Neurochemical Research

, Volume 37, Issue 7, pp 1442–1449 | Cite as

Rats With Different Thresholds to Clonic Convulsions Induced by DMCM Differ in the Binding of [3H]-MK-801 and [3H]-Ouabain in the Membranes of Brain Regions

  • Marcos Brandão Contó
  • José Gilberto Barbosa de Carvalho
  • Marco Antonio Campana Venditti
Original Paper


Considering the putative participation of N-methyl-D-aspartate (NMDA) receptors and the Na+, K+-ATPase enzymes in the susceptibility to convulsions induced by the benzodiazepine inverse agonist methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), the present study sought to determine if rats with high (HTR) and low (LTR) thresholds to clonic convulsions induced by DMCM differed in the following aspects: the binding of NMDA receptors by [3H]-MK-801, Na+, K+-ATPase activity (K+-stimulated p-nitrophenylphosphatase) and high-affinity [3H]-ouabain binding to membranes from discrete brain regions. Compared to the HTR subgroup, the LTR subgroup presented a lower binding of [3H]-MK-801 in the hippocampus, frontal cortex and striatum. The subgroups did not differ in K+-p-nitrophenylphosphatase activity, but the LTR subgroup had a lower density of isozymes with a high-affinity to ouabain in the brainstem and in the frontal cortex and a lower affinity to ouabain in the hippocampus than the HTR subgroup. These results suggest that NMDA receptors and ouabain-sensitive Na+, K+-ATPase isozymes may underlie the susceptibility to DMCM-induced convulsions.


[3H]-ouabain [3H]-MK 801 Na+, K+-ATPase Convulsion DMCM 



We thank Ricardo Marques for his technical assistance. This research was supported by Associação Fundo de Incentivo à Pesquisa (AFIP). M.B. Contó was the recipient of a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes).


  1. 1.
    Browning RA (1985) Role of the brain-stem reticular formation in tonic-clonic seizures: lesion and pharmacological studies. Fed Proc 44(8):2425–2431PubMedGoogle Scholar
  2. 2.
    Gale K (1988) Progression and generalization of seizure discharge: anatomical and neurochemical substrates. Epilepsia 29(2):S15–S34PubMedCrossRefGoogle Scholar
  3. 3.
    Kelly ME, Battye RA, McIntyre DC (1999) Cortical spreading depression reversibly disrupts convulsive motor seizure expression in amygdala-kindled rats. Neurosci 91(1):305–313CrossRefGoogle Scholar
  4. 4.
    Norden AD, Blumenfeld H (2002) The role of subcortical structures in human epilepsy. Epilepsy Behav 3:219–231PubMedCrossRefGoogle Scholar
  5. 5.
    Willoughby JO, Macckenzie L, Medvedev A, Hiscock JJ (1999) Generalized convulsive epilepsy: possible mechanisms. J Clin Neurosci 6(3):189–194PubMedCrossRefGoogle Scholar
  6. 6.
    Berkovic SF, Mulley JC, Scheffer IE, Petrou S (2006) Human epilepsies: interaction of genetic and acquired factors. Trends Neurosci 29(7):391–397PubMedCrossRefGoogle Scholar
  7. 7.
    Bateson AN (2004) The benzodiazepine site of the GABAA receptor: an old target with a new potential? Sleep Med 5(1):S9–S15PubMedCrossRefGoogle Scholar
  8. 8.
    Platt SR (2007) The role of glutamate in central nervous system health and disease—A review. Vet J 173:278–286PubMedCrossRefGoogle Scholar
  9. 9.
    Benarroch EE (2007) GABAA receptor heterogeneity, function, and implications for epilepsy. Neurology 68:612–614PubMedCrossRefGoogle Scholar
  10. 10.
    Corda MG, Orlandi M, Lecca D, Giorgi O (1992) Decrease in Gabaergic function induced by pentylenotetrazol kindling in rats: antagonism by MK-801. JPET 262(2):792–800Google Scholar
  11. 11.
    Croucher MJ, Bradford HF (1990) NMDA receptor blockade inhibits glutamate-induced kindling of the rat amygdala. Brain Res 506:349–352PubMedCrossRefGoogle Scholar
  12. 12.
    Croucher MJ, Bradford HF, Sunter DC, Watkins JC (1988) Inhibition of the development of electrical kindling of the prepyriform cortex by daily focal injections of excitatory amino acid antagonists. Eur J Pharmacol 152:29–38PubMedCrossRefGoogle Scholar
  13. 13.
    Croucher MJ, Cotterell KL, Bradford HF (1992) Competitive NMDA receptor antagonists raise electrically kindled generalized seizure thresholds. Neurochem Res 17(5):409–413PubMedCrossRefGoogle Scholar
  14. 14.
    Tsuda M, Suzuki T, Misawa M (1997) Role of the NMDA receptor complex in DMCM-induced seizure in mice. NeuroReport 8:603–606PubMedCrossRefGoogle Scholar
  15. 15.
    Tsuda M, Shimizu N, Yajima Y, Suzuki T, Misawa M (1998) Hypersusceptibility to DMCM-induced seizures during diazepam withdrawal in mice: evidence for upregulation of NMDA receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 357:309–315CrossRefGoogle Scholar
  16. 16.
    Camacho A, Massieu L (2006) Role of glutamate transporters in the clearance and release of glutamate during ischemia and its relation to neuronal death. Arch Med Res 37:11–18PubMedCrossRefGoogle Scholar
  17. 17.
    Cousin MA, Nicholls DG, Pocock JM (1995) Modulation of ion gradients and glutamate release in cultured cerebellar granule cells by ouabain. J Neurochem 64(5):2097–2104PubMedCrossRefGoogle Scholar
  18. 18.
    Li S, Stys PK (2001) Na+K+ -ATPase inhibition and depolarization induce glutamate release via reverse Na+-dependent transport in spinal cord white matter. Neurosci 107(4):675–683CrossRefGoogle Scholar
  19. 19.
    Veldhuis WB, van der Stelt M, Delmas F, Gillet B, Veldink G, Vliegenthart JFG et al (2003) In vivo excitotoxicity induced by ouabain, a Na +/K + -ATPase inhibitor. J Cereb Blood Flow Metab 23:62–74PubMedCrossRefGoogle Scholar
  20. 20.
    Therien A, Blostein R (2000) Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279:C541–C566PubMedGoogle Scholar
  21. 21.
    Yu SP (2003) Na + , K + -ATPase: the new face of an old player in pathogenesis and apoptotic/hybrid cell death. Biochem Pharmacol 66:1601–1609PubMedCrossRefGoogle Scholar
  22. 22.
    Blanco G, Mercer RW (1998) Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol 275:F633–F650PubMedGoogle Scholar
  23. 23.
    Jewell EA, Lingrel JB (1991) Comparison of the substrate dependence properties of the rat Na, K-ATPase α1, α2 and α3 isoforms expressed in HeLa Cells. J Biol Chem 266(25):16925–16930PubMedGoogle Scholar
  24. 24.
    Bignami A, Palladini G (1966) Experimentally produced cerebral status spongiosus and continuous pseudorhythmic electroencephalographic discharges with a membrane-ATPase inhibitor in the rat. Nature 209:413–414PubMedCrossRefGoogle Scholar
  25. 25.
    Pedley TA, Zuckermann EC, Glaser GH (1969) Epileptogenic effects of localized ventricular perfusion of ouabain on dorsal hippocampus. Exp Neurol 25:207–219PubMedCrossRefGoogle Scholar
  26. 26.
    Mobasheri A, Avila J, Cózar-Castellano I, Brownleader MD, Trevan M, Francis MJO et al (2000) Na + , K + -ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interaction. Biosci Rep 20(2):51–91PubMedCrossRefGoogle Scholar
  27. 27.
    Blanco G, Xie ZJ, Mercer RW (1993) Functional expression of the α2 and α3 isoforms of the Na, K-ATPase in baculovirus-infected insect cells. Proc Natl Acad Sci USA 90:1824–1828PubMedCrossRefGoogle Scholar
  28. 28.
    O’Brien WJ, Lingrel JB, Wallick ET (1994) Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate. Arch Biochem Biophys 310(1):32–39PubMedCrossRefGoogle Scholar
  29. 29.
    Ferrarese C, Cogliati C, Tortorella R, Zucca C, Bogliun G, Beghi E (1998) Diazepam binding inhibitor (DBI) in the plasma of pediatric and adult epileptic patients. Epilepsy Res 29:129–134PubMedCrossRefGoogle Scholar
  30. 30.
    Polc P, Dučić I (1991) Benzodiazepine antagonist flumazenil reduces bicuculine-induced enhancement of neuronal activity in the spinal cord. Neuropharmacol 30(1):107–111CrossRefGoogle Scholar
  31. 31.
    Polc P, Jahromi SS, Facciponte G, Pelletier MR, Zhang L, Carlen PL (1996) Benzodiazepine antagonists reduce epileptiform discharges in rat hippocampal slices. Epilepsia 37(10):1007–1014PubMedCrossRefGoogle Scholar
  32. 32.
    Pole P, Jahromi SS, Facciponte G, Pelletier MR, Zhang L, Carlen PL (1995) Benzodiazepine antagonist flumazenil reduces hippocampal epileptiform activity. Neuroreport 6(11):1549–1552CrossRefGoogle Scholar
  33. 33.
    Van Rijn C, Meinardi H (2009) Neurochemistry and epileptology. Epilepsia 50(3):17–29PubMedCrossRefGoogle Scholar
  34. 34.
    Vezzani A, Serafini R, Stasi MA, Samanin R, Ferrarese C (1991) Epileptogenic activity of two peptides derived from diazepam binding inhibitor after intrahippocampal injection in rats. Epilepsia 32(5):597–603PubMedCrossRefGoogle Scholar
  35. 35.
    Braestrup C, Schmiechen R, Neef G, Nielsen M, Petersen EN (1982) Interaction of convulsive ligands with benzodiazepine receptors. Science 216:1241–1243PubMedCrossRefGoogle Scholar
  36. 36.
    Petersen EN (1983) DMCM: a potent convulsive benzodiazepine receptor ligand. Eur J Pharmacol 94:117–124PubMedCrossRefGoogle Scholar
  37. 37.
    Contó MB, Carvalho JGB, Venditti MAC (2011) Rats with different thresholds for DMCM-induced clonic convulsions differ in the sleep-time of diazepam and [3H]-Ro 15–4513 binding. Epilepsy Res. doi: 10.1016/j.eplepsyres.2011.09.014 PubMedGoogle Scholar
  38. 38.
    Rosa RB, Schwartzbold C, Dalcin KB, Ghisleni GC, Ribeiro CAJ, Moretto MB et al (2004) Evidence that 3-hydroxyglutaric acid interacts with NMDA receptors in synaptic plasma membranes from cerebral cortex of young rats. Neurochem Int 45:1087–1094PubMedCrossRefGoogle Scholar
  39. 39.
    Bowery NG, Wong EHF, Hudson AL (1988) Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain. Br J Pharmacol 93:944–954PubMedGoogle Scholar
  40. 40.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  41. 41.
    Alves R, de Carvalho JGB, Benedito MAC (2005) High and low rearing subgroups of rats selected in the open field differ in the activity of K+-stimulated-p-nitrophenylphosphatase in the hippocampus. Brain Res 1058:178–182PubMedCrossRefGoogle Scholar
  42. 42.
    Bignotto M, Benedito MAC (2006) Repeated electroconvulsive shock induces changes in high-affinity [3H]-ouabain binding to rat striatal membranes. Neurochem Res 31:515–521PubMedCrossRefGoogle Scholar
  43. 43.
    Cheng YC, Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108PubMedCrossRefGoogle Scholar
  44. 44.
    March PA (1998) Seizures: Classification, Etiologies, and Pathophysiology. Clin Tech Small Anim Pract 13(3):119–131PubMedCrossRefGoogle Scholar
  45. 45.
    Ekonomou A, Angelatou F (1999) Upregulation of NMDA receptors in hippocampus and cortex in the pentylenetetrazol-induced “kindling” model of epilepsy. Neurochem Res 24(12):1515–1522PubMedCrossRefGoogle Scholar
  46. 46.
    Oguro K, Ito M, Tsuda H, Mutoh K, Shiraishi H, Shirasaka Y et al (1990) NMDA-sensitive L-[3H]glutamate binding in cerebral cortex of El mice. Epilepsy Res 6:211–214PubMedCrossRefGoogle Scholar
  47. 47.
    Luthman J, Humpel C (1997) Pentylenotetrazol kindling decreases N-methyl-D-aspartate and kainate but increases gamma-aminobutyric acid-A receptor binding in discrete rat brain areas. Neurosci Let 239:9–12CrossRefGoogle Scholar
  48. 48.
    Hauger R, Luu HMD, Meyer DK, Goodwin FK, Paul SM (1985) Characterization of “high-affinity” [3H]ouabain binding in the rat central nervous system. J Neurochem 44(6):1709–1715PubMedCrossRefGoogle Scholar
  49. 49.
    Pôças ESC, Costa PRR, Silva AJM, Noel F (2003) 2-Methoxy-3,8,9-trihydroxy coumestan: a new synthetic inhibitor of Na + , K + -ATPase with an original mechanism of action. Biochem Pharmacol 66:2169–2176PubMedCrossRefGoogle Scholar
  50. 50.
    Maki AA, Baskin DG, Stahl WL (1992) [3H]-Ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography. J Histochem Cytochem 40(6):771–779PubMedCrossRefGoogle Scholar
  51. 51.
    Clapcote SJ, Duffy S, Xie G, Kirshenbaum G, Bechard AR, Schack VR et al (2009) Mutation 1810N in the alfa3 isoform of Na+, K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS. PNAS 106(33):14085–14090PubMedCrossRefGoogle Scholar
  52. 52.
    Arnaiz GRL, Reinés A, Herbin T, Peña C (1998) Na + , K + -ATPase interaction with a brain endogenous inhibitor (endobain E). Neurochem Int 33:425–433CrossRefGoogle Scholar
  53. 53.
    Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T (1992) Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 44(3):377–399PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Marcos Brandão Contó
    • 1
  • José Gilberto Barbosa de Carvalho
    • 1
  • Marco Antonio Campana Venditti
    • 1
  1. 1.Departamento de PsicobiologiaUniversidade Federal de São Paulo, Escola Paulista de Medicina (UNIFESP/EPM)São PauloBrazil

Personalised recommendations