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 microM (n = 3). Iron uptake was saturable with a maximum uptake rate of 1.95 +/- 0.43 pmol . mg protein-1 . min-1 and an apparent Michaelis constant of 1.1 +/- 0.1 microM. Nonradioactive ferric citrate and citrate inhibited 59Fe 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 microM) 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.
Department of Physiology, University of Western Australia, Nedlands 6907, Western Australia, Australia.
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D015082:2,2'-Dipyridyl / Q000494:pharmacology; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D005290:Ferric Compounds / Q000493:pharmacokinetics*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007504:Iron Radioisotopes / Q000493:pharmacokinetics; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D002250:Carbon Radioisotopes; D002614:Chelating Agents; D005290:Ferric Compounds; D007504:Iron Radioisotopes; D010618:Phenanthrolines; C017049:bathophenanthroline disulfonic acid; D015082:2,2'-Dipyridyl; C025314:ferric citrate; D007501:Iron