Inhibitory mechanism of lead on transferrin-bound iron uptake by rabbit reticulocytes: A fractal analysis

Experimental data of transferrin and transferrin-bound iron uptake byrabbit reticulocytes in the presence or absence of extracellular lead isanalyzed by means of a fractal model. A highly significant correlation offractal dimension (Df) of intracellular transferrin or transferrin-boundiron uptake with varying extracellular concentrations of lead (0 ~ 25umol/L) was observed (Transferrin: r = 0.897, p = 0.015; transferrin-boundiron: r = 0.947, p = 0.004). The Df of membrane-bound transferrin (r =-0.618, p = 0.191) or transferrin-bound iron (r = 0.144, p = 0.786) did notappear to be markedly altered by lead. Further analysis shows thatinhibitory degree of lead on intracellular iron uptake is higher than thaton intracellular transferrin uptake. These results suggest that theinhibitory effect of lead on the iron uptake may occur in intracellularprocess rather than in membrane binding step, probably inhibitingtranslocation of iron across the endosomal membrane.     

Transferrin and iron uptake by rat reticulocytes

The uptake of transferrin labeled with 3H and 59Fe by rat reticulocytes was studied to clarify the characteristics of the uptake process and intracellular transport. Rat reticulocytes took up transferrin in a saturable, time- and temperature-dependent manner. Scatchard analysis of the binding parameters indicated that transferrin molecules were bound to cell-surface receptors with high affinity. Monodansyl- cadaverine, a potent inhibitor of transglutaminase, reduced the amount of internalized transferrin but has no effect on the total amount of cell-associated transferrin, suggesting that transferrin is taken up by rat reticulocytes via receptor-mediated endocytosis. About 50% of the internalized 3H label was released from the cells after reincubation for 1 h in fresh medium. In contrast, no release of 59Fe label was observed. By immunoprecipitation and subsequent SDS-PAGE the released 3H-labeled product was identified as apotransferrin. Lysosomotropic reagents and a proton ionophore reduced the uptake of 59Fe. These results indicated that iron was removed from transferrin at an intracellular site in an acidic environment. The released iron was found not to associate with any intermediate ligands before it was utilized for heme synthesis in mitochondria.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.     

Utilization of transferrin-bound iron by Listeria monocytogenes

Abstract It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes . This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.     

Effect of different durations of exercise on transferrin-bound iron uptake by rat erythroblast

This study evaluated effects of different durations of exercise on transferrin receptor (TfR) expression on the membrane of rat erythroblasts. Female rats were assigned to six groups: 3, 6 and 12 months of strenuous exercise (swimming 2 h/day, 5 days/wk) groups and their corresponding controls. At the end of experiments, the erythroblasts were isolated for Tf binding assay and transferrin-bound iron (Tf-Fe) uptake. Tissue non-heme iron and hematological iron indices were also measured. The TfR number on the cells was about 603,189 plus minus 107,562, 890,150 plus minus 164,849 and 384,695 plus minus 46,295 molecules/cell in three control groups (3, 6, 12 months) respectively. Exercise groups had significantly higher levels of TfR than those of the control groups, being 1,374,137 plus minus 243,677, 2,175,360 plus minus 462,737 and 1,012,759 plus minus 249,423 molecules/cell in 3, 6 and 12 months of exercise groups respectively (p < 0.05). After 30 min of incubation, cellular Tf approached to levels of 8.28 plus minus 1.94, 10.73 plus minus 3.30 and 6.60 plus minus 0.93 fmole/10(6) cells in 3, 6 and 12 months of exercise groups, while the corresponding control values were 3.09 plus minus 0.36, 5.03 plus minus 1.01 and 2.51 plus minus 0.88 fmole/10(6) cells respectively (all P < 0.05). The rates of cellular iron accumulation were 7.07 (3), 8.79 (6) and 5.96 (12 month) fmole/10(6) cells/min in the exercised rats and 2.91, 3.85, and 2.03 fmole/10(6) cells/min in their corresponding controls (all p < 0.05). However, no significant difference was observed in the ratios (Exercise/Corresponding control) of the increased TfR expression, Tf-Fe uptake and Tf endocytosis as well as of the decreased plasma iron and tissue non-heme iron levels induced by different periods of exercise. Furthermore, the increase in the length of exercise (6 or 12 month) did not induce a remarkable decrease in plasma hemoglobin and hematocrit. These results indicate that a true iron deficiency or 'sport anemia' can not develop even if under longer periods (6 or 12 month) of strenuous exercise.     

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.     

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

Role of membrane surface potential and other factors in the uptake of non-transferrin-bound iron by reticulocytes

Reticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non-transferrin-bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo-cytes incubated with ⁵⁹Fe-labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K⁺, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono-, di-, tri-, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley-Liss, Inc.     

Transferrin endocytosis and the mechanism of iron uptake by reticulocytes in the toad (Bufo marinus)

Reticulocytosis was induced in toads (Bufo marinus) by treatment with phenylhydrazine. Iron and transferrin uptake and transferrin endocytosis and exocytosis by these cells were measured. The mean number of transferrin receptors per cell was found to be 4.5 X 10(5) and the affinity constant of transferrin to receptors was 0.2 X 10(7) M-1. Iron and transferrin uptake were temp.-dependent processes. An inflection point occurred at 15-16 degrees C in the Arrhenius plots of endocytosis and iron uptake. The activation energies of these two processes above and below the inflection temperature were 31 and 71 kJ/mol. It is concluded that iron uptake by immature toad erythroid cells occurs by receptor-mediated endocytosis which still functions at temps as low as 5 degrees C.     

Utilization of lactoferrin-bound and transferrin-bound iron by Campylobacter jejuni

Campylobacter jejuni NCTC 11168 was capable of growth to levels comparable with FeSO₄ in defined iron-limited medium (minimal essential medium alpha [MEMα]) containing ferrilactoferrin, ferritransferrin, or ferri-ovotransferrin. Iron was internalized in a contact-dependent manner, with 94% of cell-associated radioactivity from either ⁵⁵Fe-loaded transferrin or lactoferrin associated with the soluble cell fraction. Partitioning the iron source away from bacteria significantly decreased cellular growth. Excess cold transferrin or lactoferrin in cultures containing ⁵⁵Fe-loaded transferrin or lactoferrin resulted in reduced levels of ⁵⁵Fe uptake. Growth of C. jejuni in the presence of ferri- and an excess of apoprotein reduced overall levels of growth. Following incubation of cells in the presence of ferrilactoferrin, lactoferrin became associated with the cell surface; binding levels were higher after growth under iron limitation. A strain carrying a mutation in the cj0178 gene from the iron uptake system Cj0173c-Cj0178 demonstrated significantly reduced growth promotion in the presence of ferrilactoferrin in MEMα compared to wild type but was not affected in the presence of heme. Moreover, this mutant acquired less ⁵⁵Fe than wild type when incubated with ⁵⁵Fe-loaded protein and bound less lactoferrin. Complementation restored the wild-type phenotype when cells were grown with ferrilactoferrin. A mutant in the ABC transporter system permease gene (cj0174c) showed a small but significant growth reduction. The cj0176c-cj0177 intergenic region contains two separate Fur-regulated iron-repressible promoters. This is the first demonstration that C. jejuni is capable of acquiring iron from members of the transferrin protein family, and our data indicate a role for Cj0178 in this process.     

Role of plasma membrane phospholipids in the uptake and release of transferrin and its iron by reticulocytes

Summary The involvement of membrane phospholipids in the utilization of transferrinbound iron by reticulocytes was investigated using [⁵⁹Fe]- and [¹²⁵I]-labelled transferrin and rabbit reticulocytes which had been incubated with phospholipas A. Transferrin and iron uptake and release were all inhibited by phospholipas A which produced a marked decrease in the relative abundance of phosphatidylcholine and phosphatidylethanolamine and equivalent increases in their lyso-compounds in the reticulocyte plasma membrane. There was a close correlation between the iron uptake rate and the rate and amount of transferrin uptake and the amount of the lysophospholipids in the membrane. Incubation of the cells with exogenous lysophosphatidylethanolamine or lysophosphatidylcholine also produced inhibition of iron and transferrin uptake. The reduced uptake produced by phospholipase A could be reversed if the lyso-compounds were removed by fatty acid-free bovine serum albumin or by reincubation in medium 199. Treatment with phospholipase A was shown to increase the amount of transferrin bound by specific receptors on the reticulocyte membrane but to inhibit the entry of transferrin into the cells.The present investigation provides evidence that the phospholipid composition of the cell membrane influences the interaction of transferrin with its receptors, the processes of endocytosis and exocytosis whereby transferrin enters and leaves the cells, and the mechanism by which iron is mobilized between its binding to transferrin and incorporation into heme. In addition, the results indicate that phosphatidylethanolamine is present in the outer half of the lipid bilayer of reticulocyte membrane.     

The membrane effect of 1-anilino-8-naphthalene sulfonate on transferrin and iron uptake by rabbit reticulocytes

Studies on the effect of 1-anilino-8-naphthalene sulfonate on uptake of transferrin and iron by rabbit reticulocytes show that the inhibitory action of ANS is localized at the membrane level. The intravesicular pH and cellular ATP level were not affected by this anionic probe. ANS shifted the transition temperature and reduced the enthalpy changes of iron uptake by rabbit reticulocytes. These suggested that the drug reduced the membrane fluidity. Hence, ANS disturbed the physicochemical environment of the receptor for transferrin resulting in the perturbation of receptor-mediated endocytosis.     

Effects of spermine on transferrin and iron uptake by reticulocytes: I. Action on the endocytic uptake of transferrin

Iron uptake by rabbit reticulocytes was inhibited by spermine in a concentration-dependent manner. Examination of the single-cycle endocytosis of 125I-transferrin showed that a graded reduction in the rate of exocytosis of transferrin was related to increasing extracellular spermine concentrations. This reduction could affect the recycling of transferrin receptors and resulted in the loss of membrane binding sites in spermine-treated cells. As large vacuoles were observed in cells treated with spermine, the endotubular function of these cells was probably affected. Spermine also enhanced the binding affinity of transferrin to membrane receptors. The mechanism for this enhancement was not clear.