Nitric oxide mediates iron release from ferritin

Nitric oxide (NO) synthesis by cytotoxic activated macrophages has been postulated to result in a progressive loss of iron from tumor target cells as well as inhibition of mitochondrial respiration and DNA synthesis. In the present study, the addition of an NO-generating agent, sodium nitroprusside, to the iron storage protein ferritin resulted in the release of iron from ferritin and the released iron-catalyzed lipid peroxidation. Hemoglobin, which binds NO, and superoxide anion, which reacts with NO, inhibited nitroprusside-dependent iron release from ferritin, thereby providing evidence that NO can mobilize iron from ferritin. These results suggest that NO generation in vivo could lead to the mobilization of iron from ferritin disrupting intracellular iron homeostasis and increasing the level of reactive oxygen species.     

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label‐free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron‐mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.     

Inhibitory Effect of Several Nitric Oxide-Generating Compounds on Purified Na+,K+-ATPase Activity from Porcine Cerebral Cortex

Abstract: Nitric oxide (NO)-generating compounds (NO donors) such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, S-nitroso-l -glutathione, 3-morpholinosyndnonimine (SIN-1), (dl )-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene inhibited the Na⁺,K⁺-ATPase activity purified from porcine cerebral cortex. NO-reducing or -scavenging agents, such as superoxide dismutase or N-(dithiocarbamate)-N-methyl-d -glucamine sodium salt, l -ascorbic acid, and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not α-tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, l -ascorbic acid, and α-tocopherol. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (^?NO₂), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN-1). PTIO did not potentiate, but rather attenuated, the SIN-1-induced inhibition. SIN-1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO^?). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, l -ascorbic acid, and α-tocopherol. These results suggest that NO donors may release NO or NO-derived products, presumably ^?NO₂ and ONOO^?, and may inhibit the Na⁺,K⁺-ATPase activity by interacting with a SH group at the active site of the enzyme.     

The relationship between iron release, ferritin synthesis and intracellular iron distribution in mouse peritoneal macrophages. Evidence for a reduced level of metabolically available iron in elicited macrophages

The rate of iron release from thioglycollate-elicited mouse peritoneal macrophages pulsed with 59Fe-labelled transferrin-antitransferrin immune complexes was lower than that from resident or Corynebacterium parvum-activated macrophages. Anaerobic conditions increased the rate of iron release by thioglycollate-elicited macrophages but had no effect on resident or C. parvum-activated macrophages. Thioglycollate-elicited macrophages also contained less ferritin and were deficient in their ability to synthesis ferritin. Incubation of these cells in medium containing 100 microM iron caused some increase in ferritin synthesis, but the response to iron was much less pronounced than that by resident or C. parvum-activated macrophages. In the thioglycollate-elicited macrophages, relatively less iron was incorporated into ferritin, and more into other soluble macromolecules and insoluble haemosiderin-like compounds than in the other types of macrophages. It is proposed that thioglycollate-elicited macrophages tend to divert iron to a relatively inert intracellular pool, and that this could account for their reduced ability to release iron. Such a mechanism might help to explain the reduced release of iron by liver and spleen macrophages occurring during inflammation.     

Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by ⁵⁹Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.     

Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls

NADPH-cytochrome P-450 reductase-catalyzed reduction of paraquat promoted the release of iron from ferritin. Aerobically, iron release was inhibited approximately 60% by superoxide dismutase, whereas xanthine oxidase-dependent iron release was inhibited nearly 100%. This suggests that both superoxide and the paraquat cation radical can catalyze the release of iron from ferritin. Accordingly, under anaerobic conditions, the paraquat radical mediated a very rapid, complete release of iron from ferritin. Similarly, the cation free radicals of the closely related chemicals, diquat and benzyl viologen, also promoted iron release. ESR studies demonstrated that electron transfer from the paraquat cation radical to ferritin accounts for the reductive release of iron. The ferritin structure was not altered by exposure to the paraquat radical and also retained its ability to re-incorporate iron. These studies indicate that release of iron from ferritin may be a common feature contributing to free radical-mediated toxicities.     

Characteristics and Kinetics of Iron Releasefrom the Ferritin under the EGCG reduction

The mechanism of iron release from ferritin in vivo is still unclear even though it represents a key step of the metabolism of iron in vivo. Here, both interaction intensity and binding stability between epigallocatechin gallate (EGCG) from tea and liver ferritin of Dasyatis akajei (DALF) were investigated using UV–visible, fluorescence and circular dichroism (CD) spectrometry, respectively. The results indicated that EGCG could reduce the iron within the ferritin shell directly in the absence of chemical reducers such as Na₂S₂O₄, but this process was strictly pH-dependent, and the rate of iron release is faster at low pH than at high pH. The kinetic study of iron release showed that this process fitted the law of zero order reaction, which differed from that of first order reaction by various chemical reducers such as Vitamin C. In addition, Both fluorescence and CD spectrometry were further used to study the reduction mechanism of iron release in vitro, showing that there was a slight conformation change of the ferritin shell during EGCG reduction because of a complex formation of DALF–EGCG. It appears that chemical reducers with large molecular sizes reduce the iron across the protein shell by the way of an electron transfer pathway (ETP). A novel pathway for iron release from DALF with EGCG reduction is suggested to explain for a reductive route of iron metabolism by biological reducers in vivo.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

The role of ferritin in developing primary bean leaves under various light conditions

Ferritin and ferritin-iron in the primary leaves of Phaseolus vulgaris L. were determined during growth in the dark, in the light, and during de-etiolation. The ratio ferritin protein/total protein appeared to be rather constant. In dark-grown leaves maximally 50% of the total extractable iron was found to be present in ferritin. This percentage was lower in deetiolating and light-grown leaves. In ten-day-old green leaves no ferritin-iron could be measured. The translocation of iron from cotyledons to the developing plant appears to be related to the need for iron in the leaves. These results suggest that ferritin acts as a buffer molecule for iron in plants.     

Purification,electrophoretic behavior,and kinetics of iron release of liver ferritin of Dasyatis akajei

From the liver of fish Dasyatis akajei, ferritin has been isolated by thermal denaturation and ammonium sulfate fractionation and then further purified by anion exchange chromatography and gel exclusion chromatography. The molecular weight of the liver ferritin of D. akajei (DALF) was measured to be 400 kDa by PAGE. Moreover, SDS-PAGE experimentation indicates that protein shell of DALF consists of the H and L subunits with molecular weight of 18 and 13 kDa, respectively. Using isoelectric focusing with pH ranging from 5.0 to 6.0, the ferritin purified by the PAGE exhibited three bands with different pI values in the gel slab. Diameters of the protein shell and iron core were also investigated by transmission electron microscope and determined to be 10–12 nm and 5–8 nm, respectively. A kinetic study of DALF reveals that the rate of self-regulation of the protein shell rather than the complex surface of the iron core plays an important role in forming a process for iron release with mixed orders.     

On the limited ability of superoxide to release iron from ferritin

Reductive release of iron from ferritin may catalyze cytotoxic radical reactions like the Haber-Weiss reaction. The ability of .O2- to mobilize Fe(II) from ferritin was studied by using the xanthine/xanthine oxidase reaction, with and without superoxide dismutase, and with bathophenanthroline sulphonate as the chelator. Not more than one or two Fe(II)/ferritin molecules could be released by an .O2(-)-dependent mechanism, even after repeated exposures of ferritin to bursts of .O2-. The amount of releaseable iron depended on the size and the age of the iron core, but not on the iron content of the protein shell of ferritin which was manipulated by chelators and addition of FeCl3. The kinetic characteristics of the .O2(-)-mediated iron release indicated the presence of a small pool of readily available iron at the surface of the core. The very limited .O2(-)-dependent release of iron from ferritin is compatible with a protective role of ferritin against toxic iron-catalyzed reactions.     

Investigation of the release of iron from ferritin by naturally occurring antioxidants

Ferritin is the main intracellular iron storage protein. The release of iron from ferritin in the presence of a number of phenolic based compounds of nutritional significance was studied at physiological pH. The release of iron was measured by monitoring the formation of the iron(II)-ferrozine complex. The kinetics of this process were studied in Hepes buffer (pH 7.00), at 37 degrees C. The order of ability to remove iron from ferritin is epigallocatechin>gallic acid methyl ester approximately equal to sinapic acid>ferulic acid. The presence of the oxyradical scavenger urea resulted in a slight inhibition in the release of iron from ferritin by both gallic acid methyl ester and epigallocatechin. The ability of each reagent to release iron is interpreted on the basis of their ability to (a) reduce the bound iron and (b) complex the iron with the oxidised form of the phenol, thus mobilising it from the protein. These studies indicate that some phenolic based compounds that have been epidemiologically associated with a negative effect on iron absorption in man, can individually mobilise and release iron from ferritin under suitable conditions.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

In Vitro Studies of Ferritin Iron Release and Neurotoxicity

Abstract: The increase in brain iron associated with several neurodegenerative diseases may lead to an increased production of free radicals via the Fenton reaction. Intracellular iron is usually tightly regulated, being bound by ferritin in an insoluble ferrihydrite core. The neurotoxin 6-hydroxydopamine (6-OHDA) releases iron from the ferritin core by reducing it to the ferrous form. Iron release induced by 6-OHDA and structurally related compounds and two other dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium iodide (MPP⁺) and 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), were compared, to identify the structural characteristics important for such release. 1,2,4-Trihydroxybenzene (THB) was most effective in releasing ferritin-bound iron, followed by 6-OHDA, dopamine, catechol, and hydroquinone. Resorcinol, MPP⁺, and TaClo were ineffective. The ability to release iron was associated with a low oxidation potential. It is proposed that a low oxidation potential and an ortho -dihydroxyphenyl structure are important in the mechanism by which ferritin iron is mobilized. In the presence of ferritin, both 6-OHDA and THB strongly stimulated lipid peroxidation, an effect abolished by the addition of the iron chelator deferoxamine. These results suggest that ferritin iron release contributes to free radical-induced cell damage in vivo.