Mu-opioid Receptor Down-regulation Research Articles

The Epigenetics of Neuropathic Pain: A Systematic Update.

Epigenetics deals with alterations to the gene expression that occur without change in the nucleotide sequence in the DNA. Various covalent modifications of the DNA and/or the surrounding histone proteins have been revealed, including DNA methylation, histone acetylation, and methylation, which can either stimulate or inhibit protein expression at the transcriptional level. In the past decade, an exponentially increasing amount of data has been published on the association between epigenetic changes and the pathomechanism of pain, including its most challenging form, neuropathic pain. Epigenetic regulation of the chromatin by writer, reader, and eraser proteins has been revealed for diverse protein targets involved in the pathomechanism of neuropathic pain. They include receptors, ion channels, transporters, enzymes, cytokines, chemokines, growth factors, inflammasome proteins, etc. Most work has been invested in clarifying the epigenetic downregulation of mu opioid receptors and various K+ channels, two types of structures mediating neuronal inhibition. Conversely, epigenetic upregulation has been revealed for glutamate receptors, growth factors, and lymphokines involved in neuronal excitation. All these data cannot only help better understand the development of neuropathic pain but outline epigenetic writers, readers, and erasers whose pharmacological inhibition may represent a novel option in the treatment of pain.

Intrathecal administration of the fat-mass and obesity-associated protein inhibitor mitigates neuropathic pain in female rats.

Several intracellular signals are involved in the sexual dimorphism of chronic pain. Our previous studies demonstrated that the fat-mass and obesity-associated protein (FTO), a demethylase of RNA N6-methyladenosine, in the injured dorsal root ganglion (DRG) contributed to the development and maintenance of nerve injury-induced nociceptive hypersensitivity in male rats and male mice. However, whether these effects of DRG FTO are in a sex-dependent manner is still unknown. The present study sought to investigate the effect of intrathecal administration of a specific FTO inhibitor, meclofenamic acid (MA), on chronic constriction injury (CCI)-induced nociceptive hypersensitivity in female rats. Intrathecal injection of MA attenuated the CCI-induced mechanical allodynia, heat hyperalgesia, and cold hyperalgesia in both the induction and maintenance periods, without changing acute/basal pain and locomotor function, in female rats. Intrathecal MA also blocked the CCI-induced hyperactivations of neurons and astrocytes in the ipsilateral L4 and L5 dorsal horns of female rats. Mechanistically, intrathecal MA prevented the CCI-induced increase in the histone methyltransferase G9a expression and reversed the G9a-controlled downregulation of mu-opioid receptor and Kv1.2 proteins in the ipsilateral L4 and L5 DRGs of female rats. These findings indicate that the effects of the FTO inhibitor on nerve injury-induced nociceptive hypersensitivity in female rats are similar to those in male rats reported previously. Our data also further confirm the role of DRG FTO in neuropathic pain and suggest potential clinical application of the FTO inhibitors for the prevention and treatment of this disorder in both men and women.

Maternal opioids decrease mu‐opioid receptor expression in the neonatal preBötzinger Complex

An understudied population in the opioid crisis are infants exposed to in utero opioids experiencing severe respiratory deficits. Our recent work demonstrated maternal opioids destabilized neonatal breathing, increased apneas, and blunted opioid‐induced respiratory frequency depression; however, the mechanism(s) underlying these neonatal breathing deficits remains unclear. We hypothesized deficits in neonatal breathing after maternal opioids are due to downregulation of mu‐opioid receptors on key cell types (e.g. neurons and astrocytes), essential for respiratory rhythm generation and modulation in the preBötzinger Complex. While opioid receptor expression is downregulated in the whole brain after maternal opioids, it remains unclear if opioid receptor expression changes in the preBötzinger Complex after in utero opioids to induce neonatal breathing deficits. To assess the typical developmental changes in opioid receptors in the preBötzinger Complex, mu‐opioid receptor expression was first assessed using immunohistochemistry and confocal microscopy on neonatal neurons and astrocytes in the preBötzinger Complex at three ages: the day of birth (postnatal day 0, P0) when neonates must begin robust rhythmic breathing, an early neonatal age (postnatal day 4, P4), and during a critical developmental window (postnatal day 11, P11). At all ages, mu‐opioid receptors were expressed on both neurons and astrocytes in the preBötzinger Complex; however, mu‐opioid receptor expression decreased from P0 (n=6) to P4 (n=6) and was lowest at P11 (n=6). Preliminary data after maternal opioids suggest mu‐opioid receptor expression was lower in preBötzinger Complexes at P0 (n=2) and P4 (n=2), but returned to control levels by P11 (n=2). These results are further supported by in vitro brainstem‐spinal cord recordings of the cervical rootlets (C4) from neonates after maternal opioids, demonstrating decreased opioid sensitivity of isolated respiratory control circuitry after maternal opioids (n=2). Results from these studies will contribute to our understanding of the mechanisms contributing to impaired neonatal breathing after maternal opioids and may lead to better treatments for infants suffering breathing deficits after maternal opioids.

MeCP2 Epigenetic Silencing of Oprm1 Gene in Primary Sensory Neurons Under Neuropathic Pain Conditions.

Opioids are the last option for the pharmacological treatment of neuropathic pain, but their antinociceptive effects are limited. Decreased mu opioid receptor (MOR) expression in the peripheral nervous system may contribute to this. Here, we showed that nerve injury induced hypermethylation of the Oprm1 gene promoter and an increased expression of methyl-CpG binding protein 2 (MeCP2) in injured dorsal root ganglion (DRG). The downregulation of MOR in the DRG is closely related to the augmentation of MeCP2, an epigenetic repressor, which could recruit HDAC1 and bind to the methylated regions of the Oprm1 gene promoter. MeCP2 knockdown restored the expression of MOR in injured DRG and enhanced the analgesic effect of morphine, while the mimicking of this increase via the intrathecal infusion of viral vector-mediated MeCP2 was sufficient to reduce MOR in the DRG. Moreover, HDAC1 inhibition with suberoylanilide hydroxamic acid, an HDAC inhibitor, also prevented MOR reduction in the DRG of neuropathic pain mice, contributing to the augmentation of morphine analgesia effects. Mechanistically, upregulated MeCP2 promotes the binding of a high level of HDCA1 to hypermethylated regions of the Oprm1 gene promoter, reduces the acetylation of histone H3 (acH3) levels of the Oprm1 gene promoter, and attenuates Oprm1 transcription in injured DRG. Thus, upregulated MeCP2 and HDAC1 in Oprm1 gene promoter sites, negatively regulates MOR expression in injured DRG, mitigating the analgesic effect of the opioids. Targeting MeCP2/HDAC1 may thus provide a new solution for improving the therapeutic effect of opioids in a clinical setting.

Effect of Pharmacological Inhibition of Fat-Mass and Obesity-Associated Protein on Nerve Trauma-Induced Pain Hypersensitivities.

Genetic knockout or knockdown of fat-mass and obesity-associated protein (FTO), a demethylase that participates inRNA N6-methyladenosine modification in injured dorsal root ganglion (DRG), has been demonstrated to alleviate nerve trauma-induced nociceptive hypersensitivities. However, these genetic strategies are still impractical in clinical neuropathic pain management. The present study sought to examine the effect of intrathecal administration of two specific FTO inhibitors, meclofenamic acid (MA) and N-CDPCB, on the development and maintenance of nociceptive hypersensitivities caused by unilateral L5 spinal nerve ligation (SNL) in rats. Intrathecal injection of either MA or N-CDPCB diminished dose-dependently the SNL-induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and spontaneous ongoing nociceptive responses in both development and maintenance periods, without altering acute/basal pain and locomotor function. Intrathecal MA also reduced the SNL-induced neuronal and astrocyte hyperactivities in the ipsilateral L5 dorsal horn. Mechanistically, intrathecal injection of these two inhibitors blocked the SNL-induced increase in the histone methyltransferase G9a expression and rescued the G9a-controlled downregulation of mu opioid receptor and Kv1.2 proteins in the ipsilateral L5 DRG. These findings further indicate the role of DRG FTO in neuropathic pain and suggest potential clinical application of the FTO inhibitors for management of this disorder.

Upregulation of μ-Opioid Receptor in the Rat Spinal Cord Contributes to the α2-Adrenoceptor Agonist Dexmedetomidine-Induced Attenuation of Chronic Morphine Tolerance in Cancer Pain.

BackgroundSustained morphine treatment for cancer pain has been limited due to analgesic tolerance. Opioid receptor internalization and desensitization mediated by downregulation of mu-opioid receptor (MOR) expression have been confirmed as one of the mechanisms of chronic morphine tolerance. In addition to the opiate system, the α2-adrenergic system is involved in the development of morphine tolerance. Several studies reported that co-administration of α2-adrenoceptor agonist dexmedetomidine inhibits morphine tolerance in normal or neuropathic pain animals. However, the effect of dexmedetomidine on morphine tolerance has not been studied in cancer pain. Therefore, we investigated the effect of intrathecal injection of dexmedetomidine on the development of morphine tolerance in cancer pain and on the expression of MOR in the spinal cord of morphine-tolerant cancer pain rats.MethodsThe model was established using a rat’s right hind paw injection of Walker 256 cancer cells. Subcutaneous morphine (10mg/kg) was administrated twice daily for 7 days; meanwhile, the rats received intrathecal α2-adrenoceptor agonist dexmedetomidine (10μ/kg) or antagonist MK-467 (0.25mg/kg) in test groups. Rats receiving drug vehicle served as the control group. Antinociception was detected by von Frey filaments and hot-plate tests. The expression of MOR in the spinal cord was examined through real-time reverse transcription polymerase chain reaction and Western blotting. The data were analyzed via analysis of variance followed by Student t-test with Bonferroni correction.ResultsSeven-day chronic morphine administration elicited notable analgesic tolerance in the rats with cancer pain. Co-administration of α2-adrenoceptor agonist dexmedetomidine enhanced morphine analgesia and attenuated morphine tolerance, which could be blocked by α2-adrenoceptor antagonist MK-467. Furthermore, pre-treatment of dexmedetomidine significantly upregulated MOR protein expression without a notable change in MOR mRNA expression in the spinal cord.ConclusionOur findings suggest that intrathecal injection of dexmedetomidine enhanced morphine analgesia and attenuated morphine tolerance in cancer pain, potentially by upregulating MOR expression in the spinal cord. The α2-adrenoceptor agonist may provide a more versatile analgesia option for morphine treatment for cancer pain.

Phosphatidylethanolamine‐Binding Protein Promotes Opioid Anti‐Nociception in the Brain and Spinal Cord by Reducing βarrestin2 Recruitment to the Mu Opioid Receptor

Opioids have been widely accepted as an effective treatment for chronic pain at a cost of serious side effects, including tolerance and addiction. Side effects are caused in part by desensitization and downregulation of mu opioid receptors (MOR) through recruitment of βarrestin2. Phosphatidylethanolamine‐Binding Protein (PEBP) is known to regulate either Raf Kinase or G Protein Receptor Kinase‐2 (GRK2) through phosphorylation by Protein Kinase C (PKC), and GRK2 is a major player in the recruitment of βarrestin2. However, PEBP has not been investigated for its role in MOR signaling and opioid response. We first tested this using an in vitro model of βarrestin2 recruitment in MOR expressing U2OS cells. We found that by inhibiting PEBP through siRNA knockdown or the small molecule inhibitor locostatin, we could increase the recruitment of βarrestin2 to the MOR in 2 separate assays. Conversely, we found that activating PKC via phorbol myristate acetate (PMA) led to PEBP phosphorylation/activation, and a reduction in βarrestin2 recruitment to the MOR, which could be blocked by PEBP knockdown. We then tested the role of PEBP in vivo by injection of locostatin into the brain or spinal cord, or CRISPR editing of PEBP in the spinal cord. We found that blocking PEBP in vivo led to a strong reduction in morphine‐induced tail flick anti‐nociception in both the brain and spinal cord. Together, these results support the hypothesis that PEBP acts to sequester GRK2 and block βarrestin2 recruitment downstream of the MOR both in vivo and in vitro. These results also suggest that enhanced PKC/PEBP activation by opioid drugs could further reduce opioid side effects and enhance analgesic efficacy.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EFFECTS OF ZAMZAM WATER AND METHADONE ON THE EXPRESSION OF MU-OPIOID RECEPTOR-1 GENE IN MORPHINE-DEPENDENT RATS AFTER CHRONIC MORPHINE ADMINISTRATION

Background: Sodium ion is an essential ion that is implicated in many physiological functions. Recently, sodium ion was reported to facilitate the activation of Mu-Opioid Receptor (MOR) by binding at the allosteric site of the MOR. Zamzam water is water originated from Mecca. Couple of studies proved that Zamzam water has the therapeutic effect owing to its mineral. In this study, we want to determine the ion concentration of Zamzam water and then to investigate the effects of Zamzam water and co-treatment with methadone on the regulation of MOR-1gene after chronic morphine administration.
 Materials and Methods: Zamzam water, tap water and normal mineral water were analyzed using Ion chromatography. Meanwhile, in animal study, 50 male Sprague Dawley rats were randomly divided into five groups. All group of rat were made dependence on morphine using intraperitoneal injection except for normal group. Morphine dependent rats then were treated with methadone, Zamzam water and co-treatment methadone with Zamzam water for thirty days, respectively. The Ventral Tegmental Area (VTA) of rat’s brain was dissected and subjected to real-time quantitative RT-PCR to determine the regulation of MOR-1 gene expression. The obtained data were analyzed using SPSS v.11 software, and one-way ANOVA followed by Tukey’s Post-test.
 Results: The data obtained showed that Zamzam water is significantly high in ion concentration compared to tap water and normal mineral water. Besides, the result from gene expression analysis showed co-treatment Zamzam water and methadone significantly prevented the downregulation of MOR as compared to methadone and Zamzam water treatment alone (P

Anti-nociceptive effect of patchouli alcohol: Involving attenuation of cyclooxygenase 2 and modulation of mu-opioid receptor.

To explore the anti-nociceptive effect of patchouli alcohol (PA), the essential oil isolated from Pogostemon cablin (Blanco) Bent, and determine the mechanism in molecular levels. The acetic acid-induced writhing test and formalin-induced plantar injection test in mice were employed to confirm the effect in vivo. Intracellular calcium ion was imaged to verify PA on mu-opioid receptor (MOR). Cyclooxygenase 2 (COX2) and MOR of mouse brain were expressed for determination of PA's target. Cellular experiments were carried out to find out COX2 and MOR expression induced by PA. PA significantly reduced latency period of visceral pain and writhing induced by acetic acid saline solution (P<0.01) and allodynia after intra-plantar formalin (P<0.01) in mice. PA also up-regulated COX2 mRNA and protein (P<0.05) with a down-regulation of MOR (P<0.05) both in in vivo and in vitro experiments, which devote to the analgesic effect of PA. A decrease in the intracellular calcium level (P<0.05) induced by PA may play an important role in its anti-nociceptive effect. PA showed the characters of enhancing the MOR expression and reducing the intracellular calcium ion similar to opioid effect. Both COX2 and MOR are involved in the mechanism of PA's anti-nociceptive effect, and the up-regulation of the receptor expression and the inhibition of intracellular calcium are a new perspective to PA's effect on MOR.

OPRM1 Methylation Contributes to Opioid Tolerance in Cancer Patients.

We demonstrate that epigenetic regulation of OPRM1 contributes to opioid tolerance in cancer patients, and that targeted gene therapy could treat cancer-induced nociception and opioid tolerance in a mouse cancer model.

Curvilinear relationships between mu-opioid receptor labeling and undirected song in male European starlings (Sturnus vulgaris)

Female-directed communication in male songbirds has been reasonably well studied; yet, relatively little is known about communication in other social contexts. Songbirds also produce song that is not clearly directed towards another individual (undirected song) when alone or in flocks. Although the precise functions of undirected song may differ across species, this type of song is considered important for flock maintenance, song learning or practice. Past studies show that undirected song is tightly coupled to analgesia and positive affective state, which are both mediated by opioid activity. Furthermore, labeling for the opioid met-enkephalin in the medial preoptic nucleus (POM) correlates positively with undirected song production. We propose that undirected song is facilitated and maintained by opioid receptor activity in the POM and other brain regions involved in affective state, analgesia, and social behavior. To provide insight into this hypothesis, we used immunohistochemistry to examine relationships between undirected song and mu-opioid receptors in male starlings. Polynomial regression analyses revealed significant inverted-U shaped relationships between measures of undirected song and mu-opioid receptor labeling in the POM, medial bed nucleus of the stria terminalis (BSTm), and periaqueductal gray (PAG). These results suggest that low rates of undirected song may stimulate and/or be maintained by mu-opioid receptor activity; however, it may be that sustained levels of mu-opioid receptor activity associated with high rates of undirected song cause mu-opioid receptor down-regulation. The results indicate that mu-opioid receptor activity in POM, BSTm, and PAG may underlie previous links identified between undirected song, analgesia, and affective state.

Reduced Number, G Protein Coupling, and Antinociceptive Efficacy of Spinal Mu-Opioid Receptors in Diabetic Rats Are Reversed by Nerve Growth Factor

This study investigated putative mechanisms of impaired spinal opioid antinociception such as a downregulation of mu-opioid receptor (MOR) number, coupling, and efficacy in rats with advanced (12 weeks) streptozotocin (STZ)-induced diabetes. Intravenous injection of STZ (45 mg/kg) in Wistar rats led to selective degeneration of insulin-producing pancreatic ß-cells, elevated blood glucose, and mechanical hyperalgesia. In these animals, dose-dependent and naloxone-reversible intrathecal fentanyl antinociception was significantly impaired and associated with a loss in MOR immunoreactivity of calcitonin gene-related peptide–immunoreactive (CGRP-IR) sensory nerve terminals, membrane-bound MOR binding sites, and MOR-stimulated G protein coupling within the dorsal horn of the spinal cord. Intrathecal delivery of nerve growth factor (NGF) in diabetic animals normalized spinal MOR number and G protein coupling and rescued spinal fentanyl-induced antinociception. These findings identify for the first time a loss in functional MOR on central terminals of sensory neurons as a contributing factor for the impaired spinal opioid responsiveness during advanced STZ-induced diabetes that can be reversed by NGF. Moreover, they support growing evidence of a distinct regulation of opioid responsiveness during various painful states of disease (eg, arthritis, cancer, neuropathy) and may give novel therapeutic incentives. PerspectiveIn diabetic neuropathy a loss in sensory neuron mu-opioid receptor number and coupling contributes to impaired spinal opioid antinociception that can be reversed by NGF. These findings support growing evidence of a distinct regulation of opioid responsiveness during various painful diseases and may give novel therapeutic incentives.

The effect of mitragynine on cAMP formation and mRNA expression of mu-opioid receptors mediated by chronic morphine treatment in SK–N–SH neuroblastoma cell

Ethopharmacological relevanceMitragynine is an indole alkaloid compound of Mitragyna speciosa (M. speciosa) Korth. (Rubiaceae). This plant is native to the southern regions of Thailand and northern regions of Malaysia and is frequently used to manage the withdrawal symptoms in both countries. Aim of studyTo investigate the effect of mitragynine after chronic morphine treatment on cyclic AMP (cAMP) level and mRNA expression of mu-opioid receptor (MOR) in human neuroblastoma SK–N–SH cell. Method and MaterialsMitragynine was isolated from the Mitragyna speciosa plant using the acid–base extraction method. The cAMP level upon forskolin stimulation in the cells was determined using the Calbiochem® Direct Immunoassay Kit. The mRNA expression of the MOR was carried out using quantitative RT-PCR. ResultCotreatment and pretreatment of morphine and mitragynine significantly reduced the production of cAMP level at a lower concentration of mitragynine while the higher concentration of this compound could lead to the development of tolerance and dependence as shown by the increase of the cAMP level production in foskolin stimulation. In MOR mRNA expression study, cotreatment of morphine with mitragynine significantly reduced the down-regulation of MOR mRNA expression as compared to morphine treatment only. ConclusionThese finding suggest that mitragynine could possibly avoid the tolerance and dependence on chronic morphine treatment by reducing the up-regulation of cAMP level as well as reducing the down-regulation of MOR at a lower concentration of mitragynine.

Association of Mu Opioid Receptor with Neurogenic Inflammation in Sickle Mic

Abstract 895Sickle cell disease (SCD) is accompanied by chronic inflammation and severe pain for which available therapies are unsatisfactory. We found that pain was accompanied by increased peripheral expression of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and decreased expression of mu opioid receptor (MOR) in the skin of BERK mice expressing sickle hemoglobin (HbSS) compared to control HbA-BERK mice expressing normal human hemoglobin (Kohli et al., Blood 2010). Neuropeptide modulations are key features of neurogenic inflammation in chronic pain states. Tryptase released from mast cells is known to activate the release of neuropeptides from peripheral nerve endings, which in turn act on target cells including mast cells, inflammatory cells, endothelial cells and smooth muscle cells, resulting in increased vascular flow, inflammation and plasma extravasation collectively termed neurogenic inflammation. MOR influences neuropeptide release and perhaps modulates neurogenic inflammation. We hypothesize that neurogenic inflammation contributes to chronic pain and inflammation in SCD. We first examined the quantitative expression of MOR-, SP-, CGRP- and tryptase-immunoreactivity (ir) using laser scanning confocal microscopy (LSCM) in thick skin sections of BERK sickle and HbA-BERK control mice. Significantly decreased MOR-ir (p<0.001), but increased SP-, CGRP- (p<0.01 for both) and tryptase-ir (p<0.001) was observed in the skin of BERK as compared to HbA-BERK. Decreased MOR-ir co-localized with epidermal and dermal layers in the skin of BERK, but blood vessels showed robust MOR-ir in BERK similar to HbA-BERK. Tryptase-ir was more pronounced in the epidermal layer, indicative of its proximity to peripheral nerve endings. Increased tryptase co-localized with CD117/c-kit, confirming the mast cell origin. Next, we analyzed if MOR downregulation contributes to these alterations, by using mice with deletion of mu-, delta- and kappa-opioid receptors (MOR, DOR and KOR, respectively) and wild type (WT), 129S6 mice. SP-, CGRP- and tryptase-ir were increased about 2-fold in the skin of MOR, DOR and KOR knockout as compared to WT mice, suggesting that downregulation of MOR in sickle mice may contribute to increased neuropeptide and tryptase expression. Consistent with an increase in cutaneous neuropeptides, BERK and MOR knockout mice showed ∼2.5- and ∼1.5-fold increase in dorsal skin blood flow using Laser Doppler blood perfusion monitor, as compared to HBA-BERK and WT control mice, respectively. Thermography measurements revealed a correlative increase in temperature in dorsal skin of BERK as compared to HbA-BERK (p<0.05), indicative of increased inflammation. These neurochemical and physiological alterations in the skin were accompanied by activation of dorsal root ganglion (DRG) neuron and glial cells. LSCM of whole DRG revealed increased-ir for activating transcription factor-3 (ATF3), a marker of neuropathic and inflammatory insult; and increased glial fibrillary acidic protein (GFAP)-ir, a marker of inflammation, in BERK relative to HbA-BERK mice. It is likely that chronic pain and ischemia-reperfusion injury in SCD activates ATF3 in DRG leading to the release of mast cell tryptase followed by release of neuropeptides in the DRG and their ‘antidromic’ release from the nociceptive C-fibers in the skin. In addition, recurrent/chronic pain and inflammation in SCD may cause peripheral nociceptor sensitization mediated by mast cell-tryptase, and neuropeptide release from nerve endings. Nociceptor sensitization together with decrease in MOR, in turn may activate mast cell tryptase, promoting a vicious cycle between components of central nervous system and inflammation that may exacerbate neurogenic inflammation in SCD. It is likely that the pro-inflammatory neuropeptide-enhanced microenvironment has a compounding effect on inflammation and pain in SCD. We speculate that therapies targeting neuropeptides and their release mechanism(s) may potentiate the effect of analgesics and anti-inflammatory therapies for treating pain in SCD. Disclosures:No relevant conflicts of interest to declare.

Morphine Stimulates Wound Healing Via Mu Opioid Receptor and Promotes Wound Closure in Sickle Mice

Abstract 2118Painful non-healing leg ulcers in sickle cell disease (SCD) pose major treatment challenges for which there is no satisfactory therapy. We observed that the vascular and nerve architecture are abnormal, and mu opioid receptor (MOR) expression is decreased in the skin of BERK mice expressing sickle hemoglobin, as compared to HbA-BERK mice expressing normal human hemoglobin (Kohli et al., Blood, 2010). MOR mediates morphine-induced angiogenesis and analgesia, which are critical to the healing process. We hypothesized that MOR mediates the healing process and that MOR agonists such as morphine will promote healing by stimulating angiogenesis. Ischemic wounds (6 mm diameter) with impaired blood supply were created on the dorsal skin of sickle (BERK, and S+SAntilles) and control (HbA-BERK and C57BL6, respectively) mice; and mu-, delta-, and kappa-opioid receptor knockout (MOR-, DOR- and KOR-KO, respectively) mice and their 129S6 wild type controls. BERK and S+SAntilles did not survive ischemic wound surgery, but S+SAntilles survived non-ischemic, 4 mm punch biopsies on the leg. Wounds were treated topically twice a day with morphine, 3 mg/g Eucerin cream or with PBS-Eucerin cream. Periodic wound tracings were used to quantitate wound area with Adobe Photoshop and % area healed was calculated. Ischemic wounds (100% closure on d17 in control 129S6) took significantly longer to heal as compared to punch biopsies (100% closure on d8 in control C57BL6). Morphine significantly accelerated healing as compared to PBS-cream in control, S+SAntilles, DOR- and KOR-KO, but not in MOR-KO mice, suggesting that morphine-induced healing is MOR-dependent. Of note, PBS-treated ischemic wounds in MOR-KO healed significantly slower than 129S6 control wounds, indicating that MOR is involved in wound healing. Blood flow was significantly higher in the intact unwounded dorsal skin of MOR-KO as compared to 129S6 mice (p<0.001). Significant decrease occurred after wounding in both 129S6 and MOR-KO, which was restored to baseline in morphine-treated 129S6 but not in MOR-KO wounds on d20. Thus, morphine promotes vascular function, which appears to be mediated by MOR in the healing wounds.In PBS-cream treated punch biopsy wounds, 100% wound closure occurred on day 8 as compared to day 6 with morphine treatment in both C57BL6 control and S+SAntilles sickle mice. Laser scanning confocal microscopy (LSCM) of healed wound scars 30d after wounding revealed significantly increased blood vessels (∼2-fold) in morphine-treated as compared to PBS treated in both C57BL6 and S+SAntilles mice. A significant increase was also noted in PGP 9.5-immunoreactive (ir) nerve fibers and lymphatics in morphine-treated scars. Pro-inflammatory and vaso-active neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP) were increased ∼2-fold in PBS-treated wound scars 30d post-wounding as compared to baseline in S+SAntilles mice, suggestive of increased neurogenic inflammation. However both neuropeptides returned to baseline levels in morphine-treated wounds. Similarly, both SP- and CGRP-ir in 129S6 and MOR-KO were increased significantly in PBS-treated mice but not in morphine-treated as compared to baseline before wounding. It is noteworthy, that SP- and CGRP-ir were significantly higher in the unwounded skin of MOR-, DOR- and KOR-KO as compared to 129S6 control mice, suggesting that all three ORs are involved in the regulation of neuropeptides. Therefore, even though wound healing and blood flow were not influenced by morphine in MOR-KO, neuropeptides were responsive to morphine treatment in MOR-KO. Together, these data suggest that MOR downregulation may contribute to impaired wound healing and that other ORs are critical to regulate neuropeptides to control neurogenic inflammation in the skin, which may in turn influence the healing process. Thus, morphine orchestrates wound healing by stimulating vascular function via MOR and modulating neurogenic inflammation via ORs in the skin. Since increased inflammation and vasculopathy underlie wound pathobiology in SCD, morphine may have a therapeutic effect on healing leg ulcers. Disclosures:No relevant conflicts of interest to declare.

Membrane-delimited proteolytic regulation of opioid receptors

Prolonged exposure of opioid receptors to agonists leads to their regulation by the classical process of clathrin-dependent internalization, followed by their intracellular degradation (down regulation). We have previously shown that the opioid agonist etorphine induced an additional process of down regulation of mu-opioid receptors (MOR) that occurred in intact MOR-transfected HEK-293 cells, as well as in isolated membranes. In the present study we show that etorphine similarly down regulated rat kappa-opioid receptors (KORs), which do not undergo the classical process of internalization and down regulation. This process was resistant to inhibitors of clathrin-coated pit formation (hypertonic sucrose, mono-dansyl-cadaverine) and was mainly mediated by membranous serine- and amino-peptidases. We further show that various opioid ligands, besides etorphine, induced down regulation of either KOR or MOR in isolated membranes. The ability of the various opioid ligands to induce membrane-delimited KOR or MOR down regulation did not correlate to their classical pharmacological profile, suggesting functional selectivity of the effect. Levorphanol, but not its stereoisomer dextrophan, induced membrane-delimited down regulation of both KOR and MOR, indicating that stereoselective binding to the receptor was necessary to initiate the process. Our findings that this proteolytic regulation of opioid receptors occurs not only in isolated membranes but also in intact cells and that it occurs even when the receptors are resistant to the conventional process of down regulation indicate its possible physiological role in the regulation of opioid activity.

Facilitation of μ-Opioid Receptor Activity by Preventing δ-Opioid Receptor-Mediated Codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane isa potential strategy to improve opioid analgesic therapies.

Dual regulation of mu opioid receptors in SK-N-SH neuroblastoma cells by morphine and interleukin-1β: Evidence for opioid-immune crosstalk

Treatment of SK-N-SH cells with morphine and interleukin-1beta (IL-1β) produced dual regulation of the mRNA for the human mu opioid receptor (MOR) protein. Morphine produced a decrease in the MOR mRNA while IL-1β increased it, as assessed by real-time quantitative PCR. These data were consistent with immunocytochemical studies of treated and untreated cells. Morphine-mediated down-regulation of MOR was blocked by naltrexone and IL-1β-induced up-regulation of MOR was blocked by interleukin-1 receptor type 1 antagonist. Immune-opioid crosstalk was examined by IL-1β and morphine co-treatment. These data are the first to show dual regulation of MOR in neuroblastoma cells.

Effect of agmatine on DAMGO-induced mu-opioid receptor down-regulation and internalization via activation of IRAS, a candidate for imidazoline I 1 receptor

Agmatine, an endogenous ligand for imidazoline I 1 receptor, has previously been shown to prevent opioid tolerance in rats and mice, but the cellular mechanisms remain unknown. In the present study, the effects of agmatine activation on imidazoline I 1 receptor on the desensitization, down-regulation and internalization of μ opioid receptor were investigated. Two cell lines, CHO cells transfected μ opioid receptor (CHO-μ cells) and co-transfected μ opioid receptor and imidazoline I 1 receptor antisera-selected protein (IRAS) (CHO-μ/IRAS cells), were used. In both CHO-μ cells and CHO-μ/IRAS cells, agmatine (0.01–10 μM) did not affect the desensitization of μ opioid receptor induced by [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) (10 μM) treatment for 30 min. However, agmatine (0.1–100 nM) co-pretreatment with DAMGO (1 μM) for 12 h concentration-dependently inhibited DAMGO-induced down-regulation of μ opioid receptor in CHO-μ/IRAS cells, but not in CHO-μ cells. Efaroxan, the I 1/α 2-adrenoceptors mix antagonist, completely reversed the inhibitory effect of agmatine, suggesting the participation of imidazoline I 1 receptor. In addition, agmatine (1–100 nM) inhibited DAMGO-induced internalization of μ opioid receptor in CHO-μ/IRAS cells, which was reversed by efaroxan as well. While treatment with DAMGO (1 μM) or co-treatment with agmatine (1–100 nM) for 12 h failed to affect the mRNA level of μ opioid receptor. Taken together, these results indicate that the inhibitory effect of agmatine on tolerance in vitro might be related to attenuation of the internalization and down-regulation of μ opioid receptor via activation of imidazoline I 1 receptor.

Nandrolone decreases mu opioid receptor expression in SH-SY5Y human neuroblastoma cells

Nandrolone and other anabolic androgenic steroids alter the expression and function of neurotransmitter systems and contribute to drug dependence. Nandrolone treatment (10-10 M) caused a time-dependent and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolone-treated cells. Treatment with actinomycin D (10 (-5)M), a transcription inhibitor, revealed that nandrolone might regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through posttranscriptional mechanisms requiring the androgen receptor.