Buprenorphine hydrochloride, morphine pellets (75 mg each of base), and morphine sulfate were provided by the NIDA drug supply program. DAMGO, forskolin, IBMX, naloxone hydrochloride, fentanyl, and diazepam were obtained from Sigma Aldrich, St Louis, MO.Carfentanil was obtained from Cayman Chemical C Ham-F12 and FBS were purchased from Invitrogen, Carlsbad, CA. RM1049 and RM1490 were provided by R2M Pharma, South San Francisco, CA and synthesized as previously reported (Medina et al., 2019). Naloxone, morphine and fentanyl were dissolved in 0.9% saline; stock solutions of the other compounds were prepared by dissolving in DMSO and kolliphor and then diluting with 0.9% saline to working concentrations, which contained 1% or less of DMSO and kolliphor. Vehicle control solutions consisted of 0.9% saline, 1% DMSO, and 1% kolliphor. Unless otherwise stated injection were subcutaneous (s.c.) at 3 to 5 ml/kg.
Cell Culture and in-vitro assays:MOR cAMP assays: CHO Tag-lite human MOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, MA) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, CA). Cells were then harvested using accutase (Corning, Corning, NY), centrifuged at 1300 RPM for 5min, and plated at 5000 cells per 5 μL per well in a 5x dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, MA) in white HTRF low volume 384-well plates (Cisbio, Bedford, MA). Plates were then incubated at 37°C in 5% CO 2 for 10minutes. For the This article has not been copyedited and formatted. The final version may differ from this version.