The multidrug resistance protein BMDP/ABCG2: A new and highly relevant efflux pump at the blood–brain barrier

Abstract

Combinatorial chemistry is able to generate high numbers of compounds of pharmaceutical interest. An important aspect for a new drug is to know its availability in the nervous system, which means the ability to cross the barriers between blood and brain. Thus the permeability of thousands of compounds will have to be screened in the near future. This will not be possible in vivo, so that powerful in vitro models are demanded that closely mimic the in vivo system at least with respect to barrier properties. Two barrier systems are of main interest with respect to the passage of compounds into the brain: the blood–brain barrier (BBB) of the cerebral capillaries and the blood–liquor barrier (BLB) of the choroid plexus. In this paper main focus will be on the development of an in vitro model for the BBB. The brain capillary endothelial cells (BCEC) are isolated from freshly slaughtered pigs and cultivated on permeable filters to mimic the interfacial localization between blood and CNS. Cultivating the cells in serum-free medium improves their barrier properties. Hydrocortisone supplementation additionally reinforces the BBB properties. Under these conditions we observed an up-regulation of an efflux pump that has been found earlier in a breast cancer cell line, there named BCRP (Breast Cancer Resistance Protein). This efflux pump is also known as Mitoxantrone Resistance Protein (MXR) or Placenta-specific ABC Protein (ABCP). We identified this protein for the first time at the blood–brain barrier. mRNA expression studies of different brain cells showed that this efflux pump is mainly expressed in the brain capillary endothelial cells. With RT-PCR we showed a minor expression in pericytes as well as in epithelial cells from the choroid plexus and in spinal cord astrocytes. From cDNA sequence analysis we know that this efflux pump is a homologue to the ABCG2-transporter consisting of only one ATP-binding cassette and six membrane spanning helices. For this so-called half transporter, an oligomerization in the plasma membrane is discussed. Quantitative real-time PCR analysis indicates that this pump is highly relevant for the blood–brain barrier functionality since its expression level is about 3 fold higher compared to P-gp. Northern blot analysis yielded a 30 fold higher mRNA content. Immunocytochemistry revealed that in brain endothelial cells the protein is predominantly localized in the apical plasma membrane which compares to the blood side in vivo. The transport direction for known ABCG2 substrates consistently supports this finding. This paper will discuss the importance of this newly discovered ABC transporter at the blood–brain barrier in comparison to other members of ABC subfamilies.