Production and characterization of recombinant heteropolymers of human ferritin H and L chains

Vertebrate ferritins are iron storage proteins composed by 24 subunits of one or more types. The recombinant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers with various proportions of H- and L-type chains cannot be predicted. Present study shows that unfolded human ferritin H- and L- type chains renature under similar conditions to form homopolymers indistinguishable from the native ones and that, when mixed, the unfolded H and L chains renature to form heteropolymers with restricted heterogeneity and with the expected H:L ratios. Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunological reactivity, and stability to guanidine denaturation varied as predicted, based on the homopolymers. In contrast, the rate of iron uptake, monitored by the variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; further increments in H chains had no additional effect. This finding indicates that, under the present conditions, only a limited number of H chains are needed for the maximum rate of ferritin iron uptake. Variations of L- and H-type chains in vivo may thus have biological relevance.     

Ferritin in cultured human cytotrophoblasts: synthesis and subunit distribution

The present study aims at the role of ferritin in the regulation of syncytiotrophoblast free iron levels. The differentiated cytotrophoblast cell in culture is used as a model for this maternal-fetal interface. Cytotrophoblast cells isolated from term placentae are cultured in iron-poor (Medium 199), iron-depleted [desferrioxamine(DFO)] and iron-supplemented [diferric transferrin (hTF-2Fe), ferric ammonium citrate (FAC)] medium. Distribution and de novo synthesis of isoferritins is studied, together with the cellular iron concentration and the ferritin iron saturation. Compared to ferritin isolated from total placenta, ferritin obtained from villous tissue is enriched with acidic isoforms. This observation is in agreement with measured light (L) to heavy (H) subunit ratios < 1 of de novo synthesized ferritin in cultured cytotrophoblast cells. Neither iron-poor culture medium, nor hTf-2Fe supplemented medium affects the cellular iron or ferritin concentration. FAC increased the cellular ferritin iron saturation and (by synthesis) the acidic isoferritin concentrations. The results strongly suggest, that the term syncytiotrophoblast is able to balance transferrin-mediated iron uptake and iron release. In case of FAC supplementation, the syncytiotrophoblast is unable to keep intracellular iron low, and ferritin synthesis is stimulated. The predominance of acidic ferritins and the preferential synthesis of H subunits can be functionally explained by the established fact that iron incorporation in acidic ferritins is faster due to the presence of ferroxidase centres. Damage by free iron catalysed hydroxyl radical formation is therefore minimized.     

Relationship between phospholipid transfer protein activity and HDL level and size among inbred mouse strains

Site-directed mutagenesis was used to investigate the loading of iron into rat liver ferritin by ceruloplasmin. Changes were made in the H chain to investigate the role of tyrosines involved in an inherent ferroxidase activity thought to be involved in the self-loading of iron into ferritin. Mutation Y34F affected the rate of iron loading by ceruloplasmin and incorporation of the oxidized iron into the core. Mutation Y29R (making it analogous to the L chain) had no effect on iron oxidation but slightly decreased core formation. A double mutation in the L chain, to open the alpha-helix bundle channel, and R25Y, making the protein more analogous to the H chain, increased the amount of iron incorporated into the core, again suggesting that this Tyr is involved in ligand exchange for core formation. Additional changes in the L chain involving the BC loop suggest that the entire BC loop is involved in the association of ferritin with ceruloplasmin, increasing its ferroxidase activity and the rate of iron loading into ferritin.     

Characterization of serum ferritin in iron overload: possible identity to natural apoferritin

Serum ferritins from a patient with haemochromatosis and from a patient with transfusional siderosis were compared with tissue isoferritins on the basis of their iron content, isoferritin spectrum and immunological properties. Both serum ferritins had a low iron content and corresponded to only the most basic isoferritins in liver. The serum ferritins were very similar to the natural apoferritin from liver in all respects.     

Contribution of pancreatic scintiscanning to the diagnosis of chronic pancreatitis

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.     

Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins

UV-visible spectroscopy, electrode oximetry, and pH stat were used to study Fe(II) oxidation and hydrolysis in horse spleen ferritin (HoSF) and recombinant human H-chain and L-chain ferritins (HuHF and HuLF). Appropriate test reactions and electrode responses were measured, establishing the reliability of oxygen electrode/pH stat for kinetics studies of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O2 and H+/Fe(II), and rates of oxygen uptake and proton production were simultaneously measured as a function of iron loading of the protein. The data show a clear distinction between the diiron ferroxidase site and mineral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for the first time and is given by 2Fe2+ + O2 + 3H2O --> [Fe2O(OH)2]2+ + H2O2 + 2H+ where [Fe2O(OH)2]2+ represents the hydrolyzed dinuclear iron(III) center postulated to be a mu-oxo-bridged species from UV spectrometric titration data and absorption band maxima. The transfer of iron from the ferroxidase site to the mineral core has been now established to be [Fe2O(OH)2]2+ + H2O --> 2FeOOH(core) + 2H+. Regeneration of protein ferroxidase activity with time is observed for both HoSF and HuHF, consistent with their having enzymatic properties, and is facilitated by higher pH (7.0) and temperature (37 degreesC) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given by 4Fe2+ + O2 + 6H2O --> 4FeOOH(core) + 8H+. As the protein progressively acquires iron, oxidation/hydrolysis increasingly shifts from a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.     

Light induced redox reactions involving mammalian ferritin as photocatalyst

Under excitation by visible light the iron storage protein ferritin catalyses the reduction of cytochrome c and viologens as well as the oxidation of carboxylic acids, thiol compounds, and sulfite. The photochemically active element of ferritin is its mineral ferrihydrite semiconductor core. Band-gap excitation of these microcrystals leads to generation of electron-hole pairs that are sufficiently long-lived and reactive to engage in redox reactions with components of the medium. Photoreduction of cytochrome c and viologens occurs due to electron transfer from the conduction band of the iron oxide cluster through the protein shell surrounding the ferritin core. Laser photolysis coupled with time-resolved kinetics spectroscopy showed the electron transfer to propylviologen sulfonate to proceed in the microsecond time range. In the absence of electron acceptor at pH < 7, light excitation results in photodissolution of the iron oxide cluster with concomitant formation of Fe(II). These novel findings concerning the photocatalytic activity of ferritin with its inherent biological implications are discussed.     

Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.     

Purification of a RNA-binding protein from rat liver. Identification as ferritin L chain and determination of the RNA/protein binding characteristics

In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3'-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3'-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.     

Mobilization of iron from coal fly ash was dependent upon the particle size and the source of coal

Particulate air pollution, including coal fly ash, contains iron, and some of the pathological effects after inhalation may be due to reactive oxygen species produced by iron-catalyzed reactions. The objective of this study was to determine whether iron, present in coal fly ash, was mobilized, leading to ferritin induction in human airway epithelial cells, and whether the size of the particles affected the amount of iron mobilized. Three types of coal were used to generate the three size fractions of fly ash collected. The Utah coal fly ash was generated from a bituminous b coal, the Illinois coal fly ash from a bituminous c coal, and the North Dakota coal fly ash from a lignite a coal. Three size fractions were studied to compare the amount of iron mobilized in human airway epithelial (A549) cells and by citrate in cell-free suspensions. The size fractions selected were fine (<2.5 microm) and coarse (2.5-10 microm) components of PM10, airborne particulate matter <10 microm in diameter, and the fraction greater than 10 microm. Coal fly ash samples were incubated with 1 mM citrate to determine if iron associated with coal fly ash could be mobilized. Iron was mobilized by citrate from all three size fractions of all three coal types to levels as high as 56.7 nmol of Fe/mg of coal fly ash after 24 h. With all three coal types, more iron was mobilized by citrate from the <2.5 microm fraction than from the >2.5 microm fractions. Further, the mobilized iron was in the Fe(III) form. To determine if iron associated with the coal fly ash could be mobilized by A549 cells, cells were treated with coal fly ash, and the amount of the iron storage protein ferritin was determined after 24 h. Ferritin levels were increased by as much as 11.9-fold in cells treated with coal fly ash. With two of the three types of coal studied, more ferritin was induced in cells treated with the <2.5 microm fraction than with the >2.5 microm fractions. Further, inhibition of the endocytosis of the coal fly ash by the cells resulted in ferritin levels that were near that of the untreated cells, suggesting that iron was mobilized intracellularly, not in the culture medium. The results of this study suggest that differences in particle size and speciation of iron may affect the release of iron in human airway epithelial cells.     

Iron release from human monocytes after erythrophagocytosis in vitro: an investigation in normal subjects and hereditary hemochromatosis patients

This study investigated the release of erythrocyte-derived iron from purified human monocytes obtained from healthy volunteers and hereditary hemochromatosis (HH) patients. After erythrophagocytosis of 59Fe-labeled erythrocytes, a complete transfer of iron from hemoglobin (Hb) to ferritin was observed within 24 hours in both control and HH monocytes. The iron was released from the monocytes in the form of ferritin, Hb, and as nonprotein bound low molecular weight iron (LMW-Fe). During the initial rapid phase (<1.5 hours), iron release mostly consisted of Hb and LMW-Fe, while in the later phase (>1.5 hours), it was composed of ferritin and LMW-Fe. The kinetics of iron release were identical for HH monocytes. A high percentage of the total amount of iron was released as Hb both by viable normal and HH monocytes, suggesting that iron release as Hb is a physiologic process, which may occur whenever the erythrocyte-processing capacity of macrophages is exceeded. Most remarkably, HH monocytes released twice as much iron in a LMW form as control cells. Iron released in the form of LMW-Fe readily binds to plasma transferrin and may contribute to the high transferrin saturation and the occurrence of circulating nontransferrin-bound iron observed in HH patients.     

The role of iron in the regulation of hepatic transferrin synthesis

The role of iron supply in the regulation of hepatic transferrin synthesis by the isolated perfused rat liver was studied using nutritional iron deficiency as the experimental model. The increased transferrin release encountered in iron deficiency could be equated with enhanced de novo synthesis as evidenced by the inhibitory effects of cycloheximide and measurements of intrahepatic protein pools before and after perfusion. Refeeding with iron, sufficient to restore plasma iron and hepatic ferritin iron but before correction of anaemia, promoted a reduction towards normal in the transferrin synthetic rate. This effect was not produced by transfusional correction of the anaemia, suggesting a specific response to iron supply. Phenobarbitone treatment, which produced a marked fall in hepatic ferritin iron concentration but no change in haemoglobin or plasma iron concentrations, promoted a specific enhancement of transferrin synthesis in both control and iron deficient livers. The concentration of liver iron stores appears to be a major regulatory factor in the control of hepatic transferrin synthesis.     

Ferritin effect on the transverse relaxation of water: NMR microscopy at 9.4 T

Accumulation of ferritin, the iron storage protein, has been linked recently to aging and a number of pathologies. Noninvasive detection of iron storage by MRI relies on its extremely strong effect on water relaxation. The aim of this article is to characterize the effect of ferritin on transverse water relaxation in a high magnetic field, using an imaging Carr-Purcell Meiboom-Gill (CPMG) preparation sequence. Ferritin-induced water relaxation showed quadratic dependence on the iron loading factor, implying a paramagnetic mechanism. However, an additional zero order term was found, that could be due to the initial stages of the iron core loading. Significant enhancement of ferritin contrast was obtained at very short Tau CPMG durations. This approach for enhancing ferritin contrast was demonstrated by NMR microscopy of ferritin-injected Xenopus oocytes, thus showing the feasibility of ferritin detection in a high magnetic field, even in systems with short transverse relaxation.     

Changes in biochemical indicators of iron status during iron repletion and depletion in monkeys

Different modes of iron depletion and repletion were studied in monkeys to understand the sequential changes in and the relative importance of different biochemical indicators of iron status. Six control monkeys were divided into two groups, one was fed an iron-deficient diet (group 1) and the other underwent phlebotomy in addition to receiving an iron-deficient diet (group 2). Previously iron-depleted monkeys were subdivided into 4 groups of 3 animals each. While one group was continued on the iron-deficient diet (group 3), the second group received parenteral iron (group 4), the third group (group 5) received a sufficient-iron-containing diet, and the fourth group was fed 50% of the iron requirement. All indicators of iron status like hemoglobin (Hb), erythrocyte protoporphyrin (EPP), serum transferrin saturation and serum ferritin were monitored periodically, in addition to liver and bone marrow iron. all the indicators except serum ferritin and liver iron showed a decrease in group 2. On the other hand, animals receiving parenteral iron (group 4) showed an increase in all the parameters except serum ferritin. The dietary supplementation produced an increase in Hb and a decrease in EPP only (groups 5 and 6). There was a significant positive correlation between changes in bone marrow iron and Hb concentration depending on the severity of depletion and repletion. Both serum ferritin and liver iron did not respond to changes in dietary iron. Another parameter which responded to repletion was EPP. Serum ferritin and liver iron did not respond to changes in dietary iron or was not sensitive to subclinical iron deficiency. The results indicate that change in Hb is more sensitive to detect the deficiency of iron. It was also observed that different parameters respond variably under different modes of depletion and repletion.     

Brain iron, transferrin and ferritin concentrations are altered in developing iron-deficient rats

To study the iron, transferrin, and ferritin distribution at subcellular levels in response to acute dietary iron deficiency, we tested the hypothesis that early post-weaning iron deficiency can change iron and iron regulatory protein concentrations in rat brain. Male Sprague-Dawley rats were fed diets containing either 2 or 35 micrograms iron/g for 2, 3 or 4 wk starting at 21 d of age. Brain iron, transferrin and ferritin concentrations in cytosolic and microsomal fractions of either whole brain or pons and cerebellum were then determined. After 14 d of dietary iron restriction, brain iron concentrations were 50% lower in the microsomal fraction and 30% lower in cytosol compared with controls. Brain cytosolic transferrin concentration almost doubled in the same animals. Brain ferritin concentration in fractions from rats fed the iron-deficient diet for 14 d was lower than in controls, but then remained fairly constant. Absolute brain weight and total brain protein contents were unaffected by iron restriction. This study extends previous research by demonstrating that the brain responds to changes in body iron status with a change in transferrin concentration. If the dietary restriction is quite severe, this adaptation is insufficient. This study also notes that brain ferritin decreases with decreasing body iron status, though it was less responsive than nonheme iron in liver. The concept that iron enters the brain through a highly regulated endocytotic process at the blood brain barrier, that undoubtedly involves the regulation of transferrin receptors in capillary endothelial cell, is supported by our observation of elevated transferrin concentrations in brain of iron-deficient rats.     

Altered systemic iron metabolism in Parkinson's disease

Iron deposition in the substantia nigra in Parkinson's disease has been associated with an increase in lactoferrin receptors and a reduction in ferritin concentration. This accumulation of iron in the brain may accelerate free radical formation, lipid peroxidation, and neuronal death. Remarkably, there are few data available concerning systemic iron metabolism in Parkinson's disease. We measured total iron binding capacity and circulating iron, ferritin, transferrin, and transferrin receptors; calculated transferrin saturation; and estimated dietary iron intake in patients with idiopathic Parkinson's disease and in controls. Concentrations of circulating iron, ferritin, and transferrin as well as total iron binding capacity and transferrin saturation were significantly lower in patients than controls. There were no differences in transferrin receptors or dietary intake of iron. The decrease in levels of systemic ferritin and transferrin and the total iron binding capacity parallels observations in a Parkinson's disease brain, but the reductions in serum iron concentrations and transferrin saturation do not, and were unexpected. These results suggest the existence of a defect in the systems that regulate the synthesis of the major proteins of iron metabolism in the liver as well as the brain in Parkinson's disease that may, over time, expedite entry of iron into the brain and decrease iron in the extracellular compartment.     

Right to left differences in the ankle joint complex range of motion

In most eukaryotic cells, synthesis of the iron storage protein, ferritin is regulated by iron levels and redox conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical reactions. Although iron-catalysed reactions are believed to contribute to oxidative damage and cataractogenesis, little is known about iron storage in the lens. In this study, ferritin concentration was measured in cultured canine lens epithelial cells. Baseline ferritin concentration ranged from 76-163 ng (mg protein)-1; cells cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration. The iron chelator, Desferal, significantly decreased ferritin concentration. The reducing agent dithiothreitol decreased the hemin-induced increase in ferritin levels, but not baseline levels. In contrast, ascorbic acid induced a large increase in ferritin content. Other studies have shown that induction of ferritin synthesis can protect against oxidative damage. Regulation of ferritin levels may represent a mechanism by which the lens epithelium is protected from oxidative damage. In vivo, epithelial cells are normally exposed to much lower iron concentrations than the cultured lens epithelial cells in this study. However, in pathological circumstances, the iron content and redox state of the aqueous humor is dramatically altered and may affect the steady state levels of ferritin within the lens. This remains to be determined.     

Human H-kininogen is a ferritin-binding protein

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.     

Management of hemochromatosis. Hemochromatosis Management Working Group

The complications of iron overload in hemochromatosis can be avoided by early diagnosis and appropriate management. Therapeutic phlebotomy is used to remove excess iron and maintain low normal body iron stores, and it should be initiated in men with serum ferritin levels of 300 microg/L or more and in women with serum ferritin levels of 200 microg/L or more, regardless of the presence or absence of symptoms. Typically, therapeutic phlebotomy consists of 1) removal of 1 unit (450 to 500 mL) of blood weekly until the serum ferritin level is 10 to 20 microg/L and 2) maintenance of the serum ferritin level at 50 microg/L or less thereafter by periodic removal of blood. Hyperferritinemia attributable to iron overload is resolved by therapeutic phlebotomy. When applied before iron overload becomes severe, this treatment also prevents complications of iron overload, including hepatic cirrhosis, primary liver cancer, diabetes mellitus, hypogonadotrophic hypogonadism, joint disease, and cardiomyopathy. In patients with established iron overload disease, weakness, fatigue, increased hepatic enzyme concentrations, right upper quadrant pain, and hyperpigmentation are often substantially alleviated by therapeutic phlebotomy. Patients with liver disease, joint disease, diabetes mellitus and other endocrinopathic abnormalities, and cardiac abnormalities often require additional, specific management. Dietary management of hemochromatosis includes avoidance of medicinal iron, mineral supplements, excess vitamin C, and uncooked seafoods. This can reduce the rate of iron reaccumulation; reduce retention of nonferrous metals; and help reduce complications of liver disease, diabetes mellitus, and Vibrio infection. This comprehensive approach to the management of hemochromatosis can decrease the frequency and severity of iron overload, improve quality of life, and increase longevity.     

Are bombesin-like peptides involved in the mediation of stress response?

Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress. In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 microM iron, added to the culture medium as FeCl3, increased the lysosomal iron content and their sensitivity to H2O2-induced (0.25 mM for 30 min) oxidative stress. Over time (24-72 h), however, the cells were desensitized to the cytotoxic effects of H2O2; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H2O2. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H2O2, lysosomes remained intact and were no different from control cells which were exposed to H2O2 but not iron. These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.