Sly usedC6m cells in studies of opioid signalling such as 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 have compared the capability to precipitate expression of AC sensitization and the pharmacological profiles of naltrexone and 6b-naltrexol, together with the normal opioid antagonist naloxone, the peptidic antagonist CTAP plus 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 results show that there isn’t any inherent efficacy difference involving 6b-naltrexol and naltrexone under the circumstances studied and additionally that development and manifestation of AC sensitization is not dependent around the formation of a constitutively active m-opioid receptor.MethodsCell culture and treatment options C6 rat glioma cells stably transfected with all the rat m-opioid receptor (C6m) or HEK293 cells stably transfected with all the FLAG-tagged mouse m-opioid receptor have been 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 had been grown within the presence of 10 fetal bovine serum at 37 in 5 CO2. For chronic opioid treatment, cells had been incubated overnight with ten mmol -1 DAMGO ([D-Ala2,NMe-Phe4,Glyol5]-enkephalin). C6m cells have been utilised 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 3.2 0.two pmol g-1 protein receptor and HEK cells 9.7 1.three pmol g-1 protein receptor, determined by [3H]diprenorphine binding.Membrane preparation Cells had 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 in the plate by incubation in harvesting buffer (20 mmol -1 HEPES, pH 7.four, 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 Items Inc., Bartlesville, OK). The homogenate was centrifuged at 18 000g at four for 20 min, as well as 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 1402837-79-9 Autophagy concentration was measured by the technique of Bradford (1976).[3H]Diprenorphine binding For competitive binding, cell membranes were 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 7.4 with and devoid of 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 ten mmol -1 naloxone. Assays have been stopped by fast filtration by way of glass microfiber filtermats, form GF/C (Whatman, Clifton, NJ) by using 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 each sample. Filtermats were heat sealed in polyethylene bags, and radioactivity retained on the filters was measured by liquid scintillation counting in a Wallac 1450 MicroBeta Liquid Scintillation and Luminescence Counter (Perkin Elmer, Histamine dihydrochloride In Vitro Boston, MA). [35S]GTPgS [Gu.
