in cataract formation in diabetic patients.

Topics: Aged; Aged, 80 and over; Calpain; Caspase 12; Cataract; Diabetes Mellitus; Diabetic Retinopathy; Epithelial Cells; Fluorescent Antibody Technique, Indirect; Glycated Hemoglobin; Humans; Lens, Crystalline; Microscopy, Confocal; Middle Aged; Prospective Studies

Selenite-induced cataract is associated with oxidative stress, loss of calcium homeostasis, activation of calpain enzymes, and apoptotic cell death in the lens. An evaluation of naturally occurring antioxidants that also restrict calcium influx into the lens and calpain activation and thus prevent lenticular cell death may lead to the development of safe and effective anticataractogenic drugs. This study focuses on a naturally occurring flavone, chrysin, and its efficacy in preventing cataractogenic changes in in vitro cultured Wistar rat lenses.. Lenses from Wistar rats incubated for 24 h at 37 °C in Dulbecco's modified Eagle's medium (DMEM) were categorized into four main groups: Group I (control, incubated in DMEM alone); Group II (selenite-challenged and untreated, incubated in DMEM that contained 100 µM/ml of sodium selenite only); Group III (selenite-challenged and chrysin-treated, incubated in DMEM that contained sodium selenite [100 µM/ml of DMEM] and chrysin [200 µM/ml of DMEM]); and Group IV (chrysin-treated, incubated in DMEM that contained chrysin [200 µM/ml of DMEM] only). The Group III (selenite-challenged and chrysin-treated) lenses were further categorized into five sub-groups: Group IIIa (incubated for 24 h in DMEM that contained sodium selenite and chrysin added simultaneously), Group IIIb (first incubated for 2 h in DMEM that contained chrysin only and then for up to 24 h in fresh DMEM that contained sodium selenite only), Group IIIc (first incubated for 30 min in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only), and Groups IIId and IIIe (first incubated for 1 h and 2 h, respectively, in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only).. Gross morphological assessment revealed dense opacification (Grade +++) in the selenite-challenged, untreated lenses (Group II); however, seven of the eight selenite-challenged and simultaneously chrysin-treated (Group IIIa) lenses showed no opacification (Grade 0) after 24 h incubation, while the remaining single lens exhibited only a slight degree of opacification (Grade +). In the Group IIIa lenses, the reduced glutathione, protein sulfhydryl, and malondialdehyde concentrations appeared to have been maintained at near-normal levels. The mean lenticular concentration of calcium was significantly lower in the Group IIIa lenses than that in the Group II lenses and approximated the values observed in the normal control (Group I) lenses. The Group IIIa lenses also exhibited significantly (p<0.05) higher mean lenticular activity of calpain, significantly higher mean mRNA transcript levels of genes that encode m-calpain and lenticular preferred calpain (Lp82), and significantly higher mean levels of the m-calpain and Lp82 proteins than the corresponding values in the Group II lenses. Casein zymography results suggested that chrysin prevented calpain activation and autolysis. Significantly (p<0.05) lower mean levels of mRNA transcripts of the genes that encode calcium transporter proteins (plasma membrane Ca(2+)-ATPase-1 and sarco/endoplasmic reticulum Ca(2+)-ATPase-2) and lenticular apoptotic-cascade proteins (early growth response protein-1, caspase-3, caspase-8, and caspase-9) and significantly (p<0.05) lower mean concentrations of the proteins themselves were seen in the Group IIIa rat lenses in comparison to the values noted in the Group II rat lenses.. Chrysin appears to prevent selenite-induced cataractogenesis in vitro by maintaining the redox system components at near-normal levels and by preventing the abnormal expression of several lenticular calcium transporters and apoptotic-cascade proteins, thus preventing accumulation of calcium and subsequent calpain activation and lenticular cell death in cultured Wistar rat lenses.

Topics: Animals; Antioxidants; Apoptosis Regulatory Proteins; Calcium; Calpain; Cataract; Flavonoids; Glutathione; Lens, Crystalline; Lipid Peroxidation; Malondialdehyde; Organ Culture Techniques; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sodium Selenite; TRPV Cation Channels

Increased lenticular oxidative stress and altered calcium/magnesium (Ca/Mg) homeostasis underlie cataractogenesis. We developed a liposomal formulation of magnesium taurate (MgT) and studied its effects on Ca/Mg homeostasis and lenticular oxidative and nitrosative stress in galactose-fed rats.. The galactose-fed rats were topically treated with liposomal MgT (LMgT), liposomal taurine (LTau), or corresponding vehicles twice daily for 28 days with weekly anterior segment imaging. At the end of the experimental period, the lenses were removed and subjected to analysis for oxidative and nitrosative stress, Ca and Mg levels, ATP content, Ca(2+)-ATPase, Na(+),K(+)-ATPase, and calpain II activities.. The LTau and LMgT groups showed significantly lower opacity index values at all time points compared to the corresponding vehicle groups (p<0.001). However, the opacity index in the LMgT group was lower than that in the LTau group (p<0.05). Significantly reduced oxidative and nitrosative stress was observed in the LTau and LMgT groups. The lens Ca/Mg ratio in LMgT group was decreased by 1.15 times compared to that in the LVh group. Calpain II activity in the LMgT group was decreased by 13% compared to the LVh group. The ATP level and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities were significantly increased in the LMgT group compared to the LVh group (p<0.05).. Topical liposomal MgT delays cataractogenesis in galactose-fed rats by maintaining the lens mineral homeostasis and reducing lenticular oxidative and nitrosative stress.

Topics: Animals; Calcium; Calpain; Cataract; Disease Progression; Galactose; Homeostasis; Lens, Crystalline; Liposomes; Magnesium; Nitrosation; Oxidative Stress; Particle Size; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Taurine

Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip-/-). In newborn Mip-/- lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=<0.05) and a similar number of genes was differentially regulated at P7. The most up-regulated genes (>6-fold) in the Mip-/- lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip-/- lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip-/- lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (∼1.5% of the transcriptome) were differentially regulated >2-fold in the Mip-/- lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation.

Topics: Animals; Aquaporins; Calpain; Cataract; Cell Death; DNA Fragmentation; Eye Proteins; Gene Expression Profiling; Immunoblotting; In Situ Nick-End Labeling; Lens, Crystalline; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Phenotype; Transcriptome

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca(2+) elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca(2+) elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.

Topics: Amino Acid Substitution; Animals; Calcium; Calpain; Cataract; Enzyme Activation; Eye Proteins; Gap Junctions; HeLa Cells; Humans; Lens, Crystalline; Mice; Mice, Transgenic; Mutation, Missense; Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin

Calpains belong to the family of calcium-dependent, structurally related intracellular cysteine proteases that exhibit significant functions in evolution of different types of cataracts in human as well as animal models. Application of calpain inhibitors generated through a virtual screening workflow may provide new avenues for the prevention of cataractogenesis. Hence, in the current study, compounds were first screened for potent calpain inhibitory activity by employing a structure-based approach, and the screening results were then validated through biological experiments in rat lenses. A hit compound, HTS08688, was obtained by structure-based virtual screening. A micromolar concentration of HTS08688 was found to prevent in vitro cataractogenesis in isolated Wistar rat lenses, while maintaining the antioxidant and calcium concentrations at near normal levels. Inhibition of superoxide anion generation, as observed through cytochemical localization studies, and maintenance of structural integrity, as demonstrated by histological analysis of lenticular tissue, also suggested that HTS08688 can ameliorate the cataractous condition induced by selenite in an in vitro rodent model. A cell proliferation assay was performed; the IC 50 value of the screened calpain inhibitor, HTS08688, against human lenticular epithelial cells-b3 was found to be 177 μM/mL. This combined theoretical and experimental approach has demonstrated a potent lead compound, HTS08688, that exhibits putative anticataractogenic activity by virtue of its potential to inhibit calpain.

Topics: Animals; Calcium; Calpain; Cataract; Cell Line; Crystallography, X-Ray; Cysteine Proteinase Inhibitors; Drug Discovery; Humans; Lens, Crystalline; Models, Molecular; Protein Conformation; Rats, Wistar; Selenious Acid

Carnosine (CAR) is an endogenous peptide and present in lens, but there is little evidence for its effectiveness in calpain-induced proteolysis inhibition and its differential effects toward different wavelengths of ultraviolet (UV) irradiation. This study aimed to develop three in vitro cataract models to compare the mechanisms underlying the protective activities of CAR. Crude crystallins extracted from porcine lenses were used for antiproteolysis assays, and purified γ-crystallins were used for anti-UV assays. The turbidity in those in vitro models mimics cataract formation and was assayed by measuring optical density (OD) at 405 nm. The effectiveness of CAR on calpain-induced proteolysis was studied at 37 and 58 °C. Patterns of proteins were then analyzed by SDS-PAGE. The turbidity was reduced significantly (p<0.05) at 60 min measurements with the increased concentration of CAR (10-300 mM). SDS-PAGE showed that the decreased intensities at both ∼28 and ∼30 kDa protein bands in heat-enhanced assays were ameliorated by CAR at ≥10 mM concentrations. In UV-B studies, CAR (200, 300 mM) reduced the turbidity of γ-crystallin significantly (p<0.05) at 6 h observations. The turbidity of samples containing γ-crystallins was ameliorated while incubated with CAR (100, 300 mM) significantly (p<0.05) following 4 h of exposure to UV-C. SDS-PAGE showed that the presence of CAR reduced UV-B-induced aggregation of γ-crystallins at ∼44 kDa and resulted in less loss of γ-crystallin following UV-C exposure. The result of modeling also suggests that CAR acts as an inhibitor of calpain. In conclusion, CAR protects lens proteins more readily by inhibiting proteolysis and UV-C-induced degradation than aggregation induced by UV-B irradiation.

Topics: Amino Acid Sequence; Animals; Calpain; Carnosine; Cataract; Crystallins; Glycoproteins; Humans; Lens, Crystalline; Molecular Sequence Data; Proteolysis; Sequence Alignment; Swine; Ultraviolet Rays

We used sheep with an autosomal dominant gene for cortical cataract as an animal model to evaluate novel macrocyclic calpain inhibitors with potential for the medical treatment of human cataract.. The macrocyclic aldehyde, CAT811, identified previously as a calpain inhibitor that prevents calcium-induced opacification in cultured sheep lenses, was tested for its ability to protect cytoskeletal proteins from calpain proteolysis. CAT811 and its alcohol analogue, CAT505, were formulated separately into ointments, and each was applied twice daily to the right eye of sheep with early cataracts for five months. Progress of cataracts in the sheep was determined by ophthalmologic examination and comparison with a matched sample of sheep treated similarly with ointment that did not contain the active ingredient.. The novel macrocyclic aldehyde, CAT811, was able to inhibit calpain proteolysis of lens cytoskeletal proteins at micromolar concentrations. When applied topically to the eyes of sheep, CAT811 was able to slow cataract development by 27% in the initial three months of treatment (P < 0.05). Its alcohol analogue, CAT505, was not able to slow cataractogenesis significantly.. The inherited sheep cataract provides a reproducible model of cortical cataract over a time scale of several months. The data reported here, using this model, demonstrated the potential of the macrocyclic calpain inhibitor, CAT811, to act as a therapeutic for treatment of cortical cataract.

Topics: Animals; Blotting, Western; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Genetic Predisposition to Disease; Glycoproteins; Lens, Crystalline; Sheep

In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model.. In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to analyze the integrity of crystallin samples.. PRX at 1,000 μM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 μM. Results were further confirmed by SDS-PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 μM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 μM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 μM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 μM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 μM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 μM PRX.. PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted.

Topics: Animals; Animals, Newborn; Calcium; Calpain; Cataract; Dose-Response Relationship, Drug; Drug Dosage Calculations; Electrophoresis, Polyacrylamide Gel; gamma-Crystallins; Lens, Crystalline; Ophthalmic Solutions; Oxazines; Proteolysis; Rats; Rats, Sprague-Dawley; Sodium Selenite; Spectrum Analysis; Swine; Ultraviolet Rays

Studies of 17 analoges of 3 (SJA6017) in an in silico calpain model are reconciled to measured IC(50) values against ovine calpain. The studies validate the potential of the "model" and criteria established for inhibition as a tool to select structures for synthesis to test as calpain inhibitors. Using this screening methodology of virtual libraries led us to synthesize several inhibitors including macrocycle 33, which in vitro sheep eye lens culture experiments showed to substantially slow opacification.

Topics: Amino Acid Sequence; Animals; Calpain; Catalytic Domain; Cataract; Databases, Factual; Dipeptides; Lens, Crystalline; Macrocyclic Compounds; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Sheep; Structure-Activity Relationship; Tissue Culture Techniques

To study the efficacy of Brassica oleracea var. italica (Broccoli) in the prevention of selenite induced biochemical changes and the incidence of cataractogenesis in vivo.. Eight day old Sprague-Dawley rat pups were divided into four groups: I-Control; II-Sodium selenite (4 mg/kg body weight) administered; III-Sodium selenite + quercetin; and IV-Sodium selenite + flavonoid fraction of broccoli (FFB). Treatment groups III and IV received quercetin and FFB intraperitoneally from 8th to 15th day at a concentration (2.0 mg/kg body weight). The development of cataract was assessed and graded by slit-lamp examination. Some relevant biochemical parameters-such as activities of superoxide dismutase (SOD), catalase, Ca(2+)ATPase, calpains, concentration of reduced glutathione (GSH), levels of calcium, lipid peroxidation product-thiobarbituric acid reacting substances (TBARS) and SDS-PAGE analysis of lens water soluble proteins (WSF) were analyzed.. FFB modulates selenite-induced biochemical changes in albino rats. Lenses of Group I rats were clear but in Group II, all lenses developed dense opacification (grade 5 and 6), whereas mild opacifications were observed in Group III and Group IV (grade 2). Group III and Group IV lenses exhibited significantly higher values of antioxidant enzymes, Ca(2+)ATPase, and GSH, whereas lower values were obtained for TBARS, calcium, and calpains compared to Group II. Lens protein profile of water soluble proteins showed normal levels of Group III and Group IV compared to Group II lenses.. FFB prevents selenite-induced cataractogenesis in albino rat pups, possibly by maintaining antioxidant status and ionic balance through Ca(2+) ATPase pump, inhibition of lipid peroxidation, calpain activation, and protein insolubilization, which have been reported in this article for the first time.

Topics: Animals; Animals, Newborn; Antioxidants; Brassica; Calcium; Calcium-Transporting ATPases; Calpain; Catalase; Cataract; Crystallins; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Glutathione; Lens, Crystalline; Lipid Peroxidation; Plant Extracts; Quercetin; Rats; Rats, Sprague-Dawley; Sodium Selenite; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances

Recent investigations have shown that phytochemical antioxidants can scavenge free radicals and prevent various diseases. Cataract is the leading cause of blindness and is associated with oxidative damage of the lens. Selenite-induced cataract in rat pups is an excellent mimic of oxidative stress-induced cataract. Selenite cataract is associated with oxidative stress, loss of calcium homeostasis, calpain activation and protein insolubilization in the lens. Our present study focuses on the isolation of flavonoids from Vitex negundo and to assess its efficacy in preventing these changes in the lens of selenite-induced cataract models. Eight-day-old Sprague-Dawley rat pups were used for the study and divided into four groups: Control (G I), Sodium selenite-induced (G II), Sodium selenite+quercetin treated (G III), Sodium selenite+flavonoids from Vitex negundo (FVN) (G IV). Cataract was induced by a single subcutaneous injection of Sodium selenite (4 mg/Kg body weight) on the 10th day. Treatment groups received quercetin (1.0mg/Kg body weight) and FVN (1.0mg/Kg body weight) intraperitoneally from 8th to 15th day. Cataract was visualized from the 16th day. Morphological examination of the rat lenses revealed no opacification in G I and mild opacification in G III and G IV (stage 1) whereas dense opacification in G II (stage 4-6). The activities of superoxide dismutase (SOD), catalase, Ca(2+)ATPase, concentration of reduced glutathione (GSH) and protein sulfhydryl content were significantly increased in G III and G IV compared to G II, while decreased activities of calpains, lower concentration of calcium and thiobarbituric acid reactive substances (TBARS) were observed in G III and IV as compared to G II. Lens protein profile of water soluble proteins showed normal levels of expression in treated groups compared to that of selenite-induced rats. These results indicate good antioxidant and therapeutic potential of FVN in modulating biochemical parameters against selenite-induced cataract, which have been reported in this paper for the first time.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Calpain; Cataract; Disease Models, Animal; Free Radical Scavengers; Homeostasis; Lens, Crystalline; Lipid Peroxidation; Oxidative Stress; Phytotherapy; Plant Extracts; Rats; Rats, Sprague-Dawley; Scattering, Radiation; Sodium Selenite; Thiobarbituric Acid Reactive Substances; Vitex

The design and elaboration of a series of macrocyclic templates that exhibit a propensity to adopt a beta-strand-like peptide-backbone conformation led to potent and selective inhibitors of calpain 2. Macrocycle 1 retarded calcium-induced opacification in an ovine-lens culture assay and is a lead compound for the development of a drug for cataract treatment. Cbz=carbobenzyloxy.

Topics: Animals; Calpain; Cataract; Cysteine Proteinase Inhibitors; Heterocyclic Compounds; Inhibitory Concentration 50; Lens, Crystalline; Macrocyclic Compounds; Models, Molecular; Sheep; Structure-Activity Relationship

The present study sought to determine whether acetyl-L-carnitine (ALCAR) prevents selenite cataractogenesis by mechanisms involving lenticular calpain activity, Wistar rat pups were divided into 3 groups of 15 each. Group I (normal) rats received an intraperitoneal (i.p.) injection of normal saline on postpartum day 10; Group II (cataract-untreated) rats received a single subcutaneous (s.c.) injection of sodium selenite (19micromol/kg body weight) on postpartum day 10; Group III (cataract-treated) pups received a single s.c. injection of sodium selenite on postpartum day 10 and intraperitoneal injections of acetyl-L-carnitine (200mg/kg body weight) on postpartum days 9-14. At the end of the study period (postpartum day 16), both eyes of each rat pup were examined by slit-lamp biomicroscopy. There was dense lenticular opacification in all Group II rats, minimal lenticular opacification in 33% of Group III rats, and no lenticular opacification in 67% of Group III and in all Group I rats. Group II lenses exhibited significantly lower mean values of calpain activity and Lp82 (lens-specific calpain) protein expression, decreases in relative transcript level of m-calpain mRNA and significantly higher mean Ca(2+) concentrations than Group I or Group III lenses; the values of these parameters in Group III rat lenses (ALCAR-treated) approximated those in Group I rat lenses. The results suggest that, in addition to its already-described antioxidant potential, ALCAR prevents selenite cataractogenesis by maintaining calpain activity at near normal levels. These findings may stimulate further efforts to develop ALCAR as a novel drug for prevention of cataract.

Topics: Acetylcarnitine; Animals; Calcium; Calpain; Cataract; Drug Evaluation, Preclinical; Gene Expression Regulation, Enzymologic; Lens, Crystalline; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Selenite

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

Topics: Animals; Calpain; Cataract; Cell Differentiation; Enzyme Activation; Eye Proteins; Gene Expression Regulation, Enzymologic; Lens, Crystalline; Mice; Mice, Mutant Strains; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Spectrin; Substrate Specificity

The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model.. The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens-based cell-free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high-performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67- and 97-day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit-lamp microscope.. In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non-toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but >90% bound to lens homogenate. The 67-day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary.. In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.

Topics: Animals; Calpain; Cataract; Cell Survival; Cells, Cultured; Cysteine Endopeptidases; Dipeptides; Disease Models, Animal; Glycoproteins; Humans; Models, Molecular; Ointments; Ophthalmic Solutions; Pyrroles; Sheep; Sheep Diseases

To produce two-dimensional electrophoresis (2-DE) maps for ovine crystallins and examine changes in ovine crystallins during cataract formation.. Soluble and insoluble fractions were isolated from normal, whole lenses of 26-week-old sheep, the proteins separated by 2-DE, and the spots digested with trypsin and subjected to tandem mass spectral analysis. Spot identifications were made by using mass spectrometry data from each spot digestion and data from 2-DE maps of proteins from soluble and insoluble cortices of 10-month-old ovine lens. Ovine alphaA-, alphaB-, and betaB3-crystallin cDNAs were sequenced, whereas other ovine crystallins were identified by using bovine sequences. Proteins were then isolated from whole lenses of 26-week-old lambs with mature hereditary cataracts, and the changes in the crystallins were determined by 2-DE. The masses of truncated crystallins were determined after elution from 2-DE gels.. The ovine lens contained the normal complement of crystallins and, similar to other mammalian lenses, underwent partial proteolysis of betaB1-, betaA3-, and betaB3-crystallin during maturation. Cataract development was associated with enhanced truncation of alpha- and beta-crystallins. C-terminal truncations of alphaA- and alphaB-crystallin and N-terminal truncation of betaB2-crystallin were observed as well as a loss of gamma-crystallin.. These data provide the first 2-DE gel maps for ovine lens crystallins and indicated that ovine lens crystallins are truncated during lens maturation. The differences in proteolysis appearing in normal and cataractous lenses suggested that calpain isoforms may be differentially activated during lens maturation and cataract. The ovine hereditary cataract is a useful nonrodent model to study the role of calpain proteolysis in cataract formation.

Topics: alpha-Crystallins; Animals; beta-Crystallins; Calpain; Cataract; DNA, Complementary; Electrophoresis, Gel, Two-Dimensional; gamma-Crystallins; Lens, Crystalline; Polymerase Chain Reaction; Sequence Analysis, DNA; Sheep; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To determine whether retinoic acid (RA)-mediated inhibition of deregulated calpains had any effect on the development of cataract given that accumulating evidence has demonstrated a possible relationship between cataractogenesis and inappropriate activation of calpains.. The authors examined for Ca(2+) influx and expression alteration of calpains in F9 cells with or without RAs, such as all-trans retinoic acid (ATRA), and specific stimulant of retinoic acid receptor alpha (RARalpha; Am580) in the presence of oxidative stress, such as mediated by H(2)O(2). They next examined the clinical relevance of RAs by applying these agents to a murine diabetic cataract and observed the development of the disease.. F9 cells constitute a well-established autonomous cell model for investigating retinoid signaling, partially representing the lens epithelial phenotype, as determined by the expression of aquaporin 0, a specific differentiation marker for lens cells. Treatment with ATRA and Am580 significantly decreased the influx of Ca(2+) into the cells, causally resulting in decreased mRNA expression and inhibited activation of calpains. In addition, RARalpha agonists significantly abrogated the upregulation of calpain 2 induced by H(2)O(2), which is a potential etiological contributor to the diabetic cataract, whereas H(2)O(2) had no effect on calpain 1. Importantly, this RA-mediated gene-expression alteration was sufficient for dramatically inhibiting the development of lens opacity in mice with diabetes.. Results showed that a certain type of RA inhibits Ca(2+) elevation and subsequent overactivation of calpains, suggesting the potential feasibility of calpain-targeting therapies mediated by RA for cataract.

Topics: Animals; Benzoates; Calcium; Calpain; Cataract; Diabetes Mellitus, Experimental; Hydrogen Peroxide; Lens, Crystalline; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrahydronaphthalenes; Tretinoin

The photoswitchable N-terminal diazo and triazene-dipeptide aldehydes 8a-d, 10a,b, and 17a,b present predominantly as the (E)-isomer, which purportedly binds deep in the S3 pocket of calpain. All compounds are potent inhibitors of m-calpain, with 8b being the most active (IC50 of 35 nM). The diazo-containing inhibitors 8a, 8c, and 10a were irradiated at 340 nm to give a photostationary state enriched in the (Z)-isomer, and in all cases, these were less active. The most water soluble triazene 17a (IC50 of 90 nM) retards calpain-induced cataract formation in lens culture.

Topics: Aldehydes; Animals; Azo Compounds; Calpain; Cataract; Culture Techniques; Dipeptides; Humans; Lens, Crystalline; Models, Molecular; Protein Binding; Protein Structure, Tertiary; Sheep; Stereoisomerism; Structure-Activity Relationship; Sulfonamides; Triazenes; Ultraviolet Rays

Previous studies have demonstrated that in 129alpha3Cx46-/- mice, age-related nuclear cataract is formed. In the present study, a more in vivo-relevant model was generated to test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice.. To test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice, 129alpha3Cx46-/- and CAPN3-/- mice were mated to generate homozygous double-knockout (dKO) mice. Lenses from the mice were examined by visual observation, laser scan analysis, and histologic and biochemical methods.. In the absence of the CAPN3 gene, the formation of a cataract was delayed, and its appearance was changed to a more diffuse, pulverulent type. Unlike in the 129alpha3Cx46-/- mouse, cleavage of gamma-crystallin was not detected in the dKO mouse. In both 129alpha3Cx46-/- and dKO mice, total Ca2+ increased.. The present study shows for the first time that calpain 3 is necessary for the formation of age-dependent nuclear cataracts in alpha3Cx46-/- mice. Evidence that the calpain 3 gene is directly involved in, or part of the pathway that leads to, gamma-crystallin cleavage is presented. These results are consistent with the hypothesis that the loss of alpha3Cx46 leads to increased levels of Ca2+ ions, and this increase activates the CAPN3 isoform, Lp82/85, which results in the formation of a nuclear cataract.

Topics: Aging; Animals; Blotting, Western; Body Water; Calcium; Calpain; Cataract; Connexins; Female; gamma-Crystallins; Genotype; Lens, Crystalline; Male; Mice; Mice, Knockout; Mice, Transgenic; Muscle Proteins; Organ Culture Techniques; Organ Size; Protein Isoforms

Selenite-induced cataractogenesis is mediated by oxidative stress, accumulation of calcium and activation of lenticular calpains. Calpains are a super family of Ca2+ dependent proteases, which are involved in lens protein proteolysis and insolubilization. Many inhibitors could prevent calpain-induced proteolysis of alpha- and beta-crystallins in rodent cataracts. Evaluating natural sources with antioxidant property and subsequent prevention of calpain activation may lead to the development of safer and more effective agents against cataractogenesis. There are no reports on the protective role of bioactive components against calpain-mediated proteolysis and subsequent cataractogenesis. The purpose of the study was to evaluate the role of Drevogenin D, a triterpenoid aglycone, isolated from Dregea volubilis in preventing selenite-induced, calcium-activated, calpain-mediated proteolysis in cultured rat lenses.. Lenses were extracted from Sprague-Dawley strain rats at the age of one month and were organ cultured in M-199 medium with HEPES buffer. The lenses were divided into three groups with eight lenses in each group as follows: lenses cultured in a normal medium (GI), lenses cultured in a sodium selenite supplemented medium (GII), and lenses cultured in a medium supplemented with sodium selenite and Drevogenin D-treated (GIII). Changes to transparency and opacity formation of lenses were monitored under microscopic observation. At the end of the experiment, biochemical parameters such as activity of lens superoxide dismutase (SOD), lens Ca2+ ATPase, concentration of Ca2+, levels of sulfhydryl content, and thiobarbituric acid reacting substances (TBARS) were determined. Changes to casein zymography for calpains, immunoblot for Lp82, and SDS-PAGE of lens water soluble protein fraction (WSF) were also done.. Microscopic evaluation of lens morphology showed that Drevogenin D prevented the opacification in G-III. Drevogenin D inhibited the accumulation of calcium, the activation of calpain system, and lipid peroxidation. Activity of Ca2+ ATPase, SOD, and SDS-PAGE profile of water soluble proteins was normalized following treatment with Drevogenin D.. Selenite-induced cataractogenesis is mediated by oxidative stress leading to a decrease in the activity of Ca2+ ATPase, resulting in the accumulation of calcium and the subsequent activation of lenticular calpains. The results obtained indicated that Drevogenin D treatment was effective in protecting the lens proteins by controlling stress-induced protein oxidation, maintenance of Ca2+ ATPase activity, calcium accumulation, lipid peroxidation, and prevention of calpain activation. Hence, Drevogenin D can be used as a potential therapeutic agent against oxidative stress-induced cataract.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Calpain; Cataract; Crystallins; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Immunoblotting; In Vitro Techniques; Lens, Crystalline; Oxidative Stress; Peptide Hydrolases; Plant Extracts; Rats; Rats, Sprague-Dawley; Sodium Selenite; Solubility; Sulfhydryl Compounds; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Triterpenes

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Topics: Aged; Aged, 80 and over; Animals; Apoptosis; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cataract; Eye Proteins; Humans; Immunoblotting; In Situ Nick-End Labeling; Ionophores; Lens, Crystalline; Macaca mulatta; Models, Animal; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling

To determine the involvement of calpain in ovine cataractogenesis by measuring calcium, calpain activity, proteolysis, and the effect of calpain inhibition.. Sheep with genetic cataracts were examined for cataract severity. Calcium in normal and cataract lenses was measured. The presence of calpain was detected by casein zymography and immunoblotting. Calpain activity was assayed using BODIPY-casein as a substrate. Degradation of calpain substrates spectrin and vimentin was assessed by immunoblotting. The calpain inhibitor SJA6017 was applied to the left eye of cataract lambs, leaving the right eye as an untreated control. Both eyes were monitored by slit-lamp microscopy for cataract progression.. Cortical cataracts were first observed in lambs at 1 to 2 months of age. Lens calcium concentration increased in the early stages of cataract formation and was >10-fold higher in mature cataract than normal lenses. Three calpain isoforms were detected in young lamb lenses. Calpain activity decreased as cataracts progressed. Both spectrin and vimentin were degraded with cataract maturity, which could indicate calpain proteolysis. Cataract lambs treated with SJA6017 eyedrops over a period of 4 months showed significantly smaller cataracts in the left treated eye over the right untreated eye.. The presence of calpains and calcium elevation during cataract formation suggests that proteolysis may play a role in opacification in ovine lens. This hypothesis is supported by the delay in opacification with SJA6017 treatment. The results also suggested that the ovine hereditary cataract is a useful nonrodent model to test the role of calpains in cataractogenesis.

Topics: Animals; Blood Glucose; Calcium; Calpain; Cataract; Cysteine Proteinase Inhibitors; Dipeptides; Electrophoresis, Polyacrylamide Gel; Female; Glycoproteins; Lens, Crystalline; Male; Ophthalmic Solutions; Sheep; Sheep Diseases

To study the role of calpain in the mechanism of oxidative cataract through detecting the level of intracellular free Ca(2+), the expression and proteolytic activity of calpain in the lens epithelial cells (LECs) of H(2)O(2)-induced cataract.. Rat lenses were cultured in vitro and cataract was induced by H(2)O(2). The level of intracellular free Ca(2+) was measured by fluorescence determination with fura-2/AM. The expression of m-calpain protein in LECs was detected with immunohistochemical method. The proteolytic activity in LECs was measured using a fluorogenic synthetic substrate.. There were significant differences of the level of intracellular free Ca(2+) (P=0.001, 0.000, 0.000), the expression of m-calpain (P=0.001, 0.000, 0.000) and the proteolytic activity of calpain (P=0.001, 0.000, 0.000) between H(2)O(2)-induced and control group at 6, 12 and 24 h, respectively.. H(2)O(2) can increase intracellular free Ca(2+), then enhance the expression and proteolytic activity of calpain which may play a role in the mechanism of oxidative cataract of rat.

Topics: Animals; Calcium; Calpain; Cataract; Epithelial Cells; Gene Expression; Lens, Crystalline; Oxidation-Reduction; Rats; Rats, Sprague-Dawley

Calpain inhibitors show the potential to serve as non-surgical alternatives in treating diabetic cataract and other types of these disorders. Here, we have tested the recently developed calpain inhibitor, SJA6017, for its ability to inhibit cataractogenesis in porcine lenses. These lenses were incubated in increasing levels of extralenticular calcium (Ca2+; 5-30 mM). Atomic absorption spectroscopy was used to determine total internal lens Ca2+ and a correlation between porcine lens Ca2+ uptake and levels of lens opacification were found with a total internal lens Ca2+ level of 5.8 microM Ca2+ g(-1) wet lens weight corresponding to the onset of catarctogenesis. A total internal lens Ca2+ level of 8.0 microM Ca2+ g(-1) wet lens weight corresponded to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when SJA6017 (final concentration 0.8 microM) was included in the extralenticular medium, suggesting that the Ca2+-mediated activation of calpains may be involved in the observed opacification. Supporting this suggestion atomic absorption spectroscopy showed that the effect of SJA6017 (final concentration 0.8 microM) on lens opacification was not due to the compound restricting porcine lens Ca2+ uptake. The results indicate that calpain-induced cataractogenesis is dependent on extracellular Ca2+ and the calpain inhibitor SJA6017 (0.8 microM) had no significant effect on Ca2+ uptake by lens. Its inhibitory effect on lens opacification may be due to a direct action on the activity of calpain.

Topics: Animals; Biological Transport; Calcium Chloride; Calpain; Cataract; Cysteine Proteinase Inhibitors; Dipeptides; In Vitro Techniques; Lens, Crystalline; Swine

To explore cornea permeable calpain inhibitors, four compounds displaying different characteristics were designed and synthesized based on the known potent calpain inhibitor, peptidyl aldehyde SJA6017. Two approaches were adopted; an improvement in the physicochemical properties, and conversion of the active aldehyde. The water-soluble peptidyl aldehyde 1 containing a pyridine ring at the P3 site showed a modest inhibition against calpains and an improvement of corneal permeability in comparison with SJA6017. Replacement of the aldehyde of SJA6017 by an alpha-ketoamide provided compound 2 that was approximately equipotent with SJA6017, but it was extremely water-insoluble. However, compound 3, in which the aldehyde was converted into a cyclic hemiacetal, proved to be a less potent calpain inhibitor than SJA6017, but demonstrated excellent transcorneal permeability. Further modification generating the cyclic hemiacetal 4 containing a thiourea linker between the P3 and P2 sites exhibited potent inhibitory activities, high cornea permeability and excellent efficacy in the rat lens culture cataract model.

Topics: Aldehydes; Animals; Calpain; Cataract; Chemical Phenomena; Chemistry, Physical; Cornea; Cysteine Endopeptidases; Dipeptides; Indicators and Reagents; Lens, Crystalline; Magnetic Resonance Spectroscopy; Multienzyme Complexes; Ophthalmic Solutions; Organ Culture Techniques; Permeability; Protease Inhibitors; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Solubility; Thiourea

To observe oxidative cataract inhibition of PD150606 by studying the opacity of the lens, the variation of protein and cells, and by further studying the role of calpains in the mechanism of oxidative cataract.. Rat lenses were cultured in vitro with H(2)O(2) and PD150606. The lenses were observed under the microscope. Simultaneously, photograph analysis was performed in order to detect the variation of opacity; the proportion of WSP was determined by the method of Lowry; the number of apoptotic lens epithelial cells (LEC) was measured by flow cytometry; the expression of Caspase 3 in LEC was determined by SAS-PAGE electrophoresis and immunoblotting.. Opacity of lens was enhanced in 24 hours when cultured with H(2)O(2) and PD150606. There were significant differences of relative gray scale between H(2)O(2)-induced and control group at 6, 12 and 24 hours (P < 0.05), significant differences of the proportion of WSP between H(2)O(2)-induced and control group at 12 and 24 hours (P < 0.01), significant differences of the number of apoptotic LEC at 6, 12 and 24 hours (P < 0.01) and significant differences of expression of Caspase 3 at 6, 12 and 24 hours (P = 0.000, 0.005, 0.004).. PD150606, an inhibitor of calpain, can inhibit H(2)O(2)-induced opacity of lens, proteolysis and apoptosis of LEC partially. The activated calpains can induce degradation of crystallins and apoptosis of LEC, which may play a role in the mechanism of oxidative cataract. Caspase 3-dependent pathway may contribute to apoptosis of LEC induced by calpains.

Topics: Acrylates; Animals; Apoptosis; Calpain; Cataract; Cells, Cultured; Female; Glycoproteins; Lens, Crystalline; Male; Rats; Rats, Sprague-Dawley

Acetaminophen (APAP) is biotransformed by hepatic cytochrome P450 (CYP) enzymes to the cataractogenic metabolite N-acetyl-p-benzoquinone imine (NAPQI). In the previous studies in which NAPQI was injected into the anterior chamber of mouse eye, we observed mitochondrial dysfunction and disturbances in Ca2+ homeostasis in the lens epithelium, and activation of the nonlysosomal neutral protease calpain. In this work we investigated whether intraperitoneal injection of APAP elicits similar cellular responses in the lens epithelium prior to the onset of lens opacity development. Following APAP injection, reactive oxygen species generation, intracellular free Ca2+ increase and calpain activation in the lens epithelium were determined in situ by fluorescence confocal microscopy. It was found that cellular events in the lens prior to the onset of opacification were essentially identical to those elicited by NAPQI. In addition, lens calpain activities were characterized based on their Ca2+ requirement and several calpain inhibitors were shown to prevent cataract development.

Topics: Acetaminophen; Animals; Calpain; Cataract; Glycoproteins; Lens, Crystalline; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Reactive Oxygen Species; Time Factors

To study the expression and role of calpain II in rat lens epithelial cells (LECs) of hydrogen peroxide (H(2)O(2))-induced cataract.. Rat lenses were cultured in vitro and cataract was induced by 2 mM H(2)O(2). The lenses were observed under microscope. Simultaneously, photographs and picture analyses were done in order to detect the variation of the opacity. The expression of calpain II in rat LECs was detected with immunohistochemical method and compared with a control group.. When the lenses were cultured in 2 mM H(2)O(2) for 3 hours (h), the expression of calpain II in rat LECs was increased obviously. There was significant difference between H(2)O(2)-induced and the control group (P = 0.006). After 6 h, vesicles appeared at the equator of the lenses. There was a significant difference between the result of picture analysis of H(2)O(2)-induced and control group (P = 0.013). So the expression of calpain II in rat LECs of H(2)O(2)-induced cataract was increased before cataract occurred. After induced with H(2)O(2) for 24 h, the opacity of lenses and the expression of LECs were both increased as compared with that at 6 h (P = 0.000, 0.000).. H(2)O(2) can enhance the expression of calpain II in rat LECs which may play a role in the mechanism of oxidative stress-induced cataract.

Topics: Animals; Calpain; Cataract; Disease Models, Animal; Epithelial Cells; Hydrogen Peroxide; Lens, Crystalline; Rats; Rats, Sprague-Dawley

To determine the involvement of calpains in human cataractogenesis, studies in aged animal models are needed. Aged, male WBN/Kob rats spontaneously develop cataract along with severe, persistent diabetes with hyperglycemia and nephropathy. The purpose of present experiments was to provide a biochemical mechanism for the involvement of ubiquitous calpains in cataractogenesis in WBN/Kob rats. Serum and urinary glucose were measured to confirm diabetes, and cataracts were observed by slit lamp biomicroscopy. Calcium determinations were performed on lens samples from several ages of WBN/Kob and Wistar rats. Casein zymography, immunoblot analysis for alpha-spectrin, calpain 2, and calpain 10 were performed to detect activation of calpain in lens samples. Serum glucose levels increased and cortical cataract developed in male WBN/Kob rats within 1 year, indicating diabetic cataract. Cataract was accompanied by several presumptive biochemical indicators of calpain activation, including increased calcium, proteolysis of alpha-spectrin, and decreased caseinolytic activity for calpains suggesting calpain activation followed by autolytic degradation. Activation of ubiquitous calpains may contribute to biochemical mechanism of cataractogenesis in spontaneously diabetic WBN/Kob rats. The WBN/Kob model may be useful for elucidating the roles of calpain 2 and calpain 10 in human cataractogenesis.

Topics: Age Factors; Animals; Calcium; Calpain; Caseins; Cataract; Diabetes Mellitus, Experimental; Female; Male; Rats; Rats, Wistar; Sex Factors; Spectrin

It is well known that m-calpain, a ubiquitous calpain, is involved in cataract formation in rodent lens. Involvement of Lp82, a lens-specific calpain, in the cataract formation is also suggested. However, the exact relationship between Lp82-mediated proteolysis and lens opacification has not yet been established. We therefore compared Lp82- and m-calpain-mediated proteolyses of alphaA-crystallin during cataractogenesis to clarify whether Lp82 is involved in cataract formation.. In order to analyze the Lp82- and m-calpain-mediated proteolyses, we developed antibodies exclusively specific to the proteolytic products of alphaA-crystallin produced by Lp82 and m-calpain actions, respectively. The proteolytic profiles of alphaA-crystallin by Lp82 and m-calpain during cataractogenesis in SCR lenses were analyzed by Western blotting and immunohistochemical staining.. While m-calpain-mediated proteolysis was detected predominantly in cataractous lenses, Lp82-mediated proteolysis was detected not only in cataractous but in normal lenses. The m-calpain-mediated proteolysis was observed in restricted areas developing and destined to develop opacification, i.e., the nuclear and perinuclear regions of lens. On the other hand, Lp82-mediated proteolysis was observed not only in the same regions but also in the cortical region where opacity does not develop. Unlike m-calpain-mediated proteolysis, Lp82-mediated proteolysis was not inhibited by the oral administration of aminoguanidine (AG), which acts to prevent lens opacification.. From these results, it is shown that there is no direct contribution of Lp82-mediated proteolysis to cataract formation in SCR. Rather, Lp82 may function in fiber cell development and/or fiber cell remodeling during lens maturation under physiological conditions, since Lp82-mediated proteolysis occurs in the cortical region of normal lens.

Topics: Aging; alpha-Crystallin A Chain; Animals; Antibodies; Blotting, Western; Calpain; Case-Control Studies; Cataract; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Immunohistochemistry; Peptide Fragments; Peptide Hydrolases; Rats; Rats, Inbred Strains; Rats, Wistar; Time Factors

To examine the role of calcium-dependent and -independent proteolytic activity in the globulization of isolated fiber cells and glucose-induced lens opacification.. Fiber cells from rat lens cortex were isolated, and the [Ca(2+)](i) and protease activity in the isolated fibers were determined by using a calcium binding dye and the protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (BOC-Leu-Met-CMAC). The activity of calpain in the lens cortex homogenate was determined with fluorescein-casein in the presence of Ca(2+) and that of fiber cell globulizing aminopeptidase (FCGAP) with BOC-Leu-Met-CMAC and reduced glutathione (GSH) in the absence of Ca(2+). The lens proteases-calpain and the novel aminopeptidase FCGAP were partially purified by diethylaminoethyl (DEAE) gel column chromatography. Single fiber cells were isolated from rat lens, plated on coverslips, and placed in a temperature-controlled chamber. Their globulization time was determined by the appearance of light-scattering globules in the absence and the presence of protease inhibitors including the aminopeptidase inhibitor bestatin. To investigate the effect of the protease inhibitors E-64 and bestatin on the prevention of hyperglycemic cataract, the rat lenses were cultured in medium 199 in the presence of 5.5 and 50 mM glucose and in the absence and the presence of protease inhibitors. Changes in light transmission by the lenses were determined by digital image analysis.. Normal levels of lens fiber cell [Ca(2+)](i), determined by using a cell-permeable dye were approximately 100 nM, and the protease activity determined with BOC-Leu-Met-CMAC was maximum at [Ca(2+)](i) of approximately 500 nM. A large fraction of the FCGAP that cleaves BOC-Leu-Met-CMAC was separated from calpain, which cleaves fluorescein-casein, by diethylaminoethyl (DEAE) gel column chromatography. The FCGAP did not bind to the column, whereas calpain bound to the column and was eluted by approximately 180 mM NaCl. Unlike calpain, the FCGAP did not require calcium for activation and did not cleave fluorescein-casein. However, the Ca(2+)-dependent calpain activated FCGAP, indicating that the latter may exist in pro-protease form. The FCGAP was selectively inhibited by the specific aminopeptidase inhibitor bestatin, indicating that FCGAP could be an aminopeptidase. However, the FCGAP was found to be immunologically distinct from leucine aminopeptidase and calpain. Perfusion of the isolated rat lens fiber cells with Ringer's solution led to their globulization in 30 +/- 3 minutes. Addition of 0.5 mM of the protease inhibitors E-64 and leupeptin increased the globulization time to 60 and 100 minutes, respectively, whereas no globulization of the fiber cells was observed for 4 hours in the presence of 0.05 mM bestatin. In rat lens cultured in medium containing 50 mM glucose, both E-64 and bestatin (0.05 mM each) significantly reduced the extent of opacification, indicating that an aminopeptidase, downstream to a Ca(2+)-dependent protease, may be involved in mediating cataractogenic changes.. In addition to calpain, a Ca(2+)-independent novel protease, FCGAP, a novel aminopeptidase, represents a significant fraction of the total proteolytic activity in the lens. Inhibition of FCGAP by bestatin attenuates Ca(2+)-induced globulization of the isolated fiber cells in vitro and hyperglycemia-induced opacification of cultured rat lens.

Topics: Aminopeptidases; Animals; Calcium; Calpain; Cataract; Chromatography, Ion Exchange; Glucose; Glutathione; Hyperglycemia; Lens Cortex, Crystalline; Leucine; Organ Culture Techniques; Phthalimides; Protease Inhibitors; Rats; Rats, Sprague-Dawley

Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to hydrolyze specific substrates important in calcium-regulated signaling pathways. Recently, an atypical member of the calpain family, calpain 10, was described, and genetic variation in this gene was associated with an increased risk of type II diabetes mellitus in humans. In the present report, a polyclonal antibody directed against rat calpain 10 was developed. This antibody was used to monitor the expression of calpain 10 protein in tissues from rats, mice, and humans. Calpain 10 protein was found to be present in all tissues examined by Western blotting including the lens, retina, brain, heart, and skeletal muscle. Although some calpain 10 was detectable in the water-soluble protein fraction of these tissues, it was preferentially found in the water-insoluble fraction. In the lens, immunohistochemistry revealed that calpain 10 was predominately located in the cytoplasm of epithelial and newly differentiating lens fibers at the transition zone. However, calpain 10 was found to be associated with the plasma membrane of differentiated lens fiber cells and the sarcolemma of skeletal muscle. In the lens epithelium-derived cell line, alphaTN4-1, the calpain 10 protein was found in a punctate distribution in the cell nucleus as well as the cytoplasm. After the elevation of intracellular calcium levels with ionomycin, calpain 10 protein levels in the nucleus of alphaTN4-1 cells increased markedly, whereas those in the cytoplasm decreased. In the lens, the elevation of intracellular calcium levels after selenite administration resulted in increased levels of calpain 10 RNA within 1 day and a loss of calpain 10 protein from the lens nucleus coincident with the onset of selenite cataract. In conclusion, calpain 10 seems to be a ubiquitous calpain, the expression level and subcellular distribution of which are dynamically influenced by calcium.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Animals; Baculoviridae; Blotting, Western; Brain; Calcium; Calpain; Cataract; Cell Line; Cell Nucleus; Child; Child, Preschool; Cloning, Molecular; Cytoplasm; Disease Models, Animal; DNA, Complementary; Humans; Immunoblotting; Immunohistochemistry; Ionomycin; Ionophores; Lens, Crystalline; Mice; Microscopy, Fluorescence; Middle Aged; Muscle, Skeletal; Myocardium; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Retina; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sarcolemma; Time Factors; Tissue Distribution

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

Topics: Animals; Calcium; Calpain; Cataract; Cell Communication; Connexins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gap Junctions; Lens, Crystalline; Leucine; Mice; Mice, Inbred Strains; Mice, Knockout; Organ Culture Techniques

1) To measure the amount of calpain inhibitor SJA6017 taken up by lenses of young rats after administration; and 2) To test efficacy of SJA6017 against selenite cataract in regard to amelioration of proteolysis of lens protein and prevention of lens nuclear opacity.. Selenite nuclear cataracts were produced by subcutaneous injection of an overdose of sodium selenite to 16-day-old rats. SJA6017 was administered daily using intraperitoneal injections at 100 mg/kg body weight/day for 4 days. Lenses were observed and photographed by slit lamp biomicroscopy, and scored into one of three stages. Enucleated lenses were also scored into one of four stages and lens opacities in the nuclear region were quantified by image analysis. Proteolysis of crystallins was detected by SDS-PAGE. The amount of SJA6017 taken up by the lens was detected with a column switching HPLC system.. Nuclear cataracts were visible in 31% of the animals receiving only selenite, while the frequency of nuclear cataract in the Se+SJA6017 group was reduced to only 16%. This effect of SJA6017 was confirmed by densitometric analysis as a reduction in the density of the nucleus. Similar proteolytic changes of crystallins occurred at all stages of selenite cataract formation. The amount of SJA6017 in the lens was detected at the level of 0.03 microM.. Systemic SJA6017 was taken up by the lens, and SJA6017 ameliorated in vivo selenite cataract formation. These studies are important because they partially validate the biochemical rationale for developing non-surgical, drug treatments for cataract prevention in man.

Topics: Animals; Calpain; Cataract; Chromatography, High Pressure Liquid; Dipeptides; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Lens, Crystalline; Models, Animal; Rats; Rats, Sprague-Dawley; Sodium Selenite

N-acetyl-p-benzoquinone imine (NAPQI), a semiquinone metabolite of acetaminophen, produces cataract in mice. Naphthalene is biotransformed to the cataractogenic metabolite 1,2-naphthoquinone (NQ). Intracameral injection of NAPQI elicits a rapid increase in free intracellular Ca2+ in the lens epithelium and calpain activation before lens opacification begins. In order to test whether the cellular response is a common feature of quinone-induced cataracts, we injected in this work 1,2-naphthoquinone (NA) in the anterior chamber of mouse eye and followed cellular responses in the lens prior to opacity development. A marked rise in free intracellular Ca2+ in the lens epithelium and concurrent activation of calpain were observed within 1 hr after NQ injection preceding lens opacity development. These results support the suggestion that Ca2+ release and calpain activation are involved in the mechanism of quinone-induced cataractogenesis.

Topics: Animals; Anterior Chamber; Calcium; Calpain; Cataract; Epithelial Cells; Glycoproteins; Lens, Crystalline; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Naphthoquinones; Oligopeptides

The crystallins in the lenses of ICR/f mutation rat, a known hereditary cataract model, were analyzed during cataractogenesis. Opacification of the mutant lenses was found to be accompanied by changes in crystallin structure and composition, including several deletions of the N-terminals of beta-crystallins and low molecular weight alpha- crystallins. Because similar deletions were observed when the soluble fraction of normal lens protein was incubated with calpain, we considered that calpain could be related to the deletions in mutant lenses. Although measurement of the content of calpain protein by the ELISA method revealed no significant difference between mutant and normal lenses, it was found that the concentrations of Ca2+ and K+ were different between the two lenses and that calpain activity was dependent on both ion concentrations. Endogenous m-calpain in the soluble fraction from normal lenses was activated by addition of 1 mm calcium chloride in the presence of 50 mm KCl (the same concentration as in mutant lenses), and insoluble protein was found in the fraction 1 d after calpain activation. On the other hand, the presence of 120 mm KCl (the concentration in normal lenses) inhibited calpain activity and prevented this insolubilization. These results suggest that calpain in mutant lenses is involved in the proteolysis of crystallins and the progression of cataract formation.

Topics: Animals; Calpain; Cataract; Crystallins; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Glutathione; Hydrolysis; Immunoblotting; Lens, Crystalline; Potassium; Rats; Rats, Mutant Strains; Rats, Wistar; Solubility

Our previous results showed that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly suppresses lens opacification in Shumiya cataract rat (SCR). Therefore, we examine whether iNOS is upregulated and involved in cataract formation in SCR.. The expressions of iNOS mRNA and iNOS protein in SCR lenses were examined by RT-PCR and Western blotting, respectively. Calpain-mediated alphaB-crystallin proteolysis was analyzed by Western blotting using antibody specific to the calpain-generated fragment of alphaB-crystallin. Lens opacification was analyzed using computerized image analysis software connected to the Anterior Eye Segment Analysis System (EAS-1000, Nidek). Calcium contents in lenses were measured by atomic absorption spectrophotometry.. High levels of iNOS mRNA and iNOS protein are expressed in cataractous lenses compared with normal lenses. The increases in their expression are markedly suppressed by the oral administration of AG, which acts to prevent lens opacification. The induction of iNOS protein is observed before the elevation in calcium content and the acceleration of calpain-mediated proteolysis, both of which are closely related to the development of lens opacification.. These findings strongly suggest that iNOS is involved in cataract formation in SCR. The induction of iNOS occurs prior to the elevation of calcium content and its induction is inhibited by AG-treatment. Considering our previous result that the elevation of calcium content is also prevented by AG-treatment, it is conceivable that upregulation of iNOS causes calcium influx into lens cells and the subsequent activation of calpain.

Topics: Animals; Blotting, Western; Calcium; Calpain; Cataract; Enzyme Inhibitors; Guanidines; Image Processing, Computer-Assisted; Lens, Crystalline; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Mutant Strains; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrophotometry, Atomic; Up-Regulation

The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice.. Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day-old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples.. Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of alphaA-crystallin.. Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

Topics: Amino Acid Sequence; Animals; Buthionine Sulfoximine; Calcimycin; Calcium; Calpain; Cataract; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Lens, Crystalline; Light; Mass Spectrometry; Mice; Mice, Inbred ICR; Molecular Sequence Data; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Scattering, Radiation; Spectrophotometry, Atomic

Topics: Animals; Base Sequence; Calpain; Cataract; Chemical Precipitation; Chromatography; Crystallins; Disease Models, Animal; DNA Primers; Enzyme Activation; Humans; Lens, Crystalline; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The purposes of this experiment were (1) to determine if apoptosis was accelerated during formation of selenite cataract, and (2) to determine the role of calpains and caspases in lens apoptosis. Evidence for apoptosis in selenite-injected rats included: approximately 7-8% of epithelial cells in germinative zone were positive, disappearance of the nuclear membrane, condensation of the chromatin, and breakdown of PARP. Activation of calpains was indicated by characteristic limited proteolysis of crystallins, breakdown of alpha-spectrin to 150/145 kDa fragments, hydrolysis of vimentin, and autolytic breakdown of m-calpain. Selenite cataract did not have an appreciable effect on the mRNA levels for caspase-3, calpains, and calpastatin. This indicated the increased enzyme activity of m-calpain and caspase-3 in selenite cataract occurred at the enzyme level rather than by upregulation of mRNAs. Increased calpain and caspase activity may be linked to the selenite-induced apoptosis. Such data are important because they indicate that apoptosis may be a fairly early event in selenite cataract.

Topics: Animals; Apoptosis; Base Sequence; Calpain; Caspases; Cataract; Disease Models, Animal; DNA Primers; Electrophoresis, Polyacrylamide Gel; In Situ Nick-End Labeling; Lens, Crystalline; Rats; Rats, Sprague-Dawley; Sodium Selenite

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. We found incidentally that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly inhibits the development of lens opacification in SCR. Since our previous results strongly suggested that calpain-mediated proteolysis contributes to lens opacification during cataract formation in SCR, we examined the calpain-mediated proteolysis in AG-treated SCR lenses in detail. The results show that the calpain-mediated limited proteolysis of crystallins is also inhibited by AG-treatment. However, the administration of AG has no effect on the substrate susceptibility to calpain. On the other hand, the autolytic activation of calpain in AG-treated lenses is strongly inhibited, although AG itself does not inhibit calpain activity in vitro. Then, we analyzed the effect of AG-treatment on calcium concentrations in lens, and found that the elevation in calcium concentration that should occur prior to cataractogenesis in lenses is strongly suppressed by AG-treatment. These results strengthen our previous conclusion that calpain-mediated proteolysis plays a critical role in the development of lens opacification in SCR. Moreover, our results indicate that the inhibition of calpain-mediated proteolysis by AG-treatment is due to the suppression of calcium ion influx into the lens cells.

Topics: Animals; Blotting, Western; Calcium; Calpain; Cataract; Crystallins; Disease Models, Animal; Guanidines; Lens, Crystalline; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Strains

Acetaminophen, an analgesic/antipyretic, is metabolized by hepatic cytochrome P450 to N -acetyl- p -benzoquinone imine (NAPQI), which is transported by blood circulation to the eye and induces anterior cortical cataract in mice. In this study we injected NAPQI into the anterior chamber of mouse eye and investigated time-dependent cellular responses in the lens. After a lag period of about 2 hr following NAPQI injection, lens opacification as determined by measurement of light scattering by the lens became evident and progressively increased thereafter. There was no difference in the profile of opacity development between a P450-inducer responsive mouse strain and a non-responsive strain. During the lag period, a marked increase in free intracellular Ca(2+)in the lens epithelium was observed at 1 hr by confocal fluorescence microscopy with a Ca(2+)probe. Concurrent with the free Ca(2+)increase, there was a 300% rise in the activity of the non-lysosomal neutral protease calpain in the lens at 1 hr after NAPQI injection. Evidence indicated degradation of vimentin in the lens in which calpain activity was enhanced. Co-injection of calpain inhibitors (N-Ac-Leu-Leu-norleucinol and N-Ac-Leu-Leu-methioninal) with NAPQI protected animals completely from cataract development, although a rise in free intracellular Ca(2+)in the lens epithelium was still observed. Lenses from the protected mice did not exhibit enhanced calpain activity. These results suggest the following sequence of events as a possible mechanism of NAPQI-induced cataract. NAPQI introduced in the anterior chamber of the eye enters the lens epithelial cells and disturbs Ca(2+)homeostasis with a resultant rise in free intracellular Ca(2+)which in turn activates calpain in the epithelium. The neutral protease then degrades cellular proteins (e.g. cytoskeletal proteins) and initiates anterior cortical cataract formation.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Benzoquinones; Calcium; Calpain; Cataract; Cytochrome P-450 Enzyme System; Epithelium; Lens, Crystalline; Light; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Microscopy, Fluorescence; Scattering, Radiation; Vimentin

To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses.. Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed.. At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium.. Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.

Topics: Animals; Calcimycin; Calcium; Calpain; Cataract; Chemical Precipitation; Crystallins; Cysteine Proteinase Inhibitors; Isoelectric Focusing; Lens Nucleus, Crystalline; Light; Organ Culture Techniques; Rats; Rats, Wistar; Scattering, Radiation

To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m-calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis.. Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing.. The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239, corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235.. Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.

Topics: Animals; Aquaporins; Calpain; Cataract; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Gas Chromatography-Mass Spectrometry; Lens, Crystalline; Membrane Glycoproteins; Peptide Fragments; Phosphoproteins; Phosphorylation; Rats; Sodium Selenite

To determine if oxidation enhances turbidity after proteolysis of rat lens crystallins by the calcium-activated protease calpain (EC 3.4.22.17).. Total soluble proteins from young rat lens were hydrolyzed for 24 hr by endogenous lens calpain, and the proteins were further incubated with the oxidant diamide for up to 7 days. Turbidity was measured daily at 405 nm. To measure proteolysis and turbidity in cultured lenses, rat lenses were cultured for 6 days in low calcium medium and diamide. The lenses were then photographed to assess transmission of light. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, alpha-spectrin, and activation of calpain.. Appreciable in vitro turbidity occurred in soluble proteins from young rat lenses after proteolysis of crystallins by endogenous calpain. Calpain inhibitor E64, or anti-oxidants DTE and GSH, inhibited this turbidity. On the other hand, the oxidant diamide markedly enhanced calpain-induced turbidity. Cultured rat lenses showed elevated intralenticular calcium and proteolysis of crystallins by calpain, but no nuclear cataract. Addition of diamide to the culture medium caused development of nuclear cataract.. Diamide enhanced turbidity only when crystallins were proteolyzed. Oxidation may be one of the factors promoting light scatter and insolubilization after proteolysis. These data are consistent with the hypothesis that proteolysis of crystallins from young rat lens may expose cysteine residues, which are then oxidized, become insoluble and scatter light.

Topics: Animals; Antioxidants; Calpain; Cataract; Crystallins; Culture Techniques; Cysteine Proteinase Inhibitors; Diamide; Dithioerythritol; Drug Synergism; Glutathione; Lens, Crystalline; Leucine; Oxidants; Oxidation-Reduction; Peptide Hydrolases; Rats; Rats, Sprague-Dawley

The purpose of this study was to examine changes in calcium-dependent proteolytic activity in the lens epithelium from whole rabbit lenses exposed to long-term oxidative stress at near physiological levels. Rabbit lenses, incubated in 50 microM H2O2 for 1 or 24 h, were checked for clarity and morphological changes in the epithelium. Proteolytic activity was measured in the epithelium using a fluorogenic synthetic substrate; N-succinyl-Leu-Tyr-7-amino-4-methylocoumarin, both in the presence and the absence of calcium (1 mM Ca2+ and 5 mM EDTA respectively). The effect on transparency and morphology of the epithelium following a 1-hour incubation in 100 microM H2O2 was also studied. Lenses incubated in 50 microM H2O2 were clear even after 24h. After a 1-hour incubation in 50 microM H2O2 the epithelium of the exposed lens appeared normal. However, after 24 h the epithelium cells appeared swollen and microscopical examination showed extensive intracellular and subepithelial vacuolization. Incubation in 100 microM H2O2 for 1 h caused loss of transparency; vacuole formation, globulization of the superficial lens fibers and death of the epithelial cells. There was a 55% increase in calcium-dependent proteolytic activity after 1 h in 50 microM H2O2, implying a role for the calcium-activated protease calpain in oxidatively induced cataract.

Topics: Animals; Calcium; Calpain; Cataract; Epithelium; Female; Hydrogen Peroxide; Lens, Crystalline; Organ Culture Techniques; Oxidative Stress; Rabbits

To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit.. Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm.. Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain.. Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.

Topics: Animals; Body Water; Calcimycin; Calcium; Calpain; Cataract; Crystallins; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Ionophores; Lens Nucleus, Crystalline; Light; Organ Culture Techniques; Organ Size; Rabbits; Scattering, Radiation; Spectrin

Mutations in the Drosophila melanogaster small optic lobes (sol) gene cause a sever reduction in the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein product of sol contains zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal region of unknown function. We have isolated human brain cDNA for SOLH, a human homologue of sol. The human SOLH gene consists of 14 exons distributed over more than 45 kb of genomic DNA. The encoded SOLH protein of 1086 amino acids has strong similarity to the D. melanogaster protein. The calpain-like domain and C-terminal region are highly conserved (58% identity), and similar Cys2-Cys2 zinc fingers are present in the N-terminal region. A reported Caenorhabditis elegans homologue contains the calpain domain and C-terminal region, but appears to lack the zinc finger region. A single copy of the zinc finger sequence is present in adjacent C. elegans genomic cosmid DNA sequence, and we show that it is part of the C. elegans sol-like transcript. Northern analysis of human tissues revealed a SOLH transcript of approximately 5 kb that was strongest in human brain. We have mapped the SOLH gene to chromosome 16p13.3 by in situ hybridization. SOLH is a candidate gene for CATM (hereditary cataracts with microphthalmia), which maps in this region.

Topics: Amino Acid Sequence; Animals; Brain Chemistry; Calpain; Cataract; Chromosomes, Human, Pair 16; Cloning, Molecular; DNA, Complementary; Drosophila melanogaster; Expressed Sequence Tags; Female; Gene Dosage; Genes; Humans; Male; Microphthalmos; Molecular Sequence Data; Organ Specificity; Physical Chromosome Mapping; Proteins; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Zinc Fingers

The relation between cataract and calpain proteolysis of lens fodrin was studied in two systems: elevated glucose (55.6 mM, diabetic model), and cytochalasin D (CD, 10(-2) mM, actin depolymerization-induced opacity model). Glucose treatment (48 h) caused a visible opaque layer and enzyme leakage, with a concomitant accumulation of ([Ca2+]i) around the lens equatorial cortex. CD caused both earlier and greater opacity and enzyme leakage than glucose. Lens fodrin digestion occurred in parallel with the timing and extent of calcium elevation. A calpain inhibitor peptide (CIP, 10(-2) mM) reduced the proteolysis of fodrin, opacity, and enzyme leakage in glucose-treated lenses but only partially retarded them in CD-treated lenses. These results suggest a mechanism in which calpain proteolysis of fodrin is a critical event in lens damage during opacification of cortical cataract.

Topics: Amino Acid Sequence; Animals; Calcium; Calpain; Carrier Proteins; Cataract; Culture Techniques; Cysteine Proteinase Inhibitors; Cytochalasin D; Fluoresceins; Fluorescent Dyes; Glucose; Glycoproteins; L-Lactate Dehydrogenase; Lens Cortex, Crystalline; Lens, Crystalline; Microfilament Proteins; Molecular Sequence Data; Organic Chemicals; Pyridinium Compounds; Rats

To measure changes in levels of Lp82 during maturation and selenite cataract formation in rat lens. Lp82 is a lens-specific, calcium-activated isozyme from the calpain family of cysteine proteases (EC 34.22.17).. Competitive RT-PCR was used to assess Lp82 and m-calpain mRNA concentrations. Immunoblotting and ELISA after DEAE chromatography measured Lp82 and m-calpain protein levels. Casein zymography assessed proteolytic activities in regions and whole lenses from maturing rats.. Levels of Lp82 mRNA, protein, and caseinolytic activity decreased more rapidly during maturation of rat lens than for m-calpain. Unexpectedly, the water-insoluble fraction of rat lens contained enzymatically active Lp82. Selenite injection also caused major loss of Lp82 protein during cataract formation.. Lp82 is a proteolytic enzyme likely functioning in early lens development and maturation. The rapid loss of Lp82 activity during lens maturation is probably caused by three factors: autodegradation associated with the proteolysis of soluble and insoluble proteins occurring in the rat lens nucleus, association of Lp82 with the lens insoluble fraction, and loss of Lp82 mRNA. Lp82 may function early in lens maturation along with m-calpain, which then is predominant in the latter stages of maturation. Proteolysis in selenite cataract is partially caused by over-activation of Lp82.

Topics: Aging; Animals; Calpain; Caseins; Cataract; Chromatography, DEAE-Cellulose; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Isoenzymes; Lens, Crystalline; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Selenite

The purpose of these experiments was to describe the expression of mRNA for calpain II proteolytic enzyme (EC 3.4.22.17) during normal maturation of rat lens and in cataract formation. Quantitative RT-PCR indicated that the concentration of mRNA for calpain II in whole lens was 3-24 times higher than in age-matched rat liver, kidney, lung and brain, and it was at least five times higher than in young human lens. mRNA levels for calpain II were highest in the outer regions of young rat lens at 5 x 10(6) copies microgram-1 total RNA. Early-stage experimental cataract caused increased calpain II mRNA, while mature nuclear cataract showed a 64% loss. In contrast, mRNA levels for GAPDH, beta-actin, and lens-specific structural protein beta A4 remained constant during experimental cataract formation. Unlike the lower and constant levels in rat liver, kidney and lung; calpain II mRNA levels in whole rat lens decreased with age. These data help explain the high enzymatic activity of calpain II in young rat lens, susceptibility of young rat lens to a variety of cataracts showing increased calcium and calpain-induced proteolysis, and low calpain enzyme activity in human lens. Since the up-regulation of calpain II mRNA was more dynamic than either the amounts of calpain II enzyme or proteolysis of crystallins in cortex, resulting proteolytic activity against the bulk of lens proteins seems to be regulated by post-translational factors, such as increased calcium. The precise role of the up-regulation of calpain II mRNA is unknown, but we hypothesize that it may be associated with the initial cataractogenic response in the epithelial cells or peripheral cortical fibers.

Topics: Animals; Blotting, Northern; Brain; Calpain; Cataract; Humans; Kidney; Lens Cortex, Crystalline; Lens Nucleus, Crystalline; Lens, Crystalline; Liver; Lung; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Selenite

The purposes of this experiment were to: (1), characterize the peptide aldehyde SJA6017, N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal, a newly synthesized inhibitor of calpain, and (2) test the effect of SJA6017 in preventing calcium ionophore-induced cataract in cultured rat lenses. In vitro, SJA6017 strongly inhibited purified m-calpain from porcine kidney. Casein zymography confirmed that SJA6017 reversibly bound to the active site of m-calpain. SJA6017 was also confirmed to be a cell-permeable inhibitor in Molt-4 cells. In cultured lenses, SJA6017 reduced nuclear opacity and proteolysis of crystallins and alpha-spectrin caused by calcium ionophore A23187. These results suggested that SJA6017 is a reversible and cell-permeable calpain inhibitor which may possess great efficacy against calcium-induced models of cataract.

Topics: Animals; Calpain; Cataract; Culture Techniques; Dipeptides; Enzyme Inhibitors; Hydrolysis; Rats; Rats, Sprague-Dawley

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. It was found that the proteolysis of some crystallins and cytoskeletal proteins is significantly enhanced in cataractous SCR lenses. The calcium concentrations in cataractous lenses rise markedly with age as compared with control lenses and the autolytic product of calpain is also detected in cataractous lenses. In order to provide direct evidence for the involvement of calpain in the proteolytic modification of lens proteins, we developed antibodies exclusively specific to the proteolytic products of some lens proteins produced by the action of calpain and analyzed their degradation during cataractogenesis in SCR by Western blotting and immunohistochemical staining. The results demonstrate that calpain participates in the proteolytic modification of lens proteins, at least alpha-crystallin (A and B chain), betaB1-crystallin, and alpha-fodrin. The proteolytic products formed by the action of calpain on these proteins are detected in cataractous lenses of SCR as young as 8 weeks of age and accumulate with age. It was also found that betaB1-crystallin, originally a soluble protein, is converted to an insoluble form by limited calpain proteolysis. The chaperon-like activity of alpha-crystallin from control lens is markedly reduced by calpain proteolysis in vitro, and alpha-crystallin in opaque lens that has already undergone proteolysis by calpain shows significantly reduced chaperon-like activity. Immunohistochemical studies reveal that the area where the calpain-mediated alpha-crystallin proteolysis is in progress coincides well with the area developing and destined to develop the opacification. These results strongly suggest that calpain may contribute to lens opacification during cataract formation in SCR.

Topics: Aging; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Calcium; Calpain; Carrier Proteins; Cataract; Cattle; Crystallins; Lens, Crystalline; Microfilament Proteins; Molecular Chaperones; Molecular Sequence Data; Peptide Fragments; Rats; Rats, Inbred Strains

Two dipeptide aldehyde cell permeable calpain inhibitors, cBz-Val-Phe and calpeptin, have been assessed for their ability to prevent cytoskeletal proteolysis and loss of transparency in whole rat lenses. Calcium overload, induced by ionomycin in artificial aqueous humor with 1mM calcium, resulted in lens opacification and degradation of cytoskeletal proteins including spectrin, filensin, and vimentin. No such changes resulted from incubation in ionomycin in the absence of calcium. In calcium overload lenses both inhibitors gave some protection against cytoskeletal protein degradation and loss of transparency. These experiments indicate that calpain has a role in cortical opacification in high calcium lenses and that cell penetrating calpain inhibitors do indeed enter lens cells and reduce both proteolysis and opacification.

Topics: Animals; Calcium; Calpain; Cataract; Cattle; Cell-Free System; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Eye Proteins; Intermediate Filament Proteins; Ionophores; Lens, Crystalline; Light; Rats; Rats, Wistar; Scattering, Radiation; Spectrin; Vimentin

Calpains are Ca-activated neutral proteases present in all cells together with an endogenous inhibitor, calpastatin. Proposed substrates are; cytoskeletal proteins like microtubules and actin, protein kinases such as PKC and membrane-bound enzymes like Ca-ATPase and the Ca-channel. In lenses from different species calpains have been detected in decreasing amounts from the epithelium to the cortex to the nucleus. Several substrates for calpain in the lens have been demonstrated: crystallins, vimentin, actin, beaded filaments and MP26 among others. Both studies on animal models and capsulorhexis indicate that calpains are mainly involved in cortical cataract.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cataract; Epithelium; Humans; Lens, Crystalline; Substrate Specificity

The purpose of these experiments was to study the mechanism for precipitation of lens crystallins in cataract. An in vitro model was developed to activate the endogenous protease calpain II in the soluble proteins from young rat lens by addition of calcium in the presence of 120 mM KCl. Light-scattering, insoluble proteins were produced approximately 4-6 days after calpain II activation. Results showed that proteolysis was caused by activation of lens calpain II, proteolysis preceded precipitation by several days, and alpha-crystallin acted as a molecular chaperone against precipitation of crystallins caused by proteolysis. These data supported our hypothesis that calpain-induced proteolysis of the N-terminal arms of beta-crystallin polypeptides leads to a loss of normal oligomerization of beta-crystallin polypeptides and formation of abnormal insoluble aggregates, possibly stabilized by hydrophobic interactions.

Topics: Amino Acid Sequence; Animals; Calcium; Calpain; Cataract; Crystallins; Electrophoresis, Gel, Two-Dimensional; Immunoblotting; In Vitro Techniques; Lens, Crystalline; Light; Models, Biological; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; Scattering, Radiation

To confirm the effect of a new aldose reductase inhibitor (ARI), rat lenses were cultured with xylose. ARI prevented opacities and reduced lens hydration caused by xylose. Next, cataract was produced by feeding a diet containing 50% galactose. ARI was tested for amelioration of cataract. On day 19 after feeding of galactose, nuclear cataracts were visible in 75% of the animals receiving only galactose, while nuclear cataracts were not observed in animals treated with ARI. In galactose cataract, lens hydration and calcium were significantly increased. Calpain in soluble and insoluble fractions was decreased. Alpha- and beta-crystallins were proteolyzed. These changes were inhibited by administration of ARI. These results suggested that proteolysis by calpain is an underlying mechanism in formation of sugar cataract in rat lens.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Body Water; Calcium; Calpain; Cataract; Crystallins; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Galactose; Lens, Crystalline; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Thiazines; Thiazoles; Xylose

To determine if the susceptibility of rat lenses to cataract formation in culture changes with increasing age and to investigate the regional differences in crystallin degradation and insolubilization during the formation of cataracts in cultured lenses.. Lenses from 4-week-old (young group) and 12-week-old (adult group) rats were divided into four subgroups: noncultured control, cultured control, cultured in calcium ionophore A23187, and cultured in ionophore plus calpain inhibitor E64. Lenses were cultured for 7 days, and the cortex and nucleus were homogenized and separated into water-soluble and water-insoluble fractions. Two-dimensional electrophoresis and N-terminal sequencing were then performed.. Young lenses treated with ionophore produced thin cortical and dense nuclear opacities. Adult lenses treated with ionophore also developed thin cortical opacity, but no nuclear opacity was observed, even though a large increase in the concentration of insoluble protein occurred. Two-dimensional electrophoresis and sequencing suggested that calpain caused protein degradation in the cortex region. However, unlike nuclear opacity, the formation of opacity in the cortex was not inhibited by E64 in young or adult lenses.. Calpain was activated, and crystallins were proteolyzed in the cortex of ionophore-treated lenses. However, cortical opacity was not the result of proteolysis by calpain. Maturation also decreased the susceptibility of rat lens nucleus to calcium ionophore cataract.

Topics: Aging; Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Lens, Crystalline; Leucine; Molecular Sequence Data; Organ Culture Techniques; Protein Denaturation; Rats; Rats, Sprague-Dawley; Solubility

Lens epithelium from patients with cataract was obtained during surgery and frozen. The samples were subjected to SDS-electrophoresis and Western blotting. Calpains were quantified using polyclonal antibodies against m- and mu-Calpain could be detected but not the isoenzyme mu-calpain, indicating that m-calpain is the significant most important calpain in human lens epithelium. Quantification of m-calpain showed no relationship to age or gender, but there were significant differences between different types of cataract.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Blotting, Western; Calpain; Cataract; Electrophoresis, Polyacrylamide Gel; Female; Humans; Lens Capsule, Crystalline; Male; Middle Aged; Sex Factors

Topics: Amino Acid Sequence; Animals; Buthionine Sulfoximine; Calpain; Cataract; Crystallins; Enzyme Activation; Methionine Sulfoximine; Mice; Mice, Inbred Strains; Molecular Sequence Data; Solubility

To determine if limited proteolysis of beta-crystallins is associated with insolubilization of proteins in rats lens during maturation and to test if the protease, calpain II, is involved.. Soluble and insoluble lens proteins from 4-day-old to 4-month-old rat lens cortexes and nuclei were separated by two-dimensional electrophoresis. The insoluble proteins from 4-month-old nuclei were electroblotted and the NH2 termini of proteins sequenced. Cleavage sites appearing at 4 months of age were compared to cleavage sites produced by purified calpain II and to cleavage sites appearing in cataracts induced by selenite in vivo or in lenses cultured with calcium ionophore A23187 or diamide.. In solubilization of more than 50% of proteins occurred in the nucleus of the transparent rat lens by 4 months of age. The insoluble protein that formed contained an abundance of partially degraded beta-crystallin polypeptides missing portions of their NH2 terminal extensions. In contrast, these truncated beta-crystallins were largely absent from both the cortex and soluble fraction of the nucleus. The cleavage sites in the insoluble beta-crystallins appearing during maturation in the lens nucleus were similar to cleavage sites produced by purified calpain II and also similar to cleavage sites appearing in the insoluble protein of cataractous lenses.. These results suggest that proteolysis of beta-crystallins by the protease calpain II contributes to protein insolubilization during lens maturation and that acceleration of this insolubilization process is associated with cataract formation in rodent lenses.

Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Crystallins; Diamide; Electrophoresis, Gel, Two-Dimensional; Lens, Crystalline; Molecular Sequence Data; Organ Culture Techniques; Peptide Fragments; Peptide Mapping; Rats; Rats, Sprague-Dawley; Sodium Selenite; Solubility

The purpose of these experiments was to examine the relationship between oxidation cataract and proteolysis in cultured rat lens. Hydrogen peroxide cataract showed insolubilization of protein, loss of 31 kDa beta B1-crystallin polypeptide, decreases in soluble calpain, and increases in insoluble calpain. This suggested that calpain may be activated in hydrogen peroxide treated lenses, since beta B1 is a known calpain substrate, and calpain undergoes autolysis and degradation when activated. Furthermore, the cysteine protease inhibitor E64 was partially effective in preventing development of H2O2-cataract. E64 also prevented the loss of the 31 kDa beta B1-crystallin polypeptide and decreased the loss of calpain in the lens. These results suggested that development of hydrogen peroxide induced cataract in rat lenses was associated with activation of calpain.

Topics: Animals; Calcium; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Hydrogen Peroxide; Lens, Crystalline; Leucine; Organ Culture Techniques; Rats; Rats, Sprague-Dawley

This study reports the first demonstration of a marked reduction in alpha-crystallin chaperone activity in an experimental model of cataract, and the study implicates activation of the cysteine protease calpain II (EC 3.4.22.17) as the in vivo protease responsible for decreased chaperone activity. Chaperone activity of normal alpha-crystallin from lenses of young rats was assayed by measuring attenuation of heat-induced aggregation and scattering of beta L-crystallin. alpha-Crystallin from the nucleus of lenses with selenite cataract showed specific selective proteolysis, and chaperone activity was diminished. Proteolysis of alpha-crystallin from selenite cataract lenses was mimicked by incubating normal alpha-crystallin with calpain II, and this also resulted in loss of chaperone activity. Two-dimensional gel electrophoresis and peptide mapping were used to identify four partially degraded alpha A- and alpha B-crystallin polypeptides following incubation of normal alpha-crystallin with calpain. Similar partially degraded alpha A and alpha B polypeptides were found in selenite cataract. Previous experiments indicated that alpha-crystallin chaperone activity decreases because of removal of the COOH terminus. Our experiments support this observation and suggest that calpain proteolysis of alpha-crystallin at the COOH terminus may result in a loss of chaperone activity in selenite cataract.

Topics: Amino Acid Sequence; Animals; Calpain; Cataract; Crystallins; Electrophoresis, Gel, Two-Dimensional; Endopeptidases; Hot Temperature; Light; Molecular Sequence Data; Peptide Fragments; Peptide Mapping; Rats; Rats, Sprague-Dawley; Scattering, Radiation; Selenium; Sodium Selenite; Trypsin

Incubation of soluble proteins from rat lens with the protease calpain II caused the precipitation of beta-crystallin polypeptides. Two-dimensional electrophoresis and sequence analysis identified beta-crystallin polypeptides both before and after their precipitation by calpain II. beta-crystallin polypeptides precipitated by calpain were cleaved at their NH2-terminal extensions. These cleavage sites were similar to cleavage sites occurring in beta-crystallin polypeptides precipitated during formation of experimental cataract induced by an overdose of selenite. These data suggested that calpain II caused beta-crystallin insolubilization during cataract formation, and indicated that the process can be mimicked in vitro.

Topics: Amino Acid Sequence; Animals; Calpain; Cataract; Chromatography, High Pressure Liquid; Crystallins; Electrophoresis, Gel, Two-Dimensional; In Vitro Techniques; Molecular Sequence Data; Peptide Mapping; Rats; Rats, Sprague-Dawley; Solubility

Abnormal activation of the protease calpain in the lens may be a cause of cataracts. Cataracts were induced in 10-day-old rats by a single overdose of sodium selenite. The water-insoluble protein from the opaque lens nucleus was separated by two-dimensional electrophoresis, electroblotted onto membranes, and the NH2-terminal sequence of partially degraded beta-crystallin polypeptides determined. Selenite cataractous lenses contained four major structural proteins, beta B1, beta B3, beta A3/A1, and beta A4 crystallins, missing from 5 to 49 amino acids from their NH2 termini. Incubation of intact beta-crystallins with calpain II in vitro produced identical cleavage sites. This provided further evidence for the role of calpain in the production of light scattering insoluble protein in cataractous lenses and also suggested that a similar process may lead to lens protein insolubilization during aging.

Topics: Amino Acid Sequence; Animals; Binding Sites; Calpain; Cataract; Crystallins; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Lens, Crystalline; Molecular Sequence Data; Peptide Fragments; Rats; Sequence Analysis

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Culture Techniques; Cysteine Proteinase Inhibitors; Dipeptides; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Lens, Crystalline; Leucine; Leupeptins; Molecular Sequence Data; Rats; Rats, Sprague-Dawley

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.

Topics: Animals; Calcimycin; Calcium; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Lens, Crystalline; Leucine; Organ Culture Techniques; Organ Size; Rats; Rats, Sprague-Dawley

Samples of the vitreous were analysed in order to identify changes of soluble proteins in vitreo-retinal disease. The soluble proteins of the vitreous were separated on an anion exchange column (Mono-Q). The degree of neutral proteolytic activity in vitreous body was also measured. The vitreous from cataract cases without vitreoretinal disease was characterized by its low content of soluble proteins equivalent to about 1% of that of serum. Albumin and transferrin were the major identified components and their concentrations were approximately 0.85 and 0.03 g/l, respectively. In cases with vitreoretinal disease the vitreous showed changes of total soluble protein and the appearance of additional protein peaks. In patients with PVR the albumin concentration in the vitreous was found to be three times higher as compared to the control group consisting of patients with cataract. Neutral proteolytic activity in the vitreous was relatively low in both normal and pathological vitreous.

Topics: Aged; Albumins; Calpain; Cataract; Chromatography, High Pressure Liquid; Eye Diseases; Eye Proteins; Humans; Middle Aged; Retinal Diseases; Solubility; Transferrin; Uveitis; Vitrectomy; Vitreous Body

The purpose of this experiment was to determine the contribution of calpain proteolytic enzyme (EC 3.4.22.17) in the formation of nuclear cataract during lens culture in xylose. Increased lens calcium was found to be required for formation of xylose nuclear cataract in our culture system. Inhibition of calpain by the cysteine protease inhibitor E64 was effective in slowing the formation of nuclear cataract, even though lens calcium and hydration were markedly elevated. These results showed that hydration and elevated calcium alone do not produce xylose nuclear cataract, and they indicated that calpain proteolysis may be necessary for xylose nuclear cataract in the rat lens.

Topics: Animals; Calcium; Calpain; Cataract; Cysteine Proteinase Inhibitors; Lens, Crystalline; Leucine; Organ Culture Techniques; Rats; Rats, Inbred Strains; Xylose

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca2+, it was limited to 6% of the soluble protein present and resulted from precipitation of proteolyzed beta-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble beta-crystallin polypeptides produced by calpain II were similar to insoluble beta-crystallin polypeptides found in cataractous lenses. Trypsin also caused insolubilization of beta-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from beta-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.

Topics: Animals; Calcium; Calpain; Cataract; Chromatography, High Pressure Liquid; Crystallins; In Vitro Techniques; Lens, Crystalline; Molecular Weight; Peptide Fragments; Rats; Solubility

Lenses cultured in diamide first developed outer cortical opacities followed by nuclear cataract. Lens hydration and total calcium were markedly increased by diamide. Proteolysis of crystallins were observed in nuclear cataract lenses. Calpain in the soluble fraction of lenses cultured with diamide was decreased, while calpain in the insoluble fraction was increased. Co-culture with E64d, an inhibitor of cysteine protease such as calpain, especially prevented nuclear opacities and proteolysis of crystallins, indicating that calpain was involved in cataract formation by diamide.

Topics: Animals; Blotting, Western; Calcium; Calpain; Cataract; Crystallins; Culture Techniques; Diamide; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Lens, Crystalline; Rats; Rats, Inbred Strains

Topics: Animals; Calcium; Calpain; Cataract; Cattle; Crystallins; Cytoskeletal Proteins; Cytoskeleton; In Vitro Techniques; Lens, Crystalline; Molecular Weight

Cataracts were produced in cultured rat lenses by either 10 microM calcium ionophore A23187, 25 microM sodium selenite, or 30 mM xylose. E64, an inhibitor of cysteine proteases, such as calpain (EC, 3.4.22.17), reduced severity of cataract and proteolysis of crystallins when included at a 500 microM concentration in the culture medium along with cataractogenic agents. Calpain II enzyme activity and the amount of calpain antigen were decreased in the cytosol of cataractous lens. However, E64 caused an increase in the amount of an 80-kD calpain subunit associated with the ethyleneglycol-bis-(beta-aminoethylether) tetraacetic acid/ethylenediaminetetraacetic acid-washed insoluble proteins when lenses were incubated with cataractous agents. These data indicate that E64 was at least partially effective in inhibiting lens calpain, and that activation of lens calpain may involve binding to the insoluble fraction. These results provide strong evidence for the activation of calpain in rodent cataracts and suggest testing inhibitors of calpain as anticataract drugs.

Topics: Animals; Calcimycin; Calpain; Cataract; Chromatography, High Pressure Liquid; Crystallins; Culture Techniques; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Lens, Crystalline; Leucine; Rats; Rats, Inbred Strains; Selenium; Sodium Selenite; Xylose

The purpose of this experiment was to test the effectiveness of E64 in prevention of selenite nuclear cataract in the whole animal. E64 is an inhibitor of cysteine proteases such as calpain (EC.3.4.22.17). In the whole animal, daily intraperitoneal injection of E64 was mildly effective in slowing the rate of formation of selenite nuclear cataract, although prevention was not permanent. Frequency of the nuclear cataract in selenite group at 5 days post selenite injection was significantly decreased from 40% to 17% in the selenite + E64 group, and the density of cataract in the Se + E64 group was reduced. However, crystallins and calpain were still degraded in the selenite + E64 group. E64 was more effective against selenite cataract when present continuously during lens culture, where it slowed the rate of formation of nuclear opacity. Amelioration of cataract occurred both in vitro and in vivo even though lens calcium concentrations were elevated. The results supported the idea that application of calpain inhibitor is beneficial in prevention of rodent selenite cataracts.

Topics: Animals; Body Water; Calcium; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Injections, Intraperitoneal; Lens, Crystalline; Leucine; Organ Culture Techniques; Rats; Rats, Inbred Strains; Selenious Acid; Selenium

When intact rat lenses were incubated in artificial aqueous humor in the presence of 1 mM calcium and a sulfhydryl reagent p-chloromercuriphenyl sulfonate (pCMPS) a visible annular opacity developed within 4 hours. Combined photographic and ion-sensitive microelectrode investigations of the lenses demonstrated that the subsequent linear increase in opacification was accompanied by an increase in internal free calcium. Opacities were not observed in lenses incubated in the absence of either pCMPS or calcium. Gel electrophoresis of the soluble and urea-soluble fractions from lenses exposed to 1 mM calcium for periods of up to 14 hours showed no evidence for crystallin degradation and only minor proteolysis of cytoskeletal proteins. When lenses were incubated under identical conditions, but with 5 mM calcium, the degree of opacification increased up to approximately 8 hours and then remained constant. A progressive loss in cytoskeletal proteins was observed which correlated with a further increase in free calcium such that by 14 hours of incubation, when the internal calcium approached 1 mM, most of the spectrin and vimentin present in the cortex of the lens had disappeared. An unidentified 110-kilodalton protein also disappeared from lenses incubated in 5 mM calcium. These results indicate that proteolysis by calcium-dependent enzymes such as calpain may play a significant role in cytoskeletal regulation and metabolism in the lens. A role for cytoskeleton/membrane/crystallin interaction in calcium-induced opacification is discussed.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Calcium; Calpain; Cataract; Crystallins; Cytoskeletal Proteins; Densitometry; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Lens, Crystalline; Membrane Potentials; Peptide Hydrolases; Rats; Rats, Inbred Strains

Cataracts were produced in rat lenses by either feeding a diet containing 50% galactose or by inducing diabetic condition by intravenous injection of streptozotocin. Proteolysis of crystallins, protease activity of calpain II enzyme (EC 3.4.22.17), and presence of calpain molecule (antigen) were determined at four cataract stages--I, cortical vacuoles, II, vacuoles plus hazy cortex, III, nuclear cataract, and IV, mature cataracts. Calpain activity was normal or moderately elevated at early stages of galactose and diabetic cataracts. Later stages III and IV showed proteolysis of lens crystallins, increased proportion of insoluble proteins, loss of calpain enzyme activity and calpain molecule from the soluble fraction, and reduced amounts of calpain associated with insoluble pellet. In galactose cataract, the largest increase in lens calcium were found when proteolysis was present. These results provide evidence for calpain-induced proteolysis of lens crystallins in two in vivo models of sugar cataracts in rodents.

Topics: Animals; Antigens; Calpain; Cataract; Crystallins; Diabetes Mellitus, Experimental; Dietary Carbohydrates; Disease Models, Animal; Galactose; Lens, Crystalline; Rats; Rats, Inbred Strains

The purpose of this experiment was to assess the roles of free, intracellular calcium and calcium-dependent neutral protease (calpain II, EC.34.22.17) in selenite nuclear cataract. Free calcium ion concentrations within lens nuclear fibers during selenite cataractogenesis increased to 3 microM on day 2 post-injection (clear lens) and to 108 microM at day 4 (nuclear cataract). Calpain II is known to be activated in vitro by calcium levels above 50 microM. Calpain II activity was present in the lens nucleus at time periods preceding formation of selenite cataract. These data suggested that after selenite injection, calpain II was activated by elevated free calcium in the nucleus, and that calpain II-induced proteolysis of nuclear proteins was an important mechanism in selenite cataract. Calpain II levels were also observed to decrease in the nucleus during selenite cataractogenesis, probably due to autolysis. This was supported by the finding that incubation of purified lens calpain II with 100 microM calcium caused partial inactivation of the protease.

Topics: Animals; Calcium; Calpain; Cataract; Lens, Crystalline; Rats; Rats, Inbred Strains; Selenious Acid; Selenium

Nuclear cataract resulting from an overdose of selenite was characterized by a five-fold increase in nuclear urea-soluble protein. The origin of this urea-soluble protein was examined by two-dimensional electrophoresis, immunoblotting with monospecific antisera against rat lens crystallins, and tryptic mapping. Cataractous urea-soluble protein was primarily composed of insolubilized beta- and gamma-crystallin polypeptides. Polypeptides from cataractous urea-soluble protein, and normal beta L-crystallin aggregates were compared by tryptic mapping. Approximately 19% of the urea-soluble protein from opaque nuclei was composed of 24.7 and 24.0 K polypeptides derived by limited proteolysis of 26.5 K beta L-crystallin polypeptide. Incubation of 26.5 K beta-crystallin polypeptide with purified rat lens calpain II in vitro caused production of fragments with similar molecular weights to polypeptides found in cataractous lenses. These results support the hypothesis that proteolysis may contribute to formation of urea-soluble protein in selenite cataract.

Topics: Animals; Calpain; Cataract; Cell Nucleus; Crystallins; Electrophoresis; Eye Proteins; Immunologic Techniques; Peptide Hydrolases; Peptides; Rats; Rats, Inbred Strains; Selenious Acid; Selenium; Solubility; Urea

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

Topics: Animals; Biological Transport; Calcium; Calpain; Cataract; Cattle; Crystallins; Endopeptidases; Lens, Crystalline; Membranes

The age-related changes of calpain II (high-Ca2+-requiring form of Ca2+-dependent cysteine proteinase; EC 3.4.22.17) and alpha-crystallin in the lens of hereditary cataract (Nakano; cac/cac) mouse were studied. Before the onset of the cataract formation, i.e., at the end of the 2nd week after birth, the calpain activity in Nakano mice was as high as that in the control ICR mice, but it decreased rapidly as the cataract progressed to completion during the 4th and the 12th week. Marked degradation of lens proteins ensued between the 2nd and the 4th weeks, and one of these proteins was identified, using monospecific antibodies, as B chain of alpha-crystallin. A chain of alpha-crystallin was not degraded in vivo, in contrast to its known susceptibility to calpain in vitro. The present data suggest that in Nakano mice, calpain may be involved in the onset or early stage of the cataract formation.

Topics: Aging; Animals; Calpain; Cataract; Crystallins; Eye Proteins; Lens, Crystalline; Mice; Mice, Inbred ICR; Mice, Inbred Strains

Topics: Aging; Animals; Calpain; Cataract; Crystallins; Lens, Crystalline; Mice; Mice, Inbred ICR; Mice, Inbred Strains