PKB values were calculated from shifts in m-opioid agonist concentrationeffect curves brought on by a single (one hundred nmol -1) concentration of antagonist within the cAMP accumulation assays in accordance with the equation pKB = -log[B/(dose-ratio – 1)], 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 in the DAMGO concentration ffect curves in the [35S]GTPgS assay experiments in response to 3 different concentrations on the antagonists as outlined by the Schild process (Arunlakshana and Schild, 1959). The data presented are from a minimum of three experiments performed in duplicate, with results presented as imply SEM. Information have been compared by using a two-tailed t-test, or two-way ANOVA to compare concentration esponse curves. Differences had been considered significant if P 0.05.Cell surface receptor levels HEK293-FLAG-m cells had been seeded onto poly-D-lysine coated plates (BD Biosciences, San Jose, CA) and incubated with orBritish Journal of Pharmacology (2009) 156 1044Drugs and reagents Tissue culture media, Geneticin, fetal bovine serum and trypsin had 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 (Aurora, OH). Morphine sulphate, 6b-naltrexol, L-Cysteinesulfinic acid (monohydrate) Epigenetic Reader Domain naltrexone and naloxone were obtained through the Narcotic Drug and Opioid Peptide Standard Study Center in the University of Michigan (Ann Arbor, MI). DAMGO, CTAP, GDP, GTPgS, forskolin, IBMX and all other biochemicals have 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 present from Dr Lakshmi Devi, Mt. Sinai School of Medicine, New York, NY.m-Opioid antagonists and inverse agonists MF Divin et alResultsAdenylyl cyclase sensitization On chronic therapy 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 Monensin methyl ester Biological Activity overshoot in C6m cells an roughly EC30 concentration of 10 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, had been all capable to induce a cAMP overshoot following overnight treatment of C6m cells together with the high-efficacy m-opioid agonist DAMGO (ten mmol -1; Figure 1A). All antagonists induced the identical degree of cAMP overshoot that was precisely the same as that obtained by washing cells by removing and replacing media to dissociate bound opioid agonist from the receptor (P 0.05). Making use of morphine (ten mmol -1) to induce AC sensitization gave a reduced percentage of cAMP overshoot compared with DAMGO across the antagonists, as previously reported (Liu and Prather, 2001), however the antagonists all gave a similar results with all the putative inverse agonist naltrexone providing exactly the same degree of overshoot (225 20 ) as 6b-naltrexol (248 16 ), CTAP (277 14 ) or RTI5989-25 (202 13 ). Moreover, the phosphodiesterase inhibitor IBMX present in our assays to stop cAMP breakdown has been reported to block the inverse agoni.
