Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Efflux of γ-aminobutyric acid caused by changes in ion concentrations and cell swelling simulating the effect of cerebral ischaemia

  • 48 Accesses

  • 5 Citations


The relationships among ischaemic GABA efflux from brain tissue and extracellular and intracellular concentrations of sodium, chloride and potassium ions were investigated by means of 1) transverse hippocampal slices from rat and 2) functional expression of a high affinity GABA transporter inXenopus oocytes. Brain slices were incubated for 20 min in medium where extracellular sodium and chloride were substituted with impermeant ions. Isethionate (Iseth) substitution for chloride generated a 7-fold increase in GABA efflux. Choline (Chol) but not N-methyl-D-glucamine (NMDG) substitution for sodium likewise increased GABA efflux. Reducing the osmolarity of the medium by decreasing both sodium and chloride concentrations (Hyp) increased GABA efflux 3-fold. This release was blocked by mannitol (Man). Blocking sodium channels with 1 μM of tetrodotoxin (TTX) also increased the release 3-fold. Energy deprivation (ED) increased the GABA release 50-fold. ED/Iseth left the release unchanged, ED/Chol increased the GABA efflux by 23%, whereas ED/NMDG reduced the release by 41%. Adding mannitol did not block the ED-evoked release, whereas TTX reduced it by 52%. Release of preloaded [3H]-GABA from oocytes expressing the GAT-1 GABA transporter was then examined. Depolarisation by current injection or 100 mM extracellular K+ did not increase GABA release. Sodium chloride injection, however, caused membrane depolarisation and a 100-fold increased GABA efflux from the oocytes. This release was blocked when the osmolarity was increased extracellularly by adding mannitol. These results show that 1) TTX releases GABA from brain tissue but blocks release during ED, 2) the high affinity GABA carrier must be altered in order to reverse, 3) ischaemic GABA release is sodium independent, and is modulated by large cations, 4) mannitoi blocks the reversal of high affinity carriers in oocytes, but the release from brain slices during ED is unaffected. Taken together, the results suggest that ischaemic release of GABA from brain tissue does not occur by means of reversed high affinity carriers alone, but rather that it is controlled by more complex mechanisms.

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

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.


  1. 1.

    Andersen P, Silfvenius H, Sundberg SH, Sveen O, Wigström H (1978) Functional characteristics of unmyelinated fibres in the hippocampal cortex. Brain Res 144: 11–18

  2. 2.

    Attwell D, Barbour B, Szatkowski M (1993) Nonvesicular release of neurotransmitter. Neuron 11: 401–407

  3. 3.

    Attwell D, Sarantis M, Barbour B, Brew H (1989) Electrogenic glutamate uptake in amphibian and mammalian retinal glial cells. Acta Physiol Scand 136 [Suppl 582]: 44

  4. 4.

    Barbour B, Brew H, Attwell D (1988) Electrogenic glutamate uptake in glial cells is activated by intracellular potassium. Nature 335: 433–435

  5. 5.

    Bedard E, Morris CE (1992) Channels activated by stretch in neurons of a helix snail. Can J Physiol Pharmacol 70: 207–213

  6. 6.

    Berg-Johnsen J, Grøndahl TØ, Langmoen IA, Haugstad TS, Hegstad E (1995) Changes in amino acid release and membrane potential during cerebral hypoxia and glucose deprivation. Neurol Res 17: 201–208

  7. 7.

    Bernath S (1992) Calcium-independent release of amino acid neurotransmitters: fact or artifact? Progr Neurobiol 38: 57–91

  8. 8.

    Billups B, Attwell D (1996) Modulation of non-vesicular glutamate release by pH. Nature 379: 171–174

  9. 9.

    Bormann J (1988) Electrophysiology of GABAA and GABAB receptor subtypes. Trends Neurosci 11: 112–116

  10. 10.

    Bouvier M, Szatkowski M, Amato A, Attwell D (1992) The glial cell glutamate uptake carrier countertransports pH-changing anions. Nature 360: 471–474

  11. 11.

    Brew H, Attwell D (1987) Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells. Nature 327: 707–709

  12. 12.

    Burke SP, Taylor CP (1992) Hippocampal glutamate release during “in vitro ischemia” is calcium-independent and TTX-sensitive. Soc Neurosci Abstr 17: 1267

  13. 13.

    Cammack JN, Rakhilin SV, Schwartz EA (1994) A GABA transporter operates asymmetrically and with variable stochiometry. Neuron 13: 949–960

  14. 14.

    Casado M, Bendahan A, Zafra F, Danbolt NC, Aragón C, Giménez C, Kanner BI (1993) Phosphorylation and modulation of brain glutamate transporters by protein kinase C. J Biol Chem 268: 27313–27317

  15. 15.

    Casado M, Zafra F, Aragón C, Giménez C (1991) Activation of high-affinity uptake of glutamate by phorbol esters in primary glial cell cultures. J Neurochem 57: 1185–1190

  16. 16.

    Catterall WA (1980) Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Ann Rev Parmacol Toxicol 20: 15–43

  17. 17.

    Chang DC, Liu J (1985) A comparative study of the effects of tetrodotoxin and the removal of external Na+ on the resting potential: evidence of separate pathways for the resting and excitable Na+ currents in squid axon. Cell Mol Neurobiol 5: 311–320

  18. 18.

    Erecinska M (1987) The neurotransmitter amino acid transport systems. A fresh outlook on an old problem. Biochem Pharmacol 36: 3547–3555

  19. 19.

    Fairman WA, Vandenberg RJ, Arizza JL, Kavanaugh MP, Amara SG (1995) An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature 375: 599–603

  20. 20.

    Godel H, Graser T, Földi P, Pfaender P, Fürst P (1984) Measurement of free amino acids in human biological fluids by high-performance liquid chromatography. J Chromatogr 297: 49–61

  21. 21.

    Grøndahl TØ, Langmoen IA (1993) Possible involvement of chloride ions in cerebral ischemic injury. J Cereb Blood Flow Metab 13: S88

  22. 22.

    Guastella J, Brecha N, Weigmann C, Lester HA, Davidson N (1992) Cloning, expression and localization of a rat brain highaffinity glycine transporter. Proc Natl Acad Sci USA 89: 7189–7193

  23. 23.

    Guastella J, Nelson N, Nelson H, Czyzyk L, Keynan S, Miedel MC, Davidson N, Lester HA, Kanner BI (1990) Cloning and expression of a rat brain GABA transporter. Science 249: 1303–1306

  24. 24.

    Guhary F, Sachs F (1984) Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J Physiol 352: 685–701

  25. 25.

    Hansen AJ (1985) Effect of anoxia on ion distribution in the brain. Physiol Rev 65: 101–148

  26. 26.

    Hansen AJ, Zeuthen T (1981) Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex. Acta Physiol Scand 113: 437–445

  27. 27.

    Haugstad TS, Hegstad E, Langmoen IA (1992) Calcium dependent release of γ-aminobutyric acid (GABA) from human cerebral cortex. Neurosci Lett 141: 61–64

  28. 28.

    Haugstad TS, Langmoen IA (1996) Release of brain amino acids during hyposmolar stress and energy deprivation. J Neurosurg Anesth 8: 159–168

  29. 29.

    Haugstad TS, Valø ET, Langmoen IA (1995) Changes in brain amino acid content induced by hyposmolar stress and energy deprivation. Neurol Res 17: 402–408

  30. 30.

    Hegstad E, Berg-Johnsen J, Haugstad TS, Hauglie-Hanssen E, Langmoen IA (1996) Amino-acid release from human cerebral cortex during simulated ischaemiain vitro. Acta Neurochir (Wien) 138: 234–241

  31. 31.

    Hille B (1992) Ionic channels of excitable membranes, 2nd Ed. Sinauer, Sunderland, MA

  32. 32.

    Ikeda M, Nakazawa T, Abe K, Kaneko T, Yamatsu K (1989) Extracellular accumulation of glutamate in the hippocampus induced by ischemia is not calcium dependent — in vitro and in vivo evidence. Neurosci Lett 96: 202–206

  33. 33.

    Islas L, Pasantes-Morales H, Sanchez JA (1993) Characterization of stretch-activated ion channels in cultured astrocytes. Glia 8: 87–96

  34. 34.

    Kanai Y, Hediger MA (1992) Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360: 467–471

  35. 35.

    Katayama Y, Kawamata T, Tamura T, Hovda DA, Becker DP, Tsubokawa T (1991) Calcium-dependent glutamate release concomitant with massive potassium flux during cerebral ischemia in vivo. Brain Res 558: 136–140

  36. 36.

    Katz B, Miledi R (1967) The timing of calcium action during neuromuscular transmission. J Physiol 189: 535–544

  37. 37.

    Kimelberg HK, Rose JW, Barron KD, Waniewski RA, Cragoe EJ (1989) Astrocytic swelling in traumatic-hypoxic brain injury. Mol Chem Neuropath 11: 1–31

  38. 38.

    Korf J, Klein HC, Venema K, Postema F (1988) Increase in striatal and hippocampal impedance and extracellular levels of amino acids by cardiac arrest in freely moving rats. J Neurochem 50: 1087–1095

  39. 39.

    Langmoen IA, Andersen P (1981) The hippocampal slicein vitro. A description of the technique and some examples of the opportunities it offers. In: Kerkut GA, Wheal H (eds) Electrophysiology of isolated mammalian CNS preparations. Academic Press, London, pp 51–105

  40. 40.

    Levi G, Raiteri M (1993) Carrier-mediate release of neurotransmitters. Trends Neurosci 16: 415–419

  41. 41.

    Lindroth P, Mopper K (1979) High performance liquid Chromatographie determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal Chem 51: 1667–1674

  42. 42.

    Lui Q-R, López-Corcuera B, Mandiyan S, Nelson H, Nelson N (1993) Molecular characterization of four pharmacologically distinct γ-aminobutyric acid transporters in mouse brain. J Biol Chem 268: 2106–2112

  43. 43.

    Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275

  44. 44.

    Mager S, Naeve J, Quick M, Labarca C, Davidson N, Lester H (1993) Steady states, charge movements and rates for a cloned GABA transporter expressed in Xenopus oocytes. Neuron 10: 177–188

  45. 45.

    Martinac B, Buechner M, Delcour AH, Adler J, Kung C (1987) Pressure-sensitive ion channel inEscherichia coli. Proc Natl Acad Sci USA 84: 2297

  46. 46.

    Nicholls D, Attwell D (1990) The release and uptake of excitatory amino acids. Trends Pharmacol Sci 11: 462–468

  47. 47.

    Osikowska-Evers BA, Wilhelm D, Nebel U, Hennemann P, Scheufler E, Tegtmeier F (1995) The effects of the novel neuroprotective compound lubeluzole on sodium current and veratridine induced sodium load in rat brain neurons and synaptososmes. J Cereb Blood Flow Metab 15: S380

  48. 48.

    Piccolino M, Pignatelli A (1996) Calcium-independent synaptic transmission: artifact or fact. Trends Neurosci 19: 120–125

  49. 49.

    Pines G, Danbolt NC, Bjørås M, Zhang Y, Bendahan A, Eide L, Koepsell H, Storm-Mathisen J, Seeberg E, Kanner BI (1992) Cloning and expression of a rat brain L-glutamate transporter. Nature 360: 464–467

  50. 50.

    Rothman SM (1985) The neurotoxicity of excitatory amino acids is produced by passive chloride influx. J Neurosci 5: 1483–1489

  51. 51.

    Sánchez-Olea R, Morán J, Schousboe A, Pasantes-Morales H (1991) Hyposmolarity-activated fluxes of taurine in astrocytes are mediated by diffusion. Neurosci Lett 130: 2336

  52. 52.

    Scheller D, Kolb J, Szathmary S, Zacharias E, de Ryck M, Reempts Jv, Clincke G, Tegtmeier F (1995) Extracellular changes of glutamate in the periinfarct zone. Effect of lubeluzole. J Cereb Blood Flow Metab 15: S379

  53. 53.

    Siesjö BK (1992) Pathophysiology and treatment of focal cerebral ischemia — part I: pathophysiology. J Neurosurg 77: 169–184

  54. 54.

    Snutch TP (1988) The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci 11: 250–256

  55. 55.

    Solis JM, Herranz AS, Herreras O, Lerma J, del Rio RM (1988) Does taurine act as an osmoregulatory substance in the rat brain? Neurosci Lett 91: 53–58

  56. 56.

    Sontheimer H, Fernandez-Marques E, Ullrich N, Pappas CA, Waxman SG (1994) Astrocyte Na+ channels are required for maintenance of Na+/K+-ATPase activity. J Neurosci 14: 2464–2475

  57. 57.

    Sontheimer H, Waxman S (1993) Expression of voltage-activated ion channels by astrocytes and oligodendrocytes in the hippocampal slice. J Neurophysiol 70: 1863–1873

  58. 58.

    Storck T, Schulte S, Hofmann K, Stoffel W (1992) Structure, expression and functional analysis of a Na+-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci USA 89: 10955–10959

  59. 59.

    Sukharev SI, Blount P, Martinac B, Blattner FR, Kung C (1994) A large-conductance mechanosensitive channel inE. coli encoded bymscL alone. Nature 368: 265–268

  60. 60.

    Szatkowski M, Barbour B, Attwell D (1990) Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348: 443–446

  61. 61.

    Taylor CP (1993) Na+ currents that fail to inactivate. Trends Neurol Sci 16: 455–460

  62. 62.

    Taylor CP, Weber M (1992) Effect of sodium channel modulating drugs on loss of EPSPs from hypoxial/hypoglycemia in rat hippocampal slices in vitro. Soc Neurosci Abstr 18: 1135

  63. 63.

    Thoroed SM (1992) Taurine transport and volume regulation of flounder erythrocytes under hyposmotic conditions. PhD Thesis, University of Oslo, Department of Biology, Division of General Physiology

  64. 64.

    Trotti D, Rossi D, Gjesdal O, Levy LM, Racagni G, Danbolt NC, Volterra A (1996) Peroxynitrite inhibits glutamate transporter subtypes. J Biol Chem 271: 5976–5979

  65. 65.

    Uhl GR (1992) Neurotransmitter transporters (plus): a promising new gene family. Trends Neurosci 15: 265–268

  66. 66.

    Wahizu Y (1971) Some effects of clonidine, procaine and tetrodotoxin on crayfish sensory neuron. Eur J Pharmacol 14: 384–388

  67. 67.

    Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217: 1214–1222

  68. 68.

    Aas J-E, Berg-Johnsen J, Hegstad E, Laake JH, Langmoen IA, Ottersen OP (1993) Redistribution of glutamate and glutamine in slices of human neocortex exposed to combined hypoxia and glucose deprivation in vitro. J Cereb Blood Flow Metab 13: 503–515

Download references

Author information

Correspondence to T. S. Haugstad.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Haugstad, T.S., Karlsen, H.E., Krajtči, P. et al. Efflux of γ-aminobutyric acid caused by changes in ion concentrations and cell swelling simulating the effect of cerebral ischaemia. Acta neurochir 139, 453–463 (1997).

Download citation


  • Cerebral ischaemia
  • GABA release
  • brain slice
  • GAT-1