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

1999; Elsevier BV; Volume: 90; Issue: 3 Linguagem: Inglês

10.1016/s0306-4522(98)00514-4

1873-7544

K. McConalogue, Eileen F. Grady, J. Minnis, B. Balestra, Marcello Tonini, Nicholas C. Brecha, Nigel W. Bunnett, Catia Sternini,

Pain Mechanisms and Treatments

The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal μ-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 μ-opioid receptors. Since the μ-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the μ-opioid receptor peptide agonist, [d-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged μ-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 μ-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced μ-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 (pA2∼8.4) is consistent with antagonism at the μ-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the μ-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced μ-opioid receptor activation can be visualized by receptor endocytosis.