Search

Purpose: In humans, vitrectomy is associated with development of nuclear cataracts. Iron catalyzes free radical formation causing oxidative damage, which is implicated in cataract formation. This study was designed to determine if vitreous humor, which can initiate differentiation of lens epithelial cells, would have an effect on iron-handling proteins., Methods: Cultured canine lens epithelial cells were treated with collected canine vitreous humor. Lysates of treated and control cells were separated by SDS-PAGE. Ferritin H- and L-chains, transferrin receptor 1, and aquaporin 0 were immunodetected and quantitated with specific antibodies. Morphologic changes in treated cells were assessed., Results: Treatment of lens epithelial cells with a 33% (vol/vol) solution of vitreous humor changed the morphology of lens cells and induced expression of aquaporin 0, a marker of fiber cell differentiation that was undetectable in control cells. Treatment did not modify the size of iron-handling proteins but significantly increased content of ferritin from 2.9- to 8.8-fold over control and decreased levels of transferrin receptor by 37% to 59%., Conclusions: Vitreous humor may significantly limit iron uptake by transferrin/transferrin receptor pathway, and by increasing ferritin levels could profoundly increase the iron-storage capacity of ferritin in lens cells. Vitreous humor may play a significant protective role against iron-catalyzed oxidative damage of lens epithelial cells and therefore in the formation of cataracts.

Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 μg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.

Kynuramines, metabolites of melatonin and L-tryptophan, are synthesized endogenously by oxygenases or in spontaneous reaction by an interaction with free radicals. We have reported previously that melatonin stimulates expression and phosphorylation of heat shock protein (HSP) 27, as well as production of HSP70 and HSP90αβ in pancreatic carcinoma cells (PANC-1). Based on those results, we hypothesized that above processes could have been involved in the interruption of intrinsic proapoptotic pathway. Herein, we report that incubation of PANC-1 cells with N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) or with L-kynurenine (L-KYN) lead to the overexpression of heat shock protein synthesis and these effects are partially reversed by 5-HT3 or MT1/MT2 receptor antagonists. PANC-1 cells in culture were treated with AFMK or L-KYN, with non selective MT1/MT2 receptor antagonist (luzindole), with 5-HT2 and 5-HT3 receptor antagonists (ketanserin and MDL72222), or combination of these substances. Both AFMK and L-KYN significantly decreased cytoplasmic HSP27 and this effect was presumably due to increased of its phosphorylation and consequent nuclear translocation, confirmed by immunoprecipitation of phosphorylated form of HSP27. These changes were accompanied by marked augmentation of HSP70 and HSP90αβ in the cytosolic fraction. Pretreatment of cell cultures with luzindole or MDL72222 followed by the addition of AFMK or L-KYN reversed the stimulatory effects of these substances on HSP expression in PANC-1 cells, whereas ketanserin failed to influence mentioned above phenomenon. We conclude that activation of HSPs in pancreatic carcinoma cells seems to be dependent on an interaction of AFMK or L-KYN with MT1/MT2 or/and 5-HT3 receptors.

Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides., (Copyright © 2014. Published by Elsevier Ltd.)

Purpose: Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of ⁵⁹Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs). Because lens tissue physiologically functions under low oxygen tension, we also tested effects of hypoxia on iron trafficking. Excess iron, not bound to proteins, can be damaging to cells due to its ability to catalyze formation of reactive oxygen species., Methods: LECs were labeled with ⁵⁹Fe-Tf or ⁵⁹FeCl₃ under normoxic or hypoxic conditions. Cell lysates were fractioned into mitochondria-rich, nuclei-rich, and cytosolic fractions. Iron uptake and its subcellular distribution were measured by gamma counting., Results: ⁵⁹Fe accumulation into LECs labeled with ⁵⁹Fe-Tf was 55-fold lower as compared with that of ⁵⁹FeCl₃. Hypoxia (24 hours) decreased uptake of iron from transferrin but not from FeCl₃. More iron from ⁵⁹FeCl₃ was directed to the mitochondria-rich fraction (32.6%-47.7%) compared with ⁵⁹Fe from transferrin (10.6%-12.6%). The opposite was found for the cytosolic fraction (8.7%-18.3% and 54.2%-46.6 %, respectively). Hypoxia significantly decreased iron accumulation in the mitochondria-rich fraction of LECs labeled with ⁵⁹Fe-Tf ., Conclusions: There are source-dependent differences in iron uptake and trafficking. Uptake and distribution of NTBI are not as strictly regulated as that of iron from transferrin. Excessive exposure to NTBI, which could occur in pathological conditions, may oxidatively damage organelles, particularly mitochondria.

Ceruloplasmin (Cp) is a ferroxidase important to the regulation of both systemic and intracellular iron levels. Cp has a critical role in iron metabolism in the brain and retina as shown in patients with aceruloplasminemia and in Cp-/-hep-/y mice where iron accumulates and neural and retinal degeneration ensue. We have previously shown that cultured lens epithelial cells (LEC) secrete Cp. The purpose of the current study was to determine if cultured retinal pigmented epithelial cells (RPE) also secrete Cp. In addition, the effects of exogenously added Cp on iron regulated proteins and pathways, ferritin, transferrin receptor, glutamate secretion and levels of hypoxia-inducible factor-1α in the nucleus were determined. Like LEC, RPE secrete Cp. Cp was found diffusely distributed within both cultured LEC and RPE, but the cell membranes had more intense staining. Exogenously added Cp caused an increase in ferritin levels in both cell types and increased secretion of glutamate. The Cp-induced increase in glutamate secretion was inhibited by both the aconitase inhibitor oxalomalic acid as well as iron chelators. As predicted by the canonical view of the iron regulatory protein (IRP) as the predominant controller of cellular iron status these results indicate that there is an increase in available iron (called the labile iron pool (LIP)) in the cytoplasm. However, both transferrin receptor (TfR) and nuclear levels of HIF-1α were increased and these results point to a decrease in available iron. Such confounding results have been found in other systems and indicate that there is a much more complex regulation of intracellularly available iron (LIP) and its downstream effects on cell metabolism. Importantly, the Cp increased production and secretion of the neurotransmitter, glutamate, is a substantive finding of clinical relevance because of the neural and retinal degeneration found in aceruloplasminemia patients. This finding and Cp-induced nuclear translocation of the hypoxia-inducible factor-1 (HIF1) subunit HIF-1α adds novel information to the list of critical pathways impacted by Cp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: The iron storage protein ferritin is necessary for the safe storage of iron and for protection against the production of iron-catalyzed oxidative damage. Ferritin is composed of 24 subunits of two types: heavy (H) and light (L). The ratio of these subunits is tissue specific, and alteration of this ratio can have profound effects on iron storage and availability. In the present study, siRNA for each of the chains was used to alter the ferritin H:L chain ratio and to determine the effect of these changes on ferritin synthesis, iron metabolism, and downstream effects on iron-responsive pathways in canine lens epithelial cells., Methods: Primary cultures of canine lens epithelial cells were used. The cells were transfected with custom-made siRNA for canine ferritin H- and L-chains. De novo ferritin synthesis was determined by labeling newly synthesized ferritin chains with 35S-methionine, immunoprecipitation, and separation by SDS-PAGE. Iron uptake into cells and incorporation into ferritin was measured by incubating the cells with 59Fe-labeled transferrin. Western blot analysis was used to determine the presence of transferrin receptor, and ELISA was used to determine total ferritin concentration. Ferritin localization in the cells was determined by immunofluorescence labeling. VEGF, glutathione secretion levels, and cystine uptake were measured., Results: FHsiRNA decreased ferritin H-chain synthesis, but doubled ferritin L-chain synthesis. FLsiRNA decreased both ferritin H- and L-chain synthesis. The degradation of ferritin H-chain was blocked by both siRNAs, whereas only FHsiRNA blocked the degradation of ferritin L-chain, which caused significant accumulation of ferritin L-chain in the cells. This excess ferritin L-chain was found in inclusion bodies, some of which were co-localized with lysosomes. Iron storage in ferritin was greatly reduced by FHsiRNA, resulting in increased iron availability, as noted by a decrease in transferrin receptor levels and iron uptake from transferrin. Increased iron availability also increased cystine uptake and glutathione concentration and decreased nuclear translocation of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium., Conclusions: Most of the effects of altering the ferritin H:L ratio with the specific siRNAs were due to changes in the availability of iron in a labile pool. They caused significant changes in iron uptake and storage, the rate of ferritin synthesis and degradation, the secretion of VEGF, and the levels of glutathione in cultured lens epithelial cells. These profound effects clearly demonstrate that maintenance of a specific H:L ratio is part of a basic cellular homeostatic mechanism.

Purpose: It was determined in an earlier study that ferritin-heavy (H) and -light (L) chains in lens fiber cells are modified in comparison to those in lens epithelial cells. The purpose of the present study was to determine whether changes in ferritin chain characteristics are developmental, age-related, or associated with cataractogenesis, by analyzing the distribution of modified chains throughout the lens fiber mass., Methods: After removing the capsule, noncataractous and cataractous lenses were separated into six layers of fiber cells. The content of ferritin H and L chains in each layer was determined by western blotting with chain-specific antibodies. The level of ferritin complex (450 kDa protein made up of assembled L and H chains) was determined using the enzyme-linked immunosorbent assay. The ability of ferritin complex to bind iron was assessed by in vitro labeling with (59)Fe., Results: Fiber cell ferritin L chains were 30 kDa (modified from the normal 19 kDa), and were present at the highest level in the outermost layers of both cataractous and non-cataractous lenses. The amount of modified L chains decreased gradually in the inner layers of the fiber mass, and was undetectable in the inner two layers of cataractous lenses. The ferritin H chains were also modified to 12 kDa (perhaps truncated from the normal 21 kDa size) in both cataractous and non-cataractous lenses. Similar levels of this modified H chain were found throughout the normal lens. Interestingly, in cataractous lenses, the modified H chains were found in decreasing amounts towards the interior of the lens, and were undetectable in the nucleus. However, in these cataractous lenses, the normal-sized ferritin H chains (21 kDA) appear in small quantities in the outer fiber layers, and increase in quantity and size (to 29 kDa) in the inner layers. This observation was best seen and demonstrated in advanced cataracts. Ferritin, which can bind iron, was found mainly in the outer layers of the lens fiber mass of normal lenses, but was more evenly distributed in fiber layers from cataractous lenses., Conclusions: Both ferritin H and L chains were modified in lens fiber cells from normal and cataractous canine lenses. These modifications were not age-related, and most likely occur during the differentiation of epithelial cells to fiber cells, since only normal-sized chains have been found in lens epithelial cells. In addition, there was a specific and distinct distribution of these modified chains throughout the lens fiber mass. The most striking differences between normal and cataractous lenses fiber cells were the appearance of normal-sized ferritin H chains and the relatively even distribution of iron binding capacity throughout the fiber mass of the cataractous lenses. These differences may reflect a response of the lens to increased oxidative stress during cataractogenesis.

This review article covers all aspects of iron metabolism, which include studies of iron levels within the eye and the processes used to maintain normal levels of iron in ocular tissues. In addition, the involvement of iron in ocular pathology is explored. In each section there is a short introduction to a specific metabolic process responsible for iron homeostasis, which for the most part has been studied in non-ocular tissues. This is followed by a summary of our current knowledge of the process in ocular tissues.

Purpose: To determine potential differences in the characteristics of the iron storage protein ferritin and its heavy (H) and light (L) subunits in fiber cells from cataractous and noncataractous lenses of older dogs., Methods: Lens fiber cell homogenates were analyzed by SDS-PAGE, and ferritin chains were immunodetected with ferritin chain-specific antibodies. Ferritin concentration was measured by ELISA. Immunohistochemistry was used to localize ferritin chains in lens sections., Results: The concentration of assembled ferritin was comparable in noncataractous and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was approximately 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver. The H-chain identified in cataractous fiber cells (29 kDa) differed from the 21-kDa standard canine H-chain and from the 12-kDa modified H-chain present in fiber cells of noncataractous lenses. Histologic analysis revealed that the H-chain was distributed differently throughout cataractous lenses compared with noncataractous lenses. There was also a difference in subunit makeup of assembled ferritin between the two lens types. Ferritin from cataractous lenses contained more H-chain and bound 11-fold more iron than ferritin from noncataractous lenses., Conclusions: There are significant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses compared with noncataractous lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition, the accumulation of H-chain in deeper fiber layers of the lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage.

Purpose: This study was designed to elucidate potential age-related changes in the concentration, structure, and assembly pattern of ferritin chains in lens fiber cells., Methods: Canine and human lens fiber cell homogenate proteins were separated by one-dimensional and two-dimensional SDS-PAGE. Ferritin chains were immunodetected and quantitated with ferritin chain-specific antibodies. Total ferritin concentration was measured by ELISA. Binding of iron was determined in vitro with (59)Fe., Results: Ferritin H- and L-chains in canine and human fiber cells of healthy lenses were extensively modified. The H-chain in both species was truncated, and its concentration increased with age. Canine L-chain was approximately 11 kDa larger than standard canine L-chain, whereas human L-chain was of the proper size. Two-dimensional separation revealed age-related polymorphism of human and canine lens fiber cell L-chains and human H-chains. Normal size ferritin chains were not identified in canine fiber cells, but a small amount of fully assembled ferritin was detected, and its concentration decreased with age., Conclusions: Such significantly altered ferritin chains are not likely to form functional ferritin capable of storing iron. Therefore, lens fiber cells, particularly from older lenses, may have limited ability to protect themselves against iron-catalyzed oxidative damage.

Purpose: The storage of iron by ferritin is determined by tissue-specific composition of its 24 subunits, which are designated as either heavy (H) or light (L). For a better understanding of how lens epithelial cells regulate their ferritin subunit makeup, the degradation pattern of each subunit type was analyzed. In addition, age-related changes in ferritin concentration and subunit makeup were determined., Methods: Ferritin turnover in primary cultures of canine lens epithelial cells was determined by metabolic labeling with [(35)S]-methionine. Transient transfection with vectors containing coding sequences for either H- or L-chains were used to modify ferritin subunit makeup. Ferritin concentration was measured by ELISA. Immunodetection and fluorescence immunocytochemistry were used to study age-related changes in ferritin chain concentration., Results: Inhibition of the proteasomal protein degradation pathway by clastolactacystin-beta-lactone had no effect on ferritin degradation, whereas inhibition of lysosomal degradation markedly increased ferritin levels, confirming that this system is involved in ferritin turnover. H-chain ferritin degraded at a faster rate than the L-chain. L-chain-rich ferritin in L-chain-transfected cells formed inclusion bodies that were localized to the cytosol. Similar inclusion bodies were found in older lens cells kept in cell culture for more than 8 days., Conclusions: Steady degradation of H-chain ferritin contributed to the maintenance of a constant level of this chain within the lens epithelial cells. In contrast, slower turnover of the L-chain resulted in accumulation of L-chain-enriched ferritin associated with cytoplasmic inclusion bodies. These L-chain-containing inclusion bodies were found in the cytosol of cells overexpressing L-ferritin chain and in nontransfected cells maintained in culture for 8 to 35 days. Overexpression of the L-chain has been associated with the formation of premature cataracts in humans with hereditary hyperferritinemia cataract syndrome. The formation of inclusion bodies in older lens epithelial cells, as demonstrated in the current investigation, is intriguing and could point to possible involvement of cytoplasmic L-chain-enriched ferritin aggregates in the formation of age-related cataract.

Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells, indicating that this may be a common mechanism for the regulation of glutamate production in many cell types. The activity of the iron-dependent enzyme cytosolic aconitase (c-aconitase) is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three-step process leading to glutamate formation. In the present study, iron increased c-aconitase activity, and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron-induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and retinal degeneration. Our results are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in the regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many nonneuronal tissues. We provide the first evidence that, in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter VGLUT1 and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole.

The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.

Alpha lipoic acid (LA) is a cofactor in mitochondrial dehydrogenase complexes. Previous studies have shown that when administered exogenously LA has antioxidant properties, which include free radical scavenging, metal chelation and regeneration of other antioxidants. The cells convert LA into dihydroplipoic acid (DHLA), which in the presence of iron can act as a prooxidant. In vitro DHLA reduces Fe(+3) to Fe(+2) and removes iron from ferritin, increasing the risk of Fe catalyzed free radical formation. In the present study we examined the in vivo effects of lipoic acid treatment on Fe metabolism in cultured lens epithelial cells, and found that LA decreases Fe uptake from transferrin, increases Fe deposition into ferritin and increases the concentration of this protein. When administered together with ascorbic acid, lipoic acid changes the characteristic heavy to light chain ratio of ferritin makeup. The decreased Fe uptake and increased storage diminishes the size of the cytosolic highly reactive Fe pool (LIP). These changes are associated with increased cell resistance to H(2)O(2) challenge. Therefore, LA may reduce the risk of Fe induced oxidative damage and also might be useful as a treatment of Fe overload., (Copyright 2003 Elsevier Science Ltd.)

Purpose: To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress., Methods: Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA: Fe uptake and incorporation into ferritin was determined by incubating transfected cells with (59)Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation., Results: Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected., Conclusions: Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.

The nitroxide, Tempol, can protect tissue from oxidative damage by removing superoxide and by oxidizing Fe(II) to Fe(III), thus decreasing formation of the hydroxyl radical. However, long-term exposure to Tempol can damage cells. The oxidation of Fe could have profound effects on Fe metabolism in cells, yet this has not been previously studied. In the present investigation, the effects of Tempol on the synthesis of the Fe storage protein, ferritin, and its ability to store Fe were studied in cultured lens epithelial cells (LEC). In addition, the effects of short- and long-term Tempol treatment on the resistance of LEC to oxidative stress were determined. Tempol had a clear dose-dependent inhibitory effect on ferritin synthesis noted at 6 h. By 20 h, ferritin synthesis returned toward normal levels. However, Fe incorporation into ferritin was decreased by almost 90% by the highest dose of Tempol, even at the 20-h time point. The decrease in Fe incorporation into ferritin was accompanied by a significant increase in the LMW pool of Fe. After short-term (4 h) treatment with Tempol, LEC were protected against the toxic effects of tertiary butyl hydroperoxide. However, after longer term treatment (20 h), Tempol itself had a toxic effect and did not afford protection. Indeed, at the higher doses, Tempol significantly reduced the ability of the cells to withstand oxidative stress. The redistribution of Fe within the cell after 20 h of Tempol treatment appears to render the cells more vulnerable to oxidative stress. The deleterious effects of Tempol on LEC are likely due to its effects on Fe metabolism, perhaps by reducing the availability of Fe for incorporation into ferritin and Fe-dependent enzymes as well as enlarging a low molecular weight pool of Fe which may be capable of catalyzing damaging free radical reactions.

Ferritin is the major intracellular iron storage protein which has been shown to protect cells against oxidative damage. Recent reports that an inherited abnormality in human ferritin synthesis is associated with early bilateral cataracts underscore the importance of understanding ferritin synthesis and iron storage in lens epithelial cells. We previously demonstrated that ascorbic acid greatly increases de novo synthesis of ferritin in lens epithelial cells. The objectives of the present study were to determine: (1) the effects of ascorbic acid and ferric ammonium citrate on iron uptake by canine lens epithelial cells from iron bound to transferrin and from ferric chloride and (2) the incorporation of this element into ferritin. Iron uptake by lens epithelial cells from 59ferric chloride was 20 times higher than from 59iron-transferrin and iron deposition into ferritin was 8-fold higher when 59ferric chloride was the source. Ascorbic acid had a stimulatory effect on iron uptake from transferrin and on incorporation of this element into ferritin. The ascorbic acid-induced increase of iron uptake required de novo protein synthesis but not specifically de novo ferritin biosynthesis. Although ferritin is not directly involved in iron uptake, the level of ferritin protein could control the pool of intracellular iron. The present results indicate that iron homeostasis in lens epithelial cells is affected mainly by changes in apoferritin synthesis, which is greatly stimulated by ascorbic acid, rather than by altering the rate of protein degradation, which is very slow in these cells under all circumstances. Ferric ammonium citrate activates iron uptake from transferrin in a wide range of cell lines by generation of free radicals. Ferric ammonium citrate also increased iron uptake from Tf in lens epithelial cells. Ferric ammonium citrate treated cells incorporated 5 times more iron and deposited 2 times more iron into ferritin than control cells. Increased incorporation of iron into ferritin was due to ferric ammonium citrate-induced stimulation of de novo ferritin synthesis rather than an increased rate of iron deposition into pre-existing ferritin. Ferric ammonium citrate had a different effect on iron uptake from ferric chloride; total iron uptake was not significantly increased while deposition into ferritin was significantly decreased. These results demonstrate that iron homeostasis in lens epithelial cells is regulated by ascorbic acid and by changes in the rate of de novo ferritin synthesis. In addition, the differences in iron uptake from transferrin and ferric chloride and its subsequent incorporation into ferritin suggests that the mechanisms by which iron is incorporated into ferritin are source dependent., (Copyright 1998 Academic Press.)

A previous study demonstrated that ascorbic acid increased the concentration of the iron storage protein, ferritin. In cultured lens epithelial cells. The current study was designed to determine the mechanism by which ascorbic acid exerts this effect. Ascorbic acid increased both ferritin mRNA levels (by about 30%) and translation of ferritin (de novo synthesis was increased up to 15-fold) within 6 hr. Cycloheximide completely abolished the ability of ascorbic acid to increase ferritin levels, whereas actinomycin D only decreased it by about 30%. Therefore, the ascorbic-acid induced increase in ferritin concentration is due mainly to an increase in ferritin synthesis at the translational levels. This is a novel role for ascorbic acid. Addition of iron with ascorbic acid further increased de novo synthesis of ferritin, but this additive effect was only noted at a later time point (20 hr). Factors which decrease ferritin mRNA translation, such as the reducing agent dithiothreitol or the iron chelator desferrioxamine, reduced the ascorbic acid effect on de novo ferritin synthesis. The effects of ascorbic acid on ferritin mRNA levels may be mediated by its oxidation product, H2O2, since, like ascorbic acid, H2O2 increased ferritin mRNA levels by 30%. However, in contrast to the ascorbic acid-induced increase in translation of ferritin, H2O2 substantially decreased de novo ferritin synthesis. This effect of H2O2 could have physiological significance in eyes where concentrations of H2O2 in the aqueous humor are elevated. High levels of H2O2 could decrease the concentration of ferritin within the lens. Since ferritin sequesters iron and has been shown to decrease oxidative damage by limiting the availability of iron to catalyse free radical reactions, H2O2-induced reduction in ferritin concentration in the lens could have deleterious effects. The ability of ascorbic acid to increase ferritin concentration in lens epithelial cells could provide an additional protective mechanism for this antioxidant vitamin. The importance of ferritin to normal lens functioning is underscored by the recent finding that humans with a dominantly inherited abnormality in ferritin synthesis exhibit early bilateral cataracts.

Transferrin (Tf), the plasma iron transport protein which supports cell proliferation and differentiation and has bacteriostatic, antioxidant and anti-inflammatory activity, has been found in relatively high concentrations in the intraocular fluids. Intraocular synthesis of Tf has recently been demonstrated, although the intraocular tissue(s) responsible have not been identified. We designed this study to determine whether certain ocular tissues can make and secrete transferrin. Transferrin content of aqueous and vitreous humors and whole lenses was determined by ELISA. Transferrin secretion by cultured epithelia from lens and ciliary body was also measured. In addition, Northern blots of RNA from cultured lens epithelial cells, ciliary body pigmented and non-pigmented epithelial cells, and from whole iris, ciliary body and retina were probed with riboprobes for Tf mRNA and 18S rRNA. Transferrin made up 23% and 16% of total canine aqueous and vitreous protein. All ocular tissues and cultured cells tested contained mRNA for Tf, however Tf was secreted into the bathing medium from lens epithelial cell cultures, but not from either the pigmented or non-pigmented epithelial cells of the ciliary body cultures, but not from either the pigmented or non-pigmented epithelial cells of the ciliary body Cycloheximide inhibited secretion of Tf from the lens epithelial cells. Lenses from inflamed eyes contained higher levels of Tf than their contralateral controls. This is the first experimental demonstration that an intraocular tissue can make and secrete Tf. Transferrin secretion by the lens may contribute significantly to the IOF content of this important intraocular protein.

Plasma proteins in the period of post-transfusion polycythaemia in rats. Acta Physiol. Pol., 1978, 29 (1): 47-54. The plasma protein of polycythaemic rats with strongly inhibited erythropoiesis and of normal rats was characterized with the use of ion exchanger chromatography on DEAE-cellulose in an NaCl concentration gradient and electrophoresis on polyacrylamide gel. Experimental posttransfusion polycythaemia was found to cause considerable changes in the amount and composition of plasma proteins. The amount of gamma and alpha1 globulins increased in polycythaemic animals, whereas albumin content decreased. There also appeared wide differences in the elution profiles obtained from ion exchanger chromatography of both kinds of plasma. Electrophoretic analysis of fractions obtained by chromatography demonstrated a similarity only in the composition of the globulin fractions of both the plasma kinds. The remaining fractions differed widely in their protein composition.

The aim of this study was to determine the erythropoietic activity of the plasma of polycythaemic rats and of one of its protein fractions playing a role in erythropoiesis regulation. The erythropoietin activity and that of the erythropoiesis inhibitor varied in the examined plasma samples at definite time periods after induction of polycythaemia. It was demonstrated that the most suitable time of plasma collection for the inhibitor investigation is the period between 115 and 187 h after the first transfusion, and that in some cases separation of this factor from erythropoietin present simultaneously in the plasma is indispensable in order to reveal the inhibitory activity. The erythropoiesis inhibitor administered jointly with erythropoietin was found to exert no influence on erythropoiesis either in normal or in polycythaemic recipients of the tested plasma.

The dynamics of erythropoiesis inhibition as the consequence of polycythaemia induced in rats by four to six transfusions of homologous erythrocytes was studied in detail. It was found, that, in rats with polycythaemia elicited by two transfusions of erythrocytes, erythropoiesis inhibition occurs 67h after the first transfusion and it is most pronounced in the period between 115-187 h. The hemopoietic system is not completely free from the inhibitory influence of polycythaemia up to 283 h after the first transfusion. Maintenance of a state of polycythaemia for 17 days by repeated transfusions strongly and durably inhibits the haemopoietic system. The red blood cell system remained for this whole period under the inhibitory effect.