Visser Corine C, Voorwinden L Heleen, Crommelin Daan J A, Danhof Meindert, de Boer Albertus G
Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, Division of Pharmacology, 2300 RA Leiden, The Netherlands.
Pharm Res. 2004 May;21(5):761-9. doi: 10.1023/b:pham.0000026425.69874.8e.
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.
研究脑毛细血管内皮细胞（BCECs）上转铁蛋白受体（TfR）的表达水平及其对125I-转铁蛋白（125I-Tf）的内吞作用。此外，研究铁、铁螯合剂甲磺酸去铁胺（DFO）、星形胶质细胞因子、GTP酶抑制剂（ tyrphostin-A8，T8）、脂多糖（LPS）和自由基清除剂N-乙酰-L-半胱氨酸（NAC）对TfR表达的影响，以深入了解TfR用于脑靶向给药的应用和优化。
使用原代培养的牛BCECs进行实验，在不存在或存在调节剂的情况下，将其与125I-Tf在4℃（用于确定结合）或37℃（用于确定内吞作用）下孵育。对于不存在或存在铁或DFO时的完全饱和曲线，使用NONMEM通过群体方法进行分析，使我们能够估计亲和力的单个值（Kd，50%受体占有率的浓度）和最大受体占有率的单独值（B(max)）。
在BCECs上，TfR在细胞外表达（B(max)为0.13 fmol/μg细胞蛋白），但也有大量的细胞内池（总B(max)为1.37 fmol/μg细胞蛋白），并且通过网格蛋白包被的小泡积极地内吞Tf。在4℃时，发现Kd为2.38 μg/ml，而在37℃时Kd为5.03 μg/ml。此外，DFO能够分别将细胞外以及总结合能力提高到0.63和3.67 fmol/μg细胞蛋白，而对Kd没有影响。DFO预孵育后37℃时的B(max)也从0.90增加到2.31 fmol/μg细胞蛋白。其他调节剂对TfR表达水平没有显著影响，尽管LPS在预孵育2小时后增加了细胞蛋白浓度。
TfR在BCECs上表达并积极内吞Tf，使其成为向血脑屏障和中枢神经系统给药的合适靶点。DFO上调TfR表达水平，这可能影响靶向效率。
