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. 1998 Jun 23;95(13):7748–7753. doi: 10.1073/pnas.95.13.7748

Figure 3.

Figure 3

D609 prevents glutamate but not BSO-induced GSH depletion and restores cystine uptake. (A and B) A time course analysis of intracellular GSH levels after the treatment of HT22 cells with glutamate (A) or BSO (B) in the presence or absence of the PC-PLC inhibitor, D609, and the 12-LOX inhibitor, baicalein. Results are presented relative to controls and are the mean ± SD of one typical experiment with three determinations. Error bars that are not visible are smaller than the width of the symbol. HT22 cells contain 12.4 ± 0.62 and 8.08 ± 0.08 nmol GSH/mg protein when grown in fetal calf serum (to assay for glutamate toxicity) and horse serum (to assay for BSO toxicity), respectively. Glu, 5 mM; BSO, 50 μM; D609, 50 μM; baicalein, 10 μM. (C) Effect of D609 on GSH levels in primary cortical neurons. Cells (5 × 106) were seeded in 100-mm dishes and were treated the next day with the indicated reagents for 12 h before the assay for GSH content. Results are presented relative to controls. Control 1 for glutamate treatment is 11.9 nmol GSH/mg protein. Control 2 for BSO treatment is 7.4 nmol GSH/mg protein. Glu, 5 mM; BSO, 50 μM; D609, 50 μM; baicalein, 1.5 μM. (D) Restoration of cystine uptake after treatment with D609. HT22 cells were pretreated with or without glutamate and D609 as indicated (treatment) for 8 h. The uptake of radioactive cystine then was monitored by incubating the cells with 35S-cystine for 20 min as described in Materials and Methods. In addition to 35S-cystine, the labeling mix also may contain glutamate and D609 as indicated (labeling) to distinguish cystine uptake via the cystine/glutamate antiporter from that via the other routes (see text for explanations). Glutamate, 5 mM; D609, 50 μM. Results are presented relative to controls without glutamate. The rate of cystine uptake for untreated controls is 22.7 pmol/μg protein/20 min.

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