The Mechanisms of Nonheme Iron Uptake Determined in IEC-6 Rat Intestinal Cells

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Abstract
The mechanisms of iron absorption, which control total body iron stores, are not fully understood. We have defined an in vitro model using rat small intestinal cells (IEC-6) to study nutrient uptake by the intestine and have characterized the mechanisms of nonheme iron uptake in this model. IEC-6 cells were grown to confluency, and then 59Fe solutions were layered on the cells, allowing iron uptake to occur through the apical surface. Iron uptake increased over time and comparable results were seen with either 59Fe(II) or 59Fe(III). Two types of 59Fe(II) binding sites were detected, a high affinity site with a dissociation constant of 1 × 10-8 mol/L and 5 × 105 sites per cell, and a lower affinity site with a dissociation constant of 2 × 10-6 mol/L and 2.6 × 106 sites per cell. The 59Fe uptake was inhibited by low temperature, low and high pH, prior iron loading of the cells, high osmotic concentrations, and by N,N′-dicyclohexylcarbodiimide, tannic acid, bathophenanthroline and colchicine. Uptake was not inhibited by vinblastine or verapamil. Transferrin mRNA was not detected in IEC-6 cells grown under normal growth conditions but could be induced under some other conditions. IEC-6 cell proteins that bind iron were detected in a subcellular vesicle fraction having estimated molecular weights of 120,000, 95,000, 55,000 and 35,000. These studies confirm iron uptake studies by other models and demonstrate that IEC-6 cells possess iron-binding moieties, a regulated iron uptake process, and little or no transcription of the transferrin gene under normal growth conditions.