AGONIST-SELECTIVE ENDOCYTOSIS OF MU-OPIOID RECEPTOR BY NEURONS IN-VIVO

Opiate alkaloids are potent analgesics that exert multiple pharmacolog ical effects in the nervous system by activating G protein-coupled rec eptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsive ness to ligands. Here, we investigated the agonist-induced internaliza tion of the mu opioid receptor (MOR) in vivo by using the guinea pig i leum as a model system and immunohistochemistry with an affinity-purif ied antibody to the C terminus of rat MOR, Antibody specificity was co nfirmed by the positive staining of human embryonic kidney 293 cells t ransfected with epitope-tagged MOR cDNA, by the lack of staining of ce lls transfected with the delta or kappa receptor cDNA, and by the abol ition of staining when the MOR antibody was preadsorbed with the MOR p eptide fragment, Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons , Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection o f the opiate agonist etorphine, intense MOR IR was present in vesiclel ike structures, which were identified as endosomes by confocal microsc opy, At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles, MOR-IR was still internalized at 120 min, Agonist-induced e ndocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause d etectable internalization, but it partially inhibited the etorphine-in duced MOR endocytosis. These results demonstrate the occurrence of ago nist-selective MOR endocytosis in neurons naturally expressing this re ceptor in vivo and suggest the existence of different mechanisms regul ating cellular responsiveness to ligands.