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 capability to precipitate expression of AC sensitization and also the pharmacological profiles of naltrexone and 6b-naltrexol, together with the standard opioid antagonist naloxone, the peptidic antagonist CTAP and also the recognized 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 is no inherent efficacy distinction involving 6b-naltrexol and naltrexone below the circumstances studied and additionally that development and manifestation of AC sensitization is just not dependent on the formation of a constitutively active m-opioid receptor.MethodsCell culture and therapies C6 rat glioma cells stably transfected with 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 0.5 mg L-1 or 0.8 mg L-1 Geneticin respectively. Cells had been grown in the presence of ten fetal bovine serum at 37 in 5 CO2. For chronic opioid therapy, cells have been incubated overnight with ten mmol -1 DAMGO ([D-Ala2,NMe-Phe4,Glyol5]-enkephalin). C6m cells have been utilized 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 three.two 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 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 using a 878385-84-3 supplier Tissue Tearor (Biospec Products 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 having a Tissue Tearor and recentrifuged. The final pellet was resuspended in 50 mmol -1 Tris, aliquoted and stored at -80 until use. Protein concentration was measured by the approach 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 0.2 nmol -1 [3H]diprenorphine in 50 mmol -1 Tris, pH 7.4 with and with out 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 in the presence of 10 mmol -1 naloxone. Assays had been stopped by fast filtration via 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 have been dried, and 0.1 mL Ecolume was added to each and every sample. Filtermats had been heat sealed in polyethylene bags, and radioactivity retained on the filters was measured by liquid scintillation counting within a Wallac 1450 MicroBeta Liquid Scintillation and Luminescence Counter (Perkin Elmer, Boston, MA). [35S]GTPgS [Gu.
