The mechanism of iron uptake by primary cultured rat hepatocytes

Abstract

The mechanism of iron uptake in isolated hepatocytes of Wistar strain female rats was investigated by primary monolayer culture for 24h at 37°C. The results were as follows:1) Iron uptake from diferric transferrin (diFeTf) showed different processes dependent on the ligand concentrations. Namely, the receptor-mediated transport was observed in a lower concentration of diFeTf from 0 to 0.2μmol/l, while another passive process was suggested in a higher concentration of the ligand from 15 to 32μmol/l.2) About 90% of 59Fe from transferrin was found in a whole membrane fraction of hepatocytes and the remainder was in cytosol. 59Fe and 125I activities from radiolabelled diFeTf incorporated into the whole membrane fraction were localized mainly in plasma membrane and lysosome fractions, which were identified by Percoll density gradient centrifugation and simultaneous marker enzyme analyses.3) Receptor assays for diFeTf and diferric asialotransferrin were performed. Ka and the binding number were 4.9×107M-1 and 3.85×105 (high affinity transferrin receptor), 2.2×106M-1 and 2.81×106 (low affinity transferrin receptor), 8.0×107M-1 and 5.22×105 (asialotransferrin receptor). The competitive bindings between diFeTf and diferric asialotransferrin to each receptor were recognized. These phenomena suggested that both receptors had a common binding for each ligand.4) Iron uptake into hepatocytes from ferrous citrate increased linearly according to increasing ferrous citrate concentration from 0.3 to 120μmol/l by a non-specific process. The total amount of iron incorporated into hepatocytes from ferrous citrate was about 5 fold higher than the uptake of the same concentration of transferrin.5) The percentage of ferritin 59Fe in whole activities from cytosol and heat extract of membrane fraction was 5 fold higher in diFeTf uptake than that in ferrous citrate, which was detected by immunoprecipitate against anti rat liver ferritin serum.6) Iron uptake from diFeTf was faster in the presence of ferrous citrate in the incubating medium than in diFeTf alone.