Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver.

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.     

Multidentate pyridinones inhibit the metabolism of nontransferrin-bound iron by hepatocytes and hepatoma cells.

The therapeutic effect of iron (Fe) chelators on the potentially toxic plasma pool of nontransferrin-bound iron (NTBI), often present in Fe overload diseases and in some cancer patients during chemotherapy, is of considerable interest. In the present investigation, several multidentate pyridinones were synthesized and compared with their bidentate analogue, deferiprone (DFP; L1, orally active) and desferrioxamine (DFO; hexadentate; orally inactive) for their effect on the metabolism of NTBI in the rat hepatocyte and a hepatoma cell line (McArdle 7777, Q7). Hepatoma cells took up much less NTBI than the hepatocytes (< 10%). All the chelators inhibited NTBI uptake (80-98%) much more than they increased mobilization of Fe from cells prelabelled with NTBI (5-20%). The hexadentate pyridinone, N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine showed comparable activity to DFO and DFP. There was no apparent correlation between Fe status, Fe uptake and chelator activity in hepatocytes, suggesting that NTBI transport is not regulated by cellular Fe levels. The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as at the cell membrane. In conclusion (a) rat hepatocytes have a much greater capacity to take up NTBI than the rat hepatoma cell line (Q7), (b) all chelators bind NTBI much more effectively during the uptake phase than in the mobilization of Fe which has been stored from NTBI and (c) while DFP is the most active chelator, other multidentate pyridinones have potential in the treatment of Fe overload, particularly at lower, more readily clinically available concentrations, and during cancer chemotherapy, by removing plasma NTBI.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

Subcellular localization of ferritin and iron taken up by rat hepatocytes.

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.     

The mechanism of hepatic iron uptake from native and denatured transferrin and its subcellular metabolism in the liver cell.

Hepatic iron uptake and metabolism were studied by subcellular fractionation of rat liver homogenates after injection of rats with a purified preparation of either native or denatured rat transferrin labelled with 125I and 59Fe. (1) With native transferrin, hepatic 125I content was maximal 5 min after injection and then fell. Hepatic 59Fe content reached maximum by 16 h after injection and remained constant for 14 days. Neither label appeared in the mitochondrial or lysosomal fractions. 59Fe appeared first in the supernatant and, with time, was detectable as ferritin in fractions sedimented with increasingly lower g forces. (2) With denatured transferrin, hepatic content of both 125I and 59Fe reached maximum by 30 min. Both appeared initially in the lysosomal fraction. With time, they passed into the supernatant and 59Fe became incorporated into ferritin. The study suggests that hepatic iron uptake from native transferrin does not involve endocytosis. However, endocytosis of denatured transferrin does occur. After the uptake process, iron is gradually incorporated into ferritin molecules, which subsequently polymerize; there is no incorporation into other structures over 14 days.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG

Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited ⁵⁵Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.     

Ferritin contains less iron (59Fe) in cells when the protein pores are unfolded by mutation

Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mm) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t1/2) decreased 17-fold), and (iii) decreased alpha-helix (8%). Overexpression (> 4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell 59Fe but increased chelatable iron, 16%, p < 0.01 (59Fe in the deferoxamine-containing medium), and decreased 59Fe in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (59Fe) in ferritin in vivo.     

Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.     

Release of iron from ferritin requires lysosomal activity

How ferritin-Fe becomes available for cell functions is unknown. Our previous studies with rat hepatoma cells indicated ferritin had to be degraded to release its Fe. In these studies, we investigated whether this occurs in other cell types and whether lysosomes are required. Release of ferritin-Fe was induced with desferoxamine (DFO) in ⁵⁹Fe-preloaded hepatoma, Caco2, and erythroid K562 cells and measured by rocket immunoelectrophoresis and autoradiography. The half-lives for ferritin-⁵⁹Fe and protein were parallel (23, 16, and 11 h for the hepatic, Caco2, and K562 cells, respectively). Co-treatment with 180 µM Fe, leupeptin, chymostatin, or chloroquine markedly decreased rates of ferritin-Fe release and ferritin degradation. Lactacystin had no effect except for a small one in erythroid cells. Fractionation of hepatoma cell lysates on iodixanol gradients showed rapid depletion of cytosolic ferritin by DFO treatment but no accumulation in lysosomes. We conclude that regardless of cell type, release of Fe from ferritin occurs mainly through lysosomal proteolysis. degradation; proteasomes     

The role of the membrane-bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe-citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK-Mel-28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I-labelled anti-MTf mAb-binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe-citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe-Tf, respectively, DFO had no influence on 59Fe-citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol-specific phospholipase C (PtdIns-PLC). Preincubation of melanoma cells with PtdIns-PLC reduced anti-MTf mAb binding to 3% of the control, while PtdIns-PLC only slightly reduced 59Fe uptake from 59Fe-citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe-citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.     

Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.     

Mucosal iron binding proteins and the inhibition of iron absorption by endotoxin

The uptake of iron from a tied off jejunal segment into the body after the injection of a 59Fe labeled test dose was decreased after the administration of endotoxin by about 80% in both normal and iron deficient animals.--In the iron deficient group the distribution of 59Fe in the cytosol fraction of jejunal mucosa between transferrin and ferritin was determined chromatographically; the amount of 59Fe in the ferritin fraction increased remarkably after the endotoxin treatment and the ratio of both was changed in favor of ferritin.--It is hypothesized that the association of the diversion of iron to the mucosal ferritin with the decrease of the transport of iron into the blood caused by endotoxin might be the consequence of abnormal oxidations in the mucosa measured by others in liver tissue.     

Factors involved in the regulation of iron transport through reticuloendothelial cells

The effects of various maneuvers on the handling of 59Fe-labeled heat-damaged red cells (59Fe HDRC) by the reticuloendothelial system were studied in rats. Raising the saturation of transferrin with oral carbonyl iron had little effect on splenic release of 59Fe but markedly inhibited hepatic release. Splenic 59Fe release was, however, inhibited by the prior administration of unlabeled HDRC or by the combination of carbonyl iron and unlabeled HDRC. When carbonyl iron was administered with unlabeled free hemoglobin, the pattern of 59Fe distribution was the same as that observed when carbonyl iron was given alone. 59Fe ferritin was identified in the serum after the administration of 59Fe HDRC but the size of the fraction was not affected by raising the saturation of transferrin. Sizing column analyses of tissue extracts from the spleen at various times after the administration of 59Fe HDRC revealed a progressive shift from hemoglobin to ferritin, with only small amounts present in a small molecular weight fraction. The small molecular weight fraction was greater in hepatic extracts, with the difference being marked in animals that had received prior carbonyl iron. The increased hepatic retention of 59Fe associated with a raised saturation of transferrin was reduced by a hydrophobic ferrous chelator (2,2'-bipyridine), a hydrophilic ferric chelator (desferrioxamine), and an extracellular hydrophilic ferric chelator (diethylene-triaminepentacetic acid). Transmembrane iron transport did not seem to be a rate-limiting factor in iron release, since no differences in 59Fe membrane fractions were noted in the different experimental settings. These findings are consistent with a model in which RE cells release iron from catabolized red cells at a relatively constant rate. When the saturation of transferrin is raised, a significant proportion of the iron is transported from the spleen to the liver either in small molecular weight complexes or in ferritin. Although a saturated transferrin had no effect on the release of iron from reticuloendothelial cells, prior loading with HDRC conditions them to release less iron.     

Uptake and release of ferritin iron. Surface effects and exchange within the crystalline core

The uptake and subsequent release of iron by apoferritin and ferritin was studied by using labelled iron ((59)Fe). The experimental results are consistent with predictions arising from a model system developed in the interpretation of previous experiments. In this model, uptake and release of ferritin iron is controlled by the available surface area of the small crystalline particles of hydrous ferric oxide found within the ferritin molecule. Evidence is also presented for the exchange of Fe(3+) ions among the various cation sites within these crystallites.     

Iron uptake and metabolism by hepatocytes

The hepatocytes form part of the iron storage system of the body. In serving this function they exchange iron bidirectionally with the plasma iron transport protein transferrin (Tf). Iron uptake involves binding of the iron-Tf complex to cell membrane receptors and endocytosis into low-density vesicles, where the iron is released from its carrier protein before the Tf is returned undegraded to the extracellular medium. Two components of the iron uptake process can be distinguished, one saturable at low concentrations of diferric Tf and the other not saturable by increasing the Tf concentration. Both result in net uptake of iron by the cells and both appear to depend on specific binding to the cell membrane and endocytosis. Hepatocytes also obtain some iron from haptoglobin-hemoglobin, heme-hemopexin, and ferritin (Fn), in each case by interaction with membrane receptors and endocytosis. Within the cell iron from all sources enters one or more transit pools, where it is available for exchange with the iron storage protein Fn, and for release from the cell to plasma Tf or to iron chelators administered therapeutically or experimentally. Chelator-mediated iron release occurs to the plasma and/or to the bile, depending on the nature of the chelator and the source of the iron.     

Comparison of changes in uptake and mucosal processing of iron in short and long-term iron depletion

Thirty minutes following an intragastric dose of [59]Fe, rats subjected to short-term and long-term iron depletion showed a similar increase in [59]Fe in plasma and a similar decrease in the retention of [59]Fe in mucosal cytosol compared to controls. With both low-iron groups, a two-fold increase in [59]Fe uptake by brush-border membrane vesicles and a six-fold reduction in the [59]Fe incorporated into the ferritin of the mucosal cytosol were observed. These studies indicate that short-term exposure to a low-iron diet triggers changes in both the uptake of iron by the brush-border membrane and the processing of iron within the mucosal cell prior to major changes in body iron status.     

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.     

Mobilization of ferritin iron in erythroblasts by chelating agents

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.