Internalization and trafficking of opioid receptor ligands in rat cortical neurons.
The binding, internalization, and trafficking of the fluorescently labeled opioid peptides Fluo-dermorphin and Fluo-deltorphin were quantitatively studied by confocal microscopy in primary cortical neurons in culture. Specific binding of these selective ligands to neurons naturally expressing mu (mu) and delta (delta) opioid receptors (OR), respectively, resulted in their internalization into neuronal somas and processes, as indicated by the persistence of fluorescent labeling following removal of cell surface binding by hypertonic acid wash. This internalization was receptor-specific, as the fluorescent signal was completely abolished when the cells were concomitantly incubated with the opioid receptor antagonist naloxone. It also was clathrin-dependent, as it was totally prevented by the endocytosis inhibitor phenylarsine oxide. Accordingly, internalized ligands were detected inside small, endosome-like vesicles. These labeled vesicles accumulated within nerve cell bodies between 5-30 min of incubation with the fluorescent ligands. This accumulation was abolished after treatment with the antitubular agent nocodazole, suggesting that it was due to a microtubule-dependent, retrograde transport of the internalized ligands from processes to the soma. By contrast, there was no change in the compartmentalization of internalized (mu)OR or deltaOR, as assessed by immunocytochemistry, suggesting that the latter were recycled locally. The present results provide the first demonstration of receptor-mediated internalization of opioid peptides in cultured neurons. It is proposed that their retrograde transport into target cells might be involved in mediating some of the long-term, transcriptional effects of opioids.
Lee
Mao-Cheng
MC
Department of Neurology and Neurosurgery, Montreal Neurological Institute, Montreal, Quebec, Canada.
Cahill
Catherine M
CM
Vincent
Jean-Pierre
JP
Beaudet
Alain
A
D000818:Animals; D000831:Animals, Newborn; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000187:drug effects / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D009292:Narcotic Antagonists / Q000494:pharmacology; D009294:Narcotics / Q000378:metabolism / Q000493:pharmacokinetics*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism / Q000493:pharmacokinetics*; D021381:Protein Transport / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011957:Receptors, Opioid / Q000187:drug effects* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008869:Microtubule-Associated Proteins; D009292:Narcotic Antagonists; D009294:Narcotics; D010455:Peptides; D011957:Receptors, Opioid