Fig. 1. A549 cells had ability to make nitrite and nitrate from NO.

NO donor was incubated with and the other without A549 cells. A549 cells (1×10⁶/well) were cultured in 6-well plates with regular DMEM growth medium (10% FBS and glucose) for 24 hrs. Prior to treat the cells with DETA NONOate (50 µM), the cells were washed twice with PBS and the culture medium was replaced with serum deprived DMEM medium. Cells were then placed in a regular CO₂ incubator (21% O₂, 5% CO₂) or hypoxic chamber (1% O₂, 5% CO₂) for 24 hrs after adding DETA NONOate. Nitrite (A) and nitrate (B) levels in conditioned media were then measured with Sievers 280i NOA (see Materials and Methods). The cell protein was extracted from the cytoplasmic and membrane fraction and similarly exposed to the NO donor for 24 hours and the nitrite and nitrate measured. The membrane fraction had the highest NO oxidase, converting NO to nitrite (C) and nitrate (D). Protein size fractionation suggested the highest oxidase activity was in the fraction < 100 kDa in size (E, F) *ρ < 0.05 and **ρ < 0.01 compared to respective controls.