Uptake of iron from transferrin by isolated hepatocytes (original) (raw)
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Scandinavian Journal of Clinical & Laboratory Investigation, 1987
Isolated rat hepatocytes containing 0.56-1.79 isg iron/10 6 cells and with an intraceilular ATP concentration of 3-4 mM, accumulate iron from transferrin linearly with time for at least 3 h. At 37°C the rate of uptake amounts to 0.3-0.7 pmol/mg cell protein per min. The uptake reaches a saturation level of 21-40 pmol/mg cell protein per h at 2.2 pM iron. At 50C the uptake does not increase over the time of incubation. Uptake of iron, but not binding of transferrin is increased 4-5-fold at oxygen concentrations 10-20 pM. At oxygen concentrations beyond these limits iron uptake is decreased. Iron taken up at low oxygen concentrations can be chelated by bathophenanthroline and bathophenanthroline disulphonate, but only if the chelators are present during the uptake experiments. The results suggest that iron uptake from transferrin by hepatocytes in suspension involves reductive removal of iron.
Journal of Biological Chemistry, 1988
both we (8) and others (13, 14) have shown that transferrin and at least part of the iron localizes to the endosomal compartment of hepatocytes. Consequently, if mechanisms other than receptor-mediated endocytosis are operating, they may be operating in parallel with or as part of receptor-mediated endocytosis. The aim of the present work was to study the importance of plasma membrane redox processes for the uptake of iron from transferrin by isolated hepatocytes. Our results suggest that at transferrin concentrations above that needed to saturate the transferrin receptors (0.1-0.5 PM (2, 3, 7)), isolated hepatocytes take up iron predominantly by mechanisms located to the plasma membrane. The process involves cooperative proton and electron fluxes with labilization of ferric iron from transferrin with subsequent reduction and translocation of iron through the plasma membrane. Thus, it would appear that only a minor part of iron accumulated by the cell is taken up by receptor-mediated endocytosis of transferrin. MATERIALS AND METHODS Chemicals-Collagenase (type IV), bovine serum albumin (essentially fatty acid-free), human transferrin (9855, essentially iron-free), CCCP,' chloroquine, methylamine, iodoacetate, monensin, and fluoresceine isothiocyanate (FITC) coupled to celite were from Sigma. Bolton and Hunter reagent for protein iodination (74 MBq/mmol) and 'VeCb (6.5-43.7 MBq/mmol) were from Amersham International (Buckinghamshire, United Kingdom). "C-labeled dextran (MI
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1990
The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophcnanghgoline disuffonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.
Journal of Clinical Investigation, 1985
In hemochromatosis and other disorders associated with iron overload, a significant fraction of the total iron in plasma circulates in the form of low molecular weight complexes not bound to transferrin. Efficient and unregulated clearance of this form of iron by the liver may account for the hepatic iron loading and toxicity that characterize these diseases. We tested this possibility by examining the hepatic removal process for representative iron complexes in the single-pass perfused rat liver. Hepatic uptake of both ferrous and ferric 55Fe from ultrafiltered human serum was found to be highly efficient and effectively irreversible (single-pass extraction of 1 MM iron, 58-75%). Similar high efficiencies were seen for iron complexed to specific physiologic and nonphysiologic coordinators, including histidine, citrate, fructose, oxalate and glutamate, and tricine. Because of lower plasma flow rates, single-pass extraction of these iron complexes in vivo should be even greater. Autoradiography confirmed that most iron had been removed by parenchymal cells. Hepatic removal from Krebs-tricine buffer was saturable with similar kinetic parameters for ferrous and ferric iron (apparent K., 14-22 ,uM; V.,, 24-38 umol min-' g liver-'). These findings suggest that high levels of non-transferrin-bound iron in plasma may be an important cause of hepatic iron loading in iron overload states.
The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells
AJP: Cell Physiology, 2009
Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using 59Fe-125I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe2Tf, Co2Tf, and Mn2Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co2Tf and Mn2Tf upregulated TFR1, reduced ferritin, and increa...
The process of cellular uptake of iron from transferrin. A computer simulation program
European Journal of Biochemistry, 1994
In an attempt to improve our understanding of the complex interplay between cell compartments and chemical species during cellular uptake of iron from transfenin, we designed a computer simulation program based on current models of receptor-mediated endocytosis and pinocytosis. The program calculates and visualizes, as a function of time, the changes in transfenin, apotransferrin, and iron concentrations occurring in all relevant cellular compartments during cellular iron acquisition from transferrin. Simulation of literature data showed that the program generates results that are in accordance with experimental data. Furthermore, from measurements of the uptake of [carboxyl-''C]dextran we could utilize the program to suggest rate constants characteristic for the pinocytic process in rat reticulocytes. Moreover, simulations indicate that the apparent difference in the iron uptake process observed between reticulocytes and hepatocytes may be explained by the contribution made by pinocytosis to the iron uptake process. Finally, the present program should have potential as an educational tool during introduction to the field of receptor-mediated endocytosis in general and to cellular iron metabolism in particular.
Iron mobilization from isolated hepatocytes
International Journal of Biochemistry, 1986
It is not known which message and mechanism triggers the cell to mobilize iron from ferritin. 2. In this paper we present the results of incubation experiments with ~gFe-labelled hepatocytes. Anemic serum gives a significant higher rate of iron mobilization than normal serum. 3. The involvement of apo-transferrin is ruled out because it did not increase iron mobilization. Citrate increased iron mobilization which is not the result of an increase in NADH/NAD +-ratio because addition of ethanol did not stimulate iron mobilization. 4. Desferrioxamine is used clinically in iron overloaded patients and it is known that iron removal is a very slow process. Although desferrioxamine can mobilize iron from ferritin in hepatocytes, a considerable amount remains inside the cell as a low molecular weight fraction. This fraction represents chelator bound iron and is slowly released into the circulation.
The role of Hfe in transferrin-bound iron uptake by hepatocytes
Hepatology, 2008
HFE-related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin-bound iron via transferrin receptor (Tfr) 1 and Tfr1-independent pathways (possibly Tfr2-mediated). In this study, the role of Hfe in the regulation of hepatic transferrin-bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice were measured after incubation with 50 nM 125 I-Tf-59 Fe (Tfr1 pathway) and 5 M 125 I-Tf-59 Fe (Tfr1-independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and western blotting, respectively. Tfr1-mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron-loaded wild-type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1-independent pathway was approximately 100-fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1-independent pathway. Conclusion: Tfr1-mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1-independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. Diferric transferrin upregulated hepatocyte Tfr2 protein expression but not iron uptake, suggesting that Tfr2 may have a limited role in the Tfr1-independent pathway. (HEPATOLOGY 2008;47:1737-1744.) Abbreviations: HH, hereditary hemochromatosis; mRNA, messenger RNA; SEM, standard error of the mean; TFR1, transferrin receptor 1; TFR2, transferrin receptor 2. From the