Specific, metabolism-driven transporters in excretory epithelia and barrier tissues play a important role in determining xenobiotic uptake, distribution and excretion. Along with xenobiotic metabolizing enzymes, these transporters are our first defense against chemical toxins. We use comparative models (renal proximal tubules from lower vertebrates, mammalian cells in culture derived from kidney and choroid plexus and intact mammalian and fish choroid plexus and brain microvessels) in combination with confocal microscopy, intracellular microinjection and isolated membrane vesicle techniques to define the cellular mechanisms that drive xenobiotic transport. Although work continues on the cellular and molecular biology of renal transport mechanisms, recent emphasis has been on development of imaging-based techniques to define mechanisms responsible for transport of foreign chemicals out of the central nervous system (CNS). First, we have used isolated brain capillaries and confocal microscopy to identify the transporters responsible for poor penetration of therapeutic drugs into the brain. For example, the somatostatin analogue, octreotide, is also handled by the ATP-driven export pumps, p-glycoprotein and Mrp2, but the chemotherapeutic, taxol, is only handled by p-glycoprotein. We then used this knowledge and an in vitro/in vivo approach to demonstrate that inhibition of p-glycoprotein both increased taxol entry into mouse brain and provided a remarkable therapeutic advantage in the treatment of an implanted human glioblastoma. Second, using a combination of radiotracer, GFP and confocal imaging techniques as well as knockout technology we localized Oat1, Oat3 and Oct2 to the apical (CSF-side) membrane of the epithelium, which is the correct location for these transporters to mediate uptake from the CSF of organic anions and organic cations, respectively. For the anionic herbicide, 2,4-D and the fluorescent organic anion, fluorescein, we found that uptake by intact rat plexus and isolated bovine apical plasma membrane vesicles was indirectly coupled to Na as one would expect for transport mediated by an Oat family transporter. With imaging we could visualize the entire process of transepithelial organic anion transport and examine for the first time the step from cell to blood, which proved to be both carrier-mediated and (unlike proximal tubule) potential sensitive. Since uptake of several organic anions from the apical side of the tissue appeared to be energetically coupled to Na and since Oat3 was not believed to be an anion exchanger, we were surprised to discover that FL uptake in choroid plexus from an Oat3 kockout mouse is reduced by more than 50%. We are currently investigating the mechanism by which transport on Oat3 is coupled to Na.
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