The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin

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Abstract
Hemin, which is toxic to brain cells, has been reported to be taken up by cultured astrocytes; however, the mechanism of uptake is currently unknown. The present study investigated the mechanism of hemin uptake by rat primary astrocyte cultures. In medium containing 10% fetal calf serum, cultured astrocytes failed to accumulate significant amounts of heme-iron, while in serum-free medium the accumulation of heme-iron was found to be time- and concentration-dependent. After 6 h of incubation with 24 μM hemin, cells contained 36.2 ± 2.4 nmol heme-iron/mg protein, which was 21% of the applied hemin. These results suggest that the accumulation of hemin in astrocytes does not require serum proteins such as hemopexin. A potential mechanism of hemin uptake in astrocytes involves the heme carrier protein 1 (HCP1), which is reported to mediate hemin uptake into intestinal cells. RT-PCR analysis revealed that astrocyte cultures contained HCP1 mRNA, and immunocytochemical staining and Western blot analysis confirmed the expression of HCP1 protein in cultured astrocytes. The functionality of HCP1 in astrocytes was demonstrated by incubating cells with zinc protoporphyrin IX (ZnPPIX), which is known to be transported into cells via HCP1, and ZnPPIX autofluorescence was detected in HCP1-positive astrocytes. In addition, ZnPPIX was found to attenuate the accumulation of heme-iron by astrocytes. These results are the first to demonstrate that cultured astrocytes contain functional HCP1 and that this transporter contributes to hemin uptake by astrocytes. HCP1 may therefore provide a new target for reducing hemin-related toxicity in brain cells.