A novel approach to enhancing cellular glutathione levels

Abstract

GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of toxic stress. Of the three amino acids that comprise GSH, cysteine is limiting for GSH synthesis. As extracellularly cysteine is readily oxidized to form cystine, cystine transport mechanisms are essential to provide cells with cysteine. Cystine uptake is mediated by system x(c)(-), a Na(+)-independent cystine/glutamate antiporter. Inhibition of system x(c)(-) by millimolar concentrations of glutamate, a pathway termed oxidative glutamate toxicity, results in GSH depletion and nerve cell death. Recently, we described a series of compounds derived from the conjugation of epicatechin (EC) with cysteine and cysteine derivatives that protected nerve cells in culture from oxidative glutamate toxicity by maintaining GSH levels. In this study, we characterize an additional EC conjugate, cysteamine-EC, that is 5- to 10-fold more potent than the earlier conjugates. In addition, we show that these EC conjugates maintain GSH levels by enhancing the uptake of cystine into cells through induction of a disulfide exchange reaction, thereby uncoupling the uptake from system x(c)(-). Thus, these novel EC conjugates have the potential to enhance GSH synthesis under a wide variety of forms of toxic stress.

Figures

Figure 1

(A) Dose dependent effect of Cya-EC on the survival of glutamate-treated HT22 cells. Cells were untreated or treated with increasing concentrations of Cya-EC in the presence of 5 mM glutamate. 24 hr later cell survival was measured by the MTT assay. The results are the average ± SEM of five independent experiments. (B) Cya-EC protects primary cortical cultures from oxidative glutamate toxicity. 2 day rat primary cortical cultures were treated with 5 or 10 mM glutamate alone or in the presence of 25 μM Cya-EC. 24 hr later cell survival was measured by the MTT assay. The results are the average ± SEM of three independent experiments. (C) Cya-EC protects HT22 cells from homocysteic acid (HCA) and quisqualate. Cells were treated with 1 mM HCA or 250 μM quisqualate in the absence or presence of 10 μM Cya-EC. 24 hr later cell survival was measured by the MTT assay. The results are the average ± SEM of three independent experiments.

Figure 2

(A) Dose dependent effect of Cya-EC on the maintenance of GSH levels in glutamate-treated HT22 cells. Cells were treated with 5 mM glutamate alone or in the presence of increasing concentrations of Cya-EC. GSH levels were measured after 8 hr and normalized to total protein. The results are the average ± SEM of three independent experiments. (B) Time dependent effect of Cya-EC on the maintenance of GSH levels in glutamate-treated HT22 cells. Cells were treated with 5 mM glutamate alone or in the presence of 10 μM Cya-EC. GSH levels were measured after 2-8 hr and normalized to total protein. The results are the average ± SEM of three independent experiments. (C) The glutamate cysteine ligase inhibitor, BSO, blocks the effects of Cya-EC on the maintenance of GSH levels and cell survival. Cells were treated with 5 mM glutamate alone, 5 mM glutamate + 10 μM Cya-EC or 5 mM glutamate + 10 μM Cya-EC in the presence of increasing concentrations of BSO. Cell survival was measured after 24 hr by the MTT assay. GSH levels were measured after 8 hr and normalized to total protein. The results are the average ± SEM of three independent experiments.

Figure 3

(A) Time dependent effect of Cya-EC on 35S-cystine uptake in HT22 cells. Cells were treated with 25 μM 35S-cystine alone or in the presence of 10 μM Cya-EC. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. Results are presented as cpm taken up per μg cellular protein. Similar results were obtained in three independent experiments. (B) Dose dependent effect of Cya-EC on 35S-cystine uptake in HT22 cells. Cells were treated for 50 min with 25 μM 35S-cystine alone or in the presence of increasing concentrations of Cya-EC. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. Results are presented as cpm taken up per μg cellular protein. Similar results were obtained in three independent experiments. (C) Correlation between the dose dependent effects of Cya-EC on 35S-cystine uptake and GSH maintenance. (D) Effect of pretreatment time on the Cya-EC-mediated increase in 35S-cystine uptake. Cya-EC was added to 35S-cystine immediately before addition to the cells or incubated with 35S-cystine at 37oC for 10-50 min before addition to the cells for 10 min. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. Results are the average ± SEM of three independent experiments. (E) Cya-EC does not protect from cystine deprivation. HT22 cells were treated with 5 mM glutamate or cystine-free DME alone or in the presence of 10 μM Cya-EC. 24 hr later cell survival was measured by the MTT assay. The results are the average ± SEM of three independent experiments.

Figure 4

RP-HPLC profiles of Cys-EC (A), Cya-EC (B) and the incubation mixture of cystine and Cya-EC in Hank's balanced solution at 37°C at t = 0 (C) and t = 50 min (D). Column: Kromasil 100 C18 (250 × 4 mm i.d.) 5 μm particle size. Eluents: [A] 0.1% (v/v) aqueous TFA; [B] 0.09% TFA in water-acetonitrile (1:4). Gradient elution: 8-30% [B] over 30 min, at a flow rate of 1 ml/min. Load: 90 μl. Detection at 214 nm.

Figure 5

Cysteine but not glutamate or quisqualate inhibit Cya-EC-mediated 35S-cystine uptake. Cells were treated with 25 μM 35S-cystine alone or in the presence of 10 μM Cya-EC along with either 5 mM glutamate, 250 μM quisqualate, 5 mM cysteine or 5 mM cystine, added to the labeling mixture just before addition to the cells for 10 min. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. Results are presented as the percent of uptake in the absence of glutamate, quisqualate, cysteine or cystine. Similar results were obtained in three independent experiments. * indicates significantly different from control.

Figure 6

Effects of cysteine transporter inhibitors on Cya-EC-mediated increases in 35S-cystine uptake and the maintenance of GSH levels and cell survival in glutamate-treated HT22 cells. For the measurement of 35S-cystine uptake, cells were treated with 25 μM 35S-cystine in the presence of 10 μM Cya-EC alone or along with 5 mM BCH, 5 mM d-serine, 5 mM arginine, 5 mM serine, 100 μM TBOA or 5 mM MAIBA which were added to the labeling mixture just before addition to the cells for 10 min. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. For the measurement of GSH, cells were treated with 5 mM glutamate alone or in the presence of 10 μM Cya-EC alone or along with 5 mM BCH, 5 mM d-serine, 5 mM arginine, 5 mM serine, 100 μM TBOA or 5 mM MAIBA. GSH levels were measured after 8 hr by a chemical assay and normalized to total protein. For the measurement of cell survival, cells were treated with 5 mM glutamate alone or in the presence of 10 μM Cya-EC alone or along with 5 mM BCH, 5 mM d-serine, 5 mM arginine, 5 mM serine, 100 μM TBOA or 5 mM MAIBA. Cell survival was measured after 24 hr by the MTT assay. Results are the average ± SEM of three independent experiments. * indicates significantly different from Cya-EC alone.

Figure 7

Effect of cysteamine (Cya) on cell survival (A) and 35S-cystine uptake and cell survival in glutamate-treated HT22 cells (B). For the measurement of cell survival, cells were treated with 5 mM glutamate alone or in the presence of increasing doses of (A) Cya or (B) 10 μM Cya alone or along with 5 mM BCH, 5 mM d-serine, 5 mM arginine, 5 mM serine, 100 μM TBOA or 5 mM MAIBA. Cell survival was measured after 24 hr by the MTT assay. For the measurement of 35S-cystine uptake, cells were treated with 25 μM 35S-cystine in the presence of 10 μM Cya alone or along with 5 mM BCH, 5 mM d-serine, 5 mM arginine, 5 mM serine, 100 μM TBOA or 5 mM MAIBA which were added to the labeling mixture just before addition to the cells for 10 min. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. Results are the average ± SEM of three independent experiments. * indicates significantly different from Cya alone.

Figure 8

Effect of different EC conjugates on 35S-cystine uptake (A) and the maintenance of GSH levels and cell survival in glutamate-treated HT22 cells (B). For the measurement of 35S-cystine uptake, cells were treated with 25 μM 35S-cystine in the presence of 10 μM of each of the conjugates or EC for 10 min following a 50 min pre-incubation at 37°C. After solubilization of the cells in 0.2 M NaOH, aliquots were taken for scintillation counting and protein determination. For the measurement of GSH, cells were treated with 5 mM glutamate alone or in the presence of 10 μM of each of the conjugates. GSH levels were measured after 8 hr by a chemical assay and normalized to total protein. For the measurement of cell survival, cells were treated with 5 mM glutamate alone or in the presence of 10 μM of each of the conjugates. Cell survival was measured after 24 hr by the MTT assay. Results are the average ± SEM of three independent experiments. * indicates significantly different from glutamate alone.

Figure 9

Proposed mechanism for the conversion of cystine to cysteine and a mixed disulfide by Cya-EC.