FIGURE 4.

The effects of BCNU are dependent on eNOS. Cells expressing Tet-regulatable GTPCH (GCH cells) were subject to BCNU exposure, and the presence or absence or eNOS was assessed (eNOS/GCH cells). A, a dose-dependent S-glutathionylation of eNOS was observed that was removed following exposure to DTT. There was no change in overall eNOS levels following BCNU treatment. No eNOS was detectable in GCH cells lacking the eNOS transgene. B, in both GCH and eNOS/GCH cells, BCNU exposure attenuated the GSH:GSSG ratio by 4-fold (*, p < 0.05). C, eNOS activity was decreased in a dose-dependent manner following exposure to BCNU in eNOS/GCH cells (●, solid line). *, p < 0.05; **, p < 0.01). These changes in eNOS activity do not occur in the presence of DTT (●, dotted line). eNOS activity is undetectable in GCH cells lacking eNOS (▴). D, total superoxide production is elevated in eNOS/GCH, but not GCH cells following exposure to BCNU. †, p < 0.01 (n = 4). E, this elevation in superoxide production is not evident in GCH cells, and no effect of either l-NAME or DTT was observed. F, these elevated levels of superoxide (*, p < 0.05), induced by BCNU (100 μm) are partially inhibitable with l-NAME (200 μm) and almost totally abolished in the presence of DTT (†, p < 0.05). G, the subsequent effect of this superoxide is the oxidation of BH4. Following BCNU (100 μm) treatment, the diminished BH4:BH2 ratio (*, p < 0.05) is further exacerbated in eNOS/GCH but not GCH cells (†, p < 0.05; n = 6).