MECHANISMS OF FERRIC CITRATE UPTAKE BY HUMAN HEPATOMA-CELLS

The mechanisms of uptake of non-transferrin-bound iron by human hepato ma cells (HuH7) were investigated using Fe-59-citrate and [C-14]citrat e. The amount of iron associated with the cells increased linearly wit h time, whereas citrate uptake reached a plateau after 45 min, resulti ng in a cellular accumulation of iron over citrate. The cells displaye d high-affinity membrane binding sites for citrate with maximum bindin g of 118 +/- 17 pmol citrate/mg protein and a dissociation constant of 21 +/- 2 mu M (n = 3). Iron uptake was saturable with a maximum uptak e rate of 1.95 +/- 0.43 pmol mg protein(-1).min(-1) and an apparent Mi chaelis constant of 1.1 +/- 0.1 mu M. Nonradioactive ferric citrate an d citrate inhibited Fe-59 uptake to a similar degree. This suggests th at the uptake of citrate-bound iron is dependent on either binding to specific citrate binding sites or the concentration of unbound iron. T he uptake of iron was inhibited by ferricyanide (>100 mu M) and ferrou s iron chelators but stimulated by ferrocyanide and ascorbate, suggest ing that the iron is reduced from Fe3+ to Fe2+ and transported into th e cell by an iron carrier-mediated step.