Skip to main content
SpringerLink
Log in

The Glutamate Aspartate Transporter (GLAST) Mediates l-Glutamate-Stimulated Ascorbate-Release Via Swelling-Activated Anion Channels in Cultured Neonatal Rodent Astrocytes

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, l-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by l-glutamate and l-aspartate, but not the non-excitatory amino acid l-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na+-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

AMPA:

(±) α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

AO:

Ascorbate oxidase

DDF:

1,9 dideoxyforskolin

DHA:

Dehydroascorbate

DHK:

Dihydrokanic acid

DHPG:

(S)-3,5-dihydroxyphenylglycine

EAAT:

Excitatory amino acid transporter

GLAST:

Glutamate asparate transporter

GLT-1:

Glutamate transporter 1

GLUT:

Facilitative glucose transporter

IAA-94:

R(+) indanyloxyacetic acid

iGluR:

Ionotropic glutamate receptor

mGluR:

Metabotropic glutamate receptor

NFA:

Niflumic acid

NMDA:

N-methyl-d-aspartate

TFB-TBOA:

(2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate

SVCT2:

Sodium-dependent vitamin C transporter, isoform 2

VRAC:

Volume-regulated anion channel

VSOAC:

Volume-sensitive osmolyte and anion channel

References

  1. Grünewald, R. A. (1993). Ascorbic acid in the brain. Brain Research Brain Research Reviews, 18, 123–133.

    Article  PubMed  Google Scholar 

  2. Harrison, F. E., & May, J. M. (2009). Vitamin C function in the brain: Vital role of the ascorbate transporter SVCT2. Free Radical Biology and Medicine, 46, 719–730.

    Article  PubMed  CAS  Google Scholar 

  3. Siushansian, R., Dixon, S. J., & Wilson, J. X. (1996). Osmotic swelling stimulates ascorbate efflux from cerebral astrocytes. Journal of Neurochemistry, 66, 1227–1233.

    Article  PubMed  CAS  Google Scholar 

  4. Wilson, J. X., Peters, C. E., Sitar, S. M., Daoust, P., & Gelb, A. W. (2000). Glutamate stimulates ascorbate transport by astrocytes. Brain Research, 858, 61–66.

    Article  PubMed  CAS  Google Scholar 

  5. Lane, D. J. R., & Lawen, A. (2008). Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells. Journal of Biological Chemistry, 283, 12701–12708.

    Article  PubMed  CAS  Google Scholar 

  6. Rebec, G. V., & Pierce, R. C. (1994). A vitamin as neuromodulator: Ascorbate release into the extracellular fluid of the brain regulates dopaminergic and glutamatergic transmission. Progress in Neurobiology, 43, 537–565.

    Article  PubMed  CAS  Google Scholar 

  7. Hediger, M. A. (2002). New view at C. Nature Medicine, 8, 445–446.

    Article  PubMed  CAS  Google Scholar 

  8. Du, J., Cullen, J. J., & Buettner, G. R. (2012). Ascorbic acid: Chemistry, biology and the treatment of cancer. Biochimica et Biophysica Acta. Epub ahead of print. doi:10.1016/j.bbcan.2012.06.003

  9. Castro, M. A., Beltrán, F. A., Brauchi, S., & Concha, I. I. (2009). A metabolic switch in brain: Glucose and lactate metabolism modulation by ascorbic acid. Journal of Neurochemistry, 110, 423–440.

    Article  PubMed  CAS  Google Scholar 

  10. Rice, M. E. (2000). Ascorbate regulation and its neuroprotective role in the brain. Trends in Neurosciences, 23, 209–216.

    Article  PubMed  CAS  Google Scholar 

  11. Qiu, S., Li, L., Weeber, E. J., & May, J. M. (2007). Ascorbate transport by primary cultured neurons and its role in neuronal function and protection against excitotoxicity. Journal of Neuroscience Research, 85, 1046–1056.

    Article  PubMed  CAS  Google Scholar 

  12. Rice, M. E., & Russo-Menna, I. (1998). Differential compartmentalization of brain ascorbate and glutathione between neurons and glia. Neuroscience, 82, 1213–1223.

    Article  PubMed  CAS  Google Scholar 

  13. Song, J. H., Shin, S. H., & Ross, G. M. (2001). Oxidative stress induced by ascorbate causes neuronal damage in an in vitro system. Brain Research, 895, 66–72.

    Article  PubMed  CAS  Google Scholar 

  14. Kim, E. J., Park, Y. G., Baik, E. J., Jung, S. J., Won, R., Nahm, T. S., et al. (2005). Dehydroascorbic acid prevents oxidative cell death through a glutathione pathway in primary astrocytes. Journal of Neuroscience Research, 79, 670–679.

    Article  PubMed  CAS  Google Scholar 

  15. Wilson, J. X. (1997). Antioxidant defense of the brain: A role for astrocytes. Canadian Journal of Physiology and Pharmacology, 75, 1149–1163.

    Article  PubMed  CAS  Google Scholar 

  16. Swanson, R. A., Ying, W., & Kauppinen, T. M. (2004). Astrocyte influences on ischemic neuronal death. Current Molecular Medicine, 4, 193–205.

    Article  PubMed  CAS  Google Scholar 

  17. Astuya, A., Caprile, T., Castro, M., Salazar, K., García Mde, L., Reinicke, K., et al. (2005). Vitamin C uptake and recycling among normal and tumor cells from the central nervous system. Journal of Neuroscience Research, 79, 146–156.

    Article  PubMed  CAS  Google Scholar 

  18. Lane, D. J. R., & Lawen, A. (2009). Ascorbate and plasma membrane electron transport—Enzymes vs efflux. Free Radical Biology and Medicine, 47, 485–495.

    Article  PubMed  CAS  Google Scholar 

  19. Hilgetag, C. C., & Barbas, H. (2009). Are there ten times more glia than neurons in the brain? Brain Structure and Function, 213, 365–366.

    Article  PubMed  Google Scholar 

  20. Lane, D. J. R., Robinson, S. R., Czerwinska, H., & Lawen, A. (2010). A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane. Biochemical Journal, 428, 191–200.

    Article  PubMed  CAS  Google Scholar 

  21. Clements, J. D. (1996). Transmitter timecourse in the synaptic cleft: Its role in central synaptic function. Trends in Neurosciences, 19, 163–171.

    Article  PubMed  CAS  Google Scholar 

  22. Walton, H. S., & Dodd, P. R. (2007). Glutamate-glutamine cycling in Alzheimer’s disease. Neurochemistry International, 50, 1052–1066.

    Article  PubMed  CAS  Google Scholar 

  23. Marcaggi, P., & Attwell, D. (2004). Role of glial amino acid transporters in synaptic transmission and brain energetics. Glia, 47, 217–225.

    Article  PubMed  Google Scholar 

  24. Tilleux, S., & Hermans, E. (2007). Neuroinflammation and regulation of glial glutamate uptake in neurological disorders. Journal of Neuroscience Research, 85, 2059–2070.

    Article  PubMed  CAS  Google Scholar 

  25. Bunch, L., Erichsen, M. N., & Jensen, A. A. (2009). Excitatory amino acid transporters as potential drug targets. Expert Opinion on Therapeutic Targets, 13, 719–731.

    Article  PubMed  CAS  Google Scholar 

  26. Liang, J., Takeuchi, H., Doi, Y., Kawanokuchi, J., Sonobe, Y., Jin, S., et al. (2008). Excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity. Brain Research, 1210, 11–19.

    Article  PubMed  CAS  Google Scholar 

  27. Danbolt, N. C. (2001). Glutamate uptake. Progress in Neurobiology, 65, 1–105.

    Article  PubMed  CAS  Google Scholar 

  28. Behrens, P. F., Franz, P., Woodman, B., Lindenberg, K. S., & Landwehrmeyer, G. B. (2002). Impaired glutamate transport and glutamate-glutamine cycling: Downstream effects of the Huntington mutation. Brain, 125, 1908–1922.

    Article  PubMed  CAS  Google Scholar 

  29. Sheldon, A. L., & Robinson, M. B. (2007). The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochemistry International, 51, 333–355.

    Article  PubMed  CAS  Google Scholar 

  30. Wilson, J. X., & Dragan, M. (2005). Sepsis inhibits recycling and glutamate-stimulated export of ascorbate by astrocytes. Free Radical Biology and Medicine, 39, 990–998.

    Article  PubMed  CAS  Google Scholar 

  31. May, J. M., Li, L., Hayslett, K., & Qux, Z.-c. (2006). Ascorbate transport and recycling by SH-SY5Y neuroblastoma cells: Response to glutamate toxicity. Neurochemical Research, 31, 785–794.

    Article  PubMed  CAS  Google Scholar 

  32. Davies, A. R. L., Belsey, M. J., & Kozlowski, R. Z. (2004). Volume-sensitive organic osmolyte/anion channels in cancer: Novel approaches to studying channel modulation employing proteomics technologies. Annals of the New York Academy of Sciences, 1028, 38–55.

    Article  PubMed  CAS  Google Scholar 

  33. Shimamoto, K., Sakai, R., Takaoka, K., Yumoto, N., Nakajima, T., Amara, S. G., et al. (2004). Characterization of novel L-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. Molecular Pharmacology, 65, 1008–1015.

    Article  PubMed  CAS  Google Scholar 

  34. Erichsen, M. N., Huynh, T. H. V., Abrahamsen, B., Bastlund, J. F., Bundgaard, C., Monrad, O., et al. (2010). Structure-activity relationship study of first selective inhibitor of excitatory amino acid transporter subtype 1: 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101). Journal of Medicinal Chemistry, 53, 7180–7191.

    Article  PubMed  CAS  Google Scholar 

  35. Jensen, A. A., Erichsen, M. N., Nielsen, C. W., Stensbøl, T. B., Kehler, J., & Bunch, L. (2009). Discovery of the first selective inhibitor of excitatory amino acid transporter subtype 1. Journal of Medicinal Chemistry, 52, 912–915.

    Article  PubMed  CAS  Google Scholar 

  36. Lane, D. J. R., Robinson, S. R., Czerwinska, H., Bishop, G. M., & Lawen, A. (2010). Two routes of iron accumulation in astrocytes: Ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron. Biochemical Journal, 432, 123–132.

    Article  PubMed  CAS  Google Scholar 

  37. Puschmann, T. B., Dixon, K. J., & Turnley, A. M. (2010). Species differences in reactivity of mouse and rat astrocytes in vitro. Neurosignals, 18, 152–163.

    Article  PubMed  CAS  Google Scholar 

  38. Deutsch, J. C. (2000). Dehydroascorbic acid. Journal of Chromatography A, 881, 299–307.

    Article  PubMed  CAS  Google Scholar 

  39. Siushansian, R., Tao, L., Dixon, S. J., & Wilson, J. X. (1997). Cerebral astrocytes transport ascorbic acid and dehydroascorbic acid through distinct mechanisms regulated by cyclic AMP. Journal of Neurochemistry, 68, 2378–2385.

    Article  PubMed  CAS  Google Scholar 

  40. Lane, D. J. R., & Lawen, A. (2008). A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation. Analytical Biochemistry, 373, 287–295.

    Article  PubMed  CAS  Google Scholar 

  41. Berger, U. V., & Hediger, M. A. (2000). The vitamin C transporter SVCT2 is expressed by astrocytes in culture but not in situ. NeuroReport, 11, 1395–1399.

    Article  PubMed  CAS  Google Scholar 

  42. Qiao, H., & May, J. M. (2008). Development of ascorbate transporters in brain cortical capillary endothelial cells in culture. Brain Research, 1208, 79–86.

    Article  PubMed  CAS  Google Scholar 

  43. May, J. M., & Z-c, Qu. (2009). Ascorbic acid efflux and re-uptake in endothelial cells: Maintenance of intracellular ascorbate. Molecular and Cellular Biochemistry, 325, 79–88.

    Article  PubMed  CAS  Google Scholar 

  44. Korzeniewski, C., & Callewaert, D. M. (1983). An enzyme-release assay for natural cytotoxicity. Journal of Immunological Methods, 64, 313–320.

    Article  PubMed  CAS  Google Scholar 

  45. Berridge, M. V., Herst, P. M., & Tan, A. S. (2005). Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnology Annual Review, 11, 127–152.

    Article  PubMed  CAS  Google Scholar 

  46. Wassler, M., Jonasson, I., Persson, R., & Fries, E. (1987). Differential permeabilzation of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins. Biochemical Journal, 247, 407–415.

    PubMed  CAS  Google Scholar 

  47. Chen, C.-J., Liao, S.-L., & Kuo, J.-S. (2000). Gliotoxic action of glutamate on cultured astrocytes. Journal of Neurochemistry, 75, 1557–1565.

    Article  PubMed  CAS  Google Scholar 

  48. Dringen, R., Kussmaul, L., & Hamprecht, B. (1998). Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Research Protocols, 2, 223–228.

    Article  PubMed  CAS  Google Scholar 

  49. Hansson, E., Muyderman, H., Leonova, J., Allansson, L., Sinclair, J., Blomstrand, F., et al. (2000). Astroglia and glutamate in physiology and pathology: Aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication. Neurochemistry International, 37, 317–329.

    Article  PubMed  CAS  Google Scholar 

  50. Mulligan, S. J., & MacVicar, B. A. (2006) VRACs CARVe a path for novel mechanisms of communication in the CNS. Science’s STKE, 2006, pe42.

  51. Gunnarson, E., Zelenina, M., Axehult, G., Song, Y., Bondar, A., Krieger, P., et al. (2008). Identification of a molecular target for glutamate regulation of astrocyte water permeability. Glia, 56, 587–596.

    Article  PubMed  Google Scholar 

  52. Hamilton, N. B., & Attwell, D. (2010). Do astrocytes really exocytose neurotransmitters? Nature Reviews Neuroscience, 11, 227–238.

    Article  PubMed  CAS  Google Scholar 

  53. Ye, Z.-C., Oberheim, N., Kettenmann, H., & Ransom, B. R. (2009). Pharmacological “cross-inhibition” of connexin hemichannels and swelling activated anion channels. Glia, 57, 258–269.

    Article  PubMed  Google Scholar 

  54. Sorota, S. (1994). Pharmacologic properties of the swelling-induced chloride current of dog atrial myocytes. Journal of Cardiovascular Electrophysiology, 5, 1006–1016.

    Article  PubMed  CAS  Google Scholar 

  55. Davis, K. A., Samson, S. E., Best, K., Mallhi, K. K., Szewczyk, M., Wilson, J. X., et al. (2006). Ca2+-mediated ascorbate release from coronary artery endothelial cells. British Journal of Pharmacology, 147, 131–139.

    Article  PubMed  CAS  Google Scholar 

  56. Parkerson, K. A., & Sontheimer, H. (2004). Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes. Glia, 46, 419–436.

    Article  PubMed  Google Scholar 

  57. Patneau, D. K., & Mayer, M. L. (1990). Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-d-aspartate and quisqualate receptors. Journal of Neuroscience, 10, 2385–2399.

    PubMed  CAS  Google Scholar 

  58. Honoré, T., Lauridsen, J., & Krogsgaard-Larsen, P. (1982). The binding of [3H]AMPA, a structural analogue of glutamic acid, to rat brain membranes. Journal of Neurochemistry, 38, 173–178.

    Article  PubMed  Google Scholar 

  59. Wiśniewski, K., & Car, H. (2002). (S)-3,5-DHPG: A review. CNS Drug Reviews, 8, 101–116.

    Article  PubMed  Google Scholar 

  60. Peavy, R. D., & Conn, P. J. (1998). Phosphorylation of mitogen-activated protein kinase in cultured rat cortical glia by stimulation of metabotropic glutamate receptors. Journal of Neurochemistry, 71, 603–612.

    Article  PubMed  CAS  Google Scholar 

  61. Seifert, G., & Steinhäuser, C. (2001). Ionotropic glutamate receptors in astrocytes. Progress in Brain Research, 132, 287–299.

    Article  PubMed  CAS  Google Scholar 

  62. Swanson, R. A., Liu, J., Miller, J. W., Rothstein, J. D., Farrell, K., Stein, B. A., et al. (1997). Neuronal regulation of glutamate transporter subtype expression in astrocytes. Journal of Neuroscience, 17, 932–940.

    PubMed  CAS  Google Scholar 

  63. Bridges, R. J., & Esslinger, C. S. (2005). The excitatory amino acid transporters: Pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes. Pharmacology & Therapeutics, 107, 271–285.

    Article  CAS  Google Scholar 

  64. Rebec, G. V., Barton, S. J., & Ennis, M. D. (2002). Dysregulation of ascorbate release in the striatum of behaving mice expressing the Huntington’s disease gene. Journal of Neuroscience, 22, RC202.

  65. Miller, B. R., Dorner, J. L., Bunner, K. D., Gaither, T. W., Klein, E. L., Barton, S. J., et al. (2012). Up-regulation of GLT1 reverses the deficit in cortically evoked striatal ascorbate efflux in the R6/2 mouse model of Huntington’s disease. Journal of Neurochemistry, 121, 629–638.

    Article  PubMed  CAS  Google Scholar 

  66. Rebec, G. V., Barton, S. J., Marseilles, A. M., & Collins, K. (2003). Ascorbate treatment attenuates the Huntington behavioral phenotype in mice. NeuroReport, 14, 1263–1265.

    Article  PubMed  CAS  Google Scholar 

  67. Han, B. C., Koh, S. B., Lee, E. Y., & Seong, Y. H. (2004). Regional difference of glutamate-induced swelling in cultured rat brain astrocytes. Life Sciences, 76, 573–583.

    Article  PubMed  CAS  Google Scholar 

  68. Kimelberg, H. K., Goderie, S. K., Higman, S., Pang, S., & Waniewski, R. A. (1990). Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures. Journal of Neuroscience, 10, 1583–1591.

    PubMed  CAS  Google Scholar 

  69. Poulsen, M. V., & Vandenberg, R. J. (2001). Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4. Journal of Physiology, 534, 159–167.

    Article  PubMed  CAS  Google Scholar 

  70. Ahmad, S., & Evans, W. H. (2002). Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: Implications for the assembly of gap junctions. Biochemical Journal, 365, 693–699.

    PubMed  CAS  Google Scholar 

  71. Portugal, C. C., Miya, V. S., da Costa Calaza, K., Santos, R. A. M., & Paes-de-Carvalho, R. (2009). Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells. Journal of Neurochemistry, 108, 507–520.

    Article  PubMed  CAS  Google Scholar 

  72. Schlag, B. D., Vondrasek, J. R., Munir, M., Kalandadze, A., Zelenaia, O. A., Rothstein, J. D., et al. (1998). Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. Molecular Pharmacology, 53, 355–369.

    PubMed  CAS  Google Scholar 

  73. Tilleux, S., Gorsaud, S., & Hermans, E. (2009). Selective up-regulation of GLT-1 in cultured astrocytes exposed to soluble mediators released by activated microglia. Neurochemistry International, 55, 35–40.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are thankful to Drs Lennart Bunch and Anders A. Jensen (University of Copenhagen) for the generous gift of UCPH-101, and to A/Prof. Stephen Robinson and Ms. Hania Czerwinska (Monash University) for the generous supply of astrocyte cultures and for helpful comments on the manuscript.

Author information

Author notes

  1. Darius J. R. Lane

    Present address: Iron Metabolism and Chelation Program, Department of Pathology, University of Sydney, Sydney, NSW, 2006, Australia

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne, VIC, 3800, Australia

    Darius J. R. Lane & Alfons Lawen

Authors
  1. Darius J. R. Lane
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alfons Lawen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Darius J. R. Lane or Alfons Lawen.

Additional information

D. J. R. Lane and A. Lawen contributed equally as senior authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lane, D.J.R., Lawen, A. The Glutamate Aspartate Transporter (GLAST) Mediates l-Glutamate-Stimulated Ascorbate-Release Via Swelling-Activated Anion Channels in Cultured Neonatal Rodent Astrocytes. Cell Biochem Biophys 65, 107–119 (2013). https://doi.org/10.1007/s12013-012-9404-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-012-9404-8

Keywords

Search

Navigation