Iron uptake from ferrichrome A and iron citrate in Ustilago sphaerogena.

Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromium- and gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.     

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.     

Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers

Aerobic microorganisms have evolved a variety of siderochromes, special ligands which can dissolve insoluble ferric iron and facilitate its transport into the cell. We have found that enb mutants of Salmonella typhimurium blocked in the biosynthesis of enterobactin (its natural iron carrier) are able to utilize siderochromes of different types made by other microorganisms as iron carriers. The antibiotic albomycin delta(2) was used to select mutants defective in ferrichrome-mediated iron uptake. Twelve classes of albomycin-resistant mutants, named sid, were defined on the basis of their growth responses to other siderochromes. Most of these classes have genetic lesions in loci that are cotransduced with panC (represented at 9 min on the genetic map). The locus designated sidJ is cotransduced with enb, whereas sidK and sidL are linked with neither panC nor enb. Genetic and physiological data indicate that S. typhimurium has several transport systems of high specificity for a variety of siderochromes produced by other microorganisms.     

Iron requirement and iron uptake from various iron compounds by different plant species

The Fe requirements of four monocotyledonous plant species (Avena sativa L., Triticum aestivum L., Oryza sativa L., Zea mays L.) and of three dicotyledonous species (Lycopersicum esculentum Mill., Cucumis sativus L., Glycine maxima (L.) Merr.) in hydroponic cultures were ascertained. Fe was given as NaFe-EDDHA chelate (Fe ethylenediamine di (O-hydroxyphenylacetate). I found that the monocotyledonous species required a substantially higher Fe concentration in the nutrient solution in order to attain optimum growth than did the dicotyledonous species. Analyses showed that the process of iron uptake was less efficient with the monocotyledonous species. When the results obtained by using chelated Fe were compared with those using ionic Fe, it was shown that the inefficient species were equally inefficient in utilizing Fe³⁺ ions. However, the differences between the efficient and the inefficient species disappeared when Fe²⁺ was used. This confirms the work of others who postulated that Fe³⁺ is reduced before uptake of chelated iron by the root. In addition, it was shown that reduction also takes place when Fe is used in ionic form. The efficiency of Fe uptake seems to depend on the efficiency of the root system of the particular plant species in reducing Fe³⁺. The removal of Fe from the chelate complex after reduction to Fe²⁺ seems to present no difficulties to the various plant species.     

Effects of carboxylic acids on the uptake of non-transferrin-bound iron by astrocytes

The concentrations of non-transferrin-bound iron are elevated in the brain during pathological conditions such as stroke and Alzheimer's disease. Astrocytes are specialised for sequestering this iron, however little is known about the mechanisms involved. Carboxylates, such as citrate, have been reported to facilitate iron uptake by intestinal cells. Citrate binds iron and limits its redox activity. The presence of high citrate concentrations in the interstitial fluid of the brain suggests that citrate may be an important ligand for iron transport by astrocytes. This study investigates whether iron accumulation by cultured rat astrocytes is facilitated by citrate or other carboxylates. Contrary to expectations, citrate, tartrate and malate were found to block iron accumulation in a concentration-dependent manner; α-ketoglutarate had limited effects, while fumarate, succinate and glutarate had no effect. This blockade was not due to an inhibition of ferric reductase activity. Instead, it appeared to be related to the capacity of these carboxylates to bind iron, since phosphate, which also binds iron, diminished the capacity of citrate, tartrate and malate to block the cellular accumulation of iron. These findings raise the possibility that citrate may have therapeutic potential in the management of neurodegenerative conditions that involve cellular iron overload.     

Iron uptake and iron limited growth of Escherichia coli K-12

Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 M without any of the known iron ionophores present. The growth rate increased between 0.05 and 2 M iron. Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 M iron. Under certain conditions iron uptake preceded growth of cells by more than an hour. During logarithmic growth the rate of iron uptake matched the growth rate. The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight.After consumption of the iron in the medium cells continued to grow with high rate for 1–2 generations. The iron uptake activity was increased during iron starvation.     

Iron and gallium increase iron uptake from transferrin by human melanoma cells: further examination of the ferric ammonium citrate-activated iron uptake process

Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both (59)Fe-transferrin (Tf) and (59)Fe-citrate (D.R. Richardson, E. Baker, J. Biol. Chem. 267 (1992) 13972-13979; D.R. Richardson, P. Ponka, Biochim. Biophys. Acta 1269 (1995) 105-114). This Fe uptake process was independent of the transferrin receptor and appeared to be activated by free radicals generated via the iron-catalysed Haber-Weiss reaction. To further understand this process, the present investigation was performed. In these experiments, cells were preincubated for 3 h at 37 degrees C with FAC or metal ion solutions and then labelled for 3 h at 37 degrees C with (59)Fe-Tf. Exposure of cells to FAC resulted in Fe uptake from (59)Fe-citrate that became saturated at an Fe concentration of 2.5 microM, while FAC-activated Fe uptake from Tf was not saturable up to 25 microM. In addition, the extent of FAC-activated Fe uptake from citrate was far greater than that from Tf. These results suggest a mechanism where FAC-activated Fe uptake from citrate may result from direct interaction with the transporter, while Fe uptake from Tf appears indirect and less efficient. Preincubation of cells with FAC at 4 degrees C instead of 37 degrees C prevented its effect at stimulating (59)Fe uptake from (59)Fe-Tf, suggesting that an active process was involved. Previous studies by others have shown that FAC can increase ferrireductase activity that may enhance (59)Fe uptake from (59)Fe-Tf. However, there was no difference in the ability of FAC-treated cells compared to controls to reduce ferricyanide to ferrocyanide, suggesting no change in oxidoreductase activity. To examine if activation of this Fe uptake mechanism could occur by incubation with a range of metal ions, cells were preincubated with either FAC, ferric chloride, ferrous sulphate, ferrous ammonium sulphate, gallium nitrate, copper chloride, zinc chloride, or cobalt chloride. Stimulation of (59)Fe uptake from Tf was shown (in order of potency) with ferric chloride, ferrous sulphate, ferrous ammonium sulphate, and gallium nitrate. The other metal ions examined decreased (59)Fe uptake from Tf. The fact that redox-active Cu(II) ion did not stimulate Fe uptake while redox-inactive Ga(III) did, suggests a mechanism of transporter activation not solely dependent on free radical generation. Indeed, the activation of Fe uptake appears dependent on the presence of the Fe atom itself or a metal ion with atomic similarities to Fe (e.g. Ga).     

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.     

Internal root control of iron uptake and utilization in maize genotypes

Plant and Soil - Studies were conducted to determine whether Fe uptake and transport in maize (Zea mays L.) is controlled inside or outside the root. Two maize genotypes exhibiting wide differences...     

Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron

Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed.     

Swelling and potassium uptake in cultured astrocytes

The intracellular water content of astrocytes in primary cultures shows a biphasic swelling pattern on exposure to various increased external K+ concentrations over the range of 1.5-100 mM. The two phases (physiological, 1.5-12 mM K+; pathological, 25-100 mM K+) are based on two different mechanisms. Both can be blocked by low Cl- solutions and involve intensive net uptake of K+. However, the physiological phase consists of the activation of a KCl + NaCl carrier, while the Na+ in turn is pumped out by Na+-K+ ATPase, with a resultant net accumulation of KCl. At pathological K+ concentrations the KCl + NaCl carrier is less active because the Na+ driving force, its energy source, is reduced (owing to depolarization by K+). However, the Donnan equilibrium across the cell membrane is heavily disturbed, which leads to passive KCl accumulation. The results suggest that volume changes in cultured glial cells during exposure to high K+ should be taken into consideration since they disguise K+ accumulation when only ion activity is measured.     

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.     

Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake

Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.