Apr 8, 1994 - Terry Fox Cancer Research Scientist of the National Cancer Insti-. $$ Heritage Medical Scientist of the Alberta Heritage Foundation for. Medical ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Val. 269, No. 27, Issue of July 8,pp. 17757-17760, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Communication
Printed in U.S.A.
neurotransmitter release, platelet aggregration, coronary vasodilation, and cardiac contractility (1,2). Nucleoside antimetabolites have therapeutic applications in human neoplastic AIDS’ (3, 4). and viral diseases, including leukemias and Both equilibrative and Na+-dependent nucleoside transport mechanisms are present in mammalian cells. In human erythrocytes, transport of purineand pyrimidinenucleosides is (Received forpublication, February 17, 1994, and in revised form, equilibrative (Na+-independent) and inhibited by nanomolar April 8, 1994) concentrations of NBMPR (5, 6). The erythrocyte transporter, an integral membraneglycoprotein of apparent M , 55,000 (71, Qi-Quan HuangS, SylviaY. M.YaoSS, Mabel W. L. Ritzel, AlanR. P. Patersonll, has been purified t o apparent homogeneity by a combination of Carol E. Cassll**, and JamesD. Young$* ion-exchange and immunoaffinity chromatography (8). Functionally and structurally similar equilibrative nucleoside transFrom the Membrane Dansport Group, Departments of Physiology, (Biochemistry, and Wharmacology, porters (designated es) are widely distributed in mammalian University of Alberta, Edmonton, cells and tissues(9-11). In addition,some mammalian cells and Alberta T6G 2H7, Canada tissues (9) possess Na+-independent nucleoside transport sysby NBMPR Expression screening in Xenopus oocytes was used to tems withlow (micromolar) sensitivity to inhibition isolate a cDNA from rat jejunal epithelium encoding a (designated ei). The molecular properties of ei transporters are unknown. Na+-dependent nucleoside transportprotein(named Na+-dependentnucleoside transport systems have been demcNT1). The cDNA sequence of cNTl predicts a protein of 648 aminoacids (relative molecular mass 71,000) with 14 onstrated ina variety of cell types, including intestinal (12-141, potential transmembrane domains. Data basesearches renal (15-17), and choroid plexus epithelia (18),as well as culindicate significant sequence similarity to the NUPC tured leukemia cells (19). On the basisof substrate selectivity, protodnucleoside symporter of Escherichia coli. There three principal Na+-dependent nucleoside transporters have is no sequence similarity between cNTl and proteinsof been recognized. Transport by the cifsystemis generally purinemammalianorigin.Functionally,cNTl exhibited the specific and guanosine and formycin B serve as model subtransport characteristics of the nucleosidetransport strates, whereas the cit system is generally pyrimidine-specific system cit (selective forpyrimidine nucleosides and and thymidine serves asa model substrate. Uridine and adenadenosine) and accepted both 3‘-azido-3’-deoxythymi- osine are transported by both systems. A third system, desigand 2’,3’-dideoxycytidine (ddC) as permeants nated cib, has broadspecificity for both purine andpyrimidine dine (AZT) (K,= 0.49 and 0.51m ~ respectively). , The demonstration nucleosides. No specific, high affinity inhibitors of Na+-linked of transportof AZT by cNTl expressed in Xenopus identified. In the present report, oocytes provides the first direct evidence that AZT en- nucleoside fluxeshave yet been we have employed an expression cloningstrategy usingXenopus ters cells by transporter-mediated processes,as well as by passive diffusion. Consistentwith the tissue distribu- oocytes to isolatea cDNA from rat jejunal epithelium encoding cNT1 a high affinity cit-type Na+-dependent nucleoside transporter tion of system cit transport activity, transcripts for were detected in kidney as well as jejunum. cNTlthere- (named cNT1) belonging to a potential new gene family. In addition to physiological nucleosides, cNTl was found to fore belongsto a potential new gene family and may be involved in the intestinal absorption and renal handlingtransport the antiviralpyrimidine nucleoside analogs AZT and of pyrimidine nucleoside analogs used to treat acquired ddC. Most previous studies of AZT and ddC membrane permeability have focused on nonepithelial cells. AZT is not transimmunodeficiency syndrome (AIDS). ported by equilibrative nucleoside transporters and enters human erythrocytes, lymphocytes, macrophages, and bone Natural and synthetic nucleosides have important physi- marrow progenitor cells mainly by nonfacilitated diffusion (20ologic and pharmacologic activities in humans.Adenosine, for 22). ddC is a low affinity es substrate (23,24). Rates of entry of a local hormone in regulation of lipolysis, AZT and ddC into human erythrocytes are much less (
