The cystine/glutamate antiporter or system xc- is a membrane-bound Na+-independent amino acid transporter that is structurally composed of a heavy chain subunit common to all amino acid transporters, 4F2, and a light chain specific subunit, xCT [1]. System xc- exchanges intracellular glutamate for extracellular cystine, thereby contributing to intracellular glutathione (GSH) synthesis and non-vesicular glutamate release [2]. The role of system xc- in non-vesicular hippocampal glutamate release was never investigated. We here provide the first evidence that system xc- is the major source of extracellular hippocampal glutamate in mice.

Semi-quantitative western blotting was performed on hippocampal tissue of young and old xCT+/+ and xCT-/- mice [3] using guinea pig antibody to VGLUT1 (1:2000; Millipore); mouse antibody to VGLUT2 (1:5000; Millipore); rabbit antibody to VGLUT3 (1/1000; Synaptic Systems); rabbit antibody to GLT-1 (1:30000) [4]; rabbit antibody to GLAST (1:4000)[5]; rabbit antibody to EAAC1 (1:1000; Alpha Diagnostic); rabbit antibody to xCT (1:10000) [6]; mouse antibody to synaptophysine (1:5000; Stressgen); rabbit antibody to GAPDH (1:5000; Santa Cruz); mouse antibody to GAPDH (1:15000; Millipore). In vivo microdialysis was performed in mouse hippocampus by implanting a microdialysis guide (CMA/7) 2 mm above final probe membrane location with the following coordinates relative to bregma: L +3.0, AP -2.7 and DV -1.5. Samples were collected at flow rate of 2 µl/min every 20 min. Glutamate and aspartate content were determined using HPLC.

We observed a significant effect of genotype on glutamate dialysate concentrations with significantly lower glutamate concentrations in baseline dialysis samples obtained from young as well as old xCT-/- mice (young: 0.079 ± 0.022 µM; old: 0.084 ± 0.015 µM) compared to their age-matched xCT+/+ littermates (young: 0.208 ± 0.036 µM; old: 0.191 ± 0.034 µM). We measured in the same dialysates the levels of aspartate, which is not a substrate for system xc- [7], as a negative control. No difference in extracellular aspartate levels could be observed between xCT+/+ and xCT-/- mice, independent of age. We studied possible compensatory changes in hippocampal protein expression of the major glial glutamate transporters GLT-1 and GLAST, the neuronal EAAC1 transporter that can also function as a cysteine transporter, and the vesicular glutamate transporters (VGLUT1-3) in response to xCT gene deletion. Our findings demonstrate that, although as a result of ageing all transporters related to glutamate reuptake as well as two vesicular glutamate transporters are downregulated in hippocampus, protein expression levels of all glutamate transporters are unaffected by genotype and that no compensatory up- or down-regulations are observed due to the loss of xCT protein. In conclusion these novel findings sustain that system xc- is an important source of extracellular glutamate in the hippocampus.

[1] Sato H et al. (1999) J Biol Chem 247: 11455-11468. [2] Bannai S. (1986) J Biol Chem 261: 2256-2263.
[3] Sato H et al. (2005) J Biol Chem. 280(45):37423-37429 [4] Yamada K et al. (1998) J Neurosci 18: 5706-5713.
[5] Shibata T et al. (1997) J Neurosci 17: 9212-9219. [6] Massie A et al. (2008) Neuroreport 19: 1589-1592.
[7] Patel SA et al. (2004) Neuropharmacology 46: 273-284.
Original languageEnglish
Title of host publicationVijftiende FORUM DER FARMACEUTISCHE WETENSCHAPPEN, Spa, 12-13 mei 2011
Publication statusPublished - 12 May 2011

    Research areas

  • system xc-, glutamate, glutathione, mouse hippocampus

ID: 2259054