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Neuroinflammation underlies the pathophysiology of multiple age-related neurological disorders. Microglia, the resident immune cells of the central nervous system, are critically involved in neuroinflammatory regulation and neural survival. Modulating microglial activation is thus a promising approach to alleviate neuronal injury. Our serial studies have revealed a neuroprotective role of the δ-opioid receptor (DOR) in several acute and chronic cerebral injuries by regulating neuroinflammation and cellular oxidative stress. More recently, we found an endogenous mechanism for the inhibition of neuroinflammation is closely related to DOR's modulation of microglia. Our recent studies showed that DOR activation could strongly protect neurons from hypoxia- and lipopolysaccharide (LPS)-induced injury by inhibiting microglial pro-inflammatory transformation, while knocking-down DOR or restraining DOR activity promoted microglia activation and the relevant inflammatory events with an aggravation of cell injury. This novel finding highlights a therapeutic potential of DOR in numerous age-related neurological disorders through the modulation of neuroinflammation by targeting microglia. This review summarized the current data regarding the role of microglia in neuroinflammation, oxidative stress, and age-related neurological diseases focusing on the pharmacological effects and signaling transduction of DOR in microglia. [ABSTRACT FROM AUTHOR]

This is a prospective observational study evaluating the change in β-endorphin (BE) and its receptors following exercise in patients with myasthenia gravis (MG) and their association with clinical improvement. Fifteen patients with mild to moderate MG, aged 16–70 years, who were able to do 6-Minute Walk Test (6-MWT) and had MG Quality of Life-15 (MGQoL-15) ≤ 45 without any contraindication for exercise were included. The patients walked 30 min daily for 3 months. The primary outcome at 3 months was > 50% improvement in MGQoL-15 from the baseline, and the secondary outcomes were MG Activities of Daily Living (MGADL), Hospital Anxiety and Depression Scale (HADS), number of steps, and distance covered on 6-MWT and adverse events. Plasma BE level, μ-opioid receptor (MOR), and δ-opioid receptor (DOR) were measured on admission and at 1 and 3 months. Twelve age- and gender-matched healthy controls who were not on regular exercise were included for comparison of BE, MOR, and DOR levels. Plasma BE level (P = 0.007) and DOR expression (P = 0.001) were lower in MG patients compared to the healthy controls. After 3 months of exercise, 6 patients improved. Plasma BE, MOR, and DOR levels increased in the first and decreased in the third month. MGQoL-15 (P < 0.001), HADS (P < 0.0001), number of steps (P < 0.007), distance (P = 0.030), and MGADL (P < 0.001) significantly improved compared to baseline. At 3 months, MGQoL-15 was associated with HADS score (P = 0.001), reduced depression (P = 0.013), MGADL (P = 0.035), and distance travelled on the 6-WMW test (P = 0.050). The improvement in depression was associated with higher BE level. [ABSTRACT FROM AUTHOR]

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

Despite the increasing impact of opioid use disorders on society, there is a disturbing lack of effective medications for their clinical management. An interesting innovative strategy to treat these disorders consists in the protection of endogenous opioid peptides to activate opioid receptors, avoiding the classical opioid‐like side effects. Dual enkephalinase inhibitors (DENKIs) physiologically activate the endogenous opioid system by inhibiting the enzymes responsible for the breakdown of enkephalins, protecting endogenous enkephalins and increasing their half‐lives and physiological actions. The activation of opioid receptors by the increased enkephalin levels, and their well‐demonstrated safety, suggests that DENKIs could represent a novel analgesic therapy and a possible effective treatment for acute opioid withdrawal, as well as a promising alternative to opioid substitution therapy minimizing side effects. This new pharmacological class of compounds could bring effective and safe medications avoiding the major limitations of exogenous opioids, representing a novel approach to overcome the problem of opioid use disorders. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is a chronic and systemic autoimmune disorder that affects preferentially the small intestine of individuals with a genetic predisposition. CD is promoted by the ingestion of gluten, a storage protein contained in the endosperm of the seeds of wheat, barley, rye, and related cereals. Once in the gastrointestinal (GI) tract, gluten is enzymatically digested with the consequent release of immunomodulatory and cytotoxic peptides, i.e., 33mer and p31-43. In the late 1970s a new group of biologically active peptides, called gluten exorphins (GEs), was discovered and characterized. In particular, these short peptides showed a morphine-like activity and high affinity for the δ-opioid receptor (DOR). The relevance of GEs in the pathogenesis of CD is still unknown. Recently, it has been proposed that GEs could contribute to asymptomatic CD, which is characterized by the absence of symptoms that are typical of this disorder. In the present work, GEs cellular and molecular effects were in vitro investigated in SUP-T1 and Caco-2 cells, also comparing viability effects with human normal primary lymphocytes. As a result, GEs treatments increased tumor cell proliferation by cell cycle and Cyclins activation as well as by induction of mitogenic and pro-survival pathways. Finally, a computational model of GEs interaction with DOR is provided. Altogether, the results might suggest a possible role of GEs in CD pathogenesis and on its associated cancer comorbidities. [ABSTRACT FROM AUTHOR]

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM). [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to determine whether the epidermal growth factor receptor (EGFR), which is a classical receptor tyrosine kinase, is involved in the protective effect of morphine against ischemia/reperfusion (I/R)-induced myocardial mitochondrial damage. Methods: Isolated rats hearts were subjected to global ischemia followed by reperfusion. Cardiac H9c2 cells were exposed to a simulated ischemia solution followed by Tyrode's solution to induce hypoxia/reoxygenation (H/R) injury. Triphenyltetrazolium chloride (TTC) was used to measure infarct size. The mitochondrial morphological and functional changes were determined using transmission election microscopy (TEM), mitochondrial stress assay, and mitochondrial swelling, respectively. Mitochondrial fluorescence indicator JC-1, DCFH-DA, and Mitosox Red were used to determine mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS) and mitochondrial superoxide. A TUNUL assay kit was used to detect the level of apoptosis. Western blotting analysis was used to measure the expression of proteins. Results: Treatment of isolated rat hearts with morphine prevented I/R-induced myocardial mitochondrial injury, which was inhibited by the selective EGFR inhibitor AG1478, suggesting that EGFR is involved in the mitochondrial protective effect of morphine under I/R conditions. In support of this hypothesis, the selective EGFR agonist epidermal growth factor (EGF) reduced mitochondrial morphological and functional damage similarly to morphine. Further study demonstrated that morphine may alleviate I/R-induced cardiac damage by inhibiting autophagy but not apoptosis. Morphine increased protein kinase B (Akt), extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation, which was inhibited by AG1478, and EGF had similar effects, indicating that morphine may activate Akt, ERK, and STAT-3 via EGFR. Morphine and EGF increased intracellular reactive oxygen species (ROS) generation. This effect of morphine was inhibited by AG1478, indicating that morphine promotes intracellular ROS generation by activating EGFR. However, morphine did not increase ROS generation when cells were transfected with siRNA against EGFR. In addition, EGFR activity was markedly increased by morphine, but the effect of morphine was reversed by naltrindole. These results suggest that morphine may activate EGFR via δ-opioid receptor activation. Conclusions: Morphine may prevent I/R-induced myocardial mitochondrial damage by activating EGFR through δ-opioid receptors, in turn increasing RISK and SAFE pathway activity via intracellular ROS. Moreover, morphine may reduce myocardial injury by regulating autophagy but not apoptosis. [ABSTRACT FROM AUTHOR]

Microglia are involved in the regulation of cerebral homeostasis and pathogen confrontation. There is, however, evidence showing that excessive microglia activation is implicated in various age-related cerebral diseases. On the other hand, microglia may experience complex changes of polarization in pathological insults, i.e., from a proinflammatory M1 to an anti-inflammatory M2 phenotype, which differentially contribute to the exacerbation or alleviation of cellular injury. Remolding the phenotype of microglia or inhibiting the excessive activation of microglia seems to be a promising approach against neurodegenerative pathologies. Since δ-opioid receptor (DOR) activation exhibits a strong protective capacity against various neuronal injuries, especially the hypoxic/ischemic injury, we asked if the DOR-induced neuroprotection is associated with its effect on microglia. We explored this fundamental issue by using pharmacological and genetic approaches in the BV2 cell line, a general type of microglial cells. The results showed that DOR expression significantly increased in the activated microglial M2 phenotype, but slightly decreased in the microglial M1 phenotype. Hypoxia induced dual polarizations of BV2 cells with an increase in DOR expression. Administration of a specific DOR agonist, UFP-512, largely inhibited lipopolysaccharide (LPS) or hypoxia-induced microglial M1 activation and inflammatory activity with high concentrations of UFP-512 being effective to reverse the interleukin-4 (IL4)-induced microglial activation. Consistent with these observations, inhibiting DOR or knocking-down DOR promoted the excessive activation of BV2 cells in both M1 and M2 directions, while DOR overexpression did the opposite. Furthermore, the PC12 cells exposed to the conditioned medium of BV2 cells treated by UFP-512 grew better than those treated directly with UFP-512 under LPS or hypoxic insults. DOR inhibitor naltrindole could block all the effects of DOR activation. The medium from the BV2 cells with DOR knock-down decreased the viability of PC12 cell, while the medium from the BV2 cells with DOR overexpression largely attenuated LPS or hypoxic injury in the PC12 cells. These first data suggest a close linkage between DOR expression/function and microglial polarization and a critical role of DOR in negative controlling microglial activation. Our work provides a novel clue for new protective strategies against neurodegenerative pathophysiology through DOR-mediated regulation of microglia. [ABSTRACT FROM AUTHOR]

Opioid receptors are members of the group of G protein-couple receptors, which have been proven to be effective targets for treating severe pain. The interactions between the opioid receptors and corresponding ligands and the receptor's activation by different agonists have been among the most important fields in opioid research. In this study, with compound M1, an active metabolite of tramadol, as the clue compound, several aminomethyl tetrahydronaphthalenes were designed, synthesized and assayed upon opioid receptors. With the resultant compounds FW-AII-OH-1 (Ki = 141.2 nM for the κ opioid receptor), FW-AII-OH-2 (Ki = 4.64 nM for the δ opioid receptor), FW-DI-OH-2 (Ki = 8.65 nM for the δ opioid receptor) and FW-DIII-OH-2 (Ki = 228.45 nM for the δ opioid receptor) as probe molecules, the structural determinants responsible for the subtype selectivity and activation mechanisms were further investigated by molecular modeling and molecular dynamics simulations. It was shown that Y7.43 was a key residue in determining the selectivity of the three opioid receptors, and W6.58 was essential for the selectivity of the δ opioid receptor. A detailed stepwise discovered agonist-induced signal transduction mechanism of three opioid receptors by aminomethyl tetrahydronaphthalene compounds was proposed: the 3–7 lock between TM3 and TM7, the DRG lock between TM3 and TM6 and rearrangement of I3.40, P5.50 and F6.44, which resulted in the cooperative movement in 7 TMs. Then, the structural relaxation left room for the binding of the G protein at the intracellular site, and finally the opioid receptors were activated. [ABSTRACT FROM AUTHOR]

Microglia are involved in the regulation of cerebral homeostasis and pathogen confrontation. There is, however, evidence showing that excessive microglia activation is implicated in various age-related cerebral diseases. On the other hand, microglia may experience complex changes of polarization in pathological insults, i.e., from a proinflammatory M1 to an anti-inflammatory M2 phenotype, which differentially contribute to the exacerbation or alleviation of cellular injury. Remolding the phenotype of microglia or inhibiting the excessive activation of microglia seems to be a promising approach against neurodegenerative pathologies. Since δ-opioid receptor (DOR) activation exhibits a strong protective capacity against various neuronal injuries, especially the hypoxic/ischemic injury, we asked if the DOR-induced neuroprotection is associated with its effect on microglia. We explored this fundamental issue by using pharmacological and genetic approaches in the BV2 cell line, a general type of microglial cells. The results showed that DOR expression significantly increased in the activated microglial M2 phenotype, but slightly decreased in the microglial M1 phenotype. Hypoxia induced dual polarizations of BV2 cells with an increase in DOR expression. Administration of a specific DOR agonist, UFP-512, largely inhibited lipopolysaccharide (LPS) or hypoxia-induced microglial M1 activation and inflammatory activity with high concentrations of UFP-512 being effective to reverse the interleukin-4 (IL4)-induced microglial activation. Consistent with these observations, inhibiting DOR or knocking-down DOR promoted the excessive activation of BV2 cells in both M1 and M2 directions, while DOR overexpression did the opposite. Furthermore, the PC12 cells exposed to the conditioned medium of BV2 cells treated by UFP-512 grew better than those treated directly with UFP-512 under LPS or hypoxic insults. DOR inhibitor naltrindole could block all the effects of DOR activation. The medium from the BV2 cells with DOR knock-down decreased the viability of PC12 cell, while the medium from the BV2 cells with DOR overexpression largely attenuated LPS or hypoxic injury in the PC12 cells. These first data suggest a close linkage between DOR expression/function and microglial polarization and a critical role of DOR in negative controlling microglial activation. Our work provides a novel clue for new protective strategies against neurodegenerative pathophysiology through DOR-mediated regulation of microglia.

Facilitation of fear extinction is a desirable action for the drugs to treat fear-related diseases, such as posttraumatic stress disorder (PTSD). We previously reported that a selective agonist of the δ-opioid receptor (DOP), KNT-127, facilitates contextual fear extinction in mice. However, its site of action in the brain and the underlying molecular mechanism remains unknown. Here, we investigated brain regions and cellular signaling pathways that may mediate the action of KNT-127 on fear extinction. Twenty-four hours after the fear conditioning, mice were reexposed to the conditioning chamber for 6 min as extinction training (reexposure 1). KNT-127 was microinjected into either the basolateral nucleus of the amygdala (BLA), hippocampus (HPC), prelimbic (PL), or infralimbic (IL) subregions of the medial prefrontal cortex, 30 min before reexposure 1. Next day, mice were reexposed to the chamber for 6 min as memory testing (reexposure 2). KNT-127 that infused into the BLA and IL, but not HPC or PL, significantly reduced the freezing response in reexposure 2 compared with those of control. The effect of KNT-127 administered into the BLA and IL was antagonized by pretreatment with a selective DOP antagonist. Further, the effect of KNT-127 was abolished by local administration of MEK/ERK inhibitor into the BLA, and PI3K/Akt inhibitor into the IL, respectively. These results suggested that the effect of KNT-127 was mediated by MEK/ERK signaling in the BLA, PI3K/Akt signaling in the IL, and DOPs in both brain regions. Here, we propose that DOPs play a role in fear extinction via distinct signaling pathways in the BLA and IL. [ABSTRACT FROM AUTHOR]

Abstract Hypoxic-ischemic brain damage (HIBD) is a relatively common malignant complication that occurs in newborn infants, but promising therapies remain limited. In this study, we focused on the role of miR-326 and its target gene δ-opioid receptor (DOR) in the pathogenesis of neonatal HIBD. The expression levels of miR-326 and DOR after hypoxic-ischemic injury were examined both in vivo and in vitro. The direct relationship between miR-326 and DOR was confirmed by a dual-luciferase reporter assay. Further, effects of miR-326 on cell viability and apoptosis levels under oxygen glucose deprivation (OGD) were analyzed. The expression levels of miR-326 were significantly lower and DOR levels were significantly higher in the HIBD group than the control group both in vivo and in vitro. Overexpression of miR-326 downregulated the expression of DOR, while suppression of miR-326 upregulated the expression of DOR. The dual-luciferase reporter assay further confirmed that DOR could be directly targeted and regulated by miR-326. MiR-326 knockdown improved cell survival and decreased cell apoptosis by decreasing the expression levels of Caspase-3 and Bax and increasing Bcl-2 expression in PC12 cells after exposure to OGD. Moreover, DOR knockdown rescued the effect of the improved cell survival and suppressed cell apoptosis induced by silencing miR-326. Our findings indicated that inhibition of miR-326 may improve cell survival and decrease cell apoptosis in neonatal HIBD through the target gene DOR.

Ischemic stroke is a global epidemic condition due to an inadequate supply of blood and oxygen to a specific area of brain either by arterial blockage or by narrowing of blood vessels. Despite having advancement in the use of thrombolytic and clot removal medicine, significant numbers of stroke patients are still left out without option for treatment. In this review, we summarize recent research work on the activation of δ-opioid receptor as a strategy for treating ischemic stroke-caused neuronal injury. Moreover, as activation of δ-opioid receptor by a non-peptidic δ-opioid receptor agonist also modulates the expression, maturation and processing of amyloid precursor protein and β-secretase activity, the potential role of these effects on ischemic stroke caused dementia or Alzheimer’s disease are also discussed.

Facilitation of fear extinction is a desirable action for the drugs to treat fear-related diseases, such as posttraumatic stress disorder (PTSD). We previously reported that a selective agonist of the δ-opioid receptor (DOP), KNT-127, facilitates contextual fear extinction in mice. However, its site of action in the brain and the underlying molecular mechanism remains unknown. Here, we investigated brain regions and cellular signaling pathways that may mediate the action of KNT-127 on fear extinction. Twenty-four hours after the fear conditioning, mice were reexposed to the conditioning chamber for 6 min as extinction training (reexposure 1). KNT-127 was microinjected into either the basolateral nucleus of the amygdala (BLA), hippocampus (HPC), prelimbic (PL), or infralimbic (IL) subregions of the medial prefrontal cortex, 30 min before reexposure 1. Next day, mice were reexposed to the chamber for 6 min as memory testing (reexposure 2). KNT-127 that infused into the BLA and IL, but not HPC or PL, significantly reduced the freezing response in reexposure 2 compared with those of control. The effect of KNT-127 administered into the BLA and IL was antagonized by pretreatment with a selective DOP antagonist. Further, the effect of KNT-127 was abolished by local administration of MEK/ERK inhibitor into the BLA, and PI3K/Akt inhibitor into the IL, respectively. These results suggested that the effect of KNT-127 was mediated by MEK/ERK signaling in the BLA, PI3K/Akt signaling in the IL, and DOPs in both brain regions. Here, we propose that DOPs play a role in fear extinction via distinct signaling pathways in the BLA and IL.

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

Opioid receptors are members of the group of G protein-couple receptors, which have been proven to be effective targets for treating severe pain. The interactions between the opioid receptors and corresponding ligands and the receptor’s activation by different agonists have been among the most important fields in opioid research. In this study, with compound M1, an active metabolite of tramadol, as the clue compound, several aminomethyl tetrahydronaphthalenes were designed, synthesized and assayed upon opioid receptors. With the resultant compounds FW-AII-OH-1 (Ki = 141.2 nM for the κ opioid receptor), FW-AII-OH-2 (Ki = 4.64 nM for the δ opioid receptor), FW-DI-OH-2 (Ki = 8.65 nM for the δ opioid receptor) and FW-DIII-OH-2 (Ki = 228.45 nM for the δ opioid receptor) as probe molecules, the structural determinants responsible for the subtype selectivity and activation mechanisms were further investigated by molecular modeling and molecular dynamics simulations. It was shown that Y7.43 was a key residue in determining the selectivity of the three opioid receptors, and W6.58 was essential for the selectivity of the δ opioid receptor. A detailed stepwise discovered agonist-induced signal transduction mechanism of three opioid receptors by aminomethyl tetrahydronaphthalene compounds was proposed: the 3–7 lock between TM3 and TM7, the DRG lock between TM3 and TM6 and rearrangement of I3.40, P5.50 and F6.44, which resulted in the cooperative movement in 7 TMs. Then, the structural relaxation left room for the binding of the G protein at the intracellular site, and finally the opioid receptors were activated.

Alzheimer’s disease (AD) is characterized by amyloid plaques and neurofibrillary tangles. Substantial evidence for AD pathogenesis suggests that β-site APP cleaving enzyme 1 (BACE1) and γ-secretase enzyme initiate the amyloidogenic pathway and produces toxic Aβ peptides that prone to aggregate in the brain. Therefore, the inhibition of BACE1 expression and function is an attractive strategy for AD therapy. In the present work, we made the first finding that activating δ-opioid receptors (DOR) with a specific DOR agonist significantly attenuated BACE1 expression and activity in the highly differentiated PC12 cells with mimicked AD injury, while the application of DOR inhibitor naltrindole reversed the UFP-512 effects, and even caused a major increase in BACE1 expression and activity as well as Aβ42 production in physiological conditions. Knocking-down DOR also enhanced BACE1 protein expression and its activity for APP processing, associating with a significant increase in Aβ42 production. In sharp contrast, activation of μ-opioid receptor (MOR) with DAMGO greatly promoted BACE1 expression and activity with an acceleration of APP cleavage, thus contributing to increased Aβ42 production. DADLE, a less selective DOR agonist that may bind to MOR, had no stable inhibitory effect on BACE1. Similar results were also found in APP mutant (APPswe) SH-SY5Y cell line, providing further validation of the DOR action on BACE1 regulation. Our novel data demonstrated entirely different roles of DOR and MOR in the regulation of BACE1 expression and activity with DOR being neuroprotective against AD injury. These findings provided a novel clue for new strategies of AD therapy via targeting endogenous opioid receptors.

The delta-opioid receptor (DOR) is one of three classic opioid receptors in the opioid system. It was traditionally thought to be primarily involved in modulating the transmission of messages along pain signaling pathway. Although there were scattered studies on its other neural functions, inconsistent results and contradicting conclusions were found in past literatures, especially in terms of DOR’s role in a hypoxic/ischemic brain. Taking inspiration from the finding that the turtle brain exhibits a higher DOR density and greater tolerance to hypoxic/ischemic insult than the mammalian brain, we clarified DOR’s specific role in the brain against hypoxic/ischemic injury and reconciled previous controversies in this aspect. Our serial studies have strongly demonstrated that DOR is a unique neuroprotector against hypoxic/ischemic injury in the brain, which has been well confirmed in current research. Moreover, mechanistic studies have shown that during acute phases of hypoxic/ischemic stress, DOR protects the neurons mainly by the stabilization of ionic homeostasis, inhibition of excitatory transmitter release, and attenuation of disrupted neuronal transmission. During prolonged hypoxia/ischemia, however, DOR neuroprotection involves a variety of signaling pathways. More recently, our data suggest that DOR may display its neuroprotective role via the BDNF-TrkB pathway. This review concisely summarizes the progress in this field.

Introduction: Genes often have multiple polymorphisms that interact with each other and the environment in different individuals. Variability in the opioid receptors can influence opiate withdrawal and dependence. In humans, A118G Single Nucleotide Polymorphisms (SNP) on μ-Opioid Receptor (MOR), 36 G>T in κ-Opioid Receptor (KOR), and T921C in the δ-Opioid Receptor (DOR) have been found to associate with substance dependence. Methods: To investigate the association between opioid receptors gene polymorphism and heroin addiction, 100 control subjects with no history of opioid use, and 100 heroin addicts (50% males and 50% females) in Tehran (capital of Iran), were evaluated. A118G, 36 G>T, and T921C SNPs on the MOR, KOR, DOR genes, respectively, were genotyped by sequencing. Results: We found no differences in either allele or genotype frequency for MOR, KOR and DOR genes SNPs between controls and subjects addicted to heroin. Conclusion: The relationships among polymorphisms may be important in determining the risk profile for complex diseases such as addiction, but opioid addiction is a multifactorial syndrome which is partially hereditary and partially affected by the environment.

Alzheimer's disease (AD) is characterized by amyloid plaques and neurofibrillary tangles. Substantial evidence for AD pathogenesis suggests that β-site APP cleaving enzyme 1 (BACE1) and γ-secretase enzyme initiate the amyloidogenic pathway and produces toxic Aβ peptides that prone to aggregate in the brain. Therefore, the inhibition of BACE1 expression and function is an attractive strategy for AD therapy. In the present work, we made the first finding that activating δ-opioid receptors (DOR) with a specific DOR agonist significantly attenuated BACE1 expression and activity in the highly differentiated PC12 cells with mimicked AD injury, while the application of DOR inhibitor naltrindole reversed the UFP-512 effects, and even caused a major increase in BACE1 expression and activity as well as Aβ42 production in physiological conditions. Knocking-down DOR also enhanced BACE1 protein expression and its activity for APP processing, associating with a significant increase in Aβ42 production. In sharp contrast, activation of μ-opioid receptor (MOR) with DAMGO greatly promoted BACE1 expression and activity with an acceleration of APP cleavage, thus contributing to increased Aβ42 production. DADLE, a less selective DOR agonist that may bind to MOR, had no stable inhibitory effect on BACE1. Similar results were also found in APP mutant (APPswe) SH-SY5Y cell line, providing further validation of the DOR action on BACE1 regulation. Our novel data demonstrated entirely different roles of DOR and MOR in the regulation of BACE1 expression and activity with DOR being neuroprotective against AD injury. These findings provided a novel clue for new strategies of AD therapy via targeting endogenous opioid receptors. [ABSTRACT FROM AUTHOR]

Hypoxic-ischemic brain damage (HIBD) is a relatively common malignant complication that occurs in newborn infants, but promising therapies remain limited. In this study, we focused on the role of miR-326 and its target gene δ-opioid receptor (DOR) in the pathogenesis of neonatal HIBD. The expression levels of miR-326 and DOR after hypoxic-ischemic injury were examined both in vivo and in vitro. The direct relationship between miR-326 and DOR was confirmed by a dual-luciferase reporter assay. Further, effects of miR-326 on cell viability and apoptosis levels under oxygen glucose deprivation (OGD) were analyzed. The expression levels of miR-326 were significantly lower and DOR levels were significantly higher in the HIBD group than the control group both in vivo and in vitro. Overexpression of miR-326 downregulated the expression of DOR, while suppression of miR-326 upregulated the expression of DOR. The dual-luciferase reporter assay further confirmed that DOR could be directly targeted and regulated by miR-326. MiR-326 knockdown improved cell survival and decreased cell apoptosis by decreasing the expression levels of Caspase-3 and Bax and increasing Bcl-2 expression in PC12 cells after exposure to OGD. Moreover, DOR knockdown rescued the effect of the improved cell survival and suppressed cell apoptosis induced by silencing miR-326. Our findings indicated that inhibition of miR-326 may improve cell survival and decrease cell apoptosis in neonatal HIBD through the target gene DOR. [ABSTRACT FROM AUTHOR]

Background/Aims: Hypoxic/ischemic injury to the liver is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ ischemic events, and δ-opioid receptor (DOR) is protective against hypoxic/ischemic injury, we asked if pharmacological activation of DOR can alter hypoxic events by regulating miRNA expression in the liver. As the first step, the present work aimed at testing the effect of DOR activation on hepatic miRNA expression in hypoxia. Methods: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected according to TaqMan low-density array (TLDA) data and analyzed by quantitative real-time PCR. Results: We found that: 1) 1-day hypoxia caused the upregulation of 9 miRNAs (miR-7a-5p, miR-10a-5p, miR-25-3p, miR-26b-5p, miR-122-5p, miR-128a-3p, miR-135b-5p, miR-145-5p, and miR-181a-5p) and the downregulation of 2 miRNAs (miR-34a-5p and miR-182); 2) 5 and 10-days hypoxia altered the expression of 4 miRNAs (miR-34c-5p, miR-184, miR-107-3p and miR192-5p); 3) DOR activation shifted the expression of 8 miRNAs (miR-122-5p, miR-146a-5p, miR-30e-5p, miR-128a-3p, miR-182, miR-192-5p miR-107-3p and miR-184) in normoxic condition; and 4) DOR activation modified hypoxia-induced changes in 6 miRNAs (miR-142-5p, miR-145-5p, miR-146a-5p, miR-204-5p, miR-34a-5p and miR-192-5p). Conclusion: Hypoxia significantly modifies the miRNA profile in the liver, while DOR activation can modify the hypoxic modification. Therefore, it is potentially possible to alter hypoxic/ischemic pathophysiology in the liver through DOR pharmacotherapy.

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes serious neurological disability; there are, however, currently few promising therapies for it. We have recently shown that δ-opioid receptor (DOR) is neuroprotective by downregulating TNF-α. Since hypoxia-ischemia (HI) triggers a robust inflammatory response, which exacerbates HI brain damage, we investigated, in this study, whether DOR activation could regulate inflammatory cytokine expression, thereby playing a protective effect on the neonatal brain under HI. Twenty-five neonatal rats were randomly divided into five groups: (1) control (control); (2) HI; (3) HI with saline (HI + NS); (4) DOR activation with UFP-512 (a potent and specific DOR agonist) under HI conditions (HI + U); and (5) DOR inhibition using NT treatment under HI conditions (HI + NT). The rats were sacrificed by decapitation at 24 h after HI, and their brains were rapidly removed for measurements. The protein expression of TNF-α, IL-6, ICAM-1, IL-10, IL-18, NQO-1, Nrf-2, and HO-1 was measured using Western blot. In the hemispheres exposed to HI, DOR activation significantly decreased the expressions of TNF-α, IL-6, and ICAM-1 in the cortex, while it significantly increased IL-10 and had no effect on IL-18 in the same region. In contrast, DOR had no appreciable effect on inflammatory cytokine expression in non-cortical tissues including hippocampal, subcortical, and cerebellar tissues. Moreover, HI stress triggered an upregulation of Nrf-2 nuclear protein as well as some of its downstream anti-inflammatory genes such as HO-1 and NQO-1 in the cortex, while DOR activation further augmented such a protective reaction against HI injury. DOR plays an important role in protecting against HI by regulating the expression of inflammatory and anti-inflammatory cytokines in the cortex, which is likely mediated by the Nrf-2/HO-1/NQO-1 signaling. [ABSTRACT FROM AUTHOR]

We have recently demonstrated that δ-opioid receptor (DOR) activation attenuates α-synuclein expression/aggregation induced by MPP(+) and/or severe hypoxia. Since α-synuclein plays a critical role in the pathogenesis of Parkinson's disease, DOR activation may trigger an antiparkinson pathway(s) against α-synuclein-induced injury. However, the underlying mechanism is unknown yet. In HEK293T and PC12 cells, we investigated the effects of DOR activation on the oligomer formation induced by α-synuclein overexpression and mutation in normoxic and hypoxic conditions and explored the potential signaling pathways for DOR protection. We found that (1) increased expression of both wild-type and A53T-mutant α-synuclein led to the formation of α-synuclein oligomers and cytotoxic injury; (2) DOR activation largely attenuated the formation of toxic α-synuclein oligomers induced by α-synuclein overexpression/mutation and/or hypoxia; (3) DOR activation attenuated α-synuclein-induced cytotoxicity through TORC1/SIK1/CREB, but not the phospho-CREB pathway, while DOR activation reduced hypoxic cell injury through the phospho-CREB mechanism; and (4) the interaction of α-synuclein and the DJ-1 was involved in the mechanisms for DOR-mediated protection against α-synuclein oligomer formation. Our findings suggest that DOR attenuates the formation of toxic α-synuclein oligomers through the phos-CREB pathway under hypoxic conditions, and through TORC1/SIK1/CREB pathways in the conditions of α-synuclein overexpression and mutation. The DJ-1 gene was involved in the DOR protection against parkinsonian injury. [ABSTRACT FROM AUTHOR]

In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not β-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4+ than in CD8+ T lymphocytes. CCL4-induced antinociception almost disappeared upon CD4+, but not CD8+, lymphocyte depletion with selective antibodies, thus supporting that the release of met-enk from CD4+ lymphocytes underlies the opioid antinociceptive response evoked by CCL4. [ABSTRACT FROM AUTHOR]

There is emerging evidence suggesting that neurotoxic insults and hypoxic/ischemic injury are underlying causes of Parkinson's disease (PD). Since PTEN-induced kinase 1 (PINK1) dysfunction is involved in the molecular genesis of PD and since our recent studies have demonstrated that the δ-opioid receptor (DOR) induced neuroprotection against hypoxic and 1-methyl-4-phenyl-pyridimium (MPP+) insults, we sought to explore whether DOR protects neuronal cells from hypoxic and/or MPP+ injury via the regulation of PINK1-related pathways. Using highly differentiated rat PC12 cells exposed to either severe hypoxia (0.5-1% O2) for 24-48 h or varying concentrations of MPP+, we found that both hypoxic and MPP+ stress reduced the level of PINK1 expression, while incubation with the specific DOR agonist UFP-512 reversed this reduction and protected the cells from hypoxia and/or MPP+-induced injury. However, the DOR-mediated cytoprotection largely disappeared after knocking down PINK1 by PINK1 small interfering RNA. Moreover, we examined several important signaling molecules related to cell survival and apoptosis and found that DOR activation attenuated the hypoxic and/or MPP+-induced reduction in phosphorylated Akt and inhibited the activation of cleaved caspase-3, whereas PINK1 knockdown largely deprived the cell of the DOR-induced effects. Our novel data suggests a unique mechanism underlying DOR-mediated cytoprotection against hypoxic and MPP+ stress via a PINK1-mediated regulation of signaling. [ABSTRACT FROM AUTHOR]

Background: Hypoxic/ischemic injury to the heart is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ischemic events, and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering miRNA expression in the heart under hypoxic conditions. The present study aimed to test our hypothesis. Methods: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected from TaqMan low-density array (TLDA) data and were further analyzed by quantitative real-time PCR. Results: We found that: 1) hypoxia alters the miRNA expression profiles depending on the hypoxic duration; 2) DOR activation shifts miRNA expression profiles in normoxic conditions and upregulates miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, and miR-338; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression and increases the levels of miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, miR-141, miR-200b, and miR-324-3p. For example, miR-196c-5p decreased by 50% while miR-135a-5p increased 2.9 fold after 10 days under hypoxic conditions. Moreover, DOR activation further strengthened the hypoxia-induced increase of the levels of miR-7a-5p. When DOR was activated using UFP-512, the level of miR-107-3p significantly increased 1 day after the administration of UFP-512, but gradually decreased back to normal under normoxia. Conclusion: Hypoxia significantly modifies the miRNA profile in the heart, which can be mimicked or modified by DOR activation. Defining the targeted pathways that regulate the diverse cellular and molecular functions of miRNAs may provide new insights into potential therapies for hypoxic/ischemic injury of the heart.

Recently, propranolol was suggested to prevent hyperlactatemia in a child with hypovolemic shock through β-adrenergic blockade. Though it is a known inhibitor of glycolysis, propranolol, outside this observation, has never been reported to fully protect against lactate overproduction. On the other hand, literature evidence exists for a cross-talk between β-adrenergic receptors (protein targets of propranolol) and δ-opioid receptor. In this literature context, it is hypothesized here that anti-diarrheic racecadotril (a pro-drug of thiorphan, an inhibitor of enkephalinases), which, in the cited observation, was co-administered with propranolol, might have facilitated the β-blocker-driven inhibition of glycolysis and resulting lactate production. The opioid-facilitated β-adrenergic blockade would be essentially additivity or even synergism putatively existing between antagonism of β-adrenergic receptors and agonism of δ-opioid receptor in lowering cellular cAMP and dependent functions.

The functional state of δ-opioid receptor signaling cascade in live cells exposed to a therapeutic concentration of lithium for a prolonged period of time (weeks) is not known because the previous studies of Li interference with OR were oriented to µ-OR only. The same applies to the analysis of the prolonged effect of Li on oxidative stress in context with δ-OR function. HEK293 cells stably expressing δ-OR were cultivated in the presence or absence of 1 mM LiCl for 7 or 21 days, homogenized and the post-nuclear (PNS) and plasma membrane (PM) fractions prepared from all four types of cells. Level of δ-OR in PM was determined by specific radioligand [ 3 H]DADLE binding and immunoblot assays; the functional coupling between δ-OR and G proteins was determined as DADLE-stimulated high-affinity [ 35 S]GTPγS binding. In the whole cells, general oxidative stress was monitored by fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCF) and results verified by analysis of PNS and isolated PM . Generation of 4-hydroxy-2-nonenal (4-HNE)-protein adducts and malondialdehyde (MDA) level were determined as products of lipid peroxidation. Li-treated cells exhibited the decreased amount of δ-OR. This was evidenced by both [ 3 H]DADLE binding and immunoblot assays. The δ-OR-G protein coupling efficiency was diminished. Simultaneously, in Li-treated cells, the highly increased oxidative stress measured as DCF fluorescence intensity was noticed. Importantly, this result was detected in live cells as well as PNS and PM. Accordingly, production of 4-HNE-protein adducts and MDA was clearly increased in Li-treated cells. The general significance of our work lies in presentation of novel data indicating that prolonged exposure of live HEK293 cells to the therapeutic concentration of Li results in down-regulation of δ-OR protein level and attenuation of δ-OR function in parallel with increased oxidative stress and increased level of lipid peroxidation products. [ABSTRACT FROM AUTHOR]

Introduction: Genes often have multiple polymorphisms that interact with each other and the environment in different individuals. Variability in the opioid receptors can influence opiate withdrawal and dependence. In humans, A118G Single Nucleotide Polymorphisms (SNP) on µ-Opioid Receptor (MOR), 36 G>T in κ-Opioid Receptor (KOR), and T921C in the d-Opioid Receptor (DOR) have been found to associate with substance dependence. Methods: To investigate the association between opioid receptors gene polymorphism and heroin addiction, 100 control subjects with no history of opioid use, and 100 heroin addicts (50% males and 50% females) in Tehran (capital of Iran), were evaluated. A118G, 36 G>T, and T921C SNPs on the MOR, KOR, DOR genes, respectively, were genotyped by sequencing. Results: We found no differences in either allele or genotype frequency for MOR, KOR and DOR genes SNPs between controls and subjects addicted to heroin. Conclusion: The relationships among polymorphisms may be important in determining the risk profile for complex diseases such as addiction, but opioid addiction is a multifactorial syndrome which is partially hereditary and partially affected by the environment. [ABSTRACT FROM AUTHOR]

The interaction of remifentanil with glutamate systems has an important role in remifentanil-induced thermal and mechanical hyperalgesia. A previous study by our group suggested that the trafficking and function of glutamate receptor 1 (GluR1) subunits contributes to remifentanil- induced hyperalgesia by regulating the phosphorylation of GluR1 in dorsal horn neurons. The present study demonstrated that δ opioid receptor (DOR) inhibition prevented thermal and mechanical hyperalgesia, which was induced by remifentanil infusion via attenuating GluR1 subunit trafficking and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function in dorsal horn neurons. Sprague Dawley rats received a plantar incision and remifentanil infusion to induce a model of postoperative hyperalgesia. Thermal and mechanical pain was tested at 8 different time-points. Expression of AMPAR subunits GluR1 and DOR, as well as the phosphorylation status of GluR1 were evaluated by western blot analysis. Furthermore, the function of AMPAR in the spinal dorsal horn was measured by whole-cell patch-clamp recording. Remifentanil-induced thermal and mechanical hyperalgesia appeared after the 60-min infusions, reaching a peak level on day 2 and persisting for 5 days. Remifentanil infusion led to upregulation of membrane expression of the AMPAR subunit GluR1 and DOR (P=0.003 and 0.001, respectively) no change in total GluR1 and DOR expression levels (P=0.244 and 0.531, respectively). Selective DOR inhibitor naltrindole caused a reduction of remifentanil-induced hyperalgesia, which was accompanied by downregulation of membrane levels of GluR1 in the spinal cord (P=0.0013). In addition, DOR inhibition led to downregulation of GluR1 phosphorylated at Ser845. Furthermore, the AMPAR-mediated miniature excitatory post-synaptic current was increased in frequency and in amplitude in dorsal horn neurons (P=0.002 and 0.0011, respectively), which was decreased by incubation with naltrindole. Combined behavioral, western blot and electrophysiological evidence indicated that remifentanil-induced hyperalgesia was mediated by DOR activation, followed by phosphorylation-dependent GluR1 trafficking and AMPAR function enhancement in the spinal cord. DOR appears to be required for remifentanil and incision-induced hyperalgesia development and to be a potential biochemical target for treating opioid-induced postoperative hyperalgesia. [ABSTRACT FROM AUTHOR]

Background/Aims: Hypoxic/ischemic injury to the liver is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ischemic events, and δ-opioid receptor (DOR) is protective against hypoxic/ischemic injury, we asked if pharmacological activation of DOR can alter hypoxic events by regulating miRNA expression in the liver. As the first step, the present work aimed at testing the effect of DOR activation on hepatic miRNA expression in hypoxia. Methods: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected according to TaqMan low-density array (TLDA) data and analyzed by quantitative real-time PCR. Results: We found that: 1) 1-day hypoxia caused the upregulation of 9 miRNAs (miR-7a-5p, miR-10a-5p, miR-25-3p, miR-26b-5p, miR-122-5p, miR-128a-3p, miR-135b-5p, miR-145-5p, and miR-181a-5p) and the downregulation of 2 miRNAs (miR-34a-5p and miR-182); 2) 5 and 10-days hypoxia altered the expression of 4 miRNAs (miR-34c-5p, miR-184, miR-107-3p and miR192-5p); 3) DOR activation shifted the expression of 8 miRNAs (miR-122-5p, miR-146a-5p, miR-30e-5p, miR-128a-3p, miR-182, miR-192-5p miR-107-3p and miR-184) in normoxic condition; and 4) DOR activation modified hypoxia-induced changes in 6 miRNAs (miR-142-5p, miR-145-5p, miR-146a-5p, miR-204-5p, miR-34a-5p and miR-192-5p). Conclusion: Hypoxia significantly modifies the miRNA profile in the liver, while DOR activation can modify the hypoxic modification. Therefore, it is potentially possible to alter hypoxic/ischemic pathophysiology in the liver through DOR pharmacotherapy. [ABSTRACT FROM AUTHOR]

Abstract Background Some peptides purified from the venom of the spider Phoneutria nigriventer have been identified as potential sources of drugs for pain treatment. In this study, we characterized the antinociceptive effect of the peptide PnPP-19 on the central nervous system and investigated the possible involvement of opioid and cannabinoid systems in its action mechanism. Methods Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. All drugs were administered by the intracerebroventricular route. Results PnPP-19 induced central antinociception in mice in the doses of 0.5 and 1 μg. The non-selective opioid receptor antagonist naloxone (2.5 and 5 μg), μ-opioid receptor antagonist clocinnamox (2 and 4 μg), δ-opioid receptor antagonist naltrindole (6 and 12 μg) and CB1 receptor antagonist AM251 (2 and 4 μg) partially inhibited the antinociceptive effect of PnPP-19 (1 μg). Additionally, the anandamide amidase inhibitor MAFP (0.2 μg), the anandamide uptake inhibitor VDM11 (4 μg) and the aminopeptidase inhibitor bestatin (20 μg) significantly enhanced the antinociception induced by a low dose of PnPP-19 (0.5 μg). In contrast, the κ-opioid receptor antagonist nor-binaltorphimine (10 μg and 20 μg) and the CB2 receptor antagonist AM630 (2 and 4 μg) do not appear to be involved in this effect. Conclusions PnPP-19-induced central antinociception involves the activation of CB1 cannabinoid, μ- and δ-opioid receptors. Mobilization of endogenous opioids and cannabinoids might be required for the activation of those receptors, since inhibitors of endogenous substances potentiate the effect of PnPP-19. Our results contribute to elucidating the action of the peptide PnPP-19 in the antinociceptive pathway.

The nucleus accumbens is a terminal area of the mesolimbic dopaminergic system that arises in the ventral tegmental area. Opioids are thought to enhance dopaminergic activity in the nucleus accumbens by activating δ- and μ-opioid receptors in the ventral tegmental area. However, δ- and μ-opioid receptor agonists increase extracellular levels of accumbal dopamine when infused directly into the nucleus accumbens of rats. Therefore, the roles of δ- and μ-opioid receptors in regulation of accumbal dopaminergic neural activity have been analyzed by using δ- and μ-opioid receptor ligands. This review describes the mechanisms underlying the stimulatory effects on accumbal dopamine efflux, which are induced by local administration of δ- and μ-opioid receptor agonists into the nucleus accumbens of freely moving rats. The focus of this article is neurochemical studies that use in vivo microdialysis techniques. Taken together, the in vivo neurochemical evidence from these studies indicates that δ- and μ-opioid receptor agonists increase accumbal dopamine efflux by activating naloxone-sensitive opioid receptors, and by mechanisms independent of naloxone-sensitive opioid receptors, in the nucleus accumbens. [ABSTRACT FROM AUTHOR]

Perinatal hypoxic-ischemic (HI) brain injury results in death or profound long-term neurologic disability in both children and adults. However, there is no effective pharmacological therapy due to a poor understanding of HI events, especially the initial triggers for hypoxic-ischemic injury such as disrupted ionic homeostasis and the lack of effective intervention strategy. In the present study, we showed that neonatal brains undergo a developmental increase in the disruption of K homeostasis during simulated ischemia, oxygen-glucose deprivation (OGD) and neonatal HI cortex has a triple phasic response (earlier attenuation, later enhancement, and then recovery) of disrupted K homeostasis to OGD. This response partially involves the activity of the δ-opioid receptor (DOR) since the earlier attenuation of ischemic disruption of K homeostasis could be blocked by DOR antagonism, while the later enhancement was reversed by DOR activation. Similar to DOR activation, acupuncture, a strategy to promote DOR activity, could partially reverse the later enhanced ischemic disruption of K homeostasis in the neonatal cortex. Since maintaining cellular K homeostasis and inhibiting excessive K fluxes in the early phase of hypoxic-ischemic insults may be of therapeutic benefit in the treatment of ischemic brain injury and related neurodegenerative conditions, and since many neurons and other cells can be rescued during the 'window of opportunity' after HI insults, our first findings regarding the role of acupuncture and DOR in attenuating ischemic disruption of K homeostasis in the neonatal HI brain suggest a potential intervention therapy in the treatment of neonatal brain injury, especially hypoxic-ischemic encephalopathy. [ABSTRACT FROM AUTHOR]