Opioid Receptors in Immune and Glial Cells-Implications for Pain Control

Opioid receptors comprise μ (MOP), δ (DOP), κ (KOP), and nociceptin/orphanin FQ (NOP) receptors. Opioids are agonists of MOP, DOP, and KOP receptors, whereas nociceptin/orphanin FQ (N/OFQ) is an agonist of NOP receptors. Activation of all four opioid receptors in neurons can induce analgesia in animal models, but the most clinically relevant are MOP receptor agonists (e.g., morphine, fentanyl). Opioids can also affect the function of immune cells, and their actions in relation to immunosuppression and infections have been widely discussed. Here, we analyze the expression and the role of opioid receptors in peripheral immune cells and glia in the modulation of pain. All four opioid receptors have been identified at the mRNA and protein levels in immune cells (lymphocytes, granulocytes, monocytes, macrophages) in humans, rhesus monkeys, rats or mice. Activation of leukocyte MOP, DOP, and KOP receptors was recently reported to attenuate pain after nerve injury in mice. This involved intracellular Ca2+-regulated release of opioid peptides from immune cells, which subsequently activated MOP, DOP, and KOP receptors on peripheral neurons. There is no evidence of pain modulation by leukocyte NOP receptors. More good quality studies are needed to verify the presence of DOP, KOP, and NOP receptors in native glia. Although still questioned, MOP receptors might be expressed in brain or spinal cord microglia and astrocytes in humans, mice, and rats. Morphine acting at spinal cord microglia is often reported to induce hyperalgesia in rodents. However, most studies used animals without pathological pain and/or unconventional paradigms (e.g., high or ultra-low doses, pain assessment after abrupt discontinuation of chronic morphine treatment). Therefore, the opioid-induced hyperalgesia can be viewed in the context of dependence/withdrawal rather than pain management, in line with clinical reports. There is convincing evidence of analgesic effects mediated by immune cell-derived opioid peptides in animal models and in humans. Together, MOP, DOP, and KOP receptors, and opioid peptides in immune cells can ameliorate pathological pain. The relevance of NOP receptors and N/OFQ in leukocytes, and of all opioid receptors, opioid peptides and N/OFQ in native glia for pain control is yet to be clarified

Distinct roles of exogenous opioid agonists and endogenous opioid peptides in the peripheral control of neuropathy-triggered heat pain

Neuropathic pain often results from peripheral nerve damage, which can involve immune response. Local leukocyte-derived opioid peptides or exogenous opioid agonists inhibit neuropathy-induced mechanical hypersensitivity in animal models. Since neuropathic pain can also be augmented by heat, in this study we investigated the role of opioids in the modulation of neuropathy-evoked heat hypersensitivity. We used a chronic constriction injury of the sciatic nerve in wild-type and opioid peptide-knockout mice, and tested opioid effects in heat and mechanical hypersensitivity using Hargreaves and von Frey tests, respectively. We found that although perineural exogenous opioid agonists, including peptidergic ligands, were effective, the endogenous opioid peptides β-endorphin, Met-enkephalin and dynorphin A did not alleviate heat hypersensitivity. Specifically, corticotropin-releasing factor, an agent triggering opioid peptide secretion from leukocytes, applied perineurally did not attenuate heat hypersensitivity in wild-type mice. Exogenous opioids, also shown to release opioid peptides via activation of leukocyte opioid receptors, were equally analgesic in wild-type and opioid peptide-knockout mice, indicating that endogenous opioids do not contribute to exogenous opioid analgesia in heat hypersensitivity. Furthermore, exogenously applied opioid peptides were ineffective as well. Conversely, opioid peptides relieved mechanical hypersensitivity. Thus, both opioid type and sensory modality may determine the outcome of neuropathic pain treatment

Cutaneous nociceptors lack sensitisation, but reveal \u3bc-opioid receptor-mediated reduction in excitability to mechanical stimulation in neuropathy

Background: Peripheral nerve injuries often trigger a hypersensitivity to tactile stimulation. Behavioural studies demonstrated efficient and side effect-free analgesia mediated by opioid receptors on peripheral sensory neurons. However, mechanistic approaches addressing such opioid properties in painful neuropathies are lacking. Here we investigated whether opioids can directly inhibit primary afferent neuron transmission of mechanical stimuli in neuropathy. We analysed the mechanical thresholds, the firing rates and response latencies of sensory fibres to mechanical stimulation of their cutaneous receptive fields.Results: Two weeks following a chronic constriction injury of the saphenous nerve, mice developed a profound mechanical hypersensitivity in the paw innervated by the damaged nerve. Using an in vitro skin-nerve preparation we found no changes in the mechanical thresholds and latencies of sensory fibres from injured nerves. The firing rates to mechanical stimulation were unchanged or reduced following injury. Importantly, \u3bc-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5]-ol-enkephalin (DAMGO) significantly elevated the mechanical thresholds of nociceptive A\u3b4 and C fibres. Furthermore, DAMGO substantially diminished the mechanically evoked discharges of C nociceptors in injured nerves. These effects were blocked by DAMGO washout and pre-treatment with the selective \u3bc-opioid receptor antagonist Cys2-Tyr3-Orn5-Pen7-amide. DAMGO did not alter the responses of sensory fibres in uninjured nerves.Conclusions: Our findings suggest that behaviourally manifested neuropathy-induced mechanosensitivity does not require a sensitised state of cutaneous nociceptors in damaged nerves. Yet, nerve injury renders nociceptors sensitive to opioids. Prevention of action potential generation or propagation in nociceptors might represent a cellular mechanism underlying peripheral opioid-mediated alleviation of mechanical hypersensitivity in neuropathy. \ua9 2012 Schmidt et al.; licensee BioMed Central Ltd

Mu-Opioid Receptor Agonist Induces Kir3 Currents in Mouse Peripheral Sensory Neurons - Effects of Nerve Injury

Neuropathic pain often arises from damage to peripheral nerves and is difficult to treat. Activation of opioid receptors in peripheral sensory neurons is devoid of respiratory depression, sedation, nausea, and addiction mediated in the brain, and ameliorates neuropathic pain in animal models. Mechanisms of peripheral opioid analgesia have therefore gained interest, but the role of G protein-coupled inwardly rectifying potassium (Kir3) channels, important regulators of neuronal excitability, remains unclear. Whereas functional Kir3 channels have been detected in dorsal root ganglion (DRG) neurons in rats, some studies question their contribution to opioid analgesia in inflammatory pain models in mice. However, neuropathic pain can be diminished by activation of peripheral opioid receptors in mouse models. Therefore, here we investigated effects of the selective μ-opioid receptor (MOR) agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) on potassium conductance in DRG neurons upon a chronic constriction injury (CCI) of the sciatic nerve in mice. For verification, we also tested human embryonic kidney (HEK) 293 cells transfected with MOR and Kir3.2. Using patch clamp, we recorded currents at -80 mV and applied voltage ramps in high extracellular potassium concentrations, which are a highly sensitive measures of Kir3 channel activity. We found a significantly higher rate of HEK cells responding with potassium channel blocker barium-sensitive inward current (233 +/- 51 pA) to DAMGO application in transfected than in untransfected group, which confirms successful recordings of inward currents through Kir3.2 channels. Interestingly, DAMGO induced similar inward currents (178 +/- 36-207 +/- 56 pA) in 15-20% of recorded DRG neurons from naive mice and in 4-27% of DRG neurons from mice exposed to CCI, measured in voltage clamp or voltage ramp modes. DAMGO-induced currents in naive and CCI groups were reversed by barium and a more selective Kir3 channel blocker tertiapin-Q. These data indicate the coupling of Kir3 channels with MOR in mouse peripheral sensory neuron cell bodies, which was unchanged after CCI. A comparative analysis of opioid-induced potassium conductance at the axonal injury site and peripheral terminals of DRG neurons could clarify the role of Kir3 channel-MOR interactions in peripheral nerve injury and opioid analgesia

Amelioration of injury-induced tissue acidosis by a nonsteroidal analgesic attenuates antinociceptive effects of the pH-dependent opioid agonist NFEPP

Opioid agonists are powerful drugs for managing pain. However, their central side effects are limiting their use and drugs with similar potency, but a lower risk profile are needed. (+/-)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenylpropionamide (NFEPP) is a novel opioid agonist that preferentially activates opioid receptors at acidic extracellular pH. NFEPP was designed to activate peripheral opioid receptors in injured tissue, therefore precluding side effects elicited at normal pH in brain or intestinal wall. Considering the common combination of opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) in multimodal analgesia, we investigated the interaction between NFEPP and a widely prescribed prototypical NSAID, diclofenac (DCF), in a rat model of unilateral hindpaw inflammation induced by complete Freund's adjuvant. We evaluated the effects of systemically applied DCF on the paw tissue pH, on the expression of inflammatory mediators in immune cells from inflamed paws and on the expression of opioid receptors in dorsal root ganglia. Additionally, we investigated the antinociceptive efficacy of NFEPP injected into the inflamed paws after DCF treatment. We found that DCF reduced inflammation-induced nociceptive responses and tissue acidosis, but did not change the mRNA expression of IL-1 beta, TNF-alpha, IL-6, IL-4, NGF, or of mu-, delta-, or kappa-opioid receptors. The treatment with DCF moderately reduced the antinociceptive efficacy of NFEPP, suggesting a correlation between an increase in local tissue pH and the decreased antinociceptive effect of this pH-sensitive opioid agonist

Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides

Background During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti- inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. Methods Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 105 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. Results Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1–17), and β-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the fluorescently labeled M0, M1, and M2 cells ex vivo showed that they remained in the nerve and preserved their phenotype. Conclusions Perineural transplantation of M2 macrophages resulted in opioid-mediated amelioration of neuropathy-induced mechanical hypersensitivity, while M1 macrophages did not exacerbate pain. Therefore, rather than focusing on macrophage-induced pain generation, promoting opioid-mediated M2 actions may be more relevant for pain control

Humanization of the Reaction Specificity of Mouse Alox15b Inversely Modified the Susceptibility of Corresponding Knock-In Mice in Two Different Animal Inflammation Models

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in the pathogenesis of inflammatory diseases, and its pro- and anti-inflammatory effects have been reported for different ALOX-isoforms. Human ALOX15B oxygenates arachidonic acid to its 15-hydroperoxy derivative, whereas the corresponding 8-hydroperoxide is formed by mouse Alox15b (Alox8). This functional difference impacts the biosynthetic capacity of the two enzymes for creating pro- and anti-inflammatory eicosanoids. To explore the functional consequences of the humanization of the reaction specificity of mouse Alox15b in vivo, we tested Alox15b knock-in mice that express the arachidonic acid 15-lipoxygenating Tyr603Asp and His604Val double mutant of Alox15b, instead of the arachidonic acid 8-lipoxygenating wildtype enzyme, in two different animal inflammation models. In the dextran sodium sulfate-induced colitis model, female Alox15b-KI mice lost significantly more bodyweight during the acute phase of inflammation and recovered less rapidly during the resolution phase. Although we observed significant differences in the colonic levels of selected pro- and anti-inflammatory eicosanoids during the time-course of inflammation, there were no differences between the two genotypes at any time-point of the disease. In Freund's complete adjuvant-induced paw edema model, Alox15b-KI mice were less susceptible than outbred wildtype controls, though we did not observe significant differences in pain perception (Hargreaves-test, von Frey-test) when the two genotypes were compared. our data indicate that humanization of the reaction specificity of mouse Alox15b (Alox8) sensitizes mice for dextran sodium sulfate-induced experimental colitis, but partly protects the animals in the complete Freund's adjuvant-induced paw edema model

Opioid receptor signaling, analgesic and side effects induced by a computationally designed pH-dependent agonist

Novel pain killers without adverse effects are urgently needed. Opioids induce central and intestinal side effects such as respiratory depression, sedation, addiction, and constipation. We have recently shown that a newly designed agonist with a reduced acid dissociation constant (pK(a)) abolished pain by selectively activating peripheral mu-opioid receptors (MOR) in inflamed (acidic) tissues without eliciting side effects. Here, we extended this concept in that pK(a) reduction to 7.22 was achieved by placing a fluorine atom at the ethylidene bridge in the parental molecule fentanyl. The new compound (FF3) showed pH-sensitive MOR affinity, [S-35]-GTP gamma S binding, and G protein dissociation by fluorescence resonance energy transfer. It produced injury-restricted analgesia in rat models of inflammatory, postoperative, abdominal, and neuropathic pain. At high dosages, FF3 induced sedation, motor disturbance, reward, constipation, and respiratory depression. These results support our hypothesis that a ligand's pK(a) should be close to the pH of injured tissue to obtain analgesia without side effects

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

<p>Abstract</p> <p>Background</p> <p>Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats.</p> <p>Results</p> <p>POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin.</p> <p>Conclusion</p> <p>Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.</p

Polyglycerol-opioid conjugate produces analgesia devoid of side effects

Novel painkillers are urgently needed. The activation of opioid receptors in peripheral inflamed tissue can reduce pain without central adverse effects such as sedation, apnoea, or addiction. Here, we use an unprecedented strategy and report the synthesis and analgesic efficacy of the standard opioid morphine covalently attached to hyperbranched polyglycerol (PG-M) by a cleavable linker. With its high-molecular weight and hydrophilicity, this conjugate is designed to selectively release morphine in injured tissue and to prevent blood-brain barrier permeation. In contrast to conventional morphine, intravenous PG-M exclusively activated peripheral opioid receptors to produce analgesia in inflamed rat paws without major side effects such as sedation or constipation. Concentrations of morphine in the brain, blood, paw tissue, and in vitro confirmed the selective release of morphine in the inflamed milieu. Thus, PG-M may serve as prototype of a peripherally restricted opioid formulation designed to forego central and intestinal side effects