Abstract
The brain efflux index (BEI), a measurement of blood–brain barrier (BBB) efflux transport, was estimated at 15 s, 30 s, 1 min, 3 min and 10 min after intracerebral injection of [14C]pyrimidines. An initial steep increase of the BEI values over time was observed for [14]uracil and [14C]thymine, followed by a more moderate increase after 1 min. For the corresponding nucleosides, [14C]uridine and [14C]thymidine, the increase of BEI values over time was less steep and linear between 30 s and 3 min. The apparent BBB efflux clearances for [14C]uridine, [14C]thymidine, [14C]uracil and [14C]thymine were (μl/min/g): 95.2 ± 12.1, 125.3 ± 18.4, 290.4 ± 28 and 358.5 ± 32.5, respectively, which is at least several folds higher than the predicted BBB influx clearances of uridine, uracil and thymidine. Quick depletion of brain parenchyma from brain microvasculature has revealed that [14C] radioactivity accumulated in brain microvessels after injection of nucleosides [14C]thymidine and [14C]uridine, but that was not observed when nucleobases, [14C]thymine and [14C]uracil, were injected. Reverse transcriptase-PCR revealed that the rat brain and liver (positive control) express dihydropyrimidine dehydrogenase, a key enzyme in pyrimidine nucleobase catabolism. Two bands representing spliced variants have been detected with the relative density of the bands (expressed relative to the density of glyceraldehyde3-phosphate dehydrogenase bands, mean ± SEM from 3 separate samples) 0.16 ± 0.06 and 0.04 ± 0.01 (brain) and 0.49 ± 0.1 and 0.07 ± 0.01 (liver). Overall, these results indicate that the net direction of pyrimidine BBB transport is the efflux transport; rapid BBB efflux transport and metabolic breakdown of pyrimidine nucleobases appear to be important for brain homeostasis.
Similar content being viewed by others
References
Connolly GP, Simmonds HA, Duley JA (1996) Pyrimidines and CNS regulation. Trends Pharmacol Sci 17:106–107. doi:10.1016/0165-6147(96)20001-X
Connolly PC, Duley JA (1999) Uridine and its nucleotides: biological actions, therapeutic potentials. Trends Pharmacol Sci 20:218–225. doi:10.1016/S0165-6147(99)01298-5
Barsotti C, Tozzi MG, Ipata PL (2002) Purine and pyrimidine salvage in whole rat brain. Utilization of ATP-derived ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate generated in experiments with dialyzed cell-free extracts. J Biol Chem 277:9865–9869. doi:10.1074/jbc.M111418200
Balestri F, Barsotti C, Lutzemberger L et al (2007) Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain. Neurochem Int 51:517–523. doi:10.1016/j.neuint.2007.06.007
Cao D, Leffert JJ, McCabe J et al (2005) Abnormalities in uridine homeostatic regulation and pyrimidine nucleotide metabolism as a consequence of the deletion of the uridine phosphorylase gene. J Biol Chem 280:21169–21175. doi:10.1074/jbc.M412343200
Cansev M (2006) Uridine and cytidine in the brain: their transport and utilization. Brain Res Brain Res Rev 52:389–397. doi:10.1016/j.brainresrev.2006.05.001
Zimmermann H (1996) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 49:589–618. doi:10.1016/0301-0082(96)00026-3
Sala-Newby GB, Skladanowski AC, Newby C (1999) The mechanism of adenosine formation in cells: cloning of cytosolic 5′ nucleotidase. J Biol Chem 274:17789–17793. doi:10.1074/jbc.274.25.17789
Phillips E, Newsholme EA (1979) Maximum activities, properties and distribution of 5′-nucleotidase, adenosine kinase and adenosine deaminase in rat and human brain. J Neurochem 33:553–558. doi:10.1111/j.1471-4159.1979.tb05187.x
Cheng N, Payne RC, Traut TW (1986) Regulation of uridine kinase. Evidence for a regulatory site. J Biol Chem 261:13006–13012
Ropp PA, Traut TW (1998) Uridine kinase: altered enzyme with decreased affinities for uridine and CTP. Arch Biochem Biophys 359:63–68. doi:10.1006/abbi.1998.0890
Suzuki NN, Koizumi K, Fukushima M et al (2004) Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase. Structure 12:751–764. doi:10.1016/j.str.2004.02.038
Balestri F, Giannecchini M, Sgarrella F et al (2007) Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions. Neurochem Int 50:517–523. doi:10.1016/j.neuint.2006.10.005
Isakovic AJ, Abbott JN, Redzic ZB (2004) Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat. J Neurochem 90:272–286. doi:10.1111/j.1471-4159.2004.02439.x
Spector R (1985) Uridine transport and metabolism in the central nervous system. J Neurochem 45(5):1411–1418. doi:10.1111/j.1471-4159.1985.tb07207.x
Spector R (1985) Thymidine transport and metabolism in choroid plexus: effect of diazepam and thiopental. J Pharmacol Exp Ther 235(1):16–19
Isakovic AJ, Segal MB, Milojkovic B et al (2002) The efflux of purine nucleobases and nucleosides from the rat brain. Neurosci Lett 318:65–68. doi:10.1016/S0304-3940(01)02478-8
Cornford EM, Oldendorf WH (1975) Independent blood-brain barrier transport systems for nucleic acid precursors. Biochim Biophys Acta 394:211–219. doi:10.1016/0005-2736(75)90259-X
Kakee A, Terasaki T, Sugiyama Y (1996) Brain efflux index as a novel method of analysing efflux transport at the blood brain barrier. J Pharmacol Exp Ther 277:1550–1559
Kitazawa T, Terasaki T, Suzuki H et al (1998) Efflux of taurocholic acid across the blood-brain barrier: interactions with cyclic peptides. J Pharmacol Exp Ther 286:890–895
Newman C, Hospod F, Schissel S (1991) Ischemic brain slice glucose utilization: effects of slice thickness, acidosis and K+. J Cereb Blood Flow Metab 11:398–406
Triguero D, Buciak J, Pardridge WM (1990) Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins. J Neurochem 54(6):1882–1888. doi:10.1111/j.1471-4159.1990.tb04886.x
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3
Lu Z, Zhang R, Diasio RB (1992) Purification and characterization of dihydropyrimidine dehydrogenase from human liver. J Biol Chem 267:17102–17109
Diasio RB (1998) The role of dihydropyrimidine dehydrogenase (DPD) modulation in 5-FU pharmacology. Oncology 12(10, Suppl 7):23–27
Smith QR, Momma S, Aoyagi M, Rapoport SI (1987) Kinetics of neutral amino acid transport across the blood-brain barrier. J Neurochem 49(5):1651–1658. doi:10.1111/j.1471-4159.1987.tb01039.x
Pardridge WM, Fierer G (1985) Blood brain barrier transport of butanol and water relative to N-isopropyl-p-iodoamphetamine as the internal reference. J Cereb Blood Flow Metab 5:275–281
Redzic ZB, Gasic JM, Markovic ID et al (1998) The effects of NO synthesis inhibition on the uptake of endogenous nucleosides into the rat brain. Neurosci Res Commun 20:11–20. doi 10.1002/(SICI)1520-6769(199801/02)22:1<11::AID-NRC3>3.0.CO;2-V
Hagberg H, Andersson P, Lacarewic P et al (1987) Extracellular adenosine, inosine, hypoxanthine and xanthine in relation to tissue nucleotides and purines in rat striatum during transient ischemia. J Neurochem 49:227–231. doi:10.1111/j.1471-4159.1987.tb03419.x
Gray JH, Owen RP, Giacomini KM (2004) The concentrative nucleoside transporter family, SLC28. Pflugers Arch––Eur J Physiol 447:728–734
Baldwin SA, Beal PR, Yao SY et al (2004) The equilibrative nucleoside transporter family, SLC29. Pflugers Arch––Eur J Phys 447:735–743
Ward JL, Sherali A, Mo Z et al (2000) Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. J Biol Chem 275(12):8375–8381. doi:10.1074/jbc.275.12.8375
Redzic ZB, Biringer J, Barnes K et al (2005) Polarized distribution of nucleoside transporters in rat brain endothelial and choroid plexus epithelial cells. J Neurochem 94:1420–1426. doi:10.1111/j.1471-4159.2005.03312.x
Loffler M, Fairbanks LD, Zameitat E et al (2005) Pyrimidine pathways in health and disease. Trends Mol Med 11:430–437. doi:10.1016/j.molmed.2005.07.003
Jansson O, Bohman C, Munch-Petersen B, Eriksson S (1992) Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P] dTTP of the enzyme from spleen, liver, heart and brain. Eur J Biochem 206(2):485–490. doi:10.1111/j.1432-1033.1992.tb16951.x
Lynx MD, McKee EE (2006) 3′-Azido-3′-deoxythymidine (AZT) is a competitive inhibitor of thymidine kinase 2 in isolated rat heart and liver mitochondria. Biochem Pharmacol 72(2):239–243. doi:10.1016/j.bcp.2006.04.004
Lynx MD, Kang BK, McKee EE (2008) Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines. Biochem Pharmacol 75(8):1610–1615. doi:10.1016/j.bcp.2008.01.006
Yao SY, Ng AM, Sundaram M, Cass CE et al (2001) Transport of antiviral 3′-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes. Mol Membr Biol 18(2):161–167. doi:10.1080/09687680110048318
Smith KM, Slugoski MD, Loewen SK et al (2005) The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics. J Biol Chem 280(27):25436–25449. doi:10.1074/jbc.M409454200
Parkinson FE, Ferguson J, Zamzow CR et al (2006) Gene expression for enzymes and transporters involved in regulating adenosine and inosine levels in rat forebrain neurons, astrocytes and C6 glioma cells. J Neurosci Res 84(4):801–808. doi:10.1002/jnr.20988
Acknowledgments
We acknowledge financial support of the Department of Physiology, Kuwait University and of the ICN Pharmaceuticals (their ex branch ICN Yugoslavia) for their kind support with radiolabeled nucleosides and nucleobases. We also acknowledge Mrs Nada Selakovic Bojovic for her technical assistance in the BEI experiments.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Redzic, Z.B., Malatiali, S.A., Craik, J.D. et al. Blood–Brain Barrier Efflux Transport of Pyrimidine Nucleosides and Nucleobases in the Rat. Neurochem Res 34, 566–573 (2009). https://doi.org/10.1007/s11064-008-9823-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11064-008-9823-5