DOWN-REGULATION OF MU-OPIOID RECEPTOR BY FULL BUT NOT PARTIAL AGONISTS IS INDEPENDENT OF G-PROTEIN COUPLING

In C-6 glial cells stably expressing rat mu-opioid receptor, opioid ag onist activation is negatively coupled to adenylyl cyclase through per tussis toxin-sensitive G proteins. In membranes, [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO) increases guanosine-5'-O-(3-[S-35]thio)t riphosphate (GTP[gamma-S-35]) binding by 367% with an EC50 value of 28 nM. Prolonged exposure to agonists induced desensitization of the rec eptor as estimated by a reduction in the maximal stimulation of GTP[ga mma-S-35] binding by DAMGO and rightward shifts in the dose-response c urves. In cells treated with 10 mu M concentrations of etorphine, DAMG O, beta-endorphin, morphine, and butorphanol, DAMGO-stimulated GTP[gam ma-S-35] binding was 58%, 149%, 205%, 286%, and 325%, respectively. Gu anine nucleotide regulation of agonist binding was correspondingly low er in membranes from tolerant cells. Furthermore, chronic opioid treat ment increased forskolin-stimulated adenylyl cyclase activity, and pot ency of DAMGO to inhibit cAMP accumulation was lower in morphine- and DAMGO-tolerant cells (EG(50) = 55 and 170 nM versus 18 nM for control) . Chronic treatment with agonists reduced [H-3]DAMGO binding in membra nes with the rank order of etorphine > DAMGO = beta-endorphin > morphi ne > butorphanol, and the affinity of DAMGO in alkaloid- but not pepti de-treated membranes was significantly lower in comparison with contro l. Pertussis toxin treatment of the cells before agonist treatment did not prevent the down-regulation by full agonists; DAMGO and etorphine exhibited similar to 80% internalization, whereas the ability of part ial agonists was greatly impaired. in addition to establishing this ce ll line as a good model for further studies on the mechanisms of opioi d tolerance, these results indicate important differences in the inact ivation pathways of receptor triggered by full and partial agonists.