Pregnenolone

All extraction and derivatization solvents were chromatography grade (EMD Chemicals, Billerica, MA). Monodeuterated ethanol was obtained from Acros Organics (New Jersey), the derivatizing reagent heptafluorobutyric acid anhydride (HFAA) was obtained from Fluka (Sigma-Aldrich, St. Louis, MO), and the neurosteroids pregnenolone, pregnanolone, 3α,5β-THDOC, and 3α,5β-androstanediol were obtained from Steraloids (Newport, RI). All other native neurosteroid standards were synthesized by the late Dr. Robert Purdy. Similar to Snelling et al. (2014) (link), the internal standard (ISTD) was 17β-estradiol-2,4,16,16,17-d5 (CDN Isotopes, Pointe-Claire, Quebec, Canada). Ultra-pure water (double distilled; ddH₂O) from a MilliQ system (Millipore Corporation, Bedford, MA) was used. Activated powdered charcoal (Darco-G-60) and Dextran (T-70) were obtained from Sigma-Aldrich.

Δ⁹-THC (NIDA Drug Supply Program, Bethesda, MD), SR141716/rimonabant (NIDA), PSNCBAM-1 and its analogs (synthesized in our laboratories), pregnenolone (Steraloids, Newport, RI), otenabant (Toronto Research Chemicals, Toronto, Canada), and the open ring degradant of the synthetic cannabinoid XLR-11 (1-[1-(5-fluoropentyl)-1H-indol-3-yl]-3,3,4-trimethyl-4-penten-1-one; Cayman Chemical, Ann Arbor, MI) were dissolved in a vehicle of 7.8% Polysorbate 80 N.F. (VWR, Marietta, GA) and 92.2% sterile saline USP (Butler Schein, Dublin, OH). For in vitro studies, Δ⁹-THC, CP55,940 (NIDA), [³H]SR141716 (24 Ci/mmol; NIDA), [³H]CP55,940 (81.1 Ci/mmol; NIDA) and unlabeled SR141716 were dissolved in absolute ethanol whereas PSNCBAM-1, RTICBM-15, RTICBM-28, and pregnenolone were dissolved in 100% DMSO. All drugs were stored at −80°C as 10 mM stocks and diluted to final concentration of 0.1–0.2% solvent. GDP (Sigma Aldrich, St. Louis, MO), unlabeled GTPγS (Sigma Aldrich, St. Louis, MO), and [³⁵S]GTPγS (1250 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA) were dissolved in distilled water, aliquotted and stored at −80°C. Adenosine deaminase (Sigma Aldrich, St. Louis, MO) was diluted in distilled water and stored at 4°C.

Progesterone (pregn-4-ene-3,20-dione), pregnenolone (3β-hydroxypregn-5-en-20-one), deoxycorticosterone (11-deoxycorticosterone; DOC), 5α-dihydroProgesterone (5α-pregnan-3,20-dione; 5α-DHP) and 5β-dihydroProgesterone (5β-pregnan-3,20-dione; 5β-DHP) were purchased from Steraloids (Newport, RI). Allopregnanolone (3α-hydroxy-5α-pregnan-20-one; 3α,5α-THP), pregnanolone (3α-hydroxy-5β-pregnan-20-one; 3α,5β-THP), allotetrahydrodexycorticosterone (3α, 21-dihydroxy-5α-pregnan-20-one; 3α,5α-THDOC), Progesterone-d₉, pregnenolone-d₄ and 5α-DHP-d₆ were synthesized and provided by Dr. Robert H. Purdy (in memoriam, Veterans Medical Research Foundation, San Diego, CA). DOC-d₈, and 3α,5α-THP-d₄ were purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). 3α,5α-THDOC-d₃ was purchased from Cambridge Isotope Laboratories (Tewksbury, MA).

EDS (99% purity) was obtained from Winthrop Sterling (Rensselaer, NY). Ovine LH (NIADDK-oLH 26) was a gift from the National Hormone and Pituitary Program (NIDDK Bethesda, MD). [1,2,6,7,16,17-³H(N)]Testesterone (SA, 140.9 Ci/mmol) and 25-[26,27-³H]hydroxycholesterol (SA, 75 Ci/mmol) were obtained from New England Nuclear (Wilminton, DE). 2-[1-C¹⁴]-Ketoisocaproic acid (SA, 50-62 mCi/mmol) and a cyclic AMP [³H] assay system were obtained from Amersham Corp (Arlington Heights, IL). 5-Cholesten-3β, 25-diol (25-hydroxycholesterol) was purchased from Sigma (St. Louis, MO). All other unlabeled steroids, including pregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione and testosterone were obtained from Steraloids Inc. (Wilton, NY) and were crystallized to constant melting points before used as enzyme substrates or as HPLC or RIA standards. Organic solvents for HPLC, including methanol, acetonitrile and tetrahydrofuran, were of HPLC grade and were purchased from J.T. Baker (Phillipsburg, NJ).