Delta-opioid receptor endocytosis in spinal cord after dermenkephalin activation

BACKGROUND: The delta(δ)-opioid receptors belong to the G protein-coupled receptors and in vitro studies have shown that δ-opioid receptors undergo an internalization process in response to agonist stimulation. The immediate consequence is the disappearance of receptors from the plasma membrane. This adaptation process reveals the cell's capacity to desensitize after a strong agonist stimulus. This process, if it occurs in vivo, could contribute to the tolerance phenomenon observed after opiate treatment. To study the mechanisms underlying regulation of the δ-opioid receptors in vivo, the effects of an application of the drug dermenkephalin, a potent and selective agonist of the δ-opioid receptor, were analysed in the rat spinal cord. RESULTS: Using immunocytochemistry and electron microscopy, we observed in control rats that membrane labelling was strictly localized at the interface between two neurites. Fifteen minutes after dermenkephalin stimulation, the plasma membrane labelling was associated with invaginated areas. Thirty minutes after stimulation, labelled vesicles were found in the cytoplasm confirming the internalization process. CONCLUSIONS: The present findings support the view that δ-opioid receptors are internalized in response to prolonged exposure to dermenkephalin in vivo and confirm the presynaptic localization of δ-opioid receptors in the dorsal horn of the rat spinal cord

In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists

GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs