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

Abstract

Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both 59Fe-transferrin (Tf) and 59Fe-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°C with FAC or metal ion solutions and then labelled for 3 h at 37°C with 59Fe-Tf. Exposure of cells to FAC resulted in Fe uptake from 59Fe-citrate that became saturated at an Fe concentration of 2.5 μM, while FAC-activated Fe uptake from Tf was not saturable up to 25 μM. 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°C instead of 37°C prevented its effect at stimulating 59Fe uptake from 59Fe-Tf, suggesting that an active process was involved. Previous studies by others have shown that FAC can increase ferrireductase activity that may enhance 59Fe uptake from 59Fe-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 59Fe 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 59Fe 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).