FIGURE 2.

Accumulation of nitrosothiols in RAW 264.7 cells upon GSNOR inhibition. A, effect of the length of incubation with C1–C3 on the buildup of nitrosylated species (RXNO) in RAW 264.7 cells. RAW 264.7 cells, treated or untreated with 33 μm C1–C3 for 16 h, were transferred to a fresh medium containing 500 μm GSNO alone (□) or GSNO and 33 μm C1 (○), C2 (▵), or C3 (■). At the indicated times, the lysates were prepared and analyzed for nitroso species and protein concentration, using the chemiluminescence and Bradford assays, respectively. Data correspond to means ± S.E. (n = 6–21 wells), and p < 0.0001 by t test versus GSNO-treated cells for all measurements except for cells treated with GSNO and C2 for 6 h (p < 0.01). B, effect of the increasing concentrations of the compounds on the buildup of nitrosylated species (RXNO) in RAW 264.7 cells. RAW 264.7 cells were incubated with 500 μm GSNO in the presence of increasing concentrations of C1 (○), C2 (▵), or C3 (■). After 4 h, cell lysates were prepared and analyzed for nitroso species and protein concentration. Data correspond to means ± S.E. (n = 3–6 wells). The concentration of the compounds used in the time-dependent experiment (Fig. 2A) is shown by the arrow. C, regulation of S-nitrosylation in RAW 264.7 cells by GSNOR. RAW cells were treated with 33 μm C3 for varied lengths of time (0, 2, 4, 8, and 24 h) alone or in combination with 1.1 mm l-NAME for 4 h (lane 4+N). At the indicated times, the cells were quenched, and the lysate was analyzed for S-nitrosothiol content by the biotin switch assay. Equal amounts of proteins were loaded in each lane, and the degree of biotinylation (and hence S-nitrosylation) was determined using an anti-biotin antibody.