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. 2000 Dec 1;352(Pt 2):363–372.

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain.

A Kiss 1, K Farah 1, J Kim 1, R J Garriock 1, T A Drysdale 1, J R Hammond 1
PMCID: PMC1221467  PMID: 11085929

Abstract

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5'-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [(3)H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [(3)H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.

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Selected References

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  1. Abd-Elfattah A. S., Jessen M. E., Lekven J., Wechsler A. S. Differential cardioprotection with selective inhibitors of adenosine metabolism and transport: role of purine release in ischemic and reperfusion injury. Mol Cell Biochem. 1998 Mar;180(1-2):179–191. [PubMed] [Google Scholar]
  2. Arch J. R., Newsholme E. A. The control of the metabolism and the hormonal role of adenosine. Essays Biochem. 1978;14:82–123. [PubMed] [Google Scholar]
  3. Baer H. P., Haq A., el-Soofi A., Serignese V., Paterson A. R. Potencies of mioflazine and its derivatives as inhibitors of adenosine transport in isolated erythrocytes from different species. J Pharm Pharmacol. 1990 May;42(5):367–369. doi: 10.1111/j.2042-7158.1990.tb05432.x. [DOI] [PubMed] [Google Scholar]
  4. Baldwin S. A., Mackey J. R., Cass C. E., Young J. D. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol Med Today. 1999 May;5(5):216–224. doi: 10.1016/S1357-4310(99)01459-8. [DOI] [PubMed] [Google Scholar]
  5. Belt J. A., Marina N. M., Phelps D. A., Crawford C. R. Nucleoside transport in normal and neoplastic cells. Adv Enzyme Regul. 1993;33:235–252. doi: 10.1016/0065-2571(93)90021-5. [DOI] [PubMed] [Google Scholar]
  6. Burke T., Lee S., Ferguson P. J., Hammond J. R. Interaction of 2',2'-difluorodeoxycytidine (gemcitabine) and formycin B with the Na+-dependent and -independent nucleoside transporters of Ehrlich ascites tumor cells. J Pharmacol Exp Ther. 1998 Sep;286(3):1333–1340. [PubMed] [Google Scholar]
  7. Böhm M., Weinhold C., Schwinger R. H., Müller-Ehmsen J., Böhm D., Reichenspurner H., Reichart B., Erdmann E. Studies of the nucleoside transporter inhibitor, draflazine, in the human myocardium. Br J Pharmacol. 1994 May;112(1):137–142. doi: 10.1111/j.1476-5381.1994.tb13043.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cass C. E., Young J. D., Baldwin S. A. Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem Cell Biol. 1998;76(5):761–770. doi: 10.1139/bcb-76-5-761. [DOI] [PubMed] [Google Scholar]
  9. Crawford C. R., Patel D. H., Naeve C., Belt J. A. Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J Biol Chem. 1998 Feb 27;273(9):5288–5293. doi: 10.1074/jbc.273.9.5288. [DOI] [PubMed] [Google Scholar]
  10. Deckert J., Morgan P. F., Marangos P. J. Adenosine uptake site heterogeneity in the mammalian CNS? Uptake inhibitors as probes and potential neuropharmaceuticals. Life Sci. 1988;42(14):1331–1345. doi: 10.1016/0024-3205(88)90162-2. [DOI] [PubMed] [Google Scholar]
  11. Delicado E. G., Sen R. P., Miras-Portugal M. T. Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells. Biochem J. 1991 Nov 1;279(Pt 3):651–655. doi: 10.1042/bj2790651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ely S. W., Berne R. M. Protective effects of adenosine in myocardial ischemia. Circulation. 1992 Mar;85(3):893–904. doi: 10.1161/01.cir.85.3.893. [DOI] [PubMed] [Google Scholar]
  13. Farinelli S. E., Greene L. A., Friedman W. J. Neuroprotective actions of dipyridamole on cultured CNS neurons. J Neurosci. 1998 Jul 15;18(14):5112–5123. doi: 10.1523/JNEUROSCI.18-14-05112.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flameng W., Sukehiro S., Möllhoff T., Van Belle H., Janssen P. A new concept of long-term donor heart preservation: nucleoside transport inhibition. J Heart Lung Transplant. 1991 Nov-Dec;10(6):990–998. [PubMed] [Google Scholar]
  15. Griffith D. A., Conant A. R., Jarvis S. M. Differential inhibition of nucleoside transport systems in mammalian cells by a new series of compounds related to lidoflazine and mioflazine. Biochem Pharmacol. 1990 Nov 15;40(10):2297–2303. doi: 10.1016/0006-2952(90)90726-2. [DOI] [PubMed] [Google Scholar]
  16. Griffith D. A., Jarvis S. M. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta. 1996 Oct 29;1286(3):153–181. doi: 10.1016/s0304-4157(96)00008-1. [DOI] [PubMed] [Google Scholar]
  17. Griffiths M., Beaumont N., Yao S. Y., Sundaram M., Boumah C. E., Davies A., Kwong F. Y., Coe I., Cass C. E., Young J. D. Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nat Med. 1997 Jan;3(1):89–93. doi: 10.1038/nm0197-89. [DOI] [PubMed] [Google Scholar]
  18. Griffiths M., Yao S. Y., Abidi F., Phillips S. E., Cass C. E., Young J. D., Baldwin S. A. Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem J. 1997 Dec 15;328(Pt 3):739–743. doi: 10.1042/bj3280739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hammond J. R., Clanachan A. S. Species differences in the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system in mammalian central nervous system membranes: evidence for interconvertible conformations of the binding site/transporter complex. J Neurochem. 1985 Aug;45(2):527–535. doi: 10.1111/j.1471-4159.1985.tb04020.x. [DOI] [PubMed] [Google Scholar]
  20. Hammond J. R. Comparative pharmacology of the nitrobenzylthioguanosine-sensitive and -resistant nucleoside transport mechanisms of Ehrlich ascites tumor cells. J Pharmacol Exp Ther. 1991 Nov;259(2):799–807. [PubMed] [Google Scholar]
  21. Hammond J. R. Differential uptake of [3H]guanosine by nucleoside transporter subtypes in Ehrlich ascites tumour cells. Biochem J. 1992 Oct 15;287(Pt 2):431–436. doi: 10.1042/bj2870431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hammond J. R. Interaction of a series of draflazine analogues with equilibrative nucleoside transporters: species differences and transporter subtype selectivity. Naunyn Schmiedebergs Arch Pharmacol. 2000 Apr;361(4):373–382. doi: 10.1007/s002100000214. [DOI] [PubMed] [Google Scholar]
  23. Hammond J. R., Johnstone R. M. Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells. Biochem J. 1989 Aug 15;262(1):109–118. doi: 10.1042/bj2620109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huang Q. Q., Harvey C. M., Paterson A. R., Cass C. E., Young J. D. Functional expression of Na(+)-dependent nucleoside transport systems of rat intestine in isolated oocytes of Xenopus laevis. Demonstration that rat jejunum expresses the purine-selective system N1 (cif) and a second, novel system N3 having broad specificity for purine and pyrimidine nucleosides. J Biol Chem. 1993 Sep 25;268(27):20613–20619. [PubMed] [Google Scholar]
  25. Kwong F. Y., Wu J. S., Shi M. M., Fincham H. E., Davies A., Henderson P. J., Baldwin S. A., Young J. D. Enzymic cleavage as a probe of the molecular structures of mammalian equilibrative nucleoside transporters. J Biol Chem. 1993 Oct 15;268(29):22127–22134. [PubMed] [Google Scholar]
  26. Lee C. W. Decrease in equilibrative uridine transport during monocytic differentiation of HL-60 leukaemia: involvement of protein kinase C. Biochem J. 1994 Jun 1;300(Pt 2):407–412. doi: 10.1042/bj3000407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee C. W., Sokoloski J. A., Sartorelli A. C., Handschumacher R. E. Induction of the differentiation of HL-60 cells by phorbol 12-myristate 13-acetate activates a Na(+)-dependent uridine-transport system. Involvement of protein kinase C. Biochem J. 1991 Feb 15;274(Pt 1):85–90. doi: 10.1042/bj2740085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mackey J. R., Baldwin S. A., Young J. D., Cass C. E. Nucleoside transport and its significance for anticancer drug resistance. Drug Resist Updat. 1998;1(5):310–324. doi: 10.1016/s1368-7646(98)80047-2. [DOI] [PubMed] [Google Scholar]
  29. Mani R. S., Hammond J. R., Marjan J. M., Graham K. A., Young J. D., Baldwin S. A., Cass C. E. Demonstration of equilibrative nucleoside transporters (hENT1 and hENT2) in nuclear envelopes of cultured human choriocarcinoma (BeWo) cells by functional reconstitution in proteoliposomes. J Biol Chem. 1998 Nov 13;273(46):30818–30825. doi: 10.1074/jbc.273.46.30818. [DOI] [PubMed] [Google Scholar]
  30. Mayer B. J., Eck M. J. SH3 domains. Minding your p's and q's. Curr Biol. 1995 Apr 1;5(4):364–367. doi: 10.1016/s0960-9822(95)00073-x. [DOI] [PubMed] [Google Scholar]
  31. Miras-Portugal M. T., Delicado E. G., Casillas T., Sen R. P. Control of nucleoside transport in neural cells. Effect of protein kinase C activation. Adv Exp Med Biol. 1991;309A:435–438. doi: 10.1007/978-1-4899-2638-8_100. [DOI] [PubMed] [Google Scholar]
  32. Mubagwa K., Mullane K., Flameng W. Role of adenosine in the heart and circulation. Cardiovasc Res. 1996 Nov;32(5):797–813. [PubMed] [Google Scholar]
  33. Murray A. W. The biological significance of purine salvage. Annu Rev Biochem. 1971;40:811–826. doi: 10.1146/annurev.bi.40.070171.004115. [DOI] [PubMed] [Google Scholar]
  34. Musacchio A., Saraste M., Wilmanns M. High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nat Struct Biol. 1994 Aug;1(8):546–551. doi: 10.1038/nsb0894-546. [DOI] [PubMed] [Google Scholar]
  35. Ogbunude P. O., Baer H. P. Competition of nucleoside transport inhibitors with binding of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside to intact erythrocytes and ghost membranes from different species. Biochem Pharmacol. 1990 Apr 1;39(7):1199–1204. doi: 10.1016/0006-2952(90)90263-k. [DOI] [PubMed] [Google Scholar]
  36. Paul B., Chen M. F., Paterson A. R. Inhibitors of nucleoside transport. A structure-activity study using human erythrocytes. J Med Chem. 1975 Oct;18(10):968–973. doi: 10.1021/jm00244a003. [DOI] [PubMed] [Google Scholar]
  37. Plagemann P. G., Woffendin C. Species differences in sensitivity of nucleoside transport in erythrocytes and cultured cells to inhibition by nitrobenzylthioinosine, dipyridamole, dilazep and lidoflazine. Biochim Biophys Acta. 1988 Apr 2;969(1):1–8. doi: 10.1016/0167-4889(88)90081-x. [DOI] [PubMed] [Google Scholar]
  38. Rongen G. A., Smits P., Ver Donck K., Willemsen J. J., De Abreu R. A., Van Belle H., Thien T. Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humans. Implications for its future role in cardioprotection. J Clin Invest. 1995 Feb;95(2):658–668. doi: 10.1172/JCI117711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shank R. P., Baldy W. J. Adenosine transport by rat and guinea pig synaptosomes: basis for differential sensitivity to transport inhibitors. J Neurochem. 1990 Aug;55(2):541–550. doi: 10.1111/j.1471-4159.1990.tb04168.x. [DOI] [PubMed] [Google Scholar]
  40. Soler C., Felipe A., Mata J. F., Casado F. J., Celada A., Pastor-Anglada M. Regulation of nucleoside transport by lipopolysaccharide, phorbol esters, and tumor necrosis factor-alpha in human B-lymphocytes. J Biol Chem. 1998 Oct 9;273(41):26939–26945. doi: 10.1074/jbc.273.41.26939. [DOI] [PubMed] [Google Scholar]
  41. Staub M., Sasvari-Szekely M., Solymossy M., Szikla K. Nucleoside transport and metabolism in lymphocytes, polymorphonuclear cells and cerebral synaptosomes. Adv Exp Med Biol. 1994;370:769–774. doi: 10.1007/978-1-4615-2584-4_161. [DOI] [PubMed] [Google Scholar]
  42. Sundaram M., Yao S. Y., Ng A. M., Griffiths M., Cass C. E., Baldwin S. A., Young J. D. Chimeric constructs between human and rat equilibrative nucleoside transporters (hENT1 and rENT1) reveal hENT1 structural domains interacting with coronary vasoactive drugs. J Biol Chem. 1998 Aug 21;273(34):21519–21525. doi: 10.1074/jbc.273.34.21519. [DOI] [PubMed] [Google Scholar]
  43. Van Belle H. Nucleoside transport inhibition: a therapeutic approach to cardioprotection via adenosine? Cardiovasc Res. 1993 Jan;27(1):68–76. doi: 10.1093/cvr/27.1.68. [DOI] [PubMed] [Google Scholar]
  44. Vickers M. F., Mani R. S., Sundaram M., Hogue D. L., Young J. D., Baldwin S. A., Cass C. E. Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae. Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q). Biochem J. 1999 Apr 1;339(Pt 1):21–32. [PMC free article] [PubMed] [Google Scholar]
  45. Williams J. B., Lanahan A. A. A mammalian delayed-early response gene encodes HNP36, a novel, conserved nucleolar protein. Biochem Biophys Res Commun. 1995 Aug 4;213(1):325–333. doi: 10.1006/bbrc.1995.2133. [DOI] [PubMed] [Google Scholar]
  46. Williams J. B., Rexer B., Sirripurapu S., John S., Goldstein R., Phillips J. A., 3rd, Haley L. L., Sait S. N., Shows T. B., Smith C. M. The human HNP36 gene is localized to chromosome 11q13 and produces alternative transcripts that are not mutated in multiple endocrine neoplasia, type 1 (MEN I) syndrome. Genomics. 1997 Jun 1;42(2):325–330. doi: 10.1006/geno.1997.4751. [DOI] [PubMed] [Google Scholar]
  47. Yao S. Y., Ng A. M., Muzyka W. R., Griffiths M., Cass C. E., Baldwin S. A., Young J. D. Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues. J Biol Chem. 1997 Nov 7;272(45):28423–28430. doi: 10.1074/jbc.272.45.28423. [DOI] [PubMed] [Google Scholar]
  48. von Lubitz D. K., Carter M. F., Beenhakker M., Lin R. C., Jacobson K. A. Adenosine: a prototherapeutic concept in neurodegeneration. Ann N Y Acad Sci. 1995 Sep 15;765:163–197. doi: 10.1111/j.1749-6632.1995.tb16573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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