Pregnenolone sulfate

The following were purchased from Sigma-Aldrich (St. Louis, MO, USA): precursors of CORT, including PGN, PGS, DCORT; precursor of testosterone (TS), androstenedione (ASD); and conjugated metabolites, pregnenolone-sulfate (PGN-S). Cholesterol was purchased from Wako Pure Chemical Industries (Osaka, Japan). LC-MS–grade acetonitrile and formic acid were purchased from Supelco (Bellefonte, PA, USA).

Carbachol, acetylcholine, dimethyl sulfoxide (DMSO), asolectin, T3, NaOH, allopregnanolone, and pregnenolone sulfate were purchased from Sigma Aldrich (St. Louis, MO). Isoflurane was purchased from Henry Schein Animal Health (Dublin, OH). T3 was dissolved in 0.1 M NaOH. allopregnanolone was dissolved in 0.1% DMSO. All other ligands were dissolved directly in modified Barth’s solution (88 mM NaCl; 1 mM KCl; 0.4 mM CaCl₂; 0.33 mM Ca(NO₃)₂; 0.8 mM MgSO₄; 5 mM tris(hydroxymethyl)aminomethane-HCl (Tris-HCl); 2.4 mM NaHCO₃); at low or high pHs, Tris-HCl was replaced with either 2-(N-morpholino)ethanesulfonic acid (MES) (T3/PS experiments at pH 6–6.7) or N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (T3 experiments at 8–9).

Pregnenolone sulfate (sodium salt) and BAPTA were purchased from Sigma-Aldrich (St. Louis, Missouri, USA); dibromoBAPTA from Molecular Probes / Thermo Fisher Scientific; and isosakuranetin from Extrasynthese (Genay, Rhône, France). Pregnenolone sulfate and isosakuranetin were diluted from stock solutions in DMSO (100 mM for Pregnenolone sulfate and 10 mM for isosakuranetin).

At days in vitro (div) 14, cultures were treated for 1 or 5 days (brief or prolonged, respectively) with kynurenic acid (KYNA, 150 nM), pregnenolone sulfate (PS, 50 μM), spermidine (SPD, 50 μM), or zinc (ZINC 10 μM) (all obtained from Sigma-Aldrich). We selected these concentrations to mimic their physiological levels reported in the cerebrospinal fluid [15 (link)]. As all used compounds at specified concentrations are water-soluble, we used bi-distilled water as a solvent and vehicle for control experiments. After treatments, cortical cultures were tested for cell viability and stimulation-induced (50 mM K⁺) glutamate release assays or were fixed for immunofluorescence staining and morphometry.

GSK1016790A (GSK), capsaicin, pregnenolone sulfate (PS), lysophosphatidylcholine (LPC) and ionomycin calcium salt were purchased from Sigma-Aldrich; Allyl isothiocyanate (AITC) was purchased from KANTO; Camphor was purchased from Wako; Fura-2-acetoxymethyl ester was purchased from Invitrogen.

T-REx-TRPM3 cells and HEK293-M8 cells, HEK293 cells expressing either TRPM3 or TRPM8, respectively, have been described elsewhere [31 (link),46 (link)]. HEK293 cells expressing a conditionally active B-Raf kinase mutant (HEK293-ΔB-Raf:ER cells) have been described [47 (link)]. The mutant kinase is activatable with 4-hydroxytamoxifen (4OHT). T-REx-TRPM3 cells, HEK293-M8 cells, and HEK293-ΔBRaf:ER were maintained in DMEM supplemented with 0.05% fetal calf serum for 24 h prior to stimulation. Stimulation was performed with pregnenolone sulfate (PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), icilin (1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557), or 4-hydroxytamoxifen (4OHT) (100 nM, Sigma # H7904, with ethanol as solvent, Taufkirchen, Germany) for 24 h in medium supplemented with 0.05% fetal calf serum. Cells were preincubated for 3 h with the compound ophiobolin A (1′R,2S,3S,3′S,4′R,5R,7′S,8′E,11′R)-4′-hydroxy-1′,3,4′-trimethyl-5-(2-methylprop-1-enyl)-6′-oxospiro[oxolane-2,12′-tricyclo[9.3.0.0^3,7]tetradec-8-ene]-8′-carbaldehyde, PubChem CID 5281387) (Santa Cruz # sc-2022266, dissolved in DMSO, Heidelberg, Germany) at a concentration of 250 nM. Cells were stimulated for 24 h in the presence of ophiobolin A.