Ne, naltrexone and CTAP as previously reported (Wang et al., 2001; 2007a), indicating a very higher sensitivity to agonist stimulation within this system. Sodium ions by decreasing the degree of active R receptor also reduce basal G-protein activation. Consequently, basal signalling can be improved by replacing Na+ ions with K+ ions (Szekeres and Traynor, 1997; Selley et al., 2000). Under these circumstances, basal [35S]GTPgS stimulation was pretty much doubled (14.9 fmol g-1 in NaCl, 27.2 fmol g-1 in KCl). All assays have been performed in the presence of 2.4 mmol -1 dithiothreitol with the exception of CTAP exactly where noted. Values represent indicates SEM for three to 5 experiments performed in duplicate. Basal binding values are offered as fmol g-1 protein. [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; DTT, dithiothreitol; RTI-5989-25, (+)-N-[trans-4-(2-methylphenyl)-2-butenyl]-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine. P 0.05, P 0.001, substantially distinctive from basal values.naltrexone and naloxone did not alter G-protein activation from basal values. In contrast, RTI-5989-25 and CTAP considerably decreased basal binding of [35S]GTPgS (P 0.001), suggesting inverse agonist activity in this assay. CTAP can be a cyclic peptide constrained by a disulphide bridge, and so the integrity of this structure may be compromised by the presence of your disulphide lowering agent, DTT present within the [35S]GTPgS assay buffer. FD&C RED NO. 40;CI 16035 Protocol Indeed, within the absence of DTT, CTAP no longer lowered [35S]GTPgS binding under basal values, but rather showed partial agonist activity that was considerable in the presence of Na+ ions (Table 2). This reversal of CTAP efficacy in the absence of DTT was not, however, because of breaking of your disulphide bond of CTAP, which was stable to incubation with 2.five mmol -1 DTT for 1 h at 25 as determined by mass spectrometry (data not shown), in agreement with the stability of this compound in vivo (Abbruscato et al., 1997). Furthermore, the receptor binding affinity for CTAP was not significantly various in the presence or absence of DTT (Ki: 1.52 0.31 nmol -1 within the absence of DTT; 1.75 0.41 nmol -1 in the presence of DTT) confirming stability with the peptide. Chronic agonist therapy has been reported to reveal inverse agonist activity at the level of [35S]GTPgS binding in HEK293 cells stably expressing the m-opioid receptor (Burford et al., 2000), in GH3 cells (Liu and 497-23-4 Autophagy Prather, 2001) and in brain membranes from chronically morphine-treated mice (Wang et al., 2004). Though our findings with cAMP overshoot do not support this, we examined [35S]GTPgS binding right after chronic agonist therapy. C6m cells were treated overnight with 10 mmol -1 DAMGO, which causes an eightfold shift in the potency of DAMGO and a 50 reduction in maximal impact of DAMGO to stimulate [35S]GTPgS binding in these cells (Yabaluri and Medzihradsky, 1997). [35S]GTPgS binding was then examined inside the presence of either one hundred mmol -1 NaCl or KCl (Table 2). There was no transform within the basal amount of [35S]GTPgS binding suggesting no improve in active states ofFigure 2 Cell surface receptor levels in HEK293-FLAG-m cells treated for 24 h with 10 mmol -1 6b-naltrexol, naltrexone, RTI-5989-25 (RTI) or CTAP. Values are expressed as percentage of control, vehicletreated cells and represent mean SEM of three experiments performed in duplicate. P 0.05, P 0.01, considerably unique from vehicl.
