The transporter DMT1 mediates uptake of iron from the diet by intestinal enterocytes. Recently, we have shown that DMT1 protein expression in the plasma membrane of human intestinal Caco-2 cells is decreased by exposure to iron in a dose-dependent fashion (Sharp et al. 2002). Intriguingly, whole cell levels of DMT1 do not change, suggesting that the transporter may be internalised into an intracellular compartment. In this study we have investigated the time course of the iron-dependent decease in DMT1 in Caco-2 cells to determine whether changes in expression might occur within a physiologically relevant period coincident with the digestion and processing of a meal in the normal gastrointestinal tract.

Caco-2 cells were grown in 25 cm² flasks for 21 days. Cells were incubated with 100 µM FeCl₃ for up to 24 h. At the end of the experimental period, whole cell and plasma membrane proteins were isolated and utilised for Western blotting and total RNA extracted and subjected to RT-PCR for DMT1. In some experiments, membrane proteins were biotinylated prior to exposure to iron. In these studies, at the end of the incubation period, cells were lysed and the amount of biotinylated DMT1 in the cytosol determined following immunoprecipitation with DMT1 antibody, protein separation by western blotting and visualisation with streptavidin-HRP and enhanced chemiluminescence.

Plasma membrane DMT1 was significantly reduced by 4 h exposure to iron (0 h, 91.4 ± 6.7 a.u.; 4 h, 52.9 ± 11.1 a.u., means ± S.E.M., n = 4, P = 0.025, Student’s unpaired t test). However, whole cell levels of DMT1 were unaltered by iron exposure. DMT1 mRNA levels isolated from control and iron-treated cells were not significantly different at this time. Interestingly, there was an increase in biotinylated DMT1 in the cytosol following exposure to iron (control 8.3 a.u., +Fe 23.7 a.u., n = 2). Taken together, these data suggest that the initial cellular response to elevated iron involves a decrease in apical membrane expression of DMT1, perhaps due to internalisation of the transporter into an intracellular compartment. These changes are rapid (between 1 and 4 h following exposure to iron) and could occur within the time scale for the digestion and processing of a meal.

This work was funded by BBSRC (project grant 90/D13400).