Characterization and modulation of the transferrin receptor on brain capillary endothelial cells.

Corine C Visser, L Heleen Voorwinden, Daan J A Crommelin, Meindert Danhof, Albertus G de Boer

Index: Pharm. Res. 21(5) , 761-9, (2004)

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Abstract

The expression level of the transferrin receptor (TfR) on brain capillary endothelial cells (BCECs) and the endocytosis of 125I-transferrin (125I-Tf) by this receptor was investigated. Furthermore, the influence of iron, the iron scavenger deferoxamine mesylate (DFO), astrocytic factors, a GTP-ase inhibitor (tyrphostin-A8, T8), lipopolysaccharide (LPS), and the radical scavenger N-acetyl-L-cysteine (NAC) on the TfR expression was studied to gain insight in the use and optimization of the TfR for drug targeting to the brain.Experiments were performed with primary cultured bovine BCECs that were incubated with 125I-Tf at 4 degrees C (to determine binding) or at 37 degrees C (to determine endocytosis) in the absence or presence of the modulators. For full saturation curves in the absence or presence of iron or DFO, analysis was performed with a population approach using NONMEM, allowing us to estimate a single value for affinity (Kd, concentration of 50% receptor occupancy) and separate values for maximum receptor occupancy (B(max).On BCECs, the TfR is expressed extracellularly (B(max) of 0.13 fmol/microg cell protein), but also has a large intracellular pool (total B(max) of 1.37 fmol/microg cell protein), and is actively endocytosing Tf via clathrin-coated vesicles. At 4 degrees C, a Kd of 2.38 microg/ml was found, whereas the Kd at 37 degrees C was 5.03 microg/ml. Furthermore, DFO is able to increase both the extracellular as well as the total binding capacity to 0.63 and 3.67 fmol/microg cell protein, respectively, whereas it had no influence on Kd. B(max) at 37 degrees C after DFO preincubation was also increased from 0.90 to 2.31 fmol/microg cell protein. Other modulators had no significant influence on the TfR expression levels, though LPS increased cellular protein concentrations after 2-h preincubation.The TfR is expressed on BCECs and actively endocytoses Tf, making it a suitable target for drug delivery to the bloodbrain barrier and the CNS. DFO up-regulates the TfR expression level, which may influence targeting efficiency.