FIG. 4.

ONOO⁻ inhibits the activity but increases the degradation of GTPCH1 in endothelial cells. A: The effect of ONOO⁻ on GTPCH1 activity in BAECs. Confluent BAECs were starved overnight and treated with ONOO⁻ (1, 10, or 50 μmol/L, separately) or equal volume of vehicle (0.1 mol/L NaOH) for 15 min in 0.1 mol/L HEPES buffer. HPLC was used to determine the GTPCH1 enzyme activity. n = 3. *P < 0.05 vs. control. B: BAECs were treated with SIN-1 for indicated time. n = 3. *P < 0.05 vs. control. C and D: Effect of ONOO⁻ on GTPCH1 stability. BAECs were treated with 50 μmol/L ONOO⁻ or vehicle for 15 min and then with CHX (100 μg/mL). Cells were harvested at the indicated time points in the presence of CHX, and the lysates were subjected to immunoblotting using anti-GTPCH1 and anti–β-actin antibodies. The blot shown is representative of three independent experiments.