PKB values had been calculated from shifts in m-opioid agonist concentrationeffect curves triggered by a single (one hundred nmol -1) concentration of antagonist in the cAMP accumulation assays in line with the equation pKB = -log[B/(dose-ratio – 1)], exactly where B equals the concentration of opioid receptor antagonist and doseratio represents the EC50 concentration in the presence of antagonist divided by the EC50 concentration in the absence of antagonist (Divin et al., 2008). pA2 values were determined from shifts within the DAMGO concentration ffect curves inside the [35S]GTPgS assay experiments in response to three different concentrations from the antagonists in line with the Schild strategy (Arunlakshana and Schild, 1959). The data presented are from at least 3 experiments performed in duplicate, with benefits presented as mean SEM. Data were compared by utilizing a two-tailed t-test, or two-way ANOVA to examine concentration esponse curves. Variations have been thought of considerable if P 0.05.Cell surface receptor levels HEK293-FLAG-m cells have been seeded onto poly-D-lysine coated plates (BD Biosciences, San Jose, CA) and incubated with orBritish Journal of Pharmacology (2009) 156 1044Drugs and 487-79-6 MedChemExpress reagents Tissue culture media, Geneticin, fetal bovine serum and trypsin have been from Invitrogen (Carlsbad, CA). [35S]GTPgS (1250 Ci mol-1) and [3H]diprenorphine (50 Ci mol-1) were obtained from Perkin-Elmer Life Sciences (Boston, MA). Adenosine deaminase was obtained from CalBiochem (San Diego, CA). Ecolume scintillation fluid was from ICN (520-33-2 Description Aurora, OH). morphine sulphate, 6b-naltrexol, naltrexone and naloxone had been obtained by means of the Narcotic Drug and Opioid Peptide Standard Investigation Center in the University of Michigan (Ann Arbor, MI). DAMGO, CTAP, GDP, GTPgS, forskolin, IBMX and all other biochemicals had been from Sigma (St. Louis, MO) and were of analytical grade. RTI-5989-25 was ready as previously described (Zaki et al., 2001). FLAG-tagged mouse m-opioid receptor was a kind gift from Dr Lakshmi Devi, Mt. Sinai College of Medicine, New York, NY.m-Opioid antagonists and inverse agonists MF Divin et alResultsAdenylyl cyclase sensitization On chronic remedy and subsequent rapid removal of opioid agonist, cells expressing m-opioid receptors exhibit an enhanced cAMP accumulation (overshoot) above untreated forskolin-stimulated controls (Watts and Neve, 2005). To assess cAMP overshoot in C6m cells an about EC30 concentration of ten mmol -1 forskolin was employed (Clark et al., 2004). At maximal concentration (ten mmol -1) the antagonists, 6b-naltrexol, CTAP, naltrexone, naloxone or RTI5989-25, were all capable to induce a cAMP overshoot following overnight remedy of C6m cells with all the high-efficacy m-opioid agonist DAMGO (ten mmol -1; Figure 1A). All antagonists induced the same degree of cAMP overshoot that was exactly the same as that obtained by washing cells by removing and replacing media to dissociate bound opioid agonist in the receptor (P 0.05). Using morphine (10 mmol -1) to induce AC sensitization gave a lower percentage of cAMP overshoot compared with DAMGO across the antagonists, as previously reported (Liu and Prather, 2001), however the antagonists all gave a related results together with the putative inverse agonist naltrexone providing precisely the same degree of overshoot (225 20 ) as 6b-naltrexol (248 16 ), CTAP (277 14 ) or RTI5989-25 (202 13 ). In addition, the phosphodiesterase inhibitor IBMX present in our assays to stop cAMP breakdown has been reported to block the inverse agoni.
