3h dpdpe

(±)-trans-U-50488H (U50,488 H), [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) [D-Pen2,5,P-CL-Phe4]Enkephalin (DPDPE), nor-binaltorphimine (nor-BNI), β-funaltrexamine (β-FNA), naloxone, GTPγS and GDP were obtained from Sigma-Aldrich (St. Louis, MO). [³H]DAMGO (51.5 Ci/mmol), [³H]DPDPE (57.4 Ci/mmol), [³H]U69593 (43.6 Ci/mmol) and [³⁵S]GTPγS (1250 Ci/mmol) were bought from Perkin Elmer.

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.

Human MOR, DOR, or KOR recombinant CHO cell membranes were incubated for 2 h at 25 °C in 0.25 mL of the buffer containing with various concentrations of the tested compound, 2 nM [³H] DAMGO, [³H] DPDPE, or [³H] U69,593 (PerkinElmer, Inc., MA, USA), respectively. The incubation was terminated by collecting membranes on Filtermat B filter (PerkinElmer, Inc.) using a FilterMate™ harvester (PerkinElmer, Inc.). The filters were then washed three times with 50 mM Tris–HCl buffer, pH 7.4. Then, MeltiLex scintillant (PerkinElmer, Inc.) was melted onto the dried filters. Radioactivity was determined by a MicroBeta scintillation counter (PerkinElmer, Inc.). Nonspecific binding was measured in the presence of 10 μM unlabeled DAMGO, DPDPE, or U-69,593 (PerkinElmer, Inc.). K_i and 95% CI values were calculated by Prism software (version 5.0).