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

Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both Fe-59-transferrin (Tf) and Fe-59-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 degreesC with FAC or metal ion solutions and then labelled for 3 h at 37 degreesC with Fe-59-Tf. Exposure of cells to FAC resulted in Fe uptake from 59Fe-citrate that became saturated at an Fe concentration of 2.5 muM, while FAG-activated Fe uptake from Tf was not saturable up to 25 muM. In addition, the extent of FAG-activated Fe uptake from citrate was far greater than that from Tf. These results suggest a mechanism where FAG-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 degreesC instead of 37 degreesC prevented its effect at stimulating Fe-59 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 Fe-59 uptake from Fe-59-Tf However, there was no difference in the ability of FAG-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 FAG, ferric chloride, ferrous sulphate, ferrous ammonium sulphate, gallium nitrate, copper chloride, zinc chloride, or cobalt chloride. Stimulation of Fe-59 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 Fe-59 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). (C) 2001 Elsevier Science B.V. All rights reserved.