Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes

The relationship between transferrin-free iron uptake and cellular metabolism was investigated using rabbit reticulocytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimycin A, 2,4-dinitrophenol, NaCN, NaN3 and rotenone) or substrates. Measurements were made of cellular ATP concentration and the rate of uptake of Fe(II) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into cytosolic and stromal fractions of the cells and ATP levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vmax but had no effect on the Km values for this process. It is concluded that the uptake of transferrin-free iron by reticulocytes is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated transport process. Additional studies were performed using transferrin-bound iron. The metabolic inhibitors also reduced the uptake of this form of iron but the inhibition could be accounted for entirely by reduction in the rate of transferrin endocytosis.     

The role of the transferrin receptor for the activation of human lymphocytes

The proliferative response of peripheral blood mononuclear cells (PBMC) in synthetic serum-free media depends on the presence of sufficient amounts of transferrin (Tf). In the present communication we show that the reduction of Tf concentration in culture media results in a decreased proliferation, whereas lymphokine production and the expression of activation markers (IL-2 receptor; transferrin receptor, (TfR); HLA class II) remain unchanged. To examine whether this effect is due to iron depletion we added iron chelates (ferric citrate, FeCi; ferric nitrilotriacetic acid, FeNTA) which can be internalized by cells without the requirement for Tf. The iron chelates could fully restore the proliferative response even in complete absence of Tf, suggesting that the observed inhibitory effect was indeed caused by iron depletion. Addition of a monoclonal TfR antibody, J 64, also caused a marked inhibition of proliferation of PBMC in regular serum-containing medium as well as in Tf-free synthetic medium; this effect could not be overcome by any of the tested iron chelates. Therefore, growth inhibition caused by J 64 cannot simply be attributed to iron starvation. These data suggest that J 64 may interfere with processes others than iron uptake and that the TfR might confer a necessary promoting signal for lymphocyte proliferation.     

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.     

Growth-stimulating effect of transferrin on a hybridoma cell line: Relation to transferrin iron-transporting function

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.     

Possible role of membrane gamma-glutamyltransferase activity in the facilitation of transferrin-dependent and -independent iron uptake by cancer cells.

BACKGROUND: The molecular mechanisms by which iron is physiologically transported trough the cellular membranes are still only partially understood. Several studies indicate that a reduction step of ferric iron to ferrous is necessary, both in the case of transferrin-mediated and transferrin-independent iron uptake. Recent studies from our laboratory described gamma-glutamyltransferase activity (GGT) as a factor capable to effect iron reduction in the cell microenvironment. GGT is located on the outer aspect of plasma membrane of most cell types, and is often expressed at high levels in malignant tumors and their metastases. The present study was aimed at verifying the possibility that GGT-mediated iron reduction may participate in the process of cellular iron uptake. RESULTS: Four distinct human tumor cell lines, exhibiting different levels of GGT activity, were studied. The uptake of transferrin-bound iron was investigated by using 55Fe-loaded transferrin, as well as by monitoring fluorimetrically the intracellular iron levels in calcein-preloaded cells. Transferrin-independent iron uptake was investigated using 55Fe complexed by nitrilotriacetic acid (55Fe-NTA complex).The stimulation of GGT activity, by administration to cells of the substrates glutathione and glycyl-glycine, was generally reflected in a facilitation of transferrin-bound iron uptake. The extent of such facilitation was correlated with the intrinsic levels of the enzyme present in each cell line. Accordingly, inhibition of GGT activity by means of two independent inhibitors, acivicin and serine/boric acid complex, resulted in a decreased uptake of transferrin-bound iron. With Fe-NTA complex, the inhibitory effect - but not the stimulatory one - was also observed. CONCLUSION: It is concluded that membrane GGT can represent a facilitating factor in iron uptake by GGT-expressing cancer cells, thus providing them with a selective growth advantage over clones that do not possess the enzyme.     

Iron uptake studies on erythroid cells

Iron uptake from ⁵⁵Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.     

Iron uptake studies on erythroid cells.

Iron uptake from 55Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.     

The role of iron in the growth of human leukemic cell lines

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.     

Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth

The ability of Staphylococcus epidermidis strains to grow in the presence of human transferrin and varying amounts of ferric iron was studied. At initial bacterial densities up to 10(4) cfu ml(-1), none of the three strains grew when transferrin iron saturation was below the full saturation point, whereas the bacteria grew consistently when transferrin was fully iron-saturated and there was non-transferrin-bound iron in the medium. Precultivation of the bacteria under iron-restricted conditions to induce siderophore production did not abolish the growth dependence on non-transferrin-bound iron. At initial bacterial densities of 10(6) cfu ml(-1), the bacteria proliferated consistently also in the presence of partially saturated transferrin. The results indicate that at low bacterial densities, S. epidermidis cannot utilise transferrin-bound iron for growth and that its proliferation is dependent on non-transferrin-bound iron.     

Uptake of iron from transferrin by isolated rat hepatocytes. A redox-mediated plasma membrane process?

The uptake of iron from transferrin by isolated rat hepatocytes varies in parallel with plasma membrane NADH:ferricyanide oxidoreductase activity, is inhibited by ferricyanide, ferric, and ferrous iron chelators, divalent transition metal cations, and depends on calcium ions. Iron uptake does not depend on endosomal acidification or endocytosis of transferrin. The results are compatible with a model in which iron, at transferrin concentrations above that needed to saturate the transferrin receptor, is taken up from transferrin predominantly by mechanisms located to or contiguous with the plasma membrane. The process involves labilization and reduction of transferrin-bound iron by cooperative proton and electron fluxes. A model which combines the plasma membrane mechanism and the receptor-mediated endocytosis mechanism is presented.     

Sodium ascorbate (vitamin C) induces apoptosis in melanoma cells via the down-regulation of transferrin receptor dependent iron uptake

Sodium ascorbate (vitamin C) has a reputation for inconsistent effects upon malignant tumor cells, which vary from growth stimulation to apoptosis induction. Melanoma cells were found to be more susceptible to vitamin C toxicity than any other tumor cells. The present study has shown that sodium ascorbate decreases cellular iron uptake by melanoma cells in a dose- and time-dependent fashion, indicating that intracellular iron levels may be a critical factor in sodium ascorbate-induced apoptosis. Indeed, sodium ascorbate-induced apoptosis is enhanced by the iron chelator, desferrioxamine (DFO) while it is inhibited by the iron donor, ferric ammonium citrate (FAC). Moreover, the inhibitory effects of sodium ascorbate on intracellular iron levels are blocked by addition of transferrin, suggesting that transferrin receptor (TfR) dependent pathway of iron uptake may be regulated by sodium ascorbate. Cells exposed to sodium ascorbate demonstrated down-regulation of TfR expression and this precedes sodium ascorbate-induced apoptosis. Taken together, sodium ascorbate-mediated apoptosis appears to be initiated by a reduction of TfR expression, resulting in a down-regulation of iron uptake followed by an induction of apoptosis. This study demonstrates the specific mechanism of sodium ascorbate-induced apoptosis and these findings support future clinical trial of sodium ascorbate in the prevention of human melanoma relapse.     

Iron-uptake in the Euryarchaeon Halobacterium salinarum

Iron-uptake is well studied in a plethora of pro- and eukaryotic organisms with the exception of Archaea, which thrive mainly in extreme environments. In this study, the mechanism of iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum strain JW 5 was analyzed. Under low-iron growth conditions no siderophores were detectable in culture supernatants. However, various xenosiderophores support growth of H. salinarum. In [⁵⁵Fe]–[¹⁴C] double-label experiments, H. salinarum displays uptake of iron but not of the chelator citrate. Uptake of iron was inhibited by cyanide and at higher concentrations by Ga. Furthermore, a K_M for iron uptake in cells of 2.36 μM and a V_max of approximately 67 pmol Fe/min/mg protein was determined. [⁵⁵Fe]-uptake kinetics were measured in the absence and presence of Ga. Uptake of iron was inhibited merely at very high Ga concentrations. The results indicate an energy dependent iron uptake process in H. salinarum and suggest reduction of the metal at the membrane level.     

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.     

Thyroid hormone dependent pituitary tumor cell growth in serum-free chemically defined culture. A new regulatory role for apotransferrin.

Thyroid hormone dependent GH1 rat pituitary tumor cell growth in serum-free chemically defined medium required a serum-derived mediator (i.e., thyromedin) which was identified as transferrin [Sirbasku, D.A., Stewart, B.H., Pakala, R., Eby, J.E., Sato, H., & Roscoe, J.M. (1990) Biochemistry 30, 295-304]. The transferrin isolated was consistent with the equine R or D variants and was biologically active only as apotransferrin (apoTf). To determine if other variants of horse transferrin also were thyromedins, a purification was developed which yielded seven separate forms. Initially, only four of these had activity when assayed in standard "iron salts containing" medium (ED50 values of 290-1160 nM). To further assess activity, the iron contents of all seven were altered either by saturation with ferric ammonium citrate or by citrate/acid depletion of the metal ion. Thereafter, potencies were compared in "iron salts containing" and "iron salts reduced" media. All seven variants proved to be active as apoTf. Bioassays in which apoTf was maximized showed ED50 values of 2.1-3.8 nM. Conversely, assays in which thyromedins were converted to Tf.2Fe showed no activity. Previously, the only known physiological function of apoTf was that of a carrier/detoxifier of iron; this study indicates a new role in hormone-dependent pituitary cell growth.     

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate ⁵⁵Fe³⁺ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of ⁵⁵Fe³⁺ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.     

Iron uptake by immature erythroid cells: Mechanism of dependence on metabolic energy

The mechanism by which the utilization of transferrin-bound iron is linked with cellular metabolism was investigated using rabbit reticulocytes and bone marrow cells. The rate of metabolism was altered by the use of inhibitors which act at different sites in the metabolic pathway (NaF, sodium fluoroacetate, rotenone, 2,4-dinitrophenol, NaCN) and by the addition of metabolic substrates (inosine, sodium pyruvate, sodium lactate). Measurements were made of the rates of iron and transferrin uptake and, in many of the experiments, of cellular ATP and NADH concentrations. The results showed that there was a significant correlation between the rate of iron uptake and the ATP concentration of the cells, but no correlation was found with the NADH concentration. The rate of transferrin uptake was inhibited to a lesser degree than that of iron uptake, and only when the ATP concentration had fallen below that necessary to inhibit iron uptake. It is concluded that the rate of uptake of transferrin-bound iron by immmature erythroid cells is dependent on the intracellular concentration of ATP but is independent of the NADH concentration.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.     

Calmodulin dependence of transferrin receptor recycling in rat reticulocytes.

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.