AGONIST-INDUCED FUNCTIONAL DESENSITIZATION OF THE MU-OPIOID RECEPTOR IS MEDIATED BY LOSS OF MEMBRANE-RECEPTORS RATHER THAN UNCOUPLING FROM G-PROTEIN

The effects of acute exposure of the opioid peptide [D-Ala(2),N-MePhe( 4),Gly-ol(5)]enkephalin (DAMGO) on the mu-opioid receptor were examine d in Chinese hamster ovary (CHO) K-1 and baby hamster kidney stable tr ansfectants. In the CHO cell line, acute 1-hr treatment with DAMGO dec reased the density of receptors without affecting the affinity or prop ortion of agonist-detected sites and attenuated the ability of the ago nist to inhibit forskolin-stimulated cAMP accumulation. In contrast, s imilar 1-hr treatment of baby hamster kidney cells did not affect rece ptor density or agonist ability to inhibit cAMP accumulation, but long er duration of agonist exposure resulted in a reduction in membrane re ceptor, identical to the CHO cells, These results suggested that for t he mu-opioid receptor, alteration in receptor density was the major de terminant for the observed agonist-induced desensitization. Consistent with this notion, the ratio of the DAMGO concentration yielding half- maximal occupation of the mu receptor to that yielding half-maximal fu nctional response was <1. This suggests the necessity for a high mu re ceptor occupancy rate for maximal functional response, so that any los s of cell surface opioid-binding sites was a critical determinant in r educing the maximal response. This hypothesis was further supported by the observation that irreversible inactivation of fixed proportions o f opioid-binding sites with beta-chlornaltrexamine demonstrated that t here were few spare receptors, which is in contrast to what has been r eported for other G protein-coupled receptors, including the delta-opi oid receptor. Taken together, these data suggest that the opioid agoni st DAMGO has a high affinity for the mu receptor but must occupy >70% of the available receptors to generate the maximal second messenger-li nked response.