22 r hydroxycholesterol

22(R)-hydroxycholesterol, 17β-estradiol, N6,2’-O-dibutyryladenosine 3’,5’-cyclic monophosphate (Bt₂cAMP), progesterone, 20α-OHP, 22(R)-hydroxycholesterol and fatty acid-free bovine serum albumin were supplied by Sigma-Aldrich (St. Louis, MO). 20α-Hydroxyprogesterone [1,2-³H(N)] (40–60 Ci/mmol; 1.48–2.22 TBq/mmol) and progesterone [1,2-³H] (40–60 Ci/mmol; 1.48–2.22 TBq/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Pregnenolone, [7-³H(N)] (10–25 Ci/mmol; 370–925 GBq/mmol) was supplied by PerkinElmer (Waltham, MA). StAR (D10H12) XP^® rabbit mAb, GAPDH (14C10) rabbit mAb, and MeCP2 (D4F3) XP™ rabbit mAb were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Purified anti-SF-1 polyclonal antibody (07–618) was purchased from EMD Millipore (Billerica, MA). All other chemicals used were of analytical grade.

Glomerulosa cell slices were prepared from near-term fetal bovine adrenal glands obtained from a local meat-packing plant, and the cells were dispersed from collagenase-digested slices by mechanical agitation. Freshly isolated cells were cultured overnight in Falcon Primaria dishes in a Dulbecco’s modified Eagles’s medium–Ham’s F-12 medium (1:1) containing 10% horse serum (v/v), 2% fetal bovine serum (v/v), ascorbate (100 μM), α-tocopherol (1.2 μM), Na₂SeO₃ (0.05 μM), butylated hydroxyanisole (50 μM), metyrapone (5 μM), penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin-B (0.25 μg/ml). After replacement of the serum-containing medium with serum-free medium plus 0.2% bovine serum albumin (BSA), the cells were incubated for an additional 20–24 hours before treatment. Prior to stimulation, the medium was removed and the cells washed three times with Krebs-Ringer bicarbonate-buffered (KRB⁺) solution containing sodium acetate (120 mM NaCl, 24.9 mM NaHCO₃, 3.5 mM KCl, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 1.25 mM CaCl₂, 0.1% dextrose, 0.2% BSA and 2.5mM sodium acetate) and incubated at 37°C in KRB⁺ equilibrated with 5% CO₂ for 30-60 minutes (eqKRB⁺). AngII and 22(R)-hydroxycholesterol were obtained from Sigma (St. Louis, MO) as were the inhibitors PP2 and Src-1. Ro 31-8220 was obtained from EMD Biosciences (San Diego, CA).