St impact of naltrexone (Wang et al., 1999). Inside the absence of IBMX the percentage of cAMP overshoot in cells treated overnight with ten mmol -1 DAMGO was reduced, but removing IBMX from the assay did not reveal any significant difference involving the degree of overshoot observed with naltrexone (275 13 ) compared with 6b-naltrexol (274 16 ), RTI (266 68 ) or CTAP (313 34 ). As these benefits contrasted with prior reports, we further compared the capacity of 6b-naltrexol and naltrexone to induce a cAMP overshoot in chronic DAMGO-treated cells (Figure 1B). Both compounds concentration-dependently induced an increase in cAMP in treated cells over control, vehicle-treated cells with comparable EC50 values of 28.2 five.2 nmol -1 and 22.4 6.0 nmol -1 (P 0.05) respectively. This was confirmed when cAMP overshoot was precipitated having a combination of the two antagonists. Therefore, following overnight DAMGO, one hundred nmol -1 6b-naltrexol, 100 nmol -1 naltrexone or 50 nmol -1 6b-naltrexol with 50 nmol -1 naltrexone precipitated the same amount of cAMP overshoot (P 0.05; Figure 1C). These final results demonstrate that the loss of a constitutively active receptor isn’t essential for cAMP overshoot but that removing the chronic m-opioid agonist, either by challenge with antagonist or by washing, is enough. Consequently in this assay each of the antagonists appeared operationally the same, and so we undertook a extra extensive pharmacological analysis to much better define their relative efficacies as neutral antagonists or inverse agonists.Figure 1 Adenylyl cyclase sensitization in C6 glioma (C6m) cells. C6m cells were treated overnight with 10 mmol -1 DAMGO, and cAMP overshoot was precipitated within the presence of 10 mmol -1 forskolin and 1 mmol -1 IBMX to stop hydrolysis of cAMP. (A) cAMP overshoot was induced with 10 mmol -1 6b-naltrexol (6b-N), naltrexone (NTX), naloxone (NLX), RTI-5989-25 (RTI), CTAP or by washing. (B) cAMP overshoot was precipitated with 1 nmol -110 mmol -1 6b-naltrexol or naltrexone. (C) cAMP overshoot was precipitated with one hundred nmol -1 6b-naltrexol, one hundred nmol -1 naltrexone or 50 nmol -1 6b-naltrexol with 50 nmol -1 naltrexone. cAMP accumulation is expressed as a percentage of ten mmol -1 forskolin-stimulated cAMP levels in vehicle-treated handle cells (four.7 0.5 pmol g-1 56092-82-1 Description protein). Values represent imply SEM of 3 to 5 experiments performed in duplicate. The 95 confidence interval of all slopes inside the Schild evaluation contained unity. KB values have been determined by measurement from the capacity of one hundred nmol -1 antagonist to shift the concentration 815610-63-0 Purity esponse curve for DAMGO-induced inhibition of forskolin-stimulated cAMP accumulation. Facts of all assays are within the Techniques. For comparison, pKi and pKB values had been calculated as -log(K). Values represent implies SEM of three experiments performed in duplicate. [35S]GTPgS, guanosine-5-O-(3-[35S]thio)triphosphate; CTAP, H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; DAMGO, [D-Ala2,N-MePhe4,Glyol5]-enkephalin; ND, not determined. a Affinity determined by single concentration of antagonist. P 0.05, P 0.01, compared with Tris buffer.naltrexol, CTAP, naltrexone, naloxone and RTI-5989-25 to concentration-dependently displace the binding on the nonselective opioid antagonist [3H]diprenorphine in membranes from C6m cells was measured in Tris-HCl buffer with out and with 100 mmol -1 NaCl and 10 mmol -1 GTPgS. All compounds showed higher m-opioid receptor affinity in the order RTI-5989-25 naltrexone 6b-naltrexol CTAP n.
