Sly usedC6m cells in studies of opioid signalling including AC sensitization (Clark et al., 2004; Clark and Traynor, 2006) and have shown equivalent m-opioid-mediated sensitization in HEK cells (Clark and Traynor, 2006). We have compared the ability to precipitate expression of AC sensitization and also the pharmacological profiles of naltrexone and 6b-naltrexol, along with the common opioid antagonist naloxone, the peptidic antagonist CTAP plus the identified d-opioid inverse agonist (+)N-[trans-4-(2-methylphenyl)-2-butenyl]-(3R,4R)-50-56-6 custom synthesis dimethyl-4(3-hydroxyphenyl)piperidine (RTI-5989-25; Zaki et al., 2001). The outcomes show that there is absolutely no inherent efficacy difference among 6b-naltrexol and naltrexone beneath the conditions studied and in addition that improvement and manifestation of AC sensitization will not be dependent around the formation of a constitutively active m-opioid receptor.MethodsCell culture and therapies C6 rat glioma cells stably transfected together with the rat m-opioid receptor (C6m) or HEK293 cells stably transfected using the FLAG-tagged mouse m-opioid receptor had been grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.5 mg L-1 or 0.8 mg L-1 Geneticin respectively. Cells have been grown inside the presence of 10 fetal bovine serum at 37 in five CO2. For chronic opioid remedy, cells were incubated overnight with 10 mmol -1 DAMGO ([D-Ala2,NMe-Phe4,Glyol5]-enkephalin). C6m cells have been made use of for all experiments except for the determination of cell surface receptor number, which utilized HEK cells expressing a FLAGtagged m-opioid receptor. C6m cells expressed 3.two 0.2 pmol g-1 protein receptor and HEK cells 9.7 1.3 pmol g-1 protein receptor, determined by [3H]diprenorphine binding.Membrane preparation Cells have been washed twice with ice cold phosphate-buffered saline (0.9 NaCl, 0.61 mmol -1 Na2HPO4 and 0.38 mmol -1 KH2PO4, pH 7.4), detached in the plate by incubation in harvesting buffer (20 mmol -1 HEPES, pH 7.4, 150 mmol -1 NaCl and 0.68 mmol -1 EDTA) and pelleted by centrifugation. The resulting pellet was suspended in cold 50 mmol -1 Tris buffer, pH 7.four and homogenized with a Tissue Tearor (Biospec Merchandise Inc., Bartlesville, OK). The homogenate was centrifuged at 18 000g at four for 20 min, plus the pellet resuspended in 50 mmol -1 Tris, homogenized with a Tissue Tearor and recentrifuged. The final pellet was resuspended in 50 mmol -1 Tris, aliquoted and stored at -80 till use. Protein concentration was measured by the technique of Bradford (1976).[3H]Diprenorphine binding For competitive binding, cell membranes have been incubated for 75 min at 25 with varying concentrations (0.1 nmol -11 mmol -1) of ligand and 0.2 nmol -1 [3H]diprenorphine in 50 mmol -1 Tris, pH 7.4 with and with out the presence of 100 mmol -1 NaCl and ten mmol -1 GTPgS. Non-specificBritish Journal of Pharmacology (2009) 156 1044m-Opioid antagonists and inverse agonists MF Divin et albinding was determined in the presence of 10 mmol -1 naloxone. Assays were stopped by fast filtration by way of glass microfiber filtermats, variety GF/C (Whatman, Clifton, NJ) by using a Brandell harvester (Gaithersburg, MD) followed by washing with cold 50 mmol -1 Tris buffer. Filtermats had been dried, and 0.1 mL Ecolume was added to every single sample. Filtermats were heat sealed in polyethylene bags, and radioactivity retained on the filters was measured by 1-Undecanol MedChemExpress liquid scintillation counting inside a Wallac 1450 MicroBeta Liquid Scintillation and Luminescence Counter (Perkin Elmer, Boston, MA). [35S]GTPgS [Gu.
