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.     

Intermediate steps in cellular iron uptake from transferrin. Detection of a cytoplasmic pool of iron, free of transferrin.

The uptake of transferrin-bound iron by receptor-mediated endocytosis has been the subject of extensive experimental investigation. However, the path followed by iron (Fe) after release from transferrin (Tf) remains obscure. Once Fe is released from Tf within the endosome, it must be transported across the endosomal membrane into the cell. The present investigation describes the presence of a cytoplasmic Tf-free Fe pool which is detectable only when cells are detached from their culture dishes at low temperature, after initial incorporation of diferric transferrin at 37 degrees C. This cellular iron pool was greatly reduced if incubation temperatures were maintained at 37 degrees C or if cells were treated with pronase. Human melanoma cells (SK-MEL-28) in culture were prelabeled by incubation with human 125I-59Fe-transferrin for 2 h, washed, and reincubated at 4 degrees C or 37 degrees C in balanced salt solution in the presence or absence of pronase. The cells were then mechanically detached from the plates and separated into "internalized" and supernatant fractions by centrifugation. Approximately 90% of cellular 59Fe and 20% of 125I-Tf remained internalized when this reincubation procedure was carried out in balanced salt solution at 37 degrees C. However, at 4 degrees C, cellular internalized iron was reduced to approximately 50% of the initial value. The release of this component of cellular 59Fe (approximately 40% of total cell 59Fe) at 4 degrees C was completely inhibited in the presence of pronase and other general proteinases at 4 degrees C and at 37 degrees C, without affecting internalized transferrin levels. Similar results were obtained in fibroblasts and hepatoma cells, indicating that this phenomenon is not unique to melanoma cells. The characterization of this Tf-free cellular Fe pool which is detectable at low temperature may yield valuable insights into the metabolic fate of iron following its transport across the membrane of the endocytotic vesicle.     

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.     

Specificity of hepatic iron uptake from plasma transferrin in the rat.

1. The role of specific interaction between transferrin and its receptors in iron uptake by the liver in vivo was investigated using 59Fe-125I-labelled transferrins from several animal species, and adult and 15-day rats. Transferrin-free hepatic uptake of 59Fe was measured 2 or 0.5 hr after intravenous injection of the transferrins. 2. Rat, rabbit and human transferrins gave high and approximately equal levels of hepatic iron uptake while transferrins from a marsupial (Sentonix brachyurus), lizard, crocodile, toad and fish gave very low uptake values. Chicken ovotransferrin resulted in higher uptake than with any other species of transferrin. 3. Iron uptake by the femurs (as a sample of bone marrow erythroid tissue) and, in another group of 19-day pregnant animals by the placentas and fetuses, was also measured, for comparison with the liver results. The pattern of uptake from the different transferrins was found to be similar to that of iron uptake by the liver except that with femurs, placentas and fetuses ovotransferrin gave low values comparable to those of the other non-mammalian species. 4. It is concluded that iron uptake by the liver from plasma transferrin in vivo is largely or completely dependent on specific transferrin-receptor interaction. The high hepatic uptake of iron from ovotransferrin was probably mediated by the asialoglycoprotein receptors on hepatocytes.     

The rate limiting step in the reticulocyte uptake of transferrin and transferrin iron. Effects of some incubation variables.

Reticulocytes incubated in an isotonic NaCl saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticulocytes failed to remove completely the iron from the transferrin which they incorporated. It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unsepcific attachment of transferrin. At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.     

Effects of spermine on transferrin and iron uptake by reticulocytes: II. Changes in intravesicular pH

An increase in extracellular spermine concentration brought about a progressive rise in intralysosomal pH in rabbit reticulocytes. Since intracellular release of iron from transferrin is believed to involve the protonation of the iron-transferrin complex, the rise in intralysomal pH could account for the inhibitory effect of spermine on iron uptake. The inhibition could be reversed if spermine was removed by washing. As a result of spermine treatment, more acid-labile N-terminal monoferric transferrin and less apotransferrin were released from the cell. These results are consistant with the protonation theory of iron release.     

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.     

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.     

Effect of nicotine on transferrin binding and iron uptake by cultured rat placenta

The effect of nicotine on transferrin and iron transport in placental cells has been studied. Nicotine inhibits iron uptake but has little effect on the steady-state levels of transferrin. The effect is temperature and concentration dependent and is not reversible. At a concentration of 15 mM nicotine inhibited transferrin endocytosis by 40%, while iron uptake was decreased by nearly 60%. Nicotine exerted a similar effect on reticulocytes, but other amines, either tertiary or quaternary, had little or no effect on either iron uptake or steady-state intracellular transferrin levels. The results suggest that nicotine acts by blocking uptake, probably by acting as a weak base inhibiting iron release from transferrin, and inhibiting exocytosis with a resultant block of endocytosis. The concentrations required to exert an effect are too high to implicate inhibition of iron transport in the effects of smoking on pregnancy.     

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

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

Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate.

The effects of ferric ammonium citrate (FAC) and desferrioxamine (DFO) on iron (Fe), and transferrin (Tf) uptake have been investigated using SK-MEL-28 human melanoma cells, which express the Tf homologue, melanotransferrin, in high concentrations. Previously we demonstrated two separate Fe uptake mechanisms from Tf, viz. a specific process mediated by the transferrin receptor (TfR) and a nonspecific process (Richardson, D. R., and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12). Cells exposed to DFO demonstrated up-regulation of the TfR with a concurrent increase in the rate of Fe uptake. Desferrioxamine also stimulated the nonspecific process of Fe uptake, resulting in a further increase in accumulation of Fe over Tf after saturation of the specific TfR. Ferric ammonium citrate had two effects. First, it resulted in down-regulation of the TfR. Second, and paradoxically, it markedly stimulated the rate of Fe uptake from Tf by the nonspecific process without increasing the rate of nonspecific Tf uptake. These data conclusively demonstrate that two entirely different mechanisms of iron uptake from Tf exist in melanoma cells and that ferric ammonium citrate may be a useful experimental tool to further characterize the specific and nonspecific mechanisms of Fe uptake from Tf.     

Transferrin receptors and transferrin iron uptake by cultured human blood monocytes

Transferrin receptors have been previously found on human macrophages and it has also been shown that transferrin iron is taken up by these cells. It has therefore been inferred that the uptake is receptor mediated and involves an endocytic pathway. The subject was addressed directly in the present study in which the transferrin-iron-receptor interaction was characterized in cultured human blood monocytes. Specific, saturable diferric transferrin binding was demonstrated, with a kDa of 3.6 X 10(-8) M and a calculated receptor density of 1.25-2.5 X 10(5) receptors per cell. Incubation at 4 degrees C markedly reduced transferrin binding and completely inhibited iron uptake. Chase experiments confirmed progressive cellular loading of iron, with concomitant loss of transferrin. Inhibitors of endocytic vesicle acidification (ammonium chloride and 2,4-dinitrophenol) inhibited iron unloading from endocytosed diferric transferrin, while microtubular inhibitors (colchicine and vindesine) and a microfilament inhibitor (cytochalasin B) reduced diferric transferrin uptake but had little effect on the iron unloading pathway. A similar effect was noted with a calcium ion antagonist (verapamil) and with 2 calmodulin antagonists (chlorpromazine and imipramine). These latter findings suggest the importance of cytoskeleton-membrane interactions via a calcium, calmodulin and protein kinase C mediated system. Endocytosed iron accumulated progressively as ferritin within the cultured monocytes.     

Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake

The human transferrin receptor is post-translationally modified by the addition of a fatty acyl moiety. In earlier studies, transient expression in Cos cells of human transferrin receptors in which Cys62 or Cys67 was altered to serine provided evidence that Cys62 is the major acylation site of the receptor (Jing, S., and Trowbridge, I. S. (1987) EMBO J. 6, 327-331). To determine whether acylation of the receptor is required for high efficiency endocytosis and iron uptake, wild type and mutant human transferrin receptors have been stably expressed in chick embryo fibroblasts using a helper-independent retroviral vector. In marked contrast to Cos cells, both Cys62 and Cys67 of the wild type human transferrin receptor were acylated in chick embryo fibroblasts. Moreover, their modification to serine did not abolish palmitate labeling, implying that one or both of these serine residues could serve as alternative lipid attachment sites in these cells. The relative labeling of mutant receptors with palmitate and the susceptibility of their lipid moieties to cleavage by hydroxylamine were consistent with Ser67 but not Ser62 serving as a lipid attachment site. Consequently, to obtain human transferrin receptors lacking covalently bound lipid in the chick embryo fibroblasts, it was necessary to alter Cys62 and Cys67 to alanine. Functional studies indicated that these non-acylated mutant receptors were internalized efficiently and mediated iron uptake from human transferrin at a similar rate to that of wild type receptors. We conclude, therefore, that acylation of the human transferrin receptor is not essential for endocytosis and recycling.     

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.     

Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes

Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor.     

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding

Summary A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4°C, after initial incubation with⁵⁹Fe-¹²⁵I-Tf at 37°C (Richardson and Baker, 1992a). The release of this internalized⁵⁹Fe could be markedly reduced if the cells were treated with proteases or incubated at 37°C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular⁵⁹Fe release increased linearly as a function of preincubation time with⁵⁹Fe-Tf subsequent to mechanical detachment at 4°C using a spatula. These data suggest that the⁵⁹Fe released was largely composed of end product(s) and was not an “intermediate Fe pool.” When the Fe(II) chelator, dipyridyl (DP), was incubated with⁵⁹Fe-Tf and the cells, it prevented the accumulation of⁵⁹Fe that was released following mechanical detachment at 4°C. Other chelators had much less effect on the proportion of⁵⁹Fe released. Examination of the⁵⁹Fe released showed that after a 4-h preincubation with⁵⁹Fe-Tf, approximately 50% of the⁵⁹Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4°C resulted in membrane disruptions that allow the release of high M, molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4°C resulted in pronounced membrane damage. In contrast, when cells were detached at 37°C, or at 4°C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37°C. The evidence also indicates that caution is required when interpreting data from expriments where cells have been mechanically detached at 4°C.     

Effects of EGF and calcium on adult parenchymal hepatocyte proliferation

Adult rat hepatocytes were grown in serum-free medium containing 0.05-4 mM Ca++ and 40 ng/ml EGF. After 48 hours of cultivation the mitotic index and the percentage of second division metaphases were determined. The results demonstrated a maximum proliferation response to EGF at a Ca++ concentration of 0.4 mM. With lower and higher external Ca++ concentrations the fraction of cells undergoing more than one cell division decreased. At lower Ca++ concentrations this decrease appears to result from a reduced viability. In contrast, the low response to EGF at higher Ca++ concentrations--especially in the physiological range--may reflect the influence of Ca++ on the state of hepatocyte differentiation.     

The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes

The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes has been examined. The antibodies used were as follows: T58/1.4, B3/25.4, 42/6.3, T56/14.3.1, and 43/31. The effects were the same, irrespective of the antibody. Transferrin and iron uptake were stimulated in both rat and rabbit reticulocytes. The stimulation was not due to an increase in the number or affinity of the receptors, but rather to an increase in the rate of turnover of the receptors. Electron microscopy suggested that the antibody acted by facilitating the formation of coated pits containing the transferrin-receptor complex.