Novel molecular targets of the neuroprotective/neurorescue multimodal iron chelating drug M30 in the mouse brain.

The novel multifunctional brain permeable iron, chelator M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline] was shown to possess neuroprotective activities in vitro and in vivo, against several insults applicable to various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the present study, we demonstrate that systemic chronic administration of M30 resulted in up-regulation of hypoxia-inducible factor (HIF)-1α protein levels in various brain regions (e.g. cortex, striatum, and hippocampus) and spinal cord of adult mice. Real-time RT-PCR revealed that M30 differentially induced HIF-1α-dependent target genes, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), enolase-1, transferrin receptor (TfR), heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), and glucose transporter (GLUT)-1. In addition, mRNA expression levels of the growth factors, brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) and three antioxidant enzymes (catalase, superoxide dismutase (SOD)-1, and glutathione peroxidase (GPx)) were up-regulated by M30 treatment in a brain-region-dependent manner. Signal transduction immunoblotting studies revealed that M30 induced a differential enhanced phosphorylation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), protein kinase B (PKB/Akt), and glycogen synthase kinase-3ß (GSK-3ß). Together, these results suggest that the multifunctional iron chelator M30 can up-regulate a number of neuroprotective-adaptive mechanisms and pro-survival signaling pathways in the brain that might function as important therapeutic targets for the drug in the context of neurodegenerative disease therapy.

Up-regulation of hypoxia-inducible factor (HIF)-1α and HIF-target genes in cortical neurons by the novel multifunctional iron chelator anti-Alzheimer drug, M30.

Based on a multimodal drug design paradigm, we have synthesized a multifunctional non-toxic, brain permeable iron chelator, M30, possessing the neuroprotective propargylamine moiety of the anti-Parkinsonian drug, rasagiline (Azilect) and antioxidant-iron chelator moiety of an 8-hydroxyquinoline derivative of our iron chelator, VK28. M30 was recently found to confer potential neuroprotective effects in vitro and in various preclinical neurodegenerative models and regulate the levels and processing of the Alzheimer's amyloid precursor protein and its toxic amyloidogenic derivative, Abeta. Here, we show that M30 activates the hypoxia-inducible factor (HIF)-1alpha signaling pathway, thus promoting HIF-1alpha mRNA and protein expression levels, as well as increasing transcription of HIF-1alpha-dependent genes, including vascular endothelial growth factor, erythropoietin, enolase-1, p21 and tyrosine hydroxylase in rat primary cortical cells. In addition, M30 also increased the expression levels of the transcripts of brain derived neurotrophic factor (BDNF) and growth-associated protein-43 (GAP-43). Regarding aspects of relevance to Alzheimer's disease (AD), western blotting analysis of glycogen synthase kinase- 3beta (GSK-3beta) signaling pathway revealed that M30 enhanced the levels of phospho-AKT (Ser473) and phospho- GSK-3beta (Ser9) and attenuated Tau phosphorylation. M30 was also shown to protect cultured cortical neurons against Abeta(25-35) toxicity. All these multimodal pharmacological activities of M30 might be beneficial for its potent efficacy in the prevention and treatment of neurodegenerative conditions, such as Parkinson's disease and AD in which oxidative stress and iron-mediated toxicity are involved.

Dopamine and serotonin metabolism in response to chronic administration of fluvoxamine and haloperidol combined treatment.

Treating primary 'negative symptoms' of schizophrenia with a combination of a typical antipsychotic and a selective serotonin reuptake inhibitor, is more effective than with antipsychotic alone and is similar to the effect of the atypical antipsychotic, clozapine. The mechanism of this treatment combination is unknown and may involve changes in dopaminergic and serotonin systems. We studied dopamine and serotonin metabolism in different rat brain areas at 1.5 and 24 h after the last dosage of chronic treatment (30 days), with haloperidol plus fluvoxamine, each drug alone, and clozapine. Haloperidol-fluvoxamine combination, haloperidol, and clozapine treatments increased striatal and frontal cortex dopamine turnover and reduced striatal tyrosine hydroxylase activity at 1.5 h. At 24 h both dopamine turnover and tyrosine hydroxylase activity were reduced. Thus, in chronically treated animals, release of striatal dopamine increases following a drug pulse and returns to baseline by 24 h. Serotonin and 5-hydroxyindoleacetic acid concentrations were decreased at 1.5 h in haloperidol-fluvoxamine and clozapine groups and returned to normal levels by 24 h. A limited behavioral assessment showed that treatment with haloperidol plus fluvoxamine reduced motor activity compared to haloperidol, and increased sniffing compared to haloperidol, fluvoxamine and clozapine. These findings indicate that combining antipsychotic with SSRI results in specific changes in dopaminergic and serotonergic systems and in behavior. The possibility that these may be relevant to the mechanism underlying the clinical effectiveness of augmentation treatment warrant further study.

Involvement of multiple survival signal transduction pathways in the neuroprotective, neurorescue and APP processing activity of rasagiline and its propargyl moiety.

Our recent studies aimed to elucidate the molecular and biochemical mechanism of actions of the novel anti-Parkinson's drug, rasagiline, an irreversible and selective monoamine oxidase (MAO)-B inhibitor and its propargyl moiety, propargylamine. In cell death models induced by serum withdrawal in rat PC12 cells and human SH-SY5Y neuroblastoma cells, both rasagiline and propargylamine exerted neuroprotective and neurorescue activities via multiple survival pathways, including: stimulation of protein kinase C (PKC) phosphorylation; up-regulation of protein and gene levels of PKCalpha, PKCepsilon and the anti-apoptotic Bcl-2, Bcl-xL, and Bcl-w; and up-regulation of the neurotrophic factors, BDNF and GDNF mRNAs. Rasagiline and propargylamine inhibited the cleavage and subsequent activation of pro-caspase-3 and poly ADP-ribose polymerase. Additionally, these compounds significantly down-regulated PKCgamma mRNA and decreased the level of the pro-apoptotic proteins, Bax, Bad, Bim and H2A.X. Rasagiline and propargylamine both regulated amyloid precursor protein (APP) processing towards the non-amyloidogenic pathway. These structure-activity studies have provided evidence that propargylamine promoted neuronal survival via neuroprotective/neurorescue pathways similar to that of rasagiline. In addition, recent study demonstrated that chronic low doses of rasagiline administered to mice subsequently to 1 methyl-4 phenyl 1,2,3,6 tetrahydropyridine (MPTP), rescued dopaminergic neurons in the substantia nigra pars compacta via activation of the Ras-PI3K-Akt survival pathway, suggesting that rasagiline may possess a disease modifying activity.

Reduction of iron-regulated amyloid precursor protein and beta-amyloid peptide by (-)-epigallocatechin-3-gallate in cell cultures: implications for iron chelation in Alzheimer's disease.

Brain iron dysregulation and its association with amyloid precursor protein (APP) plaque formation are implicated in Alzheimer's disease (AD) pathology and so iron chelation could be considered a rational therapeutic strategy for AD. Here we analyzed the effect of the main polyphenol constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG), which possesses metal-chelating and radical-scavenging properties, on the regulation of the iron metabolism-related proteins APP and transferrin receptor (TfR). EGCG exhibited potent iron-chelating activity comparable to that of the prototype iron chelator desferrioxamine, and dose dependently (1-10 microm) increased TfR protein and mRNA levels in human SH-SY5Y neuroblastoma cells. Both the immature and full-length cellular holo-APP were significantly reduced by EGCG, as shown by two-dimensional gel electrophoresis, without altering APP mRNA levels, suggesting a post-transcriptional action. Indeed, EGCG suppressed the translation of a luciferase reporter gene fused to the APP mRNA 5'-untranslated region, encompassing the APP iron-responsive element. The finding that Fe(2)SO(4) reversed the action of EGCG on APP and TfR proteins reinforces the likelihood that these effects are mediated through modulation of the intracellular iron pool. Furthermore, EGCG reduced toxic beta-amyloid peptide generation in Chinese hamster ovary cells overexpressing the APP 'Swedish' mutation. Thus, the natural non-toxic brain-permeable EGCG may provide a potential therapeutic approach for AD and other iron-associated disorders.

Green tea catechins as brain-permeable, non toxic iron chelators to "iron out iron" from the brain.

Evidence to link abnormal metal (iron, copper and zinc) metabolism and handling with Parkinson's and Alzheimer's diseases pathology has frequently been reported. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. Metal chelation has the potential to prevent iron-induced oxidative stress and aggregation of alpha-synuclein and beta-amyloid peptides. The efficacy of iron chelators depends on their ability to penetrate the subcellular compartments and cellular membranes where iron dependent free radicals are generated. Thus, natural, non-toxic, brain permeable neuroprotective drugs, are preferentially advocated for "ironing out iron" from those brain areas where it preferentially accumulates in neurodegenerative diseases. This review will discuss the most recent findings from in vivo and in vitro studies concerning the transitional metal (iron and copper) chelating property of green tea, and its major polyphenol, (-)-epigallocatechin-3-gallate with respect to their potential for the treatment of neurodegenerative diseases.

Limited potentiation of blood pressure response to oral tyramine by brain-selective monoamine oxidase A-B inhibitor, TV-3326 in conscious rabbits.

TV-3326 is a novel cholinesterase inhibitor that produces irreversible brain-selective inhibition of monoamine oxidase (MAO)-A and B and has antidepressant-like activity in rats after chronic oral administration. This study determined whether TV-3326 would cause less potentiation than other irreversible MAO-inhibitors of the blood pressure (BP) response to oral tyramine in conscious rabbits. Dose-response curves were established for the increase in BP induced by tyramine (5-200 mg/kg) administered orally via a naso-pharyngeal tube. From these, the dose that increased BP by 30 mmHg (ED(30)) was computed for each rabbit before and after oral administration of clorgyline, 1 mg/kg for one week, tranylcypromine 10 mg/kg, once, moclobemide, 20 mg/kg 3 times and TV-3326, 26 mg/kg for 2 weeks. Clorgyline, tranylcypromine and TV-3326 inhibited brain MAO-A by 90%; the former two inhibited intestinal MAO-A by 85-97% but TV-3326 had no effect. Tranylcypromine and clorgyline produced 6 and 20-fold increases in the pressor response to tyramine while TV-3326, like moclobemide, only potentiated it 2-fold. If TV-3326 is found to produce as little potentiation of the tyramine response in human subjects, it may be a potentially useful therapeutic agent for the treatment of Alzheimer's disease with depression.

Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration.

In the present study we demonstrate neuroprotective property of green tea extract and (-)-epigallocatechin-3-gallate in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson's disease. N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxin caused dopamine neuron loss in substantia nigra concomitant with a depletion in striatal dopamine and tyrosine hydroxylase protein levels. Pretreatment of mice with either green tea extract (0.5 and 1 mg/kg) or (-)-epigallocatechin-3-gallate (2 and 10 mg/kg) prevented these effects. In addition, the neurotoxin caused an elevation in striatal antioxidant enzymes superoxide dismutase (240%) and catalase (165%) activities, both effects being prevented by (-)-epigallocatechin-3-gallate. (-)-Epigallocatechin-3-gallate itself also increased the activities of both enzymes in the brain. The neuroprotective effects are not likely to be caused by inhibition of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine conversion to its active metabolite 1-methyl-4-phenylpyridinium by monoamine oxidase-B, as both green tea and (-)-epigallocatechin-3-gallate are very poor inhibitors of this enzyme in vitro (770 microg/mL and 660 microM, respectively). Brain penetrating property of polyphenols, as well as their antioxidant and iron-chelating properties may make such compounds an important class of drugs to be developed for treatment of neurodegenerative diseases where oxidative stress has been implicated.

UV-A-related alterations of young and adult lens water-insoluble alpha-crystallin, plasma membranous and cytoskeletal proteins.

The damaging effects of UV-A irradiation on lens water-insoluble alpha-crystallin, plasma membranous and cytoskeletal proteins derived from bovine lenses were studied. Young and adult bovine lenses were kept viable for 2 months in organ culture. After 24 h of incubation they were irradiated, and analyses of the proteins by one-dimensional and two-dimensional gel electrophoresis followed by Western blotting were carried out at several time intervals. RNA isolation, PCR and Northern blotting were also performed. We identified age-related changes in water-insoluble alpha-crystallin, the major membrane protein MP26 and the cytoskeletal proteins vimentin, phakinin and actin between control and UV-irradiated lenses. It appeared that adult lenses are more susceptible to UV light than young lenses, and protein modification occurred more frequently in adult lenses. UV-A irradiation affects not only the cytoskeletal structure, as deduced by the abnormal arrangement of actin in the fiber cells, but also leads to degradation of actin mRNA. Furthermore, analysis of the expression of hsp25 and hsp70 revealed some alteration in the protein pattern of adult lenses. We suggest that degradation of the cytoskeletal proteins following irradiation is due to, at least in part, the decreased protective ability of heat shock proteins upon aging.

In vitro filament-like formation upon interaction between lens alpha-crystallin and betaL-crystallin promoted by stress.

PURPOSE: To determine whether alpha-crystallin is capable of forming filament-like structures with other members of the crystallin family. METHODS: Water-soluble crystallins were isolated from calf lenses and fractionated into alpha-, betaH-, betaL-, and gamma-crystallins according to standard procedures. Chaperone-like activity of alpha-crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmission immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was excluded by the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril interaction test using thioflavin T (ThT) was also performed. RESULTS: At the supramolecular level heating at 60 degrees C has no effect on the morphologic appearance of alpha-crystallin as observed by transmission electron microscopy. Moreover alpha-crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alphabeta-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha-crystallin derived from UV-A-irradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to show that the UV-light treatment affected the chaperone-like capacity of alpha-crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-crystallin. To exclude the presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obtained with the ThT assay earlier applied for the detection of amyloid fibrils. CONCLUSIONS: Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called "rods," may result from stress-induced interaction between alpha-crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha-crystallin from UV-A-irradiated lenses is used as one of the two components of the hybrid, one can only speculate that this formation may be one of the factors leading to UV-A cataract.

Analysis of UVA-related alterations upon aging of eye lens proteins by mini two-dimensional polyacrylamide gel electrophoresis.

This study is a first approach to identify UVA-related alterations in situ of bovine eye lens proteins from the water-soluble and urea-soluble fractions upon aging. The fractions were obtained from irradiated long-term organ culture lenses and analyzed by mini two-dimensional gel electrophoresis. This micropreparative method followed by computer analysis allows high resolution and separation of microgram quantities of proteins and to detect spots which arose as a consequence of irradiation. To facilitate the analysis we first separated the water-soluble fraction into the major crystallin classes by gel filtration. Moreover, we immunoblotted the gel of the urea-soluble fraction with a specific antibody against the intermediate filament protein vimentin. Upon irradiation of young and adult lenses, alphaA-crystallin and vimentin showed obvious modifications. During aging the susceptibility to irradiation increased when vimentin started to degrade, whereas deamidation of alphaA-crystallin seems to occur.

Effect of UV-A light on the chaperone-like properties of young and old lens alpha-crystallin.

PURPOSE: To study the damaging effect of UV-A irradiation on the chaperone-like properties of alpha-crystallin and the subsequent recovery process of young and old bovine lenses. METHODS: Young and old bovine lenses were kept in organ culture. After 24 hours of incubation they were irradiated with UV-A at 365 nm, and optical quality measurements were performed during the experiments (192 hours). alpha-Crystallin and alpha1-, alphaA2-, alphaB1-, and alphaB2-crystallin subunits were analyzed, separated by gel filtration and cation exchange chromatography, respectively, after different culture times. Protein patterns were obtained after two-dimensional (2-D) gel electrophoresis. Chaperone-like activity was determined on the basis of insulin B-chain and betaL-crystallin aggregation assays. Aggregation of alpha-crystallin was analyzed, tryptophan fluorescence measurements were performed, and alpha-crystallin mRNA levels were determined. RESULTS: The water-soluble alpha-crystallin obtained from old lenses compared with young lenses after UV irradiation had decreased chaperone activity, a higher molecular weight, and increased loss of tryptophan fluorescence. Moreover, alpha-crystallin mRNA virtually disappeared, whereas extra spots on the 2-D protein pattern appeared, possibly because of deamidation. CONCLUSIONS: alpha-Crystallin obtained from old lenses is more affected by irradiation than alpha-crystallin derived from young lenses. Moreover, it appeared that alphaB-crystallin from UV-treated old lenses compared with control lenses was less susceptible to UV-A than alphaA-crystallin. It may well be that alphaB-crystallin protects alphaA-crystallin in vivo.

Effects of UV-A radiation on lens epithelial NaK-ATPase in organ culture.

PURPOSE: To investigate the mechanisms involved in the damage caused by UV-A irradiation at 365 nm on the eye lens. METHODS: Bovine lenses obtained from animals 1 to 5 years of age were placed in specially designed organ culture chambers for preincubation. Twenty-four hours later, the lenses were irradiated by 33 J/cm2 UV-A at 365 nm. During irradiation, the lenses were oriented in the culture so that the anterior surface faced the incident UV-A radiation source. After irradiation, lens optical quality was monitored throughout the 8 days of the culture period, and lens samples were taken for analysis of NaK-ATPase activity. RESULTS: Lens optics and NaK-ATPase activity were affected by irradiation of 33 J/cm2. The effects on lens epithelial NaK-ATPase activity were stronger at the equators than at the center. The damage to the activity at the center was reversible, as the lens optically recovered from the LW-A damage. CONCLUSIONS: Lens NaK-ATPase activity can recover from damage caused by UV-A at 365 nm. When the lenses received irradiation of 33 J/cm2, NaK-ATPase activity recovered from the damage during the culture period only at the center and not at the equators of the epithelium.

Transglutaminase involvement in UV-A damage to the eye lens.

Solar radiation is believed to be one of the major environmental factors involved in lens cataractogenesis. The purpose of the study was to investigate the mechanisms by which UV-A at 365 nm causes damage to the eye lens. Bovine lenses were placed in special culture cells for pre-incubation of 24 hr. The lenses were positioned so that the anterior surface faced the incident UV-A radiation source and were maintained in the cells during irradiation. After irradiation, lens optical quality was monitored throughout the culture period and lens epithelium, cortex and nuclear samples were taken for biochemical analysis. Transglutaminase activity in the lens was affected by the radiation. The activity of transglutaminase in lens epithelium cortex and nucleus increased as a result of the irradiation and then declined towards control levels during the culture period, as the lens recovered from the UV-A damage. Specific lens proteins, alpha B and beta B1 crystallins (the enzyme substrates) were analyzed by SDS polyacrylamide gel electrophoresis and immunoblotting with specific antibodies. Seventy-two hours after irradiation of 44.8 J cm-2 UV-A, alpha B crystallins were affected as was shown by the appearance of aggregation and degradation products. Some protein changes seem to be reversible. It appears that transglutaminase may be involved in the mechanism by which UV-A causes damage to the eye lens.

Age- and strain-related changes in tissue transglutaminase activity in murine macrophages: the effects of inflammation and induction by retinol.

We describe differences in the activity of tissue transglutaminase (TGase) between resident and inflammatory mouse peritoneal macrophages as a function of age. Our results established the following observations: (a) resident macrophages from senescent mice expressed higher basal TGase activity than those from young mice; (b) Maximal TGase activity on day 3 of thioglycollate injection was lower by 24% in inflammatory macrophages from senescent as compared to young animals; (c) in contrast, as the inflammatory response abated (days 4-6), the incremental decrease in TGase activity in old was lower than in young animals; (d) in vitro activation of resident macrophages by retinol and mouse serum was more effective in inducing TGase activity from outbred CD-1 young mice than from inbred C57BL/6J young mice (age differences were also more prominent in the CD-1 mouse strain); and (e) Retinol and mouse serum effectively inhibited the production of superoxide in young mice, thereby demonstrating an inverse correlation between TGase activity and superoxide production. In old animals, however, the production of superoxide was not decreased, nor was TGase increased. Although, paradoxically, resident macrophages from senescent mice were a priori more activated than those from young ones, it is concluded that macrophages from young mice respond better than those from old ones to stimuli they encounter, either during inflammation or under physiological stimulation.

Long-term organ culture system to study the effects of UV-A irradiation on lens transglutaminase.

The purpose of the study was to investigate the mechanisms by which UV-A at 365 nm causes damage to the eye lens. Bovine lenses were placed in special culture cells and were oriented so that the anterior surface faced the incident UV-A radiation source. The lenses were maintained in the cells during irradiation. After 5 days in culture, samples of lens epithelium, cortex, and nucleus were taken for biochemical analysis. Transglutaminase activity in the lens was affected by the radiation and showed increased activity in the lens cortex and nucleus. Specific lens protein, betaB1 crystallin (one of the enzyme substrates), was analyzed by SDS polyacrylamide gel electrophoresis and immunobloting with specific antibodies. The crystallin was affected as was shown by the appearance of degradation products. There is correlative information but no clear proof that transglutaminase is involved in the mechanisms by which UV-A causes damage to the eye lens.

Recovery of lens optics and epithelial enzymes after ultraviolet A radiation.

PURPOSE: To establish the mechanism by which ultraviolet A (UVA) radiation causes irreversible damage to the eye lens. METHODS: The authors irradiated 223 bovine lenses in organ culture with 22.4, 33.6, and 44.8 J/cm2 of UVA radiation (365 nm) and studied biochemical and optical properties of the lenses in long-term culture conditions. Each lens tested was placed in a specially designed cell. The lenses were oriented so that the anterior surface faced the incident UVA radiation source, and they were maintained in their cells during irradiation. After irradiation, lens optical quality was monitored throughout the culture period, and lens samples were taken for enzyme analysis. RESULTS: Full recovery of lens optical damage and activity of the enzymes hexokinase, catalase, and glucose-6-phosphate dehydrogenase in lens epithelium was observed after 8 days in culture after irradiation with 22.4J/cm2. After irradiation with 33.6J/cm2, partial recovery of optical damage was found, and there was between 80% to 90% recovery of the enzyme activity. No recovery of optical and enzyme activity was found after 44.8J/cm2 irradiation. CONCLUSIONS: Irradiation between 22.4J/cm2 to 33.6J/cm2 of UVA at 365 nm is the minimal level of irradiation that causes irreversible damage to lens enzymes and optics.

Long-term organ culture system to study the effect of UV-radiation on lens enzymes.

Environmental factors such as solar radiation and drug treatment are potential cataractogenic agents. It is suggested that their damaging effects accumulate with age. The purpose of the study was to isolate the effect of one factor (UV-radiation) and find out the mechanism by which UV radiation causes damage to the eye lens. We irradiated bovine lenses with UV-A (365 nm) radiation for 50, 75, 90, 100, and 120 min and followed the optical changes of the lenses in a long-term organ culture. Enzyme activities were analyzed in lens epithelium after five days of incubation in organ culture. The enzymes analyzed were ATPase, which belongs to the transport mechanism in lens epithelium cells, hexokinase, the key enzyme of the glycolysis pathway, G6PD, which provides NADPH to the glutathione system and catalase, which protects the cells from H(2)O(2). Optical damage was observed even for the minimal radiation. The same amount of radiation also affected ATPase and hexokinase activities. G6PD and catalase were affected only in lenses which received radiation for 90 min, We can conclude that enzymes involved in the transport mechanism and metabolism are more sensitive to UV-A (365 nm) radiation than enzymes involved in the defense mechanism against oxidation.

DL-propranolol inhibits lens hexokinase activity and affects lens optics.

A clinico-biochemical study indicated that the beta-blocker DL-propranolol may affect human lens epithelial hexokinase (HK) activity. In that study five key enzymes were analysed in 192 freshly excised human lens epithelia obtained during cataract surgery. In a large number of patients the epithelial HK was found to be inactive. Medical records of these patients showed widespread use of the drug DL-propranolol. In vitro experiments demonstrated a direct inhibitory effect of the drug on human lens HK activity. Lens refractive function was monitored during long term bovine lens culture experiments in which the potential cataractogenic agent was added to the culture media. DL-propranolol in a concentration of 0.1 mM reduced HK activity in bovine lens epithelium after 72 hr in organ culture and disrupted lens light focusing ability after 250 hr of incubation. Kinetic studies of HK inhibition suggested a competitive inhibitory effect of the drug on the enzyme.

Age-related alterations in superoxide anion generation in mouse peritoneal macrophages studied by repeated stimulations and heat shock treatment.

The ability of thioglycollate-elicited peritoneal macrophages (PM) from young and senescent mice to generate superoxide anions (O2-) under repeated stimulation or thermal stress was studied using either zymosan, opsonized zymosan (OZ), or phorbol myristate acetate (PMA). A diminished capacity to recover from repeated stimulation was found with aging. When stimulated for a second time 24 hours after the primary stimulation, PM from young animals generated 80% of the initial O2- responses to either zymosan, or OZ. Under the same conditions, PM from senescent mice generated 62% of the initial O2- produced in response to zymosan, and 45% in response to OZ. In both age groups the response to a second PMA stimulation comprised only 10% of the primary response. A considerably diminished capacity to generate O2- was also demonstrated in PM from senescent mice after recovery from exposure to thermal stress. Exposure to 42.5 degrees C for 20 minutes was found to be the threshold temperature for irreversible loss of activity in senescent PM, whereas at this temperature, PM from young animals recovered up to 70% of their O2- generating activity. Since NADPH oxidase and superoxide dismutase activities were only mildly affected by the hyperthermia in all age groups, they could not account for the age-related decline in the recovery from stress. Age-related alterations in signal transduction or receptor alterations could possibly play a primary role in this decline.