Nociceptin

HS-731 was prepared as previously described [29 (link)]. Radioligand [³H]nociceptin (119.4 Ci/mmol) was purchased from PerkinElmer (Boston, MA, USA). Tris(hydroxymethyl) aminomethane (Tris), bovine serum albumin (BSA) polyethylenimine (PEI) and nociceptin were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Cell culture media and supplements were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from standard commercial sources. Test compounds were prepared as 1 mM stocks in water and further diluted to working concentrations in 50 mM Tris-HCl buffer (pH 7.4).

LENART01 was prepared as described previously [59 (link)]. Radioligands [³H]DAMGO (51.7 Ci/mmol), [³H]DPDPE (47.4 Ci/mmol), [³H]U69,593 (49.3 Ci/mmol), [³H]nociceptin (119.4 Ci/mmol) and [³⁵S]GTPγS (1250 Ci/mmol) were purchased from PerkinElmer (Boston, MA, USA). Guanosine diphosphate (GDP), GTPγS, DAMGO, DPDPE, U69,593, nociceptin, formalin, polyethylenimine (PEI), tris(hydroxymethyl) aminomethane (Tris), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), bovine serum albumin (BSA) and cell culture media and supplements were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Dermorphin was obtained from MCE (Sollentuna, Sweden). Naloxone hydrochloride was kindly provided by Dr. Helmut Schmidhammer (University of Innsbruck, Innsbruck, Austria). All other chemicals were of analytical grade and obtained from standard commercial sources. Test compounds were prepared as 1 mM stocks in water for in vitro assays or dissolved in 0.9% physiological saline for in vivo assays and further diluted to working concentrations in the appropriate medium.