Transferrin-bound and transferrin-free iron uptake by cultured rat astrocytes

Previously we had demonstrated the presence of transferrin receptor (TfR) o n the plasma membrane of cultured I at cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astr ocytes were incubated with 1 mu M of double-labelled transferrin (I-125-Tf- Fe-59) in serum-free DMEM/F12 medium or Fe-59 II in isotonic sucrose soluti on at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe 1T uptake was analyzed by measuring the intracellular radioactivity with gamma counter The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowe d to a plateau level after 10 min, of incubation. The addition of either NH 4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release fro m Tf within endosomes, decreased the cellular Tf-Fe uptake but had no signi ficant effect on Tf uptake. Pre-treated cells with trypsin inhibited signif icantly the cellular uptake of Tf-Fe as well as Tf. These findings suggeste d that Tf-Fe transport across the membrane of astrocytes is mediated by Tf- TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. Th e highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantl y affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediat ed by TW and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.