NO inhibits iNOS translation in RAW264.7 macrophages. (A) RAW264.7 cells were stimulated with LPS (1 µg/ml) and IFN-γ (100 units/ml) in the presence or absence of 2 mM NMA or purified red blood cells (400 µM of hemoglobin). Nitrite + nitrate was measured in culture media as described in Materials and Methods. Data are expressed as mean ± SD; n ≥ 4. (B) Nitrite + nitrate accumulation in the culture media (solid bars) and NOS activity (hatched bars) were determined in RAW264.7 cells stimulated with LPS + IFN-γ for 12 hr. Cells were collected and lysed in Tris-HCl, pH 7.4, by three cycles of freezing and thawing. NOS activity was determined by measuring nitrite and nitrate from l-arginine in the presence of NADPH, FAD, FMN, and BH4 (see Materials and Methods). Data are expressed as mean ± SD; n ≥ 3. (C) Protein synthesis was measured in RAW264.7 cells stimulated with LPS + IFN-γ. At the indicated time points, cells were labeled with 35S-methionine for 2 hr and the labeled proteins separated on SDS-PAGE. Protein synthesis levels (upper panel) and iNOS protein (lower panel) were analyzed by exposing the membrane to X-ray film and Western blot, respectively. The ring-like iNOS protein bands detected by Western blot after the 12-hr incubation with NMA are due to the rapid exhaustion of the peroxidase substrate by the high levels of iNOS protein. (D) Abundance of iNOS mRNA and protein were analyzed by Northern and Western blot, respectively.