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. 2015:2015:269371.
doi: 10.1155/2015/269371. Epub 2015 Apr 9.

Regulation of System xc(-) by Pharmacological Manipulation of Cellular Thiols

Rebecca Albano  1 , Nicholas J Raddatz  1 , Julie Hjelmhaug  1 , David A Baker  1 , Doug Lobner  1
Affiliations

Regulation of System xc(-) by Pharmacological Manipulation of Cellular Thiols

Rebecca Albano et al. Oxid Med Cell Longev. 2015.

Abstract

The cystine/glutamate exchanger (system xc (-)) mediates the transport of cystine into the cell in exchange for glutamate. By releasing glutamate, system xc (-) can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and may protect cells against oxidative stress. We tested two different compounds that deplete primary cortical cultures containing both neurons and astrocytes of intracellular GSH, L-buthionine-sulfoximine (L-BSO), and diethyl maleate (DEM). Both compounds caused significant concentration and time dependent decreases in intracellular GSH levels. However; DEM caused an increase in radiolabeled cystine uptake through system xc (-), while unexpectedly BSO caused a decrease in uptake. The compounds caused similar low levels of neurotoxicity, while only BSO caused an increase in oxidative stress. The mechanism of GSH depletion by these two compounds is different, DEM directly conjugates to GSH, while BSO inhibits γ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. As would be expected from these mechanisms of action, DEM caused a decrease in intracellular cysteine, while BSO increased cysteine levels. The results suggest that negative feedback by intracellular cysteine is an important regulator of system xc (-) in this culture system.

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Figures

Figure 1
Figure 1
Diethyl maleate (DEM) exposure causes an increase, while buthionine sulfoximine (BSO) exposure causes a decrease, in 14C-cystine uptake in mixed cortical cultures. Cultures were exposed to varying concentrations of DEM or BSO for (a) 40 min, (b) 6 hrs, or (c) 24 hrs, after thorough washing, 14C-cystine uptake was measured for 20 minutes. Results are expressed as mean + SEM (n = 8–16) after normalizing to untreated control uptake. ∗ indicates significant difference from control; P < 0.05.
Figure 2
Figure 2
DEM induced increase in 14C-cystine uptake is mediated by system xc −. Cultures were exposed to 100 μM DEM for 40 min, 6 hrs, or 24 hrs, after thorough washing, 14C-cystine uptake was measured for 20 minutes with or without the system xc − inhibitor sulfasalazine (SSZ) present. Results are expressed as mean + SEM (n = 8–16) after normalizing to untreated control uptake. ∗ indicates significant difference from control; P < 0.05.
Figure 3
Figure 3
DEM and BSO cause similar low levels of neurotoxicity. Concentration response curve for 24 hr exposure to DEM and BSO on LDH release in primary cortical cultures. Results are expressed as mean + SEM (n = 8–16). ∗ indicates significant difference from untreated control; P < 0.05.
Figure 4
Figure 4
BSO, but not DEM, causes an increase in cellular oxidative stress as measured by DCF fluorescence after 24 hour treatment. Bars show % DCF fluorescence normalized to control fluorescence (mean + SEM, n = 8–16). ∗ indicates significant difference from control. P < 0.05.
Figure 5
Figure 5
DEM and BSO cause a concentration dependent decrease in cellular glutathione levels. Cultures were exposed to varying concentrations of DEM or BSO for (a) 40 min, (b) 6 hrs, or (c) 24 hrs, after which cellular reduced glutathione was determined by MCB fluorescence. Bars show % MCB fluorescence normalized to control fluorescence (mean + SEM, n = 8–16). ∗ indicates significant difference from control. P < 0.05.
Figure 6
Figure 6
DEM causes an early decrease in cellular cysteine levels, while BSO causes an early and late increase in cellular cysteine levels. Cultures were exposed to varying concentrations of DEM or BSO for (a) 6 hrs or (b) 24 hrs, after which cellular cysteine levels were determined by HPLC. Bars show % cellular cysteine normalized to control (mean + SEM, n = 8). ∗ indicates significant difference from control P < 0.05.
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