Down-regulation of mu-opioid receptor by full but not partial agonists is independent of G protein coupling.

In C6 glial cells stably expressing rat mu-opioid receptor, opioid agonist activation is negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. In membranes, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin (DAMGO) increases guanosine-5'-O-(3-[35S]thio)triphosphate (GTP[gamma-35S]) binding by 367% with an EC50 value of 28 nM. Prolonged exposure to agonists induced desensitization of the receptor as estimated by a reduction in the maximal stimulation of GTP[gamma-35S] binding by DAMGO and rightward shifts in the dose-response curves. In cells treated with 10 microM concentrations of etorphine, DAMGO, beta-endorphin, morphine, and butorphanol, DAMGO-stimulated GTP[gamma-35S] binding was 58%, 149%, 205%, 286%, and 325%, respectively. Guanine nucleotide regulation of agonist binding was correspondingly lower in membranes from tolerant cells. Furthermore, chronic opioid treatment increased forskolin-stimulated adenylyl cyclase activity, and potency of DAMGO to inhibit cAMP accumulation was lower in morphine- and DAMGO-tolerant cells (EC50 = 55 and 170 nM versus 18 nM for control). Chronic treatment with agonists reduced [3H]DAMGO binding in membranes with the rank order of etorphine > DAMGO = beta-endorphin > morphine > butorphanol, and the affinity of DAMGO in alkaloid- but not peptide-treated membranes was significantly lower in comparison with control. Pertussis toxin treatment of the cells before agonist treatment did not prevent the down-regulation by full agonists; DAMGO and etorphine exhibited approximately 80% internalization, whereas the ability of partial agonists was greatly impaired. In addition to establishing this cell line as a good model for further studies on the mechanisms of opioid tolerance, these results indicate important differences in the inactivation pathways of receptor triggered by full and partial agonists.