EXTRARENAL ORGANIC ANION AND ORGANIC CATION TRANSPORT

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Summary of Work: Active transport of organic anion OA) and cations (OC) from CSF to blood and/or from brain interstitial fluid to blood is a critical determinant of the concentration of potentially toxic neurotransmitter metabolites, drugs, and xenobiotics within the brain. We have have adapted both isolated membrane vesicle techniques and primary culture systems to study xenobiotic transport across the blood-CSF barrier (choroid plexus) and the blood-brain barrier (brain capillary endothelium). Morphological characteristics (e.g. brush border at the apical membrane, apical tight junctions, numerous mitochondria), immunohistochemical studies (apical Na,K-ATPase and basolateral GLUT1, and functional polarity (apical Na-dependent proline transport) indicate that the choroid plexus cultures retain the polarity of the intact tissue. In addition, the general properties of OC transport in the cultured plexus appear to be identical to those observed in vivo or in plexus fragments in vitro. We have begun to characterize the mechanism of plexus OC transport. Initial studies indicate that the apical entry step is proton-driven. This result argues that OC transport must take place by a mechanism distinct from that used in the kidney. We have also observed that during transport by the choroid plexus cultures, OCs are accumulated within intracellular vesicles. Many of these vesicles were observed to fuse with the plasma membrane and release of their contents extracellularly, suggesting that vesicular trafficking participates directly in net transepithelial secretion. Initial pharmacologic studies using nocodazol, a microtubule disrupting agent known to block trafficking in other systems, blocks basolateral release of vesicular OCs. We are attempting to characterize this phenomenon more completely and to assess its quantitative role in transepithelial transport of OCs. Parallel studies will be performed using the anionic herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) (which is handled by the renal OA system and is well transported by the choroid plexus; whereas, the model renal substrate p-aminohippurate is not) to study the mechanism of OA transport in this epithelium. Finally, we have established a cultured brain endothelial preparation in our laboratory as an in vitro model for the blood-brain barrier. Parallel studies on OA and OC transport across this barrier tissue will be performed to assess the mechanisms involved and compare them with those of the choroid plexus.