Peripheral Delta Opioid Receptors Mediate Formoterol Anti-allodynic Effect in a Mouse Model of Neuropathic Pain.

Neuropathic pain is a challenging condition for which current therapies often remain unsatisfactory. Chronic administration of β2 adrenergic agonists, including formoterol currently used to treat asthma and chronic obstructive pulmonary disease, alleviates mechanical allodynia in the sciatic nerve cuff model of neuropathic pain. The limited clinical data currently available also suggest that formoterol would be a suitable candidate for drug repurposing. The antiallodynic action of β2 adrenergic agonists is known to require activation of the delta-opioid (DOP) receptor but better knowledge of the molecular mechanisms involved is necessary. Using a mouse line in which DOP receptors were selectively ablated in neurons expressing Nav1.8 sodium channels (DOP cKO), we showed that these DOP peripheral receptors were necessary for the antiallodynic action of the β2 adrenergic agonist formoterol in the cuff model. Using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP), we established in a previous study, that mechanical allodynia is associated with a smaller percentage of DOPeGFP positive small peptidergic sensory neurons in dorsal root ganglia (DRG), with a reduced density of DOPeGFP positive free nerve endings in the skin and with increased DOPeGFP expression at the cell surface. Here, we showed that the density of DOPeGFP positive free nerve endings in the skin is partially restored and no increase in DOPeGFP translocation to the plasma membrane is observed in mice in which mechanical pain is alleviated upon chronic oral administration of formoterol. This study, therefore, extends our previous results by confirming that changes in the mechanical threshold are associated with changes in peripheral DOP profile. It also highlights the common impact on DOP receptors between serotonin noradrenaline reuptake inhibitors such as duloxetine and the β2 mimetic formoterol.journal article20192020 02 14importe

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

mu-Opioid receptor antibody reveals tissue-dependent specific staining and increased neuronal mu-receptor immunoreactivity at the injured nerve trunk in mice

Neuropathic pain is a debilitating chronic disease often resulting from damage to peripheral nerves. Activation of opioid receptors on peripheral sensory neurons can attenuate pain without central nervous system side effects. Here we aimed to analyze the distribution of neuronal mu-opioid receptors, the most relevant opioid receptors in the control of clinical pain, along the peripheral neuronal pathways in neuropathy. Hence, following a chronic constriction injury of the sciatic nerve in mice, we used immunohistochemistry to quantify the mu-receptor protein expression in the dorsal root ganglia (DRG), directly at the injured nerve trunk, and at its peripheral endings in the hind paw skin. We also thoroughly examined the mu-receptor antibody staining specificity. We found that the antibody specifically labeled mu-receptors in human embryonic kidney 293 cells as well as in neuronal processes of the sciatic nerve and hind paw skin dermis, but surprisingly not in the DRG, as judged by the use of mu/delta/kappa-opioid receptor knockout mice. Therefore, a reliable quantitative analysis of mu-receptor expression in the DRG was not possible. However, we demonstrate that the mu-receptor immunoreactivity was strongly enhanced proximally to the injury at the nerve trunk, but was unaltered in paws, on days 2 and 14 following injury. Thus, mu-opioid receptors at the site of axonal damage might be a promising target for the control of painful neuropathies. Furthermore, our findings suggest a rigorous tissue-dependent characterization of antibodies' specificity, preferably using knockout animals