Tf-Fe Uptake Research Articles

Angiotensin II inhibits uptake of transferrin-bound iron but not non-transferrin-bound iron by cultured astrocytes

The existence of all components of the renin–angiotensin system (RAS) and the iron metabolism system, and the recent findings on the functions of angiotensin II (ANGII) in peripheral iron metabolism imply that ANGII might play a role in iron homeostasis by regulating expression of iron transport proteins in the brain. Here, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes. We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as the expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes. This indicated that the inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on the expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in the cells. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis.

Treatment with nerve growth factor decreases expression of divalent metal transporter 1 and transferrin receptor in PC12 cells

Divalent metal transporter 1 (DMT1) and transferrin receptor (TfR) might play a key role in non-transferrin-bound iron (NTBI) and transferrin-bound iron (Tf-Fe) uptake by neuronal cells. Recent studies demonstrated that nerve growth factor (NGF)-treated PC12 cells (the neuronal phenotype) have higher NTBI as well as Tf-Fe uptake compared with untreated cells (the undifferentiated cells). We speculated the increased NTBI and Tf-Fe uptake induced by NGF treatment might be associated with the increased expression of DMT1 and TfR. In this study, we investigated the effect of NGF treatment on DMT1 and TfR expression in PC12 cells. Contrary to our expectation, treatment with NGF induced a significant decrease rather than increase in DMT1+IRE, DMT1−IRE and TfR expression in the cells. The data demonstrate that the increase in iron uptake is not associated with the DMT1 and TfR in NGF-treated PC12 cells. The RT-PCR findings of no change in DMT1 mRNA plus our data suggest that regulation of DMT1 expression by NGF might be at the post-transcriptional level.

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: Role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H 2O 2 and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H 2O 2- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H 2O 2- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of 55Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the “untargeted” carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.

Effects of cell proliferation on the uptake of transferrin-bound iron by human hepatoma cells

The effects of cellular proliferation on the uptake of transferrin-bound iron (Tf-Fe) and expression of transferrin receptor-1 (TfR1) and transferrin receptor-2 (TfR2) were investigated using a human hepatoma (HuH7) cell line stably transfected with TfR1 antisense RNA expression vector to suppress TfR1 expression. At transferrin (Tf) concentrations of 50 nmol/L and 5 μmol/L, when Tf-Fe uptake occurs by the TfR1- and TfR1-independent (NTfR1)-mediated process, respectively, the rate of Fe uptake by proliferating cells was approximately 250% that of stationary cells. The maximum rate of Fe uptake by the TfR1- and NTfR1-mediated process by proliferating cells was increased to 200% and 300% that of stationary cells, respectively. The maximum binding of Tf by both TfR1- and NTfR1-mediated processes by proliferating cells was increased significantly to 160% that of stationary cells. TfR1 and TfR2-α protein levels expressed by proliferating cells was observed to be approximately 300% and 200% greater than the stationary cells, respectively. During the proliferating growth phase, expression of TfR1 messenger RNA (mRNA) increased to 300% whereas TfR2-α mRNA decreased to 50% that of stationary cells. In conclusion, an increase in Tf-Fe uptake by TfR1-mediated pathway by proliferating cells was associated with increased TfR1 mRNA and protein expression. An increase in Tf-Fe uptake by NTfR1-mediated pathway was correlated with an increase in TfR2-α protein expression but not TfR2-α mRNA. In concluion, TfR2-α protein is likely to have a role in the mediation of Tf-Fe uptake by the NTfR1 process by HuH7 hepatoma cell in proliferating and stationary stages of growth. (H epatology 2003;38:967-977.)

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 ± 107,562, 890,150 ± 164,849 and 384,695 ± 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 ± 243,677, 2,175,360 ± 462,737 and 1,012,759 ± 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 ± 1.94, 10.73 ± 3.30 and 6.60 ± 0.93 fmole/10 6 cells in 3, 6 and 12 months of exercise groups, while the corresponding control values were 3.09 ± 0.36, 5.03 ± 1.01 and 2.51 ± 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.

Increased expression of transferrin receptor on membrane of erythroblasts in strenuously exercised rats.

This study investigated the effects of strenuous exercise on transferrin (Tf)-receptor (TfR) expression and Tf-bound iron (Tf-Fe) uptake in erythroblasts of rat bone marrow. Female Sprague-Dawley rats were randomly assigned to either an exercise or sedentary group. Animals in the exercise group swam 2 h/day for 3 mo in a glass swimming basin. Both groups received the same amount of handling. At the end of 3 mo, the bone marrow erythroblasts were freshly isolated for Tf-binding assay and determination of Tf-Fe uptake in vitro. Tissue nonheme iron and hematological iron indexes were measured. The number of Tf-binding sites found in erythroblasts was approximately 674,500 +/- 132,766 and 1,270,011 +/- 235,321 molecules/cell in control and exercised rats, respectively (P < 0. 05). Total Fe and Tf uptake by the cells was also significantly increased in the exercised rats after 30 min of incubation. Rates of cellular Fe accumulation were 5.68 and 2.58 fmol. 10(6) cells(-1). min(-1) in the exercised and control rats, respectively (P < 0.05). Tf recycling time and TfR affinity were not different in exercised and control rats. Increased cellular Fe was mainly located in the stromal fraction, suggesting that most of accumulated Fe was transported to the mitochondria for heme synthesis. The findings demonstrated that the increased cellular Fe uptake in exercised rats was a consequence of the increased TfR expression rather than the changes in TfR affinity and Tf recycling time. The increase in TfR expression and cellular Fe accumulation, as well as the decreased serum Fe concentration and nonheme Fe in the liver and the spleen induced by exercise, probably represented the early signs of Fe deficiency.

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells.

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was approximately 20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1.

Transferrin-bound iron uptake by the cultured cerebellar granule cells

Excessive brain iron has been found in several neurodegenerative diseases. However, little information is available about mechanism of iron uptake by different types of brain cells including neurons. In this study, transferrin-bound iron (Tf-Fe) accumulation in the cultured cerebellar granule cell was investigated in vitro. After 5 days of culture, the cells were incubated with 1 μM of double-labelled transferrin ( 125I-Tf- 59Fe) at 37°C for 60 min. The cellular Tf-Fe and transferrin (Tf) uptake was analysed. The result showed (1) Tf uptake by the cells increased rapidly at the first 5 min, reaching its maximum after about 20 min of incubation; (2) Tf-Fe uptake kept increasing in a linear manner during the whole period of incubation; (3) the addition of either NH 4Cl or CH 3NH 2, 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; (4) trypsin and unlabelled Tf-Fe inhibited the uptake rate of Tf-Fe as well as Tf. The results suggested that Tf-Fe transport across the membrane of this type of neuron, much like other mammalian cells, was mediated by Tf-TfR endocytosis. Dysfunction of Tf or TfR would possibly lead to iron irregulation in the brain and consequently cause damage to neuronal functions.

Transferrin Receptor-Dependent and -Independent Iron Transport in Gallium-Resistant Human Lymphoid Leukemic Cells

AbstractRecent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).

Transferrin Receptor-Dependent and -Independent Iron Transport in Gallium-Resistant Human Lymphoid Leukemic Cells

Recent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).

Toxicity of holotransferrin but not albumin in proximal tubule cells in primary culture.

Proteinuria has been invoked as a cause of tubulointerstitial injury in chronic renal disease, and in vivo studies have suggested indirectly the particular nephrotoxicity of one urinary protein holotransferrin (Tf-Fe). However, to date there has been no direct evidence for the nephrotoxicity of Tf-Fe. To examine the potential cytotoxicity of Tf-Fe and the mechanism involved, and to compare this to another urinary protein albumin, rat proximal tubule cells were studied in primary culture. Tf-Fe at pH 6.0 caused functional and ultrastructural injury, but no cytotoxicity was seen with cells exposed to albumin, apotransferrin (transferrin), or Tf-Fe at pH 7.4. The influence of pH on Tf-Fe-induced cytotoxicity was not due to pH per se, but could be explained by an effect on Tf-Fe uptake. At pH 6.0, uptake of 125I-Tf-Fe (3.55 +/- 0.05 versus 1.25 +/- 0.10 fmol/dish, P < 0.01) and intracellular iron concentration (1.14 +/- 0.25 versus 0.46 +/- 0.23 nmol/dish, P < 0.01) were increased compared with values at pH 7.4. In contrast, pH 6.0 did not increase iron uptake from FeCl3. Lysine (100 mM) inhibited Tf-Fe uptake, decreased intracellular iron concentration, and attenuated Tf-Fe-induced cytotoxicity. The iron chelator des-ferrioxamine (200 microM) and hydroxyl radical scavenger dimethylpyrroline N-oxide (32 mM) abolished lactate dehydrogenase leakage induced by Tf-Fe at pH 6.0. Lipid peroxidation, as assessed by production of malondialdehyde, preceded lactate dehydrogenase leakage. In summary, holotransferrin, but not albumin, is toxic to rat proximal tubule cells, a pH-dependent effect involving its uptake into tubule cells, its iron moiety, and its lipid peroxidation.

Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron

The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTB-Fe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTB-Fe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 micromol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 micromol/L) when uptake of iron is TR-mediated, a 10-fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 micromol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax) of Tf-Fe uptake but the Michaelis-Menten constant (Km) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR-mediated process occurs by the same cellular pathway, using a common iron-carrier. (Hepatology 1997 Sep;26(3):691-8)

Changes in the uptake of transferrin-free and transferrin-bound iron during reticulocyte maturation in vivo and in vitro

The uptake of non-transferrin-bound iron, Fe(II), transferrin-bound iron, Tf Fe and transferrin was studied in reticulocytes from anaemic rabbits during maturation and then synchronized regeneration in vivo (following injection of actinomycin D) and while maturing during in vitro incubation. The uptake of Fe(II) and Tf Fe decreased in parallel with each other and with the reticulocyte count and transferrin uptake during maturation in vivo and in vitro. Only during the early phase of reticulocyte regeneration in vivo was there a significant difference between the rates of Fe(II) and Tf Fe uptake. These results suggest that a membrane carrier for iron and the transferrin receptor are lost at the same rate during reticulocyte maturation, possibly because they are associated with each other in the cell membrane. During reticulocyte maturation the rate of Fe(II) uptake into heme declined more rapidly than uptake into the total cellular cytosol. The loss of transferrin receptors and the uptake of iron from transferrin during reticulocyte maturation was not associated with a change in the affinity of the receptors for transferrin, in the relative distribution of the receptors between the outer cell membrane and intracellular sites or in the ability of the transferrin molecule to donate two iron atoms to the cell with each intracellular cycle, but the average duration of the cycle increased.