Active transport of organic anion (OA) and cations (OC) from CSF to blood is a critical determinant of the concentration of potentially toxic neurotransmitter metabolites, drugs, and xenobiotics within the brain. We have used a combination of isolated membrane vesicles, tissue fragments in vitro, and primary culture to study xenobiotic transport across the blood-CSF barrier (choroid plexus). Our working hypothesis is that the transport mechanisms utilized by the plexus would parallel those of the kidney, but with the polarity reversed since the direction of transport is reversed (i.e., from CSF to blood rather than from blood to urine). Initial focus was on the mechanism and subcellular location of OA transport by the plexus. RT PCR demonstrated the presence of OATs 1-5 in CP. Furthermore, we showed that apical (CSF-side) transport of OA is mediated by OA/a-ketoglutarate (aKG) exchange -- the same mechanism used at the basolateral face of the renal epithelium. Since OAT1 was the only OAT known to be an exchanger, these results argued that OAT1 was the CP transporter. However, we have recently shown that OAT3 is also an aKG/OA exchanger and that OAT3 knockout mice show very limited transport of OA. Together these data indicate that OAT3 is the primary OA tranporter mediating the uphill step in CSF to blood transport of OA. The situation for OC is still more complicated. Initial studies of TEA transport in both isolated apical membrane vesicles from the bovine plexus or in primary cultures of choroid plexus cells from neonatal rats indicate the presence of proton/OC exchange at the apical membrane. Proton/OC exchange at also apical in kidney where the direction of transpoort is blood to urine. Since the direction of TEA transport is reversed (CSF to blood), TEA transport must take place by a mechanism distinct from that used in the kidney. The basolateral step in TEA transport is under investicgation using isolated membrane vesicles. In contrast, choline -- an important neurotransmitter precursor whose CNS concentration is regulated by CP transport -- is handled in a very differently. It is taken up across the apical membrane by a potential driven mechanism, which we demonstrated to be mediated by OCT2. We also constructed a fluorescent OCT2-GFP chimera that retained OC transport capacity. It is expressed basolaterally in kidney, but in CP its expression was exclusively apical. Thus, for choline, like OAs, the polarity of transport in the plexus is reversed relative to kidney. We have also assessed OC transport in cultured retinal pigmented epithelium (RPE) from the human eye using verapamil as substrate. As judged by its unique substrate specificity, RPE verapamil transport appears to be a novel system. Its driving force was shown to be proton/verapamil exchange. Interestingly, this system was shown to be inducable by substrate in culture, a process blocked by inhibitors of both protein and RNA synthesis. This is the first demonstration of induction of one of the OA or OC drug transporters.
| Sweet, Douglas H; Miller, David S; Pritchard, John B et al. (2002) Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice. J Biol Chem 277:26934-43 |
| Han, Y H; Sweet, D H; Hu, D N et al. (2001) Characterization of a novel cationic drug transporter in human retinal pigment epithelial cells. J Pharmacol Exp Ther 296:450-7 |
| Sweet, D H; Miller, D S; Pritchard, J B (2001) Ventricular choline transport: a role for organic cation transporter 2 expressed in choroid plexus. J Biol Chem 276:41611-9 |
| Miller, D S; Villalobos, A R; Pritchard, J B (1999) Organic cation transport in rat choroid plexus cells studied by fluorescence microscopy. Am J Physiol 276:C955-68 |
| Pritchard, J B; Sweet, D H; Miller, D S et al. (1999) Mechanism of organic anion transport across the apical membrane of choroid plexus. J Biol Chem 274:33382-7 |
