Mechanisms of ferric citrate uptake by human hepatoma cells.

The mechanisms of uptake of non-transferrin-bound iron by human hepatoma cells (HuH7) were investigated using 59Fe-citrate and [14C]citrate. The amount of iron associated with the cells increased linearly with time, whereas citrate uptake reached a plateau after 45 min, resulting in a cellular accumulation of iron over citrate. The cells displayed high-affinity membrane binding sites for citrate with maximum binding of 118 ± 17 pmol citrate/mg protein and a dissociation constant of 21 ± 2 μM ( n = 3). Iron uptake was saturable with a maximum uptake rate of 1.95 ± 0.43 pmol ⋅ mg protein-1 ⋅ min-1and an apparent Michaelis constant of 1.1 ± 0.1 μM. Nonradioactive ferric citrate and citrate inhibited59Fe uptake to a similar degree. This suggests that the uptake of citrate-bound iron is dependent on either binding to specific citrate binding sites or the concentration of unbound iron. The uptake of iron was inhibited by ferricyanide (>100 μM) and ferrous iron chelators but stimulated by ferrocyanide and ascorbate, suggesting that the iron is reduced from Fe3+ to Fe2+ and transported into the cell by an iron carrier-mediated step.

[1]  T. Spiro,et al.  Hydrolytic polymerization of ferric citrate. II. Influence of excess citrate , 1967 .

[2]  P. Sadler,et al.  Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. , 1989, The Journal of biological chemistry.

[3]  E. Morgan,et al.  Characterisation of citrate and iron citrate uptake by cultured rat hepatocytes. , 1998, Journal of hepatology.

[4]  P. Aisen,et al.  The interaction of transferin with isolated hepatocytes , 1980 .

[5]  E. Morgan,et al.  The mechanisms of iron uptake by fetal rat hepatocytes in culture , 1986, Hepatology.

[6]  H. Goldenberg,et al.  Uptake of iron by isolated rat hepatocytes from a hydrophilic impermeant ferric chelate, Fe(III)-DTPA. , 1996, Archives of biochemistry and biophysics.

[7]  L. Pape,et al.  Hydrolytic polymerization of ferric citrate. I. Chemistry of the polymer , 1967 .

[8]  O. Zak,et al.  Uptake of iron from N-terminal half-transferrin by isolated rat hepatocytes. Evidence of transferrin-receptor-independent iron uptake. , 1995, European journal of biochemistry.

[9]  D. Barisani,et al.  Evidence for a low Km transporter for non-transferrin-bound iron in isolated rat hepatocytes. , 1995, The American journal of physiology.

[10]  O. Zak,et al.  Transferrin receptor‐independent uptake of differic transferrin by human hepatoma cells with antisense inhibition of receptor expression , 1996, Hepatology.

[11]  I. Romslo,et al.  Uptake of iron from transferrin by isolated rat hepatocytes. A redox-mediated plasma membrane process? , 1988, The Journal of biological chemistry.

[12]  Arthur E. Martell,et al.  Stability constants of metal-ion complexes , 1964 .

[13]  P. Brissot,et al.  Characterization of non-transferrin-bound iron clearance by rat liver. , 1986, The Journal of biological chemistry.

[14]  E. Rachmilewitz,et al.  Non‐Specific Serum Iron in Thalassaemia: an Abnormal Serum Iron Fraction of Potential Toxicity , 1978, British journal of haematology.

[15]  J. Kaplan,et al.  The mammalian transferrin-independent iron transport system may involve a surface ferrireductase activity. , 1994, The Biochemical journal.

[16]  E. Morgan,et al.  Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture. , 1998, Biochimica et biophysica acta.

[17]  J. Glass,et al.  Transferrin binding and iron uptake in mouse hepatocytes. , 1983, Biochimica et biophysica acta.

[18]  M. Wessling-Resnick,et al.  Extracellular ferrireductase activity of K562 cells is coupled to transferrin-independent iron transport. , 1994, Biochemistry.

[19]  C. Furlani.  Stability constants of metal-ion complexes , 1965 .

[20]  C. Craven,et al.  Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[21]  F. L. Crane,et al.  Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system. , 1986, Biochemical and biophysical research communications.

[22]  O. Zak,et al.  Antisense suppression of transferrin receptor gene expression in a human hepatoma cell (HuH‐7) line , 1993, American journal of hematology.

[23]  E. Morgan,et al.  Transferrin and iron uptake by the liver in the rat. , 1974, The Australian journal of experimental biology and medical science.

[24]  E. Morgan,et al.  Transferrin and iron uptake by rat hepatocytes in culture. , 1984, The American journal of physiology.

[25]  R. Klausner,et al.  Regulating the fate of mRNA: The control of cellular iron metabolism , 1993, Cell.

[26]  P. Aisen,et al.  The interaction of transferrin with isolated hepatocytes. , 1980, Biochimica et biophysica acta.

[27]  D. Templeton,et al.  Uptake of non-transferrin-bound iron by both reductive and nonreductive processes is modulated by intracellular iron. , 1994, The Journal of biological chemistry.

[28]  I. Romslo,et al.  Uptake of iron from transferrin by isolated hepatocytes. , 1984, Biochimica et biophysica acta.

[29]  S. Sherlock,et al.  A non-transferrin-bound serum iron in idiopathic hemochromatosis , 1980, Digestive diseases and sciences.

[30]  T. Yamane,et al.  Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. , 1982, Cancer research.

[31]  E. Morgan,et al.  The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes. , 1988, Biochimica et biophysica acta.

[32]  L. Kühn,et al.  Gene transfer, expression, and molecular cloning of the human transferrin receptor gene , 1984, Cell.