Neurochemical Research

, Volume 32, Issue 11, pp 1978–1989 | Cite as

Extracellular Interconversion of Nucleotides Reveals an Ecto-Adenylate Kinase Activity in the Rat Hippocampus

Original Paper


Here, the extracellular interconversion of nucleotides and nucleosides was investigated in rat hippocampal slices and synaptosomes by an HPLC-UV technique. Adenosine 5′-triphosphate (ATP) was converted to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), adenosine, inosine, and hypoxanthine in the slices, whereas ADP elicited parallel and concentration-dependent formation of ATP and AMP. The specific adenylate kinase inhibitor diadenosine pentaphosphate decreased the rate of decomposition of ADP and inhibited the formation of ATP. No substantial changes in the interconversion of ADP to ATP and AMP were found in the presence of dipyridamole, flufenamic acid, the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium (PPADS), and the alkaline phosphatase substrate para-nitrophenylphosphate. Negligible levels of nucleotides were generated when uridine 5′-diphosphate (UDP), AMP or adenosine were used as substrates. Ecto-adenylate kinase activity was also observed in purified synaptosomes. In summary, we demonstrate the presence of an ecto-adenylate kinase activity in the hippocampus, which is a previously unrecognized pathway that influences the availability of purines in the central nervous system.


ADP ATP Hippocampus Ecto-adenylate kinase Diadenosine pentaphosphate NTPDase 



This study was supported by grants of the Hungarian Research Foundation (OTKA T037457), the Hungarian Medical Research Council (472/2003) and the Hungarian Research and Development Fund (NKFP1A/002/2004). The technical assistance of Ms Judit Őszi and Mrs Éva Szénássy is acknowledged. The authors are also grateful to Prof. Herbert Zimmermann for critical reading of the manuscript and to Dr. Ágnes Kittel for the electron microscopic evaluation of the synaptosomal preparation.


  1. 1.
    Sperlagh B (in press) ATP-mediated signalling in the nervous system. In: Hamon M, Vizi ES (eds) Handbook of neurochemistry and molecular neurobiology, neurotransmitter systems, vol 2. Springer-Verlag, HeidelbergGoogle Scholar
  2. 2.
    Burnstock G (2006) Purinergic signalling-an overview. In: Chadwick D, Goode J (eds) Novartis foundation symposium: purinergic signalling in neuron-glia interactions, vol 276. John Wiley and Sons, Chichester, pp 26–48CrossRefGoogle Scholar
  3. 3.
    Sperlágh B, Vizi ES (1996) Neuronal synthesis, storage and release of ATP. Semin Neurosci 8:175–186CrossRefGoogle Scholar
  4. 4.
    Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedeberg’s Arch Pharmacol 362:299–309CrossRefGoogle Scholar
  5. 5.
    Braun N, Lenz C, Gillardon F, Zimmermann M, Zimmermann H (1997) Focal cerebral ischemia enhances glial expression of ecto-5′-nucleotidase. Brain Res 766:213–226PubMedCrossRefGoogle Scholar
  6. 6.
    Braun N, Zhu Y, Krieglstein J, Culmsee C, Zimmermann H (1998) Upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia in the rat. J Neurosci 18:4891–4900PubMedGoogle Scholar
  7. 7.
    Bonan CD, Walz R, Pereira GS, Worm PV, Battastini AM, Cavalheiro EA, Izquierdo I, Sarkis JJ (2000) Changes in synaptosomal ectonucleotidase activities in two rat models of temporal lobe epilepsy. Epilepsy Res 39:229–238PubMedCrossRefGoogle Scholar
  8. 8.
    Kegel B, Braun N, Heine P, Maliszewski CR, Zimmermann H (1997) An ecto-ATPase, and an ecto-ATP diphosphohydrolase are expressed in rat brain. Neuropharmacology 36:1189–1200PubMedCrossRefGoogle Scholar
  9. 9.
    Bigonnesse F, Levesque SA, Kukulski F, Lecka J, Robson SC, Fernandes MJ, Sevigny J (2004) Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8. Biochemistry 43:5511–5519PubMedCrossRefGoogle Scholar
  10. 10.
    Heine P, Braun N, Heilbronn A, Zimmermann H (1999) Functional characterization of rat ecto-ATPase and ecto-ATP diphosphohydrolase after heterologous expression in CHO cells. Eur J Biochem/FEBS 262:102–107CrossRefGoogle Scholar
  11. 11.
    Vorhoff T, Zimmermann H, Pelletier J, Sevigny J, Braun N (2005) Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: predicted secondary structure and relation to other members of the E-NTPDase family and actin. Purinergic Signal 1:259–270CrossRefGoogle Scholar
  12. 12.
    Cunha RA, Sebastiao AM, Ribeiro JA (1998) Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors. J Neurosci 18:1987–1995PubMedGoogle Scholar
  13. 13.
    Dunwiddie TV, Diao L, Proctor WR (1997) Adenine nucleotides undergo rapid, quantitative conversion to adenosine in the extracellular space in rat hippocampus. J Neurosci 17:7673–7682PubMedGoogle Scholar
  14. 14.
    Khoo JC, Russell PJ (1972) Isoenzymes of adenylate kinase in human tissue. Biochim Biophys Acta 268:98–101PubMedGoogle Scholar
  15. 15.
    Yoneda T, Sato M, Maeda M, Takagi H (1998) Identification of a novel adenylate kinase system in the brain: cloning of the fourth adenylate kinase. Brain Res 62:187–195CrossRefGoogle Scholar
  16. 16.
    Wong PC, Chu DY (1989) Evidence for a synaptic plasma membrane associated adenylate kinase in the rat brain. Biochem Int 19:881–888PubMedGoogle Scholar
  17. 17.
    Nagy AK, Shuster TA, Delgado-Escueta AV (1989) Rat brain synaptosomal ATP:AMP-phosphotransferase activity. J Neurochem 53:1166–1172PubMedCrossRefGoogle Scholar
  18. 18.
    Battastini AM, da Rocha JB, Barcellos CK, Dias RD, Sarkis JJ (1991) Characterization of an ATP diphosphohydrolase (EC in synaptosomes from cerebral cortex of adult rats. Neurochem Res 16:1303–1310PubMedCrossRefGoogle Scholar
  19. 19.
    Nagy AK, Houser CR, Delgado-Escueta AV (1990) Synaptosomal ATPase activities in temporal cortex and hippocampal formation of humans with focal epilepsy. Brain Res 529:192–201PubMedCrossRefGoogle Scholar
  20. 20.
    Cascalheira JF, Sebastiao AM (1992) Adenine nucleotide analogues, including gamma-phosphate-substituted analogues, are metabolised extracellularly in innervated frog sartorius muscle. Eur J Pharmacol 222:49–59PubMedCrossRefGoogle Scholar
  21. 21.
    Kaulich M, Qurishi R, Muller CE (2003) Extracellular metabolism of nucleotides in neuroblastoma × glioma NG108–15 cells determined by capillary electrophoresis. Cell Mol Neurobiol 23:349–364PubMedCrossRefGoogle Scholar
  22. 22.
    Yegutkin GG, Henttinen T, Jalkanen S (2001) Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions. FASEB J 15:251–260PubMedCrossRefGoogle Scholar
  23. 23.
    Yegutkin GG, Henttinen T, Samburski SS, Spychala J, Jalkanen S (2002) The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells. Biochem J 367:121–128PubMedCrossRefGoogle Scholar
  24. 24.
    Sperlagh B, Erdelyi F, Szabo G, Vizi ES (2000) Local regulation of [3H]-noradrenaline release from the isolated guinea-pig right atrium by P2X-receptors located on axon terminals. Br J Pharmacol 131:1775–1783PubMedCrossRefGoogle Scholar
  25. 25.
    Cunha RA, Ribeiro JA (2000) Purinergic modulation of [3H]GABA release from rat hippocampal nerve terminals. Neuropharmacology 39:1156–1167PubMedCrossRefGoogle Scholar
  26. 26.
    Latini S, Pedata F (2001) Adenosine in the central nervous system: release mechanisms and extracellular concentrations. J Neurochem 79:463–484PubMedCrossRefGoogle Scholar
  27. 27.
    Sperlagh B, Szabo G, Erdelyi F, Baranyi M, Vizi ES (2003) Homo- and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system. Br J Pharmacol 139:623–633PubMedCrossRefGoogle Scholar
  28. 28.
    Suadicani SO, Brosnan CF, Scemes E (2006) P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci 26:1378–1385PubMedCrossRefGoogle Scholar
  29. 29.
    Stout CE, Costantin JL, Naus CC, Charles AC (2002) Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem 277:10482–10488PubMedCrossRefGoogle Scholar
  30. 30.
    Pearson RA, Dale N, Llaudet E, Mobbs P (2005) ATP released via gap junction hemichannels from the pigment epithelium regulates neural retinal progenitor proliferation. Neuron 46:731–744PubMedCrossRefGoogle Scholar
  31. 31.
    Bruno AN, Bonan CD, Wofchuk ST, Sarkis JJ, Battastini AM (2002) ATP diphosphohydrolase (NTPDase 1) in rat hippocampal slices and effect of glutamate on the enzyme activity in different phases of development. Life Sci 71:215–225PubMedCrossRefGoogle Scholar
  32. 32.
    Vizi ES, Liang SD, Sperlagh B, Kittel A, Juranyi Z (1997) Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP. Neuroscience 79:893–903PubMedCrossRefGoogle Scholar
  33. 33.
    Vizi ES, Nitahara K, Sato K, Sperlagh B (2000) Stimulation-dependent release, breakdown, and action of endogenous ATP in mouse hemidiaphragm preparation: the possible role of ATP in neuromuscular transmission. J Auton Nerv Syst 81:278–284PubMedCrossRefGoogle Scholar
  34. 34.
    Sperlagh B, Mergl Z, Juranyi Z, Vizi ES, Makara GB (1999) Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis. J Endocrinol 160:343–350PubMedCrossRefGoogle Scholar
  35. 35.
    Sperlagh B, Kittel A, Lajtha A, Vizi ES (1995) ATP acts as fast neurotransmitter in rat habenula: neurochemical and enzymecytochemical evidence. Neuroscience 66:915–920PubMedCrossRefGoogle Scholar
  36. 36.
    Zimmermann H (2001) Ecto-nucleotidases. In: Abbracchio MP, Williams M (eds) Handbook of experimental pharmacology: purinergic and pyrimidinergic signaling, vol 151/1. Springer-Verlag, Berlin, pp 209–251Google Scholar
  37. 37.
    Kukulski F, Levesque SA, Lavoie EG, Lecka J, Bigonnesse F, Knowles AF, Robson SC, Kirley TL, Sevigny J (2005) Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8. Purinergic Signal 1:193–204CrossRefGoogle Scholar
  38. 38.
    Braun N, Sevigny J, Robson SC, Enjyoji K, Guckelberger O, Hammer K, Di Virgilio F, Zimmermann H (2000) Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. Eur J Neurosci 12:4357–4366PubMedCrossRefGoogle Scholar
  39. 39.
    Shukla V, Zimmermann H, Wang L, Kettenmann H, Raab S, Hammer K, Sevigny J, Robson SC, Braun N (2005) Functional expression of the ecto-ATPase NTPDase2 and of nucleotide receptors by neuronal progenitor cells in the adult murine hippocampus. J Neurosci Res 80:600–610PubMedCrossRefGoogle Scholar
  40. 40.
    Belcher SM, Zsarnovszky A, Crawford PA, Hemani H, Spurling L, Kirley TL (2006) Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors. Neuroscience 137:1331–1346PubMedCrossRefGoogle Scholar
  41. 41.
    Kukulski F, Sevigny J, Komoszynski M (2004) Comparative hydrolysis of extracellular adenine nucleotides and adenosine in synaptic membranes from porcine brain cortex, hippocampus, cerebellum and medulla oblongata. Brain Res 1030:49–56PubMedCrossRefGoogle Scholar
  42. 42.
    Burrell HE, Wlodarski B, Foster BJ, Buckley KA, Sharpe GR, Quayle JM, Simpson AW, Gallagher JA (2005) Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface. J Biol Chem 280:29667–29676PubMedCrossRefGoogle Scholar
  43. 43.
    Champagne E, Martinez LO, Collet X, Barbaras R (2006) Ecto-F1Fo ATP synthase/F1 ATPase: metabolic and immunological functions. Curr Opin Lipid 17:279–284CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Department of Pharmacology, Laboratory of Molecular Pharmacology, Institute of Experimental MedicineHungarian Academy of SciencesBudapestHungary

Personalised recommendations