Nucleosides, nucleobases and their analogs (e.g., ganciclovir, acyclovir, dideoxyinosine, azidothymidine) have rapidly become important therapeutic agents in the treatment of a number of central nervous system viral infections including herpes-simplex encephalitis, cytomegalovirus retinitis and AIDS-related dementia complex. To exert a biological effect in the central nervous system, these compounds must penetrate either the blood brain barrier (BBB) or the blood-cerebrospinal fluid barrier (i.e., choroid plexus), the two barriers that provide a protected environment for the brain. Although the BBB has received considerable attention in the literature, it is becoming increasingly apparent that the choroid plexus plays an essential role in mediating the transport of many important compounds into the central nervous system. Notably, during the past granting period, we demonstrated the presence of a novel, broadly- selective Na+-nucleoside cotransporter (N3) in the choroid plexus. In addition, we recently obtained exciting preliminary data suggesting that there is a novel Na+-dependent transporter for nucleobases in the choroid plexus. The overall goal of studies in this competitive renewal application is to understand the mechanisms by which nucleosides, nucleobases and their structural analogs are transported in the choroid plexus. In the proposed studies, we plan to: (1) determine the structure of N3 in rabbit choroid plexus using an expression cloning strategy; (2) determine the tissue distribution in the rabbit of the mRNA transcript encoding N3; (3) determine the location (i.e., basolateral or brush border membrane) of N3 in the choroid plexus; (4) determine the mechanisms by which purine and pyrimidine nucleosides are transported in human choroid plexus; and (5) determine the mechanisms by which nucleobases are transported in the choroid plexus. Methods for determining the structure of N3 will involve expression cloning in Xenopus laevis oocytes, determining the primary sequence of the cDNA encoding N3 and deducing its amino acid sequence. To identify the location of N3 as well as of equilibrative nucleoside transporters in the choroid plexus, kinetic studies using isotopic uptake procedures will be carried out in cultured choroid plexus monolayers grown on porous filters and in isolated membrane vesicles. Isotopic uptake studies in ATP-depleted tissue slices will be used in characterizing nucleoside transport in human choroid plexus and nucleobase transport in rabbit choroid plexus. In particular, we will elucidate the interactions of clinically relevant nucleoside analogs with these nucleoside and nucleobase transporters. The information gained in these studies is critical in the rational design, delivery and targeting of clinically relevant nucleosides, nucleobases and their analogs to the central nervous system.
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