Blr 201

To measure ERE-mediated promoter activity, reporter genes were introduced into cells with the aid of adenovirus vectors as described previously [27] . Pituitary cells were infected with Ad-ERE.TK/Luc and Ad-TK/rLuc adenovirus vectors, both at 2 MOIs, when the cells were plated. Cells were harvested with 200 μL of Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's protocol. The cell lysates were centrifuged to remove cell debris and subjected to reporter assays using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Light intensity was measured with a luminometer (BLR-201; Aloka, Tokyo, Japan). Promoter activity levels measured by Ad-ERE.TK/Luc were normalized to those measured by Ad-TK/rLuc.

The activity of ASMase was measured as described previously. 39 Briefly, cell homogenates (20 μg) were incubated with 0.02 μCi of N-methyl-[ 14 C]sphingomyelin in 100 μl acidic reaction buffer containing 100 mmol l -1 sodium acetate and 0.1% Triton X-100, pH 5.0, at 37 °C for 15 min. The reaction was terminated with 1.5 ml of a 2:1 chloroform:methanol solution and 0.2 ml double-distilled water. After vortexing and centrifuging at 1000 g for 5 min to separate the two phases, the upper aqueous phase containing the N-methyl-[ 14 C]-sphingomyelin metabolite 14 lucigenin detection of endothelial O 2 • - Superoxide production in MECs was measured via lucigenin chemiluminescence as described previously. 40, 41 Briefly, cells were incubated in vials containing 5 μmol l -1 lucigenin (Sigma), and the NADPH oxidase activity was measured by lucigenin assay after the addition of NADPH (300 μmol l -1 ). The light reaction between superoxide and lucigenin was detected using a chemiluminescence reader (BLR-201, Aloka, Wallingford, CT, USA).

Cells were lysed with 400 µL Reporter Lysis Buffer or 200 µL Renilla Luciferase Assay Lysis Buffer according to the manufacturer's protocol. Luciferase assays were performed with the Luciferase Assay System and Renilla Luciferase Assay System (Promega, Madison, WI, USA). Light intensity was measured with a luminometer (BLR-201, Aloka, Tokyo, Japan). The promoter activities derived from the luciferase assays were normalized relative to those derived from Renilla luciferase assays.