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

Abstract

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

Biased Agonism of Endogenous Opioid Peptides at theμ-Opioid Receptor

“…Interestingly, endo-1 has been shown to produce antinociceptive cross-tolerance to morphine whereas endo-2 does not, suggesting potential differences in the mechanisms of tolerance produced by these two ligands. As both endo-1 and endo-2 stimulate receptor internalization (McConalogue et al, 1999), they would both be expected to produce tolerance via a similar mechanism to that of DAMGO. This suggests that tolerance produced by endo-1 and endo-2 may involve differential activation of signaling pathways unrelated to receptor internalization, or alternatively, that the mechanisms of development of tolerance to these ligands are cell type-dependent.…”

Morphine and Endomorphins Differentially Regulate μ-Opioid Receptor mRNA in SHSY-5Y Human Neuroblastoma Cells

“…However, differences between the actions of morphine and those of the endomorphins have been noted. For example, endomorphins-1 and -2 bind the MOR at the cell surface and cause rapid internalization, similar to DAMGO, a MOR-specific synthetic peptide agonist (McConalogue et al, 1999), whereas morphine treatment down-regulates MOR membrane density (Zadina et al, 1993) without rapid MOR internalization (Keith et al, 1996;Zhang et al, 1998). It has been suggested that endomorphin-induced MOR endocytosis may be the mechanism for MOR desensitization and down-regulation (Harrison et al, 2000).…”

“…However, differences between the actions of morphine and those of the endomorphins have been noted. For example, chronic morphine treatment downregulates MOR membrane density (Zadina et al, 1993) without internalization of the MOR (Keith et al, 1996;Zhang et al, 1998), whereas endomorphins-1 and -2 have been shown to cause rapid endocytosis of the MOR (McConalogue et al, 1999).…”

Internalization and trafficking of opioid receptor ligands in rat cortical neurons

“…Thus, studies from our laboratory have demonstrated that OR and ␦OR, when coexpressed in the same cells, internalized through partly distinct endocytic pathways and were sorted via different endocytic vesicles (Gaudriault et al, 1997). Endocytosis of both OR and ␦OR was shown to be clathrin-dependent, based on its sensitivity to a variety of clathrin disrupters Trapaidze et al, 1996;McConalogue et al, 1999;Burford et al, 1998). There is now abundant evidence that internalization of OR occurs in neurons as well as in epithelial cells, both in vitro (Sternini et al, 2000;Keith et al, 1998;McConalogue et al, 1999) and in vivo (Sternini et al, 1996;Eckersell et al, 1998;Trafton et al, 2000).…”