Abstract
Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects. Our objective was to discover a novel enhancer of ligand-induced MOR endocytosis and determine its effects on analgesia, tolerance and dependence. We used high-throughput screening to identify convallatoxin as an enhancer of ligand-induced MOR endocytosis with high potency and efficacy. Treatment of cells with convallatoxin enhanced morphine-induced MOR endocytosis through an adaptor protein 2 (AP2)/clathrin-dependent mechanism, attenuated morphine-induced phosphorylation of MOR, and diminished desensitization of membrane hyperpolarization. Furthermore, co-treatment with chronic convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance and dependence in morphine-tolerant mice. These findings suggest convallatoxin are potentially therapeutic for morphine side effects and open a new avenue to study MOR trafficking.
Highlights
Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects
MOR endocytosis, we screened a natural compound library (480 compounds) in the presence of morphine, using a sensitive enzyme complementation assay for MOR endocytosis in human osteosarcoma U2OS cells expressing human MOR (U2OS-MOR). [Met5]-enkephalin (ME) induced MOR internalization, serving as a positive control for the experimental system
To determine whether the enhancing effects of cardiac glycosides on morphine-induced MOR endocytosis was mediated through their inhibition of Na+/K+-ATPase, endogenous Na+/K+-ATPase expression was suppressed by short hairpin RNA against the Na+/K+-ATPase α1 subunit in U2OS-MOR cells (Supplementary Fig. 1)
Summary
Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects. Compared to wild-type mice, RMOR knock-in mice showed a potentiated analgesic effect but less tolerance and withdrawal in response to morphine, indicating a beneficial effect of MOR internalization in morphine analgesia[8]. It is hard to investigate the relationship between MOR endocytosis and morphine tolerance/dependence by two agonists mixture, due to their controversial mode of action in analgesia[11,12,13,14,15]. It is worth developing an alternative strategy to specific upregulate MOR endocytosis in response to morphine to reduce tolerance and dependence. We established a high-throughput screening assay to identify non-opioid small-molecule enhancers of MOR endocytosis and validated the identified molecules in both cell cultures and animals
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