Sly usedC6m cells in studies of opioid signalling which includes 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 plus the pharmacological profiles of naltrexone and 6b-naltrexol, in conjunction with the common opioid antagonist naloxone, the peptidic antagonist CTAP and also the known 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 between 6b-naltrexol and naltrexone under the conditions studied and in addition that development and manifestation of AC sensitization is not dependent on the formation of a constitutively active m-opioid receptor.MethodsCell culture and remedies C6 rat glioma cells stably transfected using the rat m-opioid receptor (C6m) or HEK293 cells stably transfected with 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.eight mg L-1 Geneticin respectively. Cells have been grown within the 108341-18-0 Purity & Documentation presence of 10 fetal bovine serum at 37 in five CO2. For chronic opioid therapy, cells were incubated overnight with 10 mmol -1 DAMGO ([D-Ala2,NMe-Phe4,Glyol5]-enkephalin). C6m cells have been employed 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 were 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 Chlorsulfuron Purity pellet was suspended in cold 50 mmol -1 Tris buffer, pH 7.4 and homogenized having a Tissue Tearor (Biospec Items Inc., Bartlesville, OK). The homogenate was centrifuged at 18 000g at 4 for 20 min, and also 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 approach 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.two nmol -1 [3H]diprenorphine in 50 mmol -1 Tris, pH 7.four with and without the need of the presence of one hundred mmol -1 NaCl and 10 mmol -1 GTPgS. Non-specificBritish Journal of Pharmacology (2009) 156 1044m-Opioid antagonists and inverse agonists MF Divin et albinding was determined within the presence of 10 mmol -1 naloxone. Assays have been stopped by speedy filtration through glass microfiber filtermats, sort 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 had been heat sealed in polyethylene bags, and radioactivity retained around the filters was measured by liquid scintillation counting in a Wallac 1450 MicroBeta Liquid Scintillation and Luminescence Counter (Perkin Elmer, Boston, MA). [35S]GTPgS [Gu.
