Opioid receptors are known to undergo complex regulatory changes in response to ligand exposure. In the present study, we examined the effect of morphine on the in vitro and in vivo density and trafficking of delta opioid receptors (deltaORs). Prolonged exposure (48 hr) of cortical neurons in culture to morphine (10 microm) resulted in a robust increase in the internalization of Fluo-deltorphin, a highly selective fluorescent deltaOR agonist. This effect was mu-mediated because it was entirely blocked by the selective mu opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) and was reproduced using the selective mu agonist fentanyl citrate. Immunogold electron microscopy revealed a marked increase in the cell surface density of deltaORs in neurons exposed to morphine, indicating that the increase in Fluo-deltorphin internalization was caused by increased receptor availability. Prolonged morphine exposure had no effect on deltaOR protein levels, as assessed by immunocytochemistry and Western blotting, suggesting that the increase in bioavailable deltaORs was caused by recruitment of reserve receptors from intracellular stores and not from receptor neosynthesis. Complementary in vivo studies demonstrated that chronic treatment of adult rats with morphine (5-15 mg/kg, s.c., every 12 hr) similarly augmented targeting of deltaORs to neuronal plasma membranes in the dorsal horn of the spinal cord. Furthermore, this treatment markedly potentiated intrathecal d-[Ala(2)]deltorphin II-induced antinociception. Taken together, these results demonstrate that prolonged stimulation of neurons with morphine markedly increases recruitment of intracellular deltaORs to the cell surface, both in vitro and in vivo. We propose that this type of receptor subtype cross-mobilization may widen the transduction repertoire of G-protein-coupled receptors and offer new therapeutic strategies.

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 (μ) and delta (δ) 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 μOR or δOR, 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. Synapse 43:102–111, 2002. © 2001 Wiley‐Liss, Inc.