System xc- is an important source of extracellular glutamate in mouse hippocampus.

Joeri Van Liefferinge, Anneleen Schallier, Hideyo Sato, Yvette Michotte, Ann Massie, Ilse Julia Smolders

Onderzoeksoutput: Meeting abstract (Book)

Samenvatting

Aim: 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]. We here provide the first evidence that system xc- is the major source of extracellular hippocampal glutamate in mice.

Methods: Semi-quantitative western blotting was performed on hippocampal tissue of young and old xCT+/+ and xCT-/- mice [2] 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) [3]; rabbit antibody to GLAST (1:4000)[4]; rabbit antibody to EAAC1 (1:1000; Alpha Diagnostic); rabbit antibody to xCT (1:10000) [5]; 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 [6].

Results: We observed 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). Our findings demonstrate that protein expression levels of the major glial glutamate transporters GLT-1 and GLAST, the neuronal EAAC1 transporter and the vesicular glutamate transporters (VGLUT1-3) are unaffected by genotype and that no compensatory up- or down-regulations are observed due to the loss of xCT protein.

Conclusion: These novel findings sustain that system xc- is an important source of extracellular glutamate in mouse hippocampus.

[1] Sato H et al. (1999) J Biol Chem 247: 11455-11468. [2] Sato H et al. (2005) J Biol Chem. 280(45):37423-37429 [3] Yamada K et al. (1998) J Neurosci 18: 5706-5713. [4] Shibata T et al. (1997) J Neurosci 17: 9212-9219. [5] Massie A et al. (2008) Neuroreport 19: 1589-1592. [6] Van Hemelrijck A et al. (2005); 144(1):63-71.
Originele taal-2English
TitelBelgian Society of Fundamental and Clinical Physiology and Pharmacology, Brussels, Belgium, March 16, 2012
Pagina'sP-14
StatusPublished - 16 mrt 2012
EvenementBelgian Society of Fundamental and Clinical Physiology and Pharmacology - Brussels, Belgium
Duur: 16 mrt 2012 → …

Conference

ConferenceBelgian Society of Fundamental and Clinical Physiology and Pharmacology
Land/RegioBelgium
StadBrussels
Periode16/03/12 → …

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