FIG. 7.

Effect of organic nitrates on the activity of the 11-kb human HO-1 promoter in the dependence of NRF2 and induction of GCH-I by HO-1 products. A: DLD-1-HO-1-prom cells were washed with PBS and incubated with Dulbecco’s modified Eagle’s medium containing 2 mmol/L l-glutamine in the absence of serum and phenol red. After 16 h, cells were incubated with 50 μmol/L PETN (or DMSO as a solvent control) or 50 μmol/L ISMN (or water as a solvent control) for 8 h, lyzed in 1× passive lysis buffer, and protein concentrations were measured using the Bradford reagent. Luciferase activity in the extracts was determined using the luciferase assay system. The light units of the firefly luciferase were normalized to the protein concentration of the cell extracts. The relative luciferase activity level of cells treated with the solvent control was set to 100%. B: DLD-1-HO-1-prom cells were transiently transfected with an anti-NRF2 siRNA (siNRF2), which is shown to downregulate NRF2 expression, or a negative control siRNA (siCtr) by lipofection with HiPerFect HTS Reagent according to the manufacturer’s recommendations. After 48 h, cells were treated as described above to analyze PETN-induced human HO-1-11kb promoter activity. The relative luciferase activity level of cells treated with DMSO and siCtr was set to 100%. C: Confluent human endothelial cells (EA.hy 926) were treated in six-well plates with solvent (0.1% DMSO = Ctr), bilirubin (BR; 10 μmol/L), the carbon monoxide–releasing compound (CORM = CO; 50 μmol/L), or PETN (P; 50 μmol/L) for 15 h in the incubator. Two wells were pooled for GCH-I protein analysis (1:2,500; Dr. E. Werner, Innsbruck, Austria). Data are the means ± SEM of 6–10 (A and B) and 3–9 (C) independent experiments. *P < 0.05 vs. control; #P < 0.05 vs. PETN (siCtr).