Activation and internalization of the mu-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 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.
McConalogue
K
K
Department of Surgery, University of California, San Francisco 94143-0660, USA.
Grady
E F
EF
Minnis
J
J
Balestra
B
B
Tonini
M
M
Brecha
N C
NC
Bunnett
N W
NW
Sternini
C
C
D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology; D005434:Flow Cytometry; D006168:Guinea Pigs; D007082:Ileum / Q000187:drug effects / Q000294:innervation / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D009197:Myenteric Plexus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D051381:Rats; D017450:Receptors, Opioid, mu / Q000187:drug effects* / Q000378:metabolism*; D014018:Tissue Distribution / Q000502:physiology
D000701:Analgesics, Opioid; D004745:Enkephalins; D009842:Oligopeptides; D017450:Receptors, Opioid, mu; C105293:endomorphin 1; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C105294:endomorphin 2