Abstract
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.
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.
| Original language | English |
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| Title of host publication | Belgian Society of Fundamental and Clinical Physiology and Pharmacology, Brussels, Belgium, March 16, 2012 |
| Pages | P-14 |
| Publication status | Published - 16 Mar 2012 |
| Event | Belgian Society of Fundamental and Clinical Physiology and Pharmacology - Brussels, Belgium Duration: 16 Mar 2012 → … |
Conference
| Conference | Belgian Society of Fundamental and Clinical Physiology and Pharmacology |
|---|---|
| Country/Territory | Belgium |
| City | Brussels |
| Period | 16/03/12 → … |
Keywords
- system xc-
- glutamate
- glutathione
- mouse hippocampus