Stimulated Iron Uptake Research Articles

Desferrioxamine E produced by an indigenous salt tolerant Pseudomonas stutzeri stimulates iron uptake of Triticum aestivum L.

Siderophore are iron-scavenging biomolecules secreted by a variety of microorganisms in the environment that promote plant growth. In this study, we have identified a hydroxamate type siderophore desferrioxamine E produced by an indigenous salt tolerant Pseudomonas stutzeri strain SGM-1 under iron deficient condition. This bacterial strain was previously isolated from the saline-sodic soil sample on the basis of its salt tolerance through media enrichment. This strain could withstand at increasing salt concentrations (up to 12% w/v NaCl) and tolerated stress of acid as well as alkalinity, even grew over wide temperature ranges. Under iron deficient condition this isolate produced siderophore which was determined qualitatively by universal Chrome Azurol-Sulphonate (CAS) assay and quantitatively by spectrophotometry from the broth supernatant, also, its chemical type was determined by various type detection tests. Identification by liquid chromatography and mass spectrometry showed the hydroxamate type siderophore was desferrioxamine E. The efficacy of the desferrioxamine E was assessed for increasing the iron content of wheat (Triticum aestivum) plant, validated through Microwave Plasma Atomic Emission Spectroscopy (MP-AES). It is suggested that this strain is a useful plant growth promoting bioinoculant for enhancing iron content under stressed conditions for sustainable agricultural practices.

A Review of Nutrients and Compounds, Which Promote or Inhibit Intestinal Iron Absorption: Making a Platform for Dietary Measures That Can Reduce Iron Uptake in Patients with Genetic Haemochromatosis.

Objective To provide an overview of nutrients and compounds, which influence human intestinal iron absorption, thereby making a platform for elaboration of dietary recommendations that can reduce iron uptake in patients with genetic haemochromatosis. Design Review. Setting. A literature search in PubMed and Google Scholar of papers dealing with iron absorption. Results The most important promoters of iron absorption in foods are ascorbic acid, lactic acid (produced by fermentation), meat factors in animal meat, the presence of heme iron, and alcohol which stimulate iron uptake by inhibition of hepcidin expression. The most important inhibitors of iron uptake are phytic acid/phytates, polyphenols/tannins, proteins from soya beans, milk, eggs, and calcium. Oxalic acid/oxalate does not seem to influence iron uptake. Turmeric/curcumin may stimulate iron uptake through a decrease in hepcidin expression and inhibit uptake by complex formation with iron, but the net effect has not been clarified. Conclusions In haemochromatosis, iron absorption is enhanced due to a decreased expression of hepcidin. Dietary modifications that lower iron intake and decrease iron bioavailability may provide additional measures to reduce iron uptake from the foods. This could stimulate the patients' active cooperation in the treatment of their disorder and reduce the number of phlebotomies.

Zinc induces iron egress from intestinal Caco-2 cells via induction of Hephaestin: A role for PI3K in intestinal iron absorption

In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders.

Intraspinal TLR4 activation promotes iron storage but does not protect neurons or oligodendrocytes from progressive iron-mediated damage

Iron is essential for basic cellular functions but in excess is highly toxic. For this reason, free iron and iron storage are controlled in the periphery by elaborate regulatory mechanisms. In contrast, iron regulation in the central nervous system (CNS) is not well defined. Given that excess iron is present after trauma, hemorrhagic stroke and neurodegeneration, understanding normal iron regulation and promoting iron uptake in CNS pathology is crucial. Peripherally, toll-like receptor 4 (TLR4) activation promotes iron sequestration by macrophages. Notably, iron-rich sites of CNS pathology typically contain TLR4 agonists, which may promote iron uptake. Indeed, our recent work showed impaired iron storage after acute spinal cord injury in mice with TLR4 deficiency. Here we used a reductionist model to ask if TLR4 activation in the CNS stimulates iron uptake and promotes neuroprotection from iron-induced toxicity. For this, we measured the ability of microglia/macrophages to sequester exogenous iron and prevent pathology with and without concomitant intraspinal TLR4 activation. Results show that, similar to the periphery, activating intraspinal TLR4 via focal LPS injection increased mRNA encoding iron uptake and storage proteins and promoted iron sequestration into ferritin-expressing macrophages. However, this did not prevent oligodendrocyte and neuron loss. Moreover, replacement of oligodendrocytes by progenitor cells – a normally robust response to in vivo macrophage TLR4 activation – was significantly reduced if iron was present concomitant with TLR4 activation. Thus, while TLR4 signaling promotes CNS iron uptake, future work needs to determine ways to enhance iron removal without blocking the reparative effects of innate immune receptor signaling.

La rizobacteria promotora del crecimiento vegetal Arthrobacter agilis UMCV2 coloniza endofíticamente a Medicago truncatula

Arthrobacter agilis UMCV2 is a rhizosphere bacterium that promotes legume growth by solubilization of iron, which is supplied to the plant. A second growth promotion mechanism produces volatile compounds that stimulate iron uptake activities. Additionally, A. agilis UMCV2 is capable of inhibiting the growth of phytopathogens. A combination of quantitative polymerase chain reaction and fluorescence in situ hybridization techniques were used here to detect and quantify the presence of the bacterium in the internal tissues of the legume Medicago truncatula. Our results demonstrate that A. agilis UMCV2 behaves as an endophytic bacterium of M. truncatula, particularly in environments where iron is available.

Identification of bean polyphenols that inhibit and enhance iron uptake by Caco‐2 cells

Polyphenolic compounds present in bean seed coats are known to be inhibitors of Fe bioavailability; however, the specific polyphenols in bean seed coats that inhibit Fe bioavailability have not been identified. In addition, the antioxidative properties of some polyphenols may actually promote Fe absorption. The objective of this study was to identify the major polyphenols that affect Fe bioavailability in black beans. Extracts of white beans and black beans were evaluated for effect on Fe uptake using a Caco‐2 cell model. LC/MS was then used to identify compounds present in active extracts. As expected, Iron uptake was inhibited by extracts from black bean seed coats relative to that of white beans. Examination of the effect of individual polyphenols present in black bean seed coats showed varying dose related Fe uptake responses, ranging from inhibition to stimulation. Myricetin, quercetin and quercetin 3‐glucoside inhibited iron uptake; ferulic acid and salicylic acid had no effect; and caffeic acid, epicatechin, gallic acid and kaempferol stimulated iron uptake in Caco‐2 cells. The results clearly indicate that not all polyphenols inhibit iron uptake and that some are promoters. Targeting these compounds in breeding and or processing of beans could yield improvements of Fe bioavailability. Funded by USDA‐ARS.

Chemicals That Stimulate Iron Uptake – A Novel Approach to Treatment of Iron Deficiency,

Abstract 3180 Iron (Fe) is an essential nutrient required for all cells, especially for erythrocyte hemoglobin synthesis which requires absorption of 1–2 mg of iron from the gastrointestinal tract. Iron deficiency as a result of inadequate dietary uptake has multiple consequences including anemia and abnormal neurologic development in children and is a global public health concern. Enterocytes in the duodenum, the site of iron absorption, can extract about 10% of dietary Fe. Nonetheless for numerous reasons simple iron supplementation has not solved the worldwide epidemic of iron deficiency. We hypothesized that small molecules which could potentiate iron uptake into cells would allow enterocytes to absorb an increased amount of dietary iron and could be beneficial in limiting iron deficiency. To identify molecules that would accelerate Fe uptake we used a high through-put screening system in conjunction with a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. Small molecules that stimulated Fe uptake were defined as causing increased calcein fluorescence quenching compared to Fe alone. K562 cells were exposed to 0.1 μM calcein for 10 minutes, thoroughly washed, and 1 × 105 cells plated into each well of multiple 96-well plates. After equilibration of the plates at 37° C, aliquots of the individual components of an in-house chemical library of ∼12,000 compounds dissolved in DMSO were screened in duplicate or triplicate and fluorescence measurements made at 0 and 30 min after addition of 10 μM FeNH4SO4 in a Synergy IV plate reader. 30 chemicals were identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose response curves and by assaying the effect on non-transferrin bound 55Fe uptake. None of the stimulators were cytotoxic for up to at least 3 days. The lead compound, LS081, had an IC50 = 1.22 ± 0.48 μM for 55Fe uptake in K562 cells compared to controls. LS081 was also used to examine the iron uptake in Caco2 cells grown in bicameral chambers, a model system to study intestinal iron absorption. LS081 significantly increased 55Fe uptake into Caco2 cells with a very rapid influx of 55Fe in the first 5 min after Fe was offered to the apical surface followed by a ∼ 4-fold increased uptake over the next 90 min. 55Fe transport across the basolateral surface into the basal chamber also increased ∼ 4 fold. The increased 55Fe transport in caco2 cells is more prominent at lower pH of 5.5 compare to pH 7.5 suggesting LS081 acted on a common divalent metal uptake pathway. Mice treated with LS081 + ferric ammonium citrate via oral gavage for two weeks significantly increased (p < 0.001 by unpaired t-test compared to ferric ammonium citrate alone) the level of ferritin, the iron storage protein, in the liver, demonstrating the absorption of LS081 from intestinal cells. In summary, using high through-put screening technique we identified small molecules that stimulate iron uptake and could be used as a drug for iron deficiency. Disclosures: No relevant conflicts of interest to declare.

Abstract 3530: Chemicals that stimulate iron uptake: a novel approach to cancer treatment

Abstract Cancer cells have increased levels of transferrin receptor and lower levels of ferritin, an iron deficient phenotype that has led to the use of iron chelators to further deplete cells of iron to limit growth. Chelators have not proven, however, to be successful anti-cancer agents. As cancer cells also have increased reactive oxygen species (ROS) we hypothesized that a contrarian approach of enhancing iron entry would allow for further increased generation of ROS causing oxidative damage and cell death. Molecules that accelerate iron uptake were identified using high through-put screening of a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. A small molecule library consisting of ∼11,000 compounds was screened and a class of compounds identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose-dependent 55Fe uptake in various cancer cell lines. The lead compound LS081 exhibited an IC50 = 1.22 ± 0.48 μM and also increased the uptake of Fe from transferrin (Tf). LS081 decreased PC-3 cell growth in the presence of Fe2+ by 64 ± 8% compared to 14 ± 1% decrease with LS081 alone (mean decrease ± SEM, n = 3) with minimal effects on the immortalized, non-malignant prostate cell line 267B1 cells. LS081 plus Fe increased DNA strand breaks and induced cell cycle arrest at the G1 phase of PC-3 cells. In addition, LS081 markedly decreased HIF-1α and HIF-2α levels in the DU-145 prostate cancer and the MDA-MB-231 breast cancer cell lines. LS081 also stimulated ROS production by nearly 3-fold and decreased clonogenicity by greater than 50% in PC-3 cells. In summary, we have developed a high through-put screening technique that identified small molecules that stimulate iron uptake both from ferri-Tf and non-Tf bound iron. These molecules have properties that suggest that these iron facilitator compounds may serve as anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2011-3530

Anticancer activity of the iron facilitator LS081

BackgroundCancer cells have increased levels of transferrin receptor and lower levels of ferritin, an iron deficient phenotype that has led to the use of iron chelators to further deplete cells of iron and limit cancer cell growth. As cancer cells also have increased reactive oxygen species (ROS) we hypothesized that a contrarian approach of enhancing iron entry would allow for further increased generation of ROS causing oxidative damage and cell death.MethodsA small molecule library consisting of ~11,000 compounds was screened to identify compounds that stimulated iron-induced quenching of intracellular calcein fluorescence. We verified the iron facilitating properties of the lead compound, LS081, through 55Fe uptake and the expression of the iron storage protein, ferritin. LS081-induced iron facilitation was correlated with rates of cancer cell growth inhibition, ROS production, clonogenicity, and hypoxia induced factor (HIF) levels.ResultsCompound LS081 increased 55Fe uptake in various cancer cell lines and Caco2 cells, a model system for studying intestinal iron uptake. LS081 also increased the uptake of Fe from transferrin (Tf). LS081 decreased proliferation of the PC-3 prostate cancer cell line in the presence of iron with a lesser effect on normal prostate 267B1 cells. In addition, LS081 markedly decreased HIF-1α and -2α levels in DU-145 prostate cancer cell line and the MDA-MB-231 breast cancer cell lines, stimulated ROS production, and decreased clonogenicity.ConclusionsWe have developed a high through-put screening technique and identified small molecules that stimulate iron uptake both from ferriTf and non-Tf bound iron. These iron facilitator compounds displayed properties suggesting that they may serve as anti-cancer agents.

Effects of ATP and ADP on iron uptake in rat heart mitochondria

Iron uptake in mitochondria and fractionated mitochondria compartments was studied to understand iron transport in heart mitochondria. The inner membrane is most active in iron uptake. Mitochondrial uptake was dependent on iron concentration and the amount of mitochondria. Iron transport was inversely proportional to pH in the range of 6.0 to 8.0. Iron transport reached a maximum after 30 min of incubation at 37°C. Iron uptake was inhibited by 1 mM ATP and stimulated by 1 mM ADP. The oxidative phosphorylation inhibitor oligomycin inhibited iron uptake, but rotenone and antimycin A did not. The divalent ions Mg2+, Cu2+, Mn2+, and Zn2+ suppressed iron uptake at 10 µM and stimulated it at 1 mM. The divalent ion Ca2+ stimulated iron uptake at 10 µM and suppressed it at 1 mM, competing with iron. The uptake of calcium was stimulated by 10 to 1000 µM ATP, while iron uptake was stimulated reciprocally by 10 to 1000 µM ADP, suggesting that these ions have movements similar to those of ATP and ADP.

Iron transport by proteoliposomes containing mitochondrial F 1F o ATP synthase isolated from rat heart

In this work, we present evidence of Fe 2+ transport by rat heart mitochondrial F 1F o ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 °C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the α, β, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg +2, Ca +2, Mn +2, Zn +2, Cu +2, Fe +3, and Al +3 was observed. Moreover, Cu +2, even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H +.

High through-Put Screening of Chemicals That Stimulate Iron Uptake: A Novel Approach to Discovery of Anti-Cancer Drugs

Iron (Fe) is an essential nutrient required for growth of all cells. Actively growing cells appear to have a greater need for iron at least as measured by increased expression of the transferrin receptor (TfR), the major route by which iron enters cells. Various attempts have been made to use iron chelators to deplete cells of iron as a means for decreasing the growth of cancer cells. We hypothesized that a contrary approach of enhancing iron entry would allow for generation of increased reactive oxygen species which could then potentiate radiation and radiomimetic drugs. To identify small molecules that would accelerate iron uptake we used a high through-put screening system in conjunction with a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. Small molecules that stimulate Fe uptake were defined as causing an increase of calcein fluorescence quenching compared to Fe alone. K562 cells were exposed to 0.1 μM calcein for 10 minutes, thoroughly washed, and 1 × 105 cells plated into each well of multiple 96-well plates. After equilibration of the plates at 37° C, aliquots of the individual components of an in-house chemical library of ~10,000 compounds dissolved in DMSO were screened in duplicate or triplicate and fluorescence measurements made at 0 and 30 min after addition of 10 μM FeNH4SO4 in a Synergy IV plate reader. 30 chemicals were identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose response curves and the lack of toxicity noted by having no significant cell death after exposure for up to 5 days. One of the compounds, LS-0108076, at 3 or 10 μM stimulated iron uptake from FeNH4SO4 by 50% in K562 cells. LS-0108076 also facilitated uptake from transferrinbound iron in a prostate cancer line as evidenced by a 2-fold increase in ferritin levels when PC-3 cells were cultured with 10% serum. LS-0108076 (3μM) also significantly increased generation of ROS from 100±0% to 150±18% as measured with DCFDA but only in the serum-free Hepes buffer with 2 μM FeNH4citrate. LS-0108076 was further examined in prostate cancer and normal prostate cells for the ability to enhance cell killing by bleomycin, a radiation mimetic anti-cancer drug that chelates Fe and causes cell death by interchelating with DNA and producing DNA strand breaks. In both the PC3 and DU145 prostate cancer cell lines, LS-0108076 enhanced bleomycin cell killing in serum-free medium with further potentiation by the addition of 2 μM Fe3+ to LS-0108076. Similarly, LS-0108076 potentiated radiation induced cell death with the effects also enhanced by the presence of Fe3+and with marked increase of DNA strand breaks as detected by a comet assay. In normal prostate cells LS-0108076 had no affect on bleomycin cell toxicity. In summary, we have developed a high through-put screening technique that identified small molecules that stimulate iron uptake and demonstrated that compounds which facilitate iron uptake increase cell sensitivity to radiation therapy and chemotherapy and can be used to potentiate anti-cancer agents.

Duodenal Cytochrome B Expression Stimulates Iron Uptake by Human Intestinal Epithelial Cells

Duodenal cytochrome B (Dcytb) is localized principally in the apical membrane of the enterocyte. It is thought to act as a ferric reductase that furnishes Fe(II), the specific and selective iron species transported by divalent metal transporter 1 (DMT1) in the duodenal enterocytes. Expression of both genes is strongly iron regulated and is thought to be required for transcellular iron trafficking in concert in response to physiological requirements. We tested this hypothesis by expressing Dcytb in Caco-2 cells, a human cell line model often used to mimic intestinal enterocytes. Iron uptake (59Fe) was significantly higher in Dcytb-transfected Caco-2 cells than in cells transfected with empty vector as a control. Fe(III) reductase activity of Dcytb was measured with ferrozine, a strong chelator of Fe(II) species. Cells expressing Dcytb exhibited enhanced ferric reductase activity as well as increased59Fe uptake compared with cells transfected with empty vector as a control. Ferrozine blocked iron uptake and preincubation of cells with dehydroascorbate (to increase cellular ascorbate levels) stimulated iron uptake. Cotransfection of Dcytb and DMT1 resulted in an additive increase in iron uptake by the cells. The results confirm Dcytb can act as a ferric reductase that stimulates iron uptake in Caco-2 cells.

Oh, NO! The Ironic Route to Neurotoxicity

Cheah et al. investigated the role of Dexras1, a Ras-family guanosine triphosphatase, and uncovered a pathway linking activation of the N -methyl-D-aspartate-type glutamate receptor (NMDAR) to regulation of iron homeostasis and implicating changes in iron uptake in NMDAR-mediated neurotoxicity. Both neuronal nitric oxide synthase (nNOS) and Dexras1 bind to the adaptor protein CAPON; NMDAR stimulation leads to the calcium-dependent activation of nNOS, enabling the S -nitrosylation and activation of Dexras1. The consequences of Dexras1 activation, however, have been unclear. Cheah et al. identified peripheral benzodiazepine receptor (PBR)-associated protein (PAP7) as a potential Dexras1 binding partner through a screen of a rat whole-brain cDNA library. The relationship was confirmed with pulldown assays using human embryonic kidney (HEK) 293 cells (which have little endogenous PAP7 and Dexras1) transfected with glutathione S -transferase-PAP7 and Myc-tagged Dexras1. PAP7 bound the iron importer divalent metal transporter (DMT1), and coimmunoprecipitation analysis revealed that Dexras1, PAP7, and DMT1 existed as a ternary complex in mouse brain. Overexpression of Dexras1 in HEK293 cells enhanced iron uptake, an effect that was markedly enhanced by cotransfection with PAP7. Exposure of cells to nitric oxide (NO) donors elicited S -nitrosylation of Dexras1 and stimulated Dexras1-dependent iron uptake in undifferentiated PC12 cells (which have abundant endogenous PAP7 and Dexras1). Furthermore, NMDA stimulated iron uptake in primary cortical neurons from wild-type but not nNOS knockout mice. Intriguingly, a cell-permeable iron chelator inhibited an NMDA-dependent increase in the formation of reactive oxygen species as well as NMDA-mediated neurotoxicity. Thus, the authors conclude that NMDA stimulates NO-dependent increase in iron uptake, which seems to play a role in NMDA-dependent neuronal death. J. H. Cheah, S. F. Kim, L. D. Hester, K. W. Clancy, S. E. Patterson III, V. Papadopoulos, S. H. Snyder, NMDA receptor-nitric oxide transmission mediates neuronal iron homeostasis via the GTPase Dexras1. Neuron 51 , 431-440 (2006). [PubMed]

The iron transporter DMT1

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482–488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.

Role of Ceruloplasmin in Cellular Iron Uptake: Addendum

We recently reported that ceruloplasmin (Cp) stimulates iron uptake by HepG2 cells by a transferrin-independent mechanism ([1][1]). We are grateful to Ian Trowbridge, at the Salk Institute, for a comment about our use of the anti-transferrin receptor monoclonal antibody H68.4 in this study. He

The surface of rat hepatocytes can transfer iron from stable chelates to external acceptors.

The chelator diethylenetriaminepentaacetate (DTPA) forms a stable complex with iron that does not donate iron to transferrin under physiological conditions, i.e., pH above 7 and isotonic milieu. It does, however, deliver iron to hepatocytes. This uptake is initiated by a mobilization of the metal from the complex by the cell surface. When an external chelator is added simultaneously, it can bind the iron and inhibit its accumulation by the cells. This is shown here with the impermeant siderophore conjugate hydroxyethyl-starch coupled desferrioxamine, as well as with apotransferrin. We also demonstrate exchange of iron between DTPA and holo-transferrin, or at least movement from the chelator to the protein, which may have lost its iron to the cell in advance, providing new binding sites for mobilized iron. The efficient hepatocyte iron donor lactoferrin greatly stimulates iron uptake from DTPA, apparently by binding iron and transferring it into the cells by endocytosis. Ferritin is unable to do this; therefore, the mobilization of iron is not caused by a reducing activity at the cell surface, because iron is readily transferred from DTPA to ferritin by the reductant ascorbic acid. The transfer process is dependent on the temperature, the time, and the amount of cells present, and is partly inhibited by sulfhydryl reagents. We conclude that this activity represents a hitherto unidentified first step in the movement of iron through the cell membrane and may be relevant for transferrin-bound, as well as for non-transferrin-bound, iron uptake by hepatocytes.

Chlorpromazine stimulatory effect on iron uptake by rat brain synaptosomes

Clinical long-term neuroleptic administration induces extrapyramidal motor side-effects, of which tardive dyskinesia is the most important. Experimentally, dopamine D 2 supersensitivity is observed after phenothiazine and butyrophenone treatment. Neuroleptic-induced tardive dyskinesia and D 2 modulation have been linked to impaired iron homeostasis in the central nervous system. Increased nonheme iron levels found in the basal ganglia of patients with extrapyramidal symptomology support the connection between iron and neuronal dopaminergic modulation. We now report the effect of chlorpromazine on iron uptake by synaptosomes of rat brain from two different iron donors: [ 55Fe]citrate and [ 55Fe]transferrin. Iron uptake from both donors by cortical synaptosomes was stimulated by Ca 2+ and enhanced by chlorpromazine in a saturable fashion. Synaptosomes from the striatum also showed increased (60%) iron uptake from [ 55Fe]citrate in the presence of chlorpromazine. Chlorpromazine stimulated iron uptake by cortical synaptosomes more efficiently than Ca 2+, at physiological levels, from both [ 55Fe]transferrin (50%) and [ 55Fe]citrate (68%). Calcium potentiated the effect of chlorpromazine upon cortical synaptosomal iron uptake from [ 55Fe]citrate, but had no apparent effect on the uptake from [ 55Fe]transferrin. Chlorpromazine-stimulated iron uptake from the latter was observed without addition of Ca 2+. Moreover, fluorescence measurement of Ca 2+ uptake by cortical synaptosomes showed intensified uptake in the presence of 50 μM chlorpromazine (42%). Visible spectral studies of chlorpromazine in the presence of Fe 3+-citrate and diferric-transferrin did not reveal iron displacement by chlorpromazine from either of the two donors. These data suggest that chlorpromazine may increase iron uptake by neurons, and may be involved in the development of tardive dyskinesia and other extrapyramidal disorders.

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with 59Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly associated with exchange of Na+. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed.

Isolation of a novel siderophore from Pseudomonas cepacia.

A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.