Functional and Molecular Aspects of Xenobiotic Transport in Choroid Plexus

Abstract

The choroid plexus (CP) epithelium forms the blood-cerebrospinal fluid (CSF) barrier, which along with the blood-brain barrier (BBB) capillary endothelium maintains the fluid environment of the brain. The CP not only secretes CSF, but also transports potentially toxic xenobiotics and waste products of neural metabolism to the blood for eventual clearance in kidney and liver. For several decades it has been known that the CP is actively involved in removing organic anions and other organic compounds from the extra-cellular fluid (Pappenheimer et al., 1961; Villalobos et al., 2002; Miller et al., 2002; Breen et al., 2004; Baehr et al., in press). However, studying CP is difficult, due to complex morphology, anatomical location and small size of the tissue and little is known about the molecular mechanisms, functional complexity and hormonal regulation of organic anion secretion. To further elucidate the underlying mechanisms of organic anion transport across CP epithelium, a primary porcine CP cell culture model was established and characterized on a molecular and functional basis. All cultures were free of contaminating cells, developed intact monolayers and were fully differentiated. Expression of marker protein prealbumin was demonstrated in isolated CP epithelial cells (CPEC) using RT-PCR, immunostaining and Western blot analyses. Alkaline phosphatase and -glutamyl transferase were quantified and cerebrospinal fluid secretion was measured. In addition, two active transport proteins, the MDR1 gene product P-glycoprotein (Pgp) and the multidrug-resistance associated protein 1, were assessed by RT-PCR, immunohistological staining and in Western blots of isolated membrane fractions. Integrity of fully differentiated monolayers was ensured by TEER measurements as well as permeability analyses using marker compounds. Further, functional analyses of Pgp were carried out. Organic anion transport was studied in a mammalian (rat), a comparative elasmobranch and in the in vitro porcine CPEC culture model, using the model compound fluorescein-methotrexate (FL-MTX). FL-MTX transport was shown to be a specific and concentrative, Na+-dependent and metabolism-dependent two-step process. Finally, for the first time, these studies demonstrate that organic anion transport is regulated by protein kinase C (PKC) and PKA. Responsible hormones remain to be identified.