International Journal of Oral Biology

The Korean Academy of Oral Biology (대한구강생물학회)
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L-trans-pyrrolidine-2,4-dicarboxylate (PDC) induces Excitotoxic and Oxidative Neuronal Death in Cultured Cortical Neurons

  • Choi, Seung-Joon (Department of Pharmacology and Center for Biomedical Human Resources (BK 21 Project) Chonnam National University Medical School) ;
  • Hwang, Shin-Ae (Department of Pharmacology and Center for Biomedical Human Resources (BK 21 Project) Chonnam National University Medical School) ;
  • Kim, Do-Kyung (Department of Oral Physiology, Chosun University School of Dentistry) ;
  • Kim, Jong-Keun (Department of Pharmacology and Center for Biomedical Human Resources (BK 21 Project) Chonnam National University Medical School)
  • Published : 2009.06.30
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Abstract

L-trans-pyrrolidine-2,4-dicarboxylate (PDC) is a potent inhibitor of glutamate transporters. In our current study, we investigated whether the neuronal death induced by PDC involves mechanisms other than excitotoxicity in mixed mouse cortical cultures. Cortical cultures at 13-14 days in vitro were used and cell death was assessed by measuring the lactate dehydrogenase efflux into bathing media. Glutamate and PDC both induced neuronal death in a concentration-dependent manner but the neurotoxic effects of glutamate were found to be more potent than those of PDC. Treatment with 10, 100 and 200 ${\mu}$M PDC equally potentiated 50 ${\mu}$M glutamate-induced neuronal death. The neuronal death induced by 75 ${\mu}$M glutamate was almost abolished by treatment with the NMDA antagonists, MK-801 and AP-5, but was unaffected by NBQX (an AMPA antagonist), trolox (antioxidant), BDNF or ZVAD-FMK (a pan-caspase inhibitor). However, the neuronal death induced by 200 ${\mu}$M PDC was partially but significantly attenuated by single treatments with MK-801, AP-5, trolox, BDNF or ZVAD-FMK but not NBQX. Combined treatments with MK-801 plus trolox, MK-801 plus ZVAD-FMK or MK-801 plus BDNF almost abolished neuronal death, whereas combined treatments with trolox plus ZVADFMK, trolox plus BDNF or ZVAD-FMK plus BDNF did not enhance the inhibitory action of any single treatment with these drugs. These results demonstrate that the neuronal death induced by PDC involves not only in the excitotoxicity induced by the accumulation of glutamate but also the oxidative stress induced by free radical generation. This suggests that apoptotic neuronal death plays a role in PDCinduced oxidative neuronal injury.

Keywords

References

  1. Bamji SX, Majdan M, Pozniak CD, Belliveau DJ, Aloyz R, Kohn J, Causing CG, Miller FD. The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J Cell Biol. 1998;140: 911-23 https://doi.org/10.1083/jcb.140.4.911
  2. Blitzblau R, Gupta S, Djali S, Robinson MB, Rosenberg PA. The glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures. Eur J Neurosci. 1996;8:1840-52 https://doi.org/10.1111/j.1460-9568.1996.tb01328.x
  3. Choi DW. Excitotoxic cell death. J Neurobiology. 1992;23:1261-76 https://doi.org/10.1002/neu.480230915
  4. Chow HC, Lynch III JJ, Rose K, Choi DW. Trolox attenuates cortical neuronal injury induced by iron, ultraviolet light, glucose deprivation, or AMPA. Brain Res. 1994;639:102-8 https://doi.org/10.1016/0006-8993(94)91769-8
  5. Ekert PG, Silke J, Vaux DL. Caspase inhibitors. Cell Death Differentiation. 1999;6:1081-6 https://doi.org/10.1038/sj.cdd.4400594
  6. Estevez AG, Spear N, Manuel SM, Radi R, Henderson CE, Barbeito L, Beckman JS. Nitric oxide and superoxide contribute to motor neuron apoptosis induced by trophic factor deprivation. J Neurosci. 1998;18:923-31
  7. Griffiths R, Dunlop J, Gorman A, Senior J, Grieve A. L-transpyrrolidine-2,4- dicarboxylate and cis-1-aminocyclobutane-1,3-dicarboxylate behave as transportable, competitive inhibitors of the high-affinity glutamate transporters. Biochem Pharmacol. 1994;47:267-74 https://doi.org/10.1016/0006-2952(94)90016-7
  8. Harris ME, Wang Y, Pedigo NJ, Hensley K, Butter.eld DA, Carney JM. Amyloid beta peptide (25-35) inhibits Nadependent glutamate uptake in rat hippocampal astrocyte cultures. J Neurochem. 1996;67:277-86 https://doi.org/10.1046/j.1471-4159.1996.67010277.x
  9. Himi T, Ikeda M, Yasuhara T, Murota SI. Oxidative neuronal death caused by glutamate uptake inhibition in cultured hippocampal neurons. J Neurosci Res. 2003;71:679-88 https://doi.org/10.1002/jnr.10510
  10. Huang PP, Esquenazi S, Le Roux PD. Cerebral cortical neuron apoptosis after mild excitotoxic injury in vitro: different roles of mesencephalic and cortical astrocytes. Neurosurgery. 1999;45:1413-22 https://doi.org/10.1097/00006123-199912000-00029
  11. Lin CL, Bristol LA, Jin L, Dykes HM, Crawford T, Clawson L, Rothstein JD. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998;20:589-602 https://doi.org/10.1016/S0896-6273(00)80997-6
  12. Lievens J-C, Dutertre M, Forni C, Salin P, Goff LK-L. Continuous administration of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate produces striatal lesion. Brain Res Mol Brain Res. 1997;50:181-9 https://doi.org/10.1016/S0169-328X(97)00182-4
  13. Massieu L, Moralesvillagran A, Tapia R. Accumulation of extracellular glutamate by inhibition of its uptake is not sufficient for inducing neuronal damage: an in vivo microdialysis study. J Neurochem. 1995;64:2262-72 https://doi.org/10.1046/j.1471-4159.1995.64052262.x
  14. Montiel T, Camacho A, Estrada-Sánchez AM, Massieu L. Differential effects of the substrate inhibitor l-transpyrrolidine-2,4-dicarboxylate (PDC) and the non-substrate inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) of glutamate transporters on neuronal damage and extracellular amino acid levels in rat brain in vivo. Neuroscience. 2005;133:667-78 https://doi.org/10.1016/j.neuroscience.2004.11.020
  15. Nakagawa T, Otsubo Y, Yatani Y, Shirakawa H, Kaneko S. Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial-neuronal cultures. Eur J Neurosci. 2008;28:1719-30 https://doi.org/10.1111/j.1460-9568.2008.06494.x
  16. Nicholls D, Attwell D. The release and uptake of excitatory amino acids. Trends Pharmacol Sci. 1990;11:462-8 https://doi.org/10.1016/0165-6147(90)90129-V
  17. Padmanabhan J, Park DS, Greene LA, Shelanski ML. Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis. J Neurosci. 1999;19: 8747-56
  18. Robinson MB, Djali S, Buchhalter JR. Inhibition of glutamate uptake with L-trans-pyrrolidine-2,4- dicarboxylate potentiates glutamate toxicity in primary hippocampal cultures. J Neurochem. 1993;61:2099-103 https://doi.org/10.1111/j.1471-4159.1993.tb07447.x
  19. Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischemia is mainly by reversed uptake. Nature. 2000;403:316-21 https://doi.org/10.1038/35002090
  20. Schousboe A, Sonnewald U, Civenni G, Gegelashvili G. Role of astrocytes in glutamate homeostasis. Implications for excitotoxicity. Adv Exp Med Biol. 1997;429:195-206
  21. Son Y, Chun SW. Effects of hydrogen peroxide on neuronal excitability and synaptic transmission in rat substantia gelatinosa neurons. Int J Oral Biol. 2007;32:153-160
  22. Springer JE, Azbill RD, Mark RJ, Begley JG, Waeg G, Mattson MP. 4-hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake. J Neurochem. 1997;68:2469-76 https://doi.org/10.1046/j.1471-4159.1997.68062469.x
  23. Tan S, Wood M, Maher P. Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells. J Neurochem. 1998;71:95-105 https://doi.org/10.1046/j.1471-4159.1998.71010095.x
  24. Thornberry NA. Caspases: a decade of death research. Cell Death Differentiation. 1999;6:1023-7 https://doi.org/10.1038/sj.cdd.4400607
  25. Velasco I, Tapia R, Massieu L. Inhibition of glutamate uptake induces progressive accumulation of extracellular glutamate and neuronal damage in rat cortical cultures. J Neurosci Res. 1996;44:551-61 https://doi.org/10.1002/(SICI)1097-4547(19960615)44:6<551::AID-JNR5>3.0.CO;2-A
  26. Volterra A, Bezzi P, Rizzini BL, Trotti D, Ullensvang K, Danbolt NC, Racagni G. The competitive transporter Ltrans-pyrrolidine-2,4-dicarboxylate triggers excitotoxicity in rat cortical neuron-astrocyte co-cultures via glutamate release rather than uptake inhibition. Eur J Neurosci. 1996;8:2019-28 https://doi.org/10.1111/j.1460-9568.1996.tb01345.x
  27. Wang GJ, Chung HJ, Schnuer J, Pratt K, Zable AC, Kavanaugh MP, Rosenberg PA. High affinity glutamate transport in rat cortical neurons in culture. Mol Pharmacol. 1998;53:88-96 https://doi.org/10.1124/mol.53.1.88