Activation and internalization of the μ-opioid receptor by the newly discovered endogenous agonists, endomorphin-1 and endomorphin-2

The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal mu-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for mu-opioid receptors. Since the mu-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the mu-opioid receptor peptide agonist, [D-Ala(2),MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged mu-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the mu-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced mu-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA(2) - 8.4) is consistent with antagonism at the mu-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the mu-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced mu-opioid receptor activation can be visualized by receptor endocytosis. (C) 1999 IBRO. Published by Elsevier Science Ltd.