Down-regulation of μ-Opioid Receptor by Full but Not Partial Agonists Is Independent of G Protein Coupling

Abstract

In C₆ glial cells stably expressing rat μ-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[γ-³⁵S]) binding by 367% with an EC₅₀value of 28 nm. Prolonged exposure to agonists induced desensitization of the receptor as estimated by a reduction in the maximal stimulation of GTP[γ-³⁵S] binding by DAMGO and rightward shifts in the dose-response curves. In cells treated with 10 μm concentrations of etorphine, DAMGO, β-endorphin, morphine, and butorphanol, DAMGO-stimulated GTP[γ-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 nmversus 18 nm for control). Chronic treatment with agonists reduced [³H]DAMGO binding in membranes with the rank order of etorphine > DAMGO = β-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 ∼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.