Biological Trace Element Research

, Volume 107, Issue 3, pp 231–245 | Cite as

Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

  • Lysette Mutkus
  • Judy L. Aschner
  • Tore Syversen
  • Michael Aschner
Original Articles


In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na+-dependent EAA transporters glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. Mehg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-3H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p<0.001) increased following treatments with 5 or 10 μM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p<0.001, both at 5 and 10 μM MeHg) increased and GLT-1 transporter protein levels significantly (p<0.001) decreased followign MeHg exposure (5 μM). (3) MeHg exposure led to significant inhibition (p<0.05) of glutamate uptake by GLAST (both 5 and 10 μM MeHg), whereas GLT-1 transporter activity was significantly (p<0.01) increased following exposure to 5 and 10 μM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.

Index Entries

Methylmercury glutamate transport GLAST GLT-1 neurotoxicity 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    F. Bakir, S. F. Damluji, L. Amin-Zaki, et al., Methylmercury poisoning in Iraq. Science 181, 230–241 (1973).PubMedCrossRefGoogle Scholar
  2. 2.
    T. Takeuchi, Biological reactions and pathological changes in human beings and animals caused by organic mercury contamination, in Environmental Mercury Contamination, R. Hartung and B. D. Dinman, eds., Ann Arbor Science, Ann Arbor, MI, pp 247–289 (1972).Google Scholar
  3. 3.
    J. S. Charleston, R. L. Body, R. P. Bolander, et al., Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure, Neurotoxicology 17, 127–138 (1996).PubMedGoogle Scholar
  4. 4.
    K. J. Mullaney, M. N. Fehm, D. Vitarella, et al., The role of −SH groups in methylmercuric chloride-induced d-aspartate and rubidium release from rat primary astrocyte cultures, Brain Res. 641, 1–9 (1994).PubMedCrossRefGoogle Scholar
  5. 5.
    J. W. Allen, L. A. Mutkus, and M. Aschner, Methylmercury-mediated inhibition of 3H-d-asparatate transport in cultured astrocytes is reversed by the antioxidant catalase, Brain Res. 902, 92–100 (2001).PubMedCrossRefGoogle Scholar
  6. 6.
    G. Shanker, J. L. Aschner, T. Syversen, et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128, 48–57 (2004).PubMedCrossRefGoogle Scholar
  7. 7.
    G. Shanker, T. Syversen, J. L. Aschner, et al., Modulatory effect of glutathione status and antioxidants on methylmercury induced free radical formation in primary cultures of cerebral astrocytes, Brain Res. Mol. Brain Res. 137, 11–22 (2005).PubMedCrossRefGoogle Scholar
  8. 8.
    J. D. Rothstein, L. Martin, A. I. Levey, et al., Localization of neuronal and glial glutamate transporters, Neuron 13, 713–725 (1996).CrossRefGoogle Scholar
  9. 9.
    T. Storck, S. Schulte, K. Hofmann, et al., Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain, Proc. Natl. Acad. Sci. USA 89, 10,955–10,959 (1992).CrossRefGoogle Scholar
  10. 10.
    G. Pines, N. C. Danbolt, M. Bjoras, et al., Cloning and expression of a rat brain l-glutamate transporter, Nature 360, 464–467 (1992).PubMedCrossRefGoogle Scholar
  11. 11.
    Y. Kanai and M. A. Hediger, Primary structure and functional characterization of a high-affinity glutamate transporter, Nature 360, 467–471 (1992).PubMedCrossRefGoogle Scholar
  12. 12.
    W. A. Fairman and S. G. Amara, Functional diversity of excitatory amino acid transporters: ion channel and transport modes, Am. J. Physiol. 277, F481-F486 (1999).PubMedGoogle Scholar
  13. 13.
    J. L. Arriza, S. Eliasof, M. P. Kavanaugh, et al., Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl. Acad. Sci. USA 94, 4155–4160 (1997).PubMedCrossRefGoogle Scholar
  14. 14.
    Y. Kanai, Family of neutral and acidic amino acid transporters: molecular biology, physiology and medical implications, Curr. Opin. Cell Biol. 9, 565–572 (1997).PubMedCrossRefGoogle Scholar
  15. 15.
    O. Haugeto, K. Ullensvang, L. M. Levy, et al., Brain glutamate transporter proteins from homomultimers, J. Biol. Chem. 271, 27,715–27,722 (1996).Google Scholar
  16. 16.
    N. C. Danbolt, G. Pines, and B. I. Kanner, Purification and reconstitution of the sodium-and potassium-coupled glutamate transport glycoprotein from rat brain, Biochemistry 29, 6734–6740 (1990).PubMedCrossRefGoogle Scholar
  17. 17.
    K. D. Sims and M. B. Robinson, Expression patterns and regulation of glutamate transporters in the developing and adult nervous system, Crit. Rev. Neurobiol. 13, 169–197 (1999).PubMedGoogle Scholar
  18. 18.
    K. P. Lehre, L. M. Levy, O. P. Ottersen, et al., Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations, J. Neurosci. 15, 1835–1853 (1995).PubMedGoogle Scholar
  19. 19.
    R. Torp, D. Lekieffre, L. M. Levy, et al., Reduced postischemic expression of a glial glutamate transporter, GLT1 in the rat hippocampus, Exp. Brain Res. 103, 51–58 (1995).PubMedCrossRefGoogle Scholar
  20. 20.
    D. V. Pow, N. L. Barnett, and P. Penfold, Are neuronal transporters relevant in retinal glutamate homeostasis? Neurochem. Int. 37, 191–198 (2000).PubMedCrossRefGoogle Scholar
  21. 21.
    A. E. Fray, P. G. Ince, S. J. Banner, et al., The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: and immunohistochemical study. Eur. J. Neurosci. 10, 2481–2489 (1998).PubMedCrossRefGoogle Scholar
  22. 22.
    S. Sasaki, T. Komori, and M. Iwata, Excitatory amino acid transporter 1 and 2 immunore activity in the spinal cord in amyotrophic lateral sclerosis, Acta Neuropathol. Berlin 100, 138–144 (2000).CrossRefGoogle Scholar
  23. 23.
    B. S. Meldrum, The role of glutamate in epilepsy and other CNS disorders, Neurology 44, S14-S23 (1994).PubMedGoogle Scholar
  24. 24.
    K. Miyamoto, H. Nakanishi, S. Moriguchi, et al., Involvement of enhanced sensitivity of N-methyl-d-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity, Brain Res. 901, 252–258 (2001).PubMedCrossRefGoogle Scholar
  25. 25.
    K. Miyamoto, K. Murao, J. Wakamiya, et al., Protective effect of MK-801 in methylmercury-induced neuronal injury, in Mercury as a Global Pollutant. 5th International Conference, p. 376 (1999).Google Scholar
  26. 26.
    M. Lafon-Cazal, S. Pietri, M. Culcasi, et al., NMDA-dependent superoxide production and neurotoxicity, Nature 364, 535–537 (1993).PubMedCrossRefGoogle Scholar
  27. 27.
    W. H. Hughes, A physiochemical rationale for the biological activity of mercury and its compounds, Ann. NY Acad. Sci. 65, 454–460 (1957).PubMedCrossRefGoogle Scholar
  28. 28.
    R. P. Igo and J. F. Ash, New mutations and phenotypes associated with glutamate and aspartate transport in Chinese Hamster ovary (CHO-K1) cells, Somat. Cell. Mol. Genet. 22, 87–103 (1996).PubMedCrossRefGoogle Scholar
  29. 29.
    J. Albrecht, M. Talbot, H. K. Kimelberg, et al., The role of sulfhydryl groups and calcium in the mercuric chloride-induced inhibition of glutamate uptake in rat primary astrocyte cultures, Brain Res. 607, 249–254 (1993).PubMedCrossRefGoogle Scholar
  30. 30.
    M. Aschner, N. B. Eberle, K. Miller, et al., Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling, Brain Res. 530, 245–250 (1990).PubMedCrossRefGoogle Scholar
  31. 31.
    N. Brooks and D. A. Kristt, Inhibition of amino acid transport and protein synthesis by HgCl2 and methylmercury in astrocytes: selectivity and reversibility, J. Neurochem. 53, 1228–1237 (1989).CrossRefGoogle Scholar
  32. 32.
    L. Mutkus, J. L. Aschner, T. Syversen, et al., Mercuric chloride (HgCl2) inhibitis the in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells, Biol. Trace Element Res. To be published. (2005).Google Scholar
  33. 33.
    L. A. Mutkus, J. L. Aschner, T. Syversen, et al., The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative, thimerosal, Biol. Trace Element Res. 105, 71–86 (2005).CrossRefGoogle Scholar
  34. 34.
    O. J. Birot, A. Peinnequin, N. Simler, et al., Vascular endothelial growth factor expression in heart of rats exposed to hypobaric hypoxia: differential response between mRNA and protein, J. Cell Physiol. 200, 107–115 (2004).PubMedCrossRefGoogle Scholar
  35. 35.
    S. Duan, C. M. Anderson, B. A. Stein, et al., Glutamate induces rapid upregulation of astrocyte glutamate transport and cell-surface expression of GLAST. J. Neurosci. 19, 10,193–10,200 (1999).Google Scholar
  36. 36.
    Y. Qian, A. Galli, S. Ramamoorthy, et al., Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression, J. Neurosci. 17, 45–57 (1997).PubMedGoogle Scholar
  37. 37.
    K. E. Davis, D. J. Straff, E. A. Weinstein, et al., Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma, J. Neurosci., 18, 2475–2485 (1998).PubMedGoogle Scholar
  38. 38.
    V. Petronilli, P. Costantini, L. Scorrano, et al., The voltage sensor of the mitochondrial permeability transition pore is tuned by the oxidation-reduction state of vicinal thiols. Increase of the gating potential by oxidants and its reversal by reducing agents, J. Biol. Chem. 269, 16,638–16,642 (1994).Google Scholar
  39. 39.
    Z. H. Pan, R. Bahring, R., Grantyn, et al., Differential modulation by sulfhydryl redox agents and glutathione of GABA- and glycine-evoked currents in rat retinal ganglion cells, J. Neurosci. 15, 1384–1391 (1995).PubMedGoogle Scholar
  40. 40.
    R. P. Seal and S. G. Amara, A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation. Neuron 21, 1487–1498 (1998).PubMedCrossRefGoogle Scholar
  41. 41.
    M. Grunewald, A. Bendahan, and B. I. Kanner, Biotinylation of single cysteine mutant of the glutamate transporter GLT-1 from rat brain reveals its unusual topolgy, Neuron 21, 623–632 (1998).PubMedCrossRefGoogle Scholar
  42. 42.
    R. Zarbiv, M. Grunewald, M. P. Kavanaugh, et al., Cysteine scanning of the surroundings of an alkali-ion binding site of the glutamate transporter GLT-1 reveals a conformationally sensitive residue. J. Biol. Chem. 273, 14,231–14,237 (1998).CrossRefGoogle Scholar
  43. 43.
    D. Trotti, B. L. Rizzini, D. Rossi, et al., Neuronal and glial glutamate transporters possess and SH-based redox regulatory mechanism, Eur. J. Neurosci. 9, 1236–1243 (1997).PubMedCrossRefGoogle Scholar
  44. 44.
    D. Trotti, N. C. Danbolt, and A. Volterra, Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol. Sci. 8, 328–334 (1998).CrossRefGoogle Scholar
  45. 45.
    N. Zerangue, and M. P. Kavanaugh, Flux coupling in a neuronal glutamate transporter, Nature 383, 634–637 (1996).PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2005

Authors and Affiliations

  • Lysette Mutkus
    • 1
  • Judy L. Aschner
    • 2
  • Tore Syversen
    • 4
  • Michael Aschner
    • 2
    • 3
  1. 1.Department of Physiology and PharmacologyWake Forest University School of MedicineWinston-Salem
  2. 2.Department of PediatricsVanderbilt University Medical CenterNashville
  3. 3.Kennedy CenterVanderbilt University Medical CenterNashville
  4. 4.Department of Clinical NeuroscienceNorwegian University of Science and TechnologyTrondheimNorway

Personalised recommendations