Mechanisms and regulation of transferrin and iron transport in a model blood–brain barrier system
Section snippets
Cell culture
Cow eyes were obtained from a local abattoir and the BRECs isolated and processed according to a previously published procedure (Gardiner et al., 1995). BRECs were grown in MCDB-131 media (Sigma, St. Louis, MO, USA) supplemented with 10% FBS, 10 ng/mL EGF, 0.2 mg/mL ENDO GRO (VEC Technologies, Inc., Rensselaer, NY, USA), 0.09 mg/ml heparin, and antibiotic/antimycotic (Gibco, Rockville, MD, USA). For the transport experiments, the BRECs were gently trypsinized and grown to confluence on Costar
Results
The mode of iron transport across a model BBB was determined using a monolayer of BRECs that were grown to confluence on porous culture inserts. Rhodamine-labeled dextran was simultaneously loaded into the wells with fluorescein-Tf. There was no significant difference in dextran flux into the basal chamber (P>0.05) when BRECs were treated with FAC, DFO, NH4Cl, or filipin (data not shown). Thus, none of the treatments increased paracellular transport.
Relative accumulation of fluorescein-Tf was
Discussion
We have used BRECs as a model of the BBB to examine the mechanism of Tf and iron transcytosis and to determine if iron status of the endothelial cells will influence the transport of iron and Tf. Dextran was used to control for paracellular transport and none of the conditions utilized in this study altered dextran transport. Thus, the changes in iron and Tf transport in this study reflect changes in specific transport. Our results demonstrate that, in the normal condition, iron is transported
Acknowledgements
This work was supported by funds from the Restless Legs Syndrome Foundation.
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