Reagents. N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norleucinal (MG132), LPS, L-[³H]arginine, DTT, methionine, cysteine, Triton X-100, clotrimazole, and miconazole were from Sigma. Tween 20 prestained molecular mass standards, were from Bio-Rad. Monoclonal antibody 1E8-B8 (Research and Diagnostic Antibodies) and polyclonal antibody 06-573 (Upstate Biotechnology) are specific for inducible nitric oxide synthase (iNOS) isoform. BBS-1 iNOS dimerization inhibitor was a gift from John F. Parkinson (Berlex). All other reagents were purchased from Fisher or the sources stated in the text.

Cell Culture. Human embryonic kidney (HEK) 293, A549 (human alveolar type II epithelium-like lung carcinoma cell line), RT4 (human bladder transitional cell papilloma), and murine macrophage RAW 264.7 cell lines were purchased from American Type Culture Collection. Cells were cultured in manufacturer-recommended media. Each medium was supplemented with 2 mM glutamine and 10% heat-inactivated FBS (HyClone).

Primary Bronchial Epithelial Cells. Normal human tracheobronchial epithelial cells, purchased from Clonetics, were cultured at the air–liquid interface in a serum-free, 1:1 mixture of DMEM (Invitrogen) and bronchial epithelium growth medium (BEGM; Clonetics), containing insulin (5 m g/ml), transferrin (10 m g/ml), epinephrine (0.5 m g/ml), triiodothyronine (6.5 ng/ml), epidermal growth factor (0.5 ng/ml), BSA (0.5 mg/ml), bovine pituitary extract (1% vol/vol), gentamycine (50 m g/ml), amphotericin B (50 ng/ml), and retinoic acid (5 × 10^–8 M). The cultures were grown submerged for the first 7 days, at which time the air–liquid interface was created by removing media from the apical compartment of the cultures. The culture media were changed every other day until the air–liquid interface was created, thereafter replacing fresh media only to the basal compartment daily. Cultures were maintained at 37°C in a humidified 5% CO₂ for further 4 weeks after confluence until they reached the fully differentiated mucociliary plateau phase.

Transfection and Cell Lysis. Cationic lipid-mediated transient transfection was done by using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. After gently rinsing twice with PBS, the cell layer was lysed on ice for 30 min in 40 mM Bis-Tris propane buffer (pH 7.7), 150 mM NaCl, 10% glycerol, and 1% Triton-X100 in the presence of protease inhibitors [1 mM phenylmethylsulfonyl fluoride/10 mg/ml pepstatin-A/10 mg/ml leupeptin/10 mg/ml aprotinin/10 mg/ml phenanthroline/16 mg/ml benzamidine HCl (Pharmingen)].

iNOS Induction. Primary bronchial epithelial cells and RT4 cells were incubated with or without a mixture of IFN-g (100 units/ml), IL-1β (0.5 ng/ml), TNF-a (10 ng/ml), and IL-6 (200 units/ml). Induction of iNOS in A549 cells was done as above except that IFN-g was used at 300 units/ml concentration, and IL-6 was omitted.

Western Analysis. Aliquots of cell lysates or immunoprecipitated proteins were mixed with a one-third volume of 4´ Laemmli sample buffer (200 mM Tris•HCl, pH 6.8/8% SDS/0.004% bromophenol blue/40% glycerol/400 mM DTT) and heated at 95°C for 5 min. Proteins were resolved on SDS-polyacrylamide gels at 125 V and transferred to nitrocellulose membranes. Immunoreactive bands were visualized with the use of an enhanced chemiluminescence system (SuperSignal West Pico, Pierce). Images were acquired by using a cooled charge-coupled device camera (Eagle Eye II Still Video System, Stratagene).

Immunoprecipitation. To immunoprecipitate iNOS, cell lysates (0.5 mg) were incubated at 4°C with anti-iNOS antibody 06-573 in a total volume of 1,000 m l of lysis buffer for 90 min. Protein A-Sepharose beads (100 m l of 10% solution) were added to the samples. After further incubation for 1 h at 4°C, beads were washed three times (1 ml each) in ice-cold lysis buffer. Immunoprecipitated proteins were eluted by heating at 85°C for 5 min in Laemmli sample buffer.

iNOS-Activity Assays. iNOS activity was assayed for intact cells by measuring nitrite accumulation in the culture media and for cell lysates by measuring the conversion of L-[³H]arginine to L-[³H]citrulline. To measure the amount nitrite in the cell medium, 100 ml of culture medium was mixed with 100 ml of Griess reagent for 10 min at room temperature, and absorbance at 543 nm was recorded in a microplate reader. Serial dilutions of sodium nitrite were used as standards. The conversion of ³H-labeled L-arginine to ³H-labeled L-citrulline was done by adding 30 m l of cell lysates and 1 ml of 1.05 mCi/ml (1 Ci = 37 GBq) L-[³H]arginine to 69 ml of buffer containing 30 mM Tris•HCl (pH 7.4), 4 mM FAD, 4 mM FMN, 4 mM tetrahydrobiopterin, 1 mM DTT, 0.5 mM L-arginine, and 0.1 mM NADPH. After incubation for 30 min at 37°C, reactions were terminated with 400 ml of 50 mM Hepes (pH 5.5) and 10 mM EGTA and were applied to 1-ml columns (Bio-Rad) of cationic exchange resin AG 50WX (Na⁺ form) that were eluted with 1 ml of water. L-[³H]citrulline was quantified by a liquid scintillation counter (Beckman).