Sly usedC6m cells in studies of opioid signalling which includes AC sensitization (Clark et al., 2004; Clark and Traynor, 2006) and have shown related m-opioid-mediated sensitization in HEK cells (Clark and Traynor, 2006). We’ve compared the ability to precipitate expression of AC sensitization and the pharmacological profiles of naltrexone and 6b-naltrexol, along with the standard opioid antagonist naloxone, the peptidic antagonist CTAP and the identified d-opioid inverse agonist (+)N-[trans-4-(2-methylphenyl)-2-butenyl]-(3R,4R)-dimethyl-4(3-hydroxyphenyl)piperidine (RTI-5989-25; Zaki et al., 2001). The outcomes show that there is absolutely no inherent efficacy difference involving 6b-naltrexol and naltrexone beneath the situations studied and furthermore that development and manifestation of AC sensitization will not be dependent on the formation of a constitutively active m-opioid receptor.MethodsCell culture and treatments C6 rat glioma cells stably transfected with all the rat m-opioid receptor (C6m) or HEK293 cells stably transfected using the FLAG-tagged mouse m-opioid receptor were grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.five mg L-1 or 0.8 mg L-1 Geneticin respectively. Cells were grown in the presence of 10 fetal bovine serum at 37 in 5 CO2. For chronic opioid treatment, cells were incubated overnight with ten mmol -1 DAMGO ([D-Ala2,NMe-Phe4,Glyol5]-enkephalin). C6m cells had been used for all experiments except for the determination of cell surface receptor quantity, which utilized HEK cells expressing a FLAGtagged m-opioid receptor. C6m cells expressed three.2 0.two 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.four), detached from 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 745017-94-1 web pellet was suspended in cold 50 mmol -1 Tris buffer, pH 7.4 and homogenized using a Tissue Tearor (Biospec Goods Inc., Bartlesville, OK). The homogenate was centrifuged at 18 000g at 4 for 20 min, along with 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 system of Bradford (1976).[3H]Diprenorphine binding For competitive binding, cell membranes had been incubated for 75 min at 25 with varying concentrations (0.1 nmol -11 mmol -1) of ligand and 0.two nmol -1 [3H]diprenorphine in 50 mmol -1 Tris, pH 7.four with and with out the presence of one hundred 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 inside the presence of 10 mmol -1 naloxone. Assays have been stopped by fast filtration by way of glass microfiber filtermats, type GF/C (Monobenzone site Whatman, Clifton, NJ) by utilizing a Brandell harvester (Gaithersburg, MD) followed by washing with cold 50 mmol -1 Tris buffer. Filtermats have been dried, and 0.1 mL Ecolume was added to every single sample. Filtermats had been heat sealed in polyethylene bags, and radioactivity retained on the filters was measured by liquid scintillation counting inside a Wallac 1450 MicroBeta Liquid Scintillation and Luminescence Counter (Perkin Elmer, Boston, MA). [35S]GTPgS [Gu.