8 br cyclic amp

Bovine serum albumin (BSA), N-2-hydroxy-ethylpiperazine-N’-2-ethane-sulphonic acid (HEPES), Hank’s balanced salt solution (HBSS), glucose, collagenase, nifedipine, ACTH, 8-Br-cyclic AMP, 25-hydroxy-cholesterol, and deoxycorticosterone were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Nimodipine was purchased from RBI/Sigma (Natick, MA, USA). Tetrandrine was purchased from Sigma-Aldrich. Trilostane was a gift from Sanofi-Synthelabo, Inc. (Malvern, PA, USA). [³H]-corticosterone, [³H]-pregnenolone and [³H]-progesterone were obtained from Amersham International Plc. (Bucks, UK). MC2-R (ACTH receptor, sc-13107) was purchased from Santa Cruz (Santa Cruz, CA, USA). Cytochrome P450 side-chain-cleavage enzyme (P450scc, bs-3608R) was purchased from Bioss Inc. (Woburn, MA, USA). The anti-steroidogenic acute regulatory protein (StAR) as a gift and kindly provided by Dr. Douglas Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA.

Cholesterol efflux assays were carried out essentially as described [37 (link)]. Thioglycollate-elicited peritoneal macrophages were seeded in 24-well plates at a density of 3 x 10⁵ cells/well overnight in Macrophage Growth Medium (MGM; DMEM supplemented with 10% FBS and 10% L-929 conditioned medium). Non-adherent cells were removed, and adherent cells were washed twice with PBS and cultured in MGM. The cells were incubated with 50 μg/mL oxidized LDL (Alfa Aesar) and 1 μCi/mL [1,2-³H(N)]-cholesterol (PerkinElmer) in MGM for 24 hours at 37°C. Following incubation, the cells were washed twice with PBS, incubated in low serum growth medium (DMEM supplemented with 1% FBS, penicillin, and streptomycin), and treated with or without 8-Br-cyclic AMP (Sigma) for 16–18 hours. To induce efflux, cells were washed twice with PBS and incubated in DMEM containing 50 μg/mL HDL (Intracel). At the indicated time points, the medium was collected and centrifuged at maximum speed for 10 minutes and cells were harvested by addition of NP-40 lysis buffer. Radioactivity in the medium and cell lysate was quantified by liquid scintillation counting, and percent cholesterol efflux was calculated as follows: (cpm_Media/(cpm_Media + cpm_Cell)) x 100 after subtracting background efflux (efflux in the absence of HDL).