Phenotype-dependent regulation of the system x_C- cystine/glutamate exchanger by glutathione levels in rat astrocyte primary cultures

Author

Todd Seib, The University of Montana

Year of Award

2010

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Richard J. Bridges

Commitee Members

Barrell Jackson, Fernando Cardozo-Paelez, Jean Pfau, Michele McGuirl

Keywords

Camp, glia, thiols, transport

Abstract

The system x_C- (Sx_C-) transporter functions under normal physiological conditions in astrocytes to mediate the exchange of extracellular cystine (L-Cys₂) for intracellular glutamate (L-Glu). The internalized L-Cys₂ serves as a rate-limiting precursor in the biosynthesis of glutathione (GSH), while the externalized L-Glu can access EAA receptors and contribute to either excitatory signaling or excitotoxcity. Although the regulation of Sx_C- has been studied in a variety of cells, particularly as related to xenobiotic exposure, much less is known about its regulation in astrocytes. In the present study the influence of phenotype (culturing in the presence of dibutyryl-cAMP) and GSH levels on the expression of Sx_C- was investigated in cultures of primary astrocytes prepared from neonatal rats. We report that Sx_C- activity in the dbcAMP-treated cells was nearly 7-fold greater than in untreated astrocytes (100 ± 21 and 15 ± 4 pmol/min/mg protein respectively) and that this uptake was further upregulated (~3-fold) in these cells following the depletion of intracellular GSH levels with buthionine sulfoximine (BSO, 500 μM, 24 hrs). Changes in Sx_C- activity correlated with: increases in both protein and mRNA levels of the xCT subunit of the Sx_C- heterodimer, an increase in the Vmax for Sx_C---mediated L-Glu uptake (147 ± 5 in untreated astrocytes to 350 ± 15 pmol/min/mg protein in dbcAMP-treated), and was linked temporally to alterations in GSH levels. The GSH depletion-induced induction of Sx_C- was not mimicked by tBHQ or non-specific oxidants and was partially preventable by the co-administration of cell permeant thiols GSH-ethyl ester (5 mM) and N-acetylcysteine (NAC). These findings demonstrate that the expression of Sx_C- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx_C- activity in CNS injury and disease.

Recommended Citation

Seib, Todd, "Phenotype-dependent regulation of the system x_C- cystine/glutamate exchanger by glutathione levels in rat astrocyte primary cultures" (2010). Graduate Student Theses, Dissertations, & Professional Papers. 747.
https://scholarworks.umt.edu/etd/747