Background and Purpose: The role of peripheral mu-opioid receptors (MOPs) in chronic pain conditions is not well understood. Here, we used a combination of mouse genetics, behavioral assays, and pharmacological interventions to investigate the contribution of MOPs on primary afferent neurons to nociceptive, inflammatory, and neuropathic pain as well as to opioid analgesia. Experimental Approach: We generated conditional knockout mice in which MOPs were selectively deleted in primary sensory neurons. Inflammatory and neuropathic pain states were induced in mutant and control wild-type mice and their behavioral responses to noxious stimuli compared. Gross motor function was also evaluated. Immunohistochemistry was used to assess MOP expression in the dorsal root ganglia, periaqueductal gray, and small intestine. Behavioral assays and whole-cell patch-clamp recordings were used to evaluate the inhibitory effects of mu-opioid agonists, DALDA and morphine, on pain behavior and neurophysiologic effects in DRG neurons, respectively. Key Results: Conditional MOP knockouts and control mice exhibited similar behavioral responses to acute nociceptive stimuli and developed similar inflammation-induced hypersensitivity. Nerve injury in animals lacking peripheral MOPs induced enhanced, bilateral mechanical allodynia. DALDA administered subcutaneously was unable to decrease the hypersensitivity induced by inflammation and nerve injury in MOP knockout animals, and morphine’s antinociceptive effects were significantly attenuated in the absence of peripheral MOPs. Conclusion and Implication: MOPs in primary sensory neurons contribute to the modulation of neuropathic pain behavior and opioid analgesia. Our observations highlight the clinical potential of peripherally acting opioid agonists in the management of chronic inflammatory and neuropathic pain.

Background and Purpose: Agonists to subtype C of the Mas-related G-protein-coupled receptors (MrgC) induce neuropathic pain inhibition after intrathecal (i.t.) administration in rodent models of nerve injury. Here, we investigated whether tolerance develops after repeated drug treatments and examined the underlying mechanisms. Experimental Approach: In male rats at 4-5 weeks after an L5 spinal nerve ligation (SNL), we conducted behavior tests to examine whether pain inhibition by JHU58, a dipeptide MrgC agonist, diminishes after repeated administration. We then examined agonist-induced endocytosis of MrgC in HEK293T cells, and the role of receptor ubiquitination in tolerance to JHU58-induced pain inhibition. Key Results: The inhibition of mechanical and heat hypersensitivity by JHU58 (0.1 mM, 10 μL, i.t.) decreased in SNL rats after repeated treatments with 0.5 mM JHU58 (10 μL, i.t., twice/day for 3 days). In HEK293T cells, acute treatment with JHU58 or BAM8-22 (a large peptide MrgC agonist) led to MrgC endocytosis from the cell membrane, and later sorting to the membrane for reinsertion. However, chronic exposure to JHU58 increased the coupling of MrgC to β-arrestin-2 and led to the ubiquitination and degradation of MrgC. Importantly, co-treatment with TAK-243 (0.2 mM, 5 μL, i.t.), a small-molecule inhibitor of the ubiquitin activating enzyme, during tolerance induction attenuated the development of JHU58 tolerance in SNL rats. Conclusion and Implications: These findings suggest that tolerance can develop to MrgC agonist-induced pain inhibition after repeated intrathecal administrations. This tolerance development may involve increased coupling of MrgC to β-arrestin-2 and ubiquitin-mediated receptor degradation.