The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the ß-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-ß-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of ß-arrestin-1 to the PER2 promoter. Furthermore, we observed that ß-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances ß-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via ß-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

Small molecule NOP agonists reverse locomotor sensitization induced by cocaine in male C57BL/6 mice.

Orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the nociceptin opioid receptor (NOP) has been shown to block cocaine-induced locomotor sensitization in mice and rats, and also reverses this phenomenon when injected intracerebroventricularly in animals with an established sensitized response. In the present study, we determined whether small-molecule NOP agonists would recapitulate this effect after systemic administration. Male C57BL/6 mice treated with cocaine (15 mg/kg) on days 1-3 and showed locomotor sensitization to the same dose of cocaine on day 8 were injected with vehicle or one of the two NOP agonists (AT-202 and AT-524) (but not cocaine) on days 9-11. On day 15, locomotor sensitization was assessed after a cocaine challenge (15 mg/kg). Subchronic administration of the two NOP agonists to sensitized mice significantly decreased the sensitized response on day 15. In a separate experiment conducted in male and female mice lacking NOP and their wildtype littermates, AT-524 reversed sensitization in male wildtype but not in mice lacking NOP. Further, co-administration of the NOP agonist with cocaine for three days on days 16-18 prevented the development of locomotor sensitization from this cocaine treatment in wild-type but not in NOP knockout mice. However, none of these effects of the NOP agonist was observed in female mice. Together, these results suggest that subchronic repeated administration of small-molecule NOP agonists may reverse adaptive behavioral changes associated with repeated intermittent cocaine treatment in male but not female mice.

The role of bidirectional communication between the adipokines and the endogenous opioid system in an experimental mouse model of colitis-associated colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of death globally. Multiple factors may contribute to the pathogenesis of CRC, including the abnormalities in the functioning of the endogenous opioid system (EOS) or adiponectin-related signaling. The aim of our study was to evaluate if differences in the expression of opioid receptors (ORs) influence the development of CRC and if modulation of adiponectin receptors using AdipoRon, a selective AdipoR1 receptor agonist, affects colorectal carcinogenesis. METHODS: Naltrexone, an opioid receptor antagonist, was injected intraperitoneally every second day for 2 weeks, at the dose of 1 mg/kg in healthy Balb/C mice to induce changes in ORs expression. CRC was induced by a single intraperitoneal injection of azoxymethane (AOM) and the addition of dextran sodium sulfate (DSS) into drinking water in three-week cycles. The development of CRC was assessed using macro- and microscopic scoring and molecular analysis (RT qPCR, ELISA) after 14 weeks. RESULTS: Naltrexone significantly increased the mRNA expression of Oprm1, Oprd1, and Oprk1 in the mouse colon and in the brain (non-significantly). The pretreatment of mice with naltrexone aggravated the course of CRC (as indicated by tumor area, colon thickness, and spleen weight). The level of circulatory adiponectin was lowered in mice with CRC and increased in the colon as compared with healthy mice. The ß-endorphin level was increased in the plasma of mice with CRC and decreased in the colon as compared to healthy mice. AdipoRon, AdipoR1 agonist, worsened the CRC development, and pretreatment with naltrexone enhanced this negative effect in mice. CRC did not affect the expression of the Adipor1 gene, but the Adipor1 level was increased in mice pretreated with naltrexone (AOM/DSS and healthy mice). AdipoRon did not influence the expression of opioid receptors at the mRNA level in the colon of mice with CRC. The mRNA expression of Ptgs2, Il6, Nos2, Il1b, Il18, Gsdmd, and Rela was increased in mice with CRC as compared to the healthy colon. AdipoRon significantly decreased mRNA expression of Ptgs2, Il6, Il1b, and Il18 as compared to CRC mice. CONCLUSION: EOS and adiponectin-related signaling may play a role in the pathogenesis of CRC and these systems may present some additivity during carcinogenesis.

Chronic intermittent ethanol exposure-induced m6A modifications around mRNA stop codons of opioid receptor genes.

Chronic alcohol consumption may alter mRNA methylation and expression levels of genes related to addiction and reward in the brain, potentially contributing to alcohol tolerance and dependence. Neuron-like (SH-SY5Y) and non-neuronal (SW620) cells were utilized as models to examine chronic intermittent ethanol (CIE) exposure-induced global m6A RNA methylation changes, as well as m6A mRNA methylation changes around the stop codon of three opioid receptor genes (OPRM1, OPRD1, and OPRK1), which are known to regulate pain, reward, and addiction behaviours. CIE exposure for three weeks significantly increased global RNA methylation levels in both SH-SY5Y (t = 3.98, P = 0.007) and SW620 (t = 2.24, P = 0.067) cells. However, a 3-week CIE exposure resulted in hypomethylation around mRNA stop codon regions of OPRM1 and OPRD1 in both cell lines [OPRM1(SH-SY5Y): t = -5.05, P = 0.0005; OPRM1(SW620): t = -3.19, P = 0.013; OPRD1(SH-SY5Y): t = -13.43, P < 0.00001; OPRD1(SW620): t = -4.00, P = 0.003]. Additionally, mRNA expression levels of OPRM1, OPRD1, and OPRK1 were downregulated (corresponding to mRNA hypomethylation) in both SH-SY5Y and SW620 cells after a 3-week CIE exposure. The present study demonstrated that chronic ethanol exposure altered global RNA methylation levels, as well as mRNA methylation and expression levels of opioid receptor genes in both neuron-like and non-neuronal cells. Our findings suggest a potential epitranscriptomic mechanism by which chronic alcohol consumption remodels the expression of reward-related and alcohol responsive genes in the brain, thus increasing the risk of alcohol use disorder development.Abbreviations: OPRM1: the µ-opioid receptor; OPRD1: the δ-opioid receptor; OPRK1: the κ-opioid receptor; CIE: chronic intermittent ethanol exposure; CIE+WD: chronic intermittent ethanol exposure followed by a 24-hr withdrawal; SH-SY5Y: human neuroblastoma cell Line; SW620: human colon carcinoma cell line; RT-qPCR: reverse transcription followed by quantitative polymerase reaction; MazF-RT-qPCR: MazF digestion followed by RT-qPCR.

Heterodimerization of apelin and opioid receptor-like 1 receptors mediates apelin-13-induced G protein biased signaling.

The apelin receptor (APJ) and the opioid-related nociceptin receptor 1 (ORL1) are family A G protein-coupled receptors that participate in a variety of physiological processes. The distribution and function of APJ and ORL1 in the nervous system and peripheral tissues are similar; however, the detailed mechanism of how these two receptors modulate signaling and physiological effects remains unclear. Here, we examined whether APJ and ORL1 form dimers, and investigated signal transduction pathways. The endogenous co-expression of APJ and ORL1 in SH-SY5Y cells was confirmed by western blotting and RT-PCR. Bioluminescence and fluorescence resonance energy transfer assays, as well as a proximity ligation assay and co-immunoprecipitation experiments, demonstrated that APJ and ORL1 heterodimerize in HEK293 cells. We found that the APJ-ORL1 heterodimer is selectively activated by apelin-13, which causes the dimer to couple to Gαi proteins and reduce the recruitment of GRKs and ß-arrestins to the dimer. We showed that the APJ-ORL1 dimer exhibits biased signaling, in which G protein-dependent signaling pathways override ß-arrestin-dependent signaling pathways. Our results demonstrate that the structural interface of the APJ-ORL1 dimer switches from transmembrane domain TM1/TM2 in the inactive state to TM5 in the active state. We used mutational analysis and BRET assays to identify key residues in TM5 (APJ L2185.55, APJ I2245.61, and ORL1 L2295.52) responsible for the receptor-receptor interaction. These results provide important information on the APJ-ORL1 heterodimer and may assist the design of new drugs targeting biased signaling pathways for treatment of pain and cardiovascular and metabolic diseases.

Molecular and Epigenetic Aspects of Opioid Receptors in Drug Addiction and Pain Management in Sport.

Opioids are substances derived from opium (natural opioids). In its raw state, opium is a gummy latex extracted from Papaver somniferum. The use of opioids and their negative health consequences among people who use drugs have been studied. Today, opioids are still the most commonly used and effective analgesic treatments for severe pain, but their use and abuse causes detrimental side effects for health, including addiction, thus impacting the user's quality of life and causing overdose. The mesocorticolimbic dopaminergic circuitry represents the brain circuit mediating both natural rewards and the rewarding aspects of nearly all drugs of abuse, including opioids. Hence, understanding how opioids affect the function of dopaminergic circuitry may be useful for better knowledge of the process and to develop effective therapeutic strategies in addiction. The aim of this review was to summarize the main features of opioids and opioid receptors and focus on the molecular and upcoming epigenetic mechanisms leading to opioid addiction. Since synthetic opioids can be effective for pain management, their ability to induce addiction in athletes, with the risk of incurring doping, is also discussed.

Morphine ameliorates pentylenetetrazole-induced locomotor pattern in zebrafish embryos; mechanism involving regulation of opioid receptors, suppression of oxidative stress, and inflammation in epileptogenesis.

Zebrafish (Danio rerio) is becoming an increasingly important model in epilepsy research. Pentylenetetrazole (PTZ) is a convulsant agent that induces epileptic seizure-like state in zebrafish and zebrafish embryos and is most commonly used in antiepileptic drug discovery research to evaluate seizure mechanisms. Classical antiepileptic drugs, such as valproic acid (VPA) reduce PTZ-induced epileptiform activities. Opioid system has been suggested to play a role in epileptogenesis. The aim of our study is to determine the effects of morphine in PTZ-induced epilepsy model in zebrafish embryos by evaluating locomotor activity and parameters related to oxidant-antioxidant status, inflammation, and cholinergic system as well as markers of neuronal activity c-fos, bdnf, and opioid receptors. Zebrafish embryos at 72 hpf were exposed to PTZ (20 mM), VPA (1 mM), and Morphine (MOR) (100 µM). MOR and VPA pretreated groups were treated with either MOR (MOR + PTZ) or VPA (VPA + PTZ) for 20 min before PTZ expoure. Locomotor activity was quantified as total distance moved (mm), average speed (mm/sec) and exploration rate (%) and analyzed using ToxTrac tracking programme. Oxidant-antioxidant system parameters, acetylcholinesterase activity, and sialic acid leves were evaluated using spectrophotometric methods. The expression of c-fos, bdnf, oprm1, and oprd1 were evaluated by RT-PCR. MOR pretreatment ameliorated PTZ-induced locomotor pattern as evidenced by improved average speed, exploration rate and distance traveled. We report the restoration of inflammatory and oxidant-antioxidant system parameters, c-fos, bdnf, and opioid receptor oprm1 as the possible mechanisms involved in the ameliorative effect of MOR against PTZ-induced epileptogenic process in zebrafish embryos.

Evaluating the expression pattern of the opioid receptor in pituitary neuroendocrine tumors (PitNET) and the role of morphine and naloxone in the regulation of pituitary cell line growth and apoptosis.

PURPOSE: The expression pattern of the opioid receptor (MOR) in pituitary neuroendocrine tumors (PitNET) and the possible effect of morphine and naloxone on GH3 cell growth and apoptosis were evaluated. METHODS: The 114 pituitary tissues including non-functioning, GH-producing and ACTH-producing PitNET and healthy cadaver pituitary tissues were included. The expression level of the MOR gene and protein was assessed using real-time PCR and Western blot. The association with patient demographic characteristics was assessed. Morphine and naloxone were applied to assess their possible pharmacological role in GH3 pituitary adenoma cell death. The cytotoxic effect, the apoptosis rate, the cell cycle distribution, the content of reactive oxygen species and the caspase 3 activity were measured. RESULTS: MOR gene levels increased significantly in pituitary neuroendocrine tumors (PitNET) compared to the healthy pituitary samples. The increased level of MOR gene expression was prominent in invasive functional and non-functional pituitary tumors. A consistent expression pattern was demonstrated for MOR protein levels in PitNET samples. A dose- and time-dependent reduction in the rate of GH3 pituitary cells was observed after morphine treatment with an IC50 of 483 µM after 24 h of incubation. Morphine induced early apoptosis, accumulation of cells in sub-G1 phase, increase in cellular ROS levels and caspase-3 activity. The observed effects of morphine were reversed after MOR blockade using 10 and 25 µM naloxone. CONCLUSION: The possible contributing role of the MOR in pituitary tumor cell growth and the putative pharmaceutical effect of morphine in pituitary neuroendocrine tumor cell death (PitNET) is illustrated.

Epigenetic Modulation of Opioid Receptors by Drugs of Abuse.

Chronic exposure to drugs of abuse produces profound changes in gene expression and neural activity associated with drug-seeking and taking behavior. Dysregulation of opioid receptor gene expression is commonly observed across a variety of abused substances including opioids, cocaine, and alcohol. Early studies in cultured cells showed that the spatial and temporal gene expression of opioid receptors are regulated by epigenetic mechanisms including DNA and histone modifications and non-coding RNAs. Accumulating evidence indicate that drugs of abuse can modulate opioid receptor gene expression by targeting various epigenetic regulatory networks. Based on current cellular and animal models of substance use disorder and clinical evidence, this review summarizes how chronic drug exposure alters the gene expression of mu, delta, kappa, and nociceptin receptors via DNA and histone modifications. The influence of drugs of abuse on epigenetic modulators, such as non-coding RNAs and transcription factors, is also presented. Finally, the therapeutic potential of manipulating epigenetic processes as an avenue to treat substance use disorder is discussed.

Mechanisms Underlining Inflammatory Pain Sensitivity in Mice Selected for High and Low Stress-Induced Analgesia-The Role of Endocannabinoids and Microglia.

In this work we strived to determine whether endocannabinoid system activity could account for the differences in acute inflammatory pain sensitivity in mouse lines selected for high (HA) and low (LA) swim-stress-induced analgesia (SSIA). Mice received intraplantar injections of 5% formalin and the intensity of nocifensive behaviours was scored. To assess the contribution of the endocannabinoid system, mice were intraperitoneally (i.p.) injected with rimonabant (0.3-3 mg/kg) prior to formalin. Minocycline (45 and 100 mg/kg, i.p.) was administered to investigate microglial activation. The possible involvement of the endogenous opioid system was investigated with naloxone (1 mg/kg, i.p.). Cannabinoid receptor types 1 and 2 (Cnr1, Cnr2) and opioid receptor subtype (Oprm1, Oprd1, Oprk1) mRNA levels were quantified by qPCR in the structures of the central nociceptive circuit. Levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled with the mass spectrometry method (LC-MS/MS). In the interphase, higher pain thresholds in the HA mice correlated with increased spinal anandamide and 2-AG release and higher Cnr1 transcription. Downregulation of Oprd1 and Oprm1 mRNA was noted in HA and LA mice, respectively, however no differences in naloxone sensitivity were observed in either line. As opposed to the LA mice, inflammatory pain sensitivity in the HA mice in the tonic phase was attributed to enhanced microglial activation, as evidenced by enhanced Aif1 and Il-1ß mRNA levels. To conclude, Cnr1 inhibitory signaling is one mechanism responsible for decreased pain sensitivity in HA mice in the interphase, while increased microglial activation corresponds to decreased pain thresholds in the tonic inflammatory phase.

A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine.

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

The Role of Inflammation, Hypoxia, and Opioid Receptor Expression in Pain Modulation in Patients Suffering from Obstructive Sleep Apnea.

Obstructive sleep apnea (OSA) is a relatively common disease in the general population. Besides its interaction with many comorbidities, it can also interact with potentially painful conditions and modulate its course. The association between OSA and pain modulation has recently been a topic of concern for many scientists. The mechanism underlying OSA-related pain connection has been linked with different pathophysiological changes in OSA and various pain mechanisms. Furthermore, it may cause both chronic and acute pain aggravation as well as potentially influencing the antinociceptive mechanism. Characteristic changes in OSA such as nocturnal hypoxemia, sleep fragmentation, and systemic inflammation are considered to have a curtailing impact on pain perception. Hypoxemia in OSA has been proven to have a significant impact on increased expression of proinflammatory cytokines influencing the hyperalgesic priming of nociceptors. Moreover, hypoxia markers by themselves are hypothesized to modulate intracellular signal transduction in neurons and have an impact on nociceptive sensitization. Pain management in patients with OSA may create problems arousing from alterations in neuropeptide systems and overexpression of opioid receptors in hypoxia conditions, leading to intensification of side effects, e.g., respiratory depression and increased opioid sensitivity for analgesic effects. In this paper, we summarize the current knowledge regarding pain and pain treatment in OSA with a focus on molecular mechanisms leading to nociceptive modulation.

Hypomethylation of the opioid receptor delta 1 gene combined with high opioid receptor delta 1 protein levels indicates increased risk of gout.

BACKGROUND: The purpose of this study was to identify biomarkers for the diagnosis of gout in Chinese Han males using methylation microarray profiling. METHODS: We screened for differentially methylated genes (DMGs) in gout using a methylation microarray and analyzed the functions of the DMGs using gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. We verified gene methylation levels by pyrosequencing and protein levels by enzyme-linked immunosorbent assays (ELISAs). Statistical analyses were performed using SPSS. Two-sided p values <0.05 were deemed to be statistically significant for all analyses. RESULTS: We identified 20,426 significant differential methylation sites (5719 high-methylation sites and 14,707 low-methylation sites). Bioinformatics analysis showed that the DMGs were mainly involved in 43 biological functions, 13 cellular components, 18 molecular functions, and 35 KEGG pathways. We selected opioid receptor delta 1 (OPRD1) for verification of methylation levels between 50 gout patients and 50 controls. The methylation levels of OPRD1 (Chr1:29,139,121) were significantly lower in the gout group (p < 0.05), while OPRD1 protein levels were significantly higher in the gout group (p < 0.05). In addition, the AUC of the combination of OPRD1 (Chr1:29,139,121) methylation and OPRD1 protein levels was 0.796 (0.710, 0.883) with a high sensitivity of 82% and a specificity of 68% (p < 0.001). CONCLUSIONS: The combination of OPRD1 (Chr1:29,139,121) hypomethylation and high levels of OPRD1 protein is a potential biomarker for gout diagnosis.

[Mechanisms of liver-soothing and mind-regulating acupuncture mediating opioid receptor participated in the treatment of migraine].

OBJECTIVE: To observe the effect of "Shugan Tiaoshen"(liver-soothing and mind-regulating) acupuncture on behavior reactions, opioid receptor expressions in the anterior cingulate cortex tissue and inflammatory factors in the serum in migraine rats, in order to explore its mechanism underlying improvement of migraine. METHODS: In the first part of this study, forty male Wistar rats were randomized into control, model, routine acupuncture and "Shugan Tiaoshen" acupuncture groups (n=10/group), and in the second part, other 40 more male Wistar rats were randomized into low, medium and high dosage of blocker of µopioid receptor (OPRM）CTOP5 and PBS groups (n=10/group, for validating the involvement of opioid receptor in the effect of "Shugan Tiaoshen"). The migraine model was established by subcutaneous injection of glyceryl trinitrate. Routine acupuncture was applied to "Baihui" (GV20) and bilateral"Fengchi" (GB20), and "Shugan Tiaoshen" acupuncture applied to GV20, and bila-teral GB20, "Neiguan" (PC6) and "Taichong" (LR3), with the needles retained for 30 min. Behavior responses (head scratching, tail biting, cage climbing and number of going there and back) were scaled. Serum IL-1ß, IL-6 and TNF-α were detected by ELISA, and the expression levels of opioid receptor µ, δ and κ (OPRM, OPRD, OPRK) mRNAs and proteins in the anterior cingulate cortex were detected by fluorescence quantitative PCR and Western blot separately. In the second part of this study, CTOP solution (5µL at concentrations of 20µg/µL,10µg/µL and 5µg/µL) or PBS was injected into the bilateral rostral portions of anterior cingulate cortex 30 min before every "Shugan Tiaoshen" acupuncture intervention, followed by observing the behavioral changes and assaying the contents of serum IL-1ß, IL-6 and TNF-α. RESULTS: After modeling, the behavioral score, serum IL-1ß, IL-6 and TNF-α contents were significantly increased in the model group relevant to the control group (P<0.05), and the beha-vioral score had no significant difference among the model and two acupuncture groups before intervention (P>0.05). Whereas the expression levels of OPRM, OPRD and OPRK mRNAs and proteins had a slight increase in the model group (P>0.05). After the intervention, the behavioral score, serum IL-1ß, IL-6 and TNF-α contents were significantly decreased and the expression levels of OPRM, OPRD and OPRK mRNAs (2.150, 1.066 and 0.805 folds in the "Shugan Tiaoshen" group) and proteins (2.273, 0.901 and 0.893 folds in the "Shugan Tiaoshen" group) notably up-regulated in both "Shugan Tiaoshen" and routine acupuncture groups relevant to the model group (P<0.01, P<0.05), showing that the biggest up-regulation of mRNA expression was OPRM. Comparison between two acupuncture groups showed that the behavioral score, and serum IL-1ß, IL-6 and TNF-α contents were significantly lower, and the expression levels of OPRM, OPRD and OPRK mRNAs and proteins obviously higher in the "Shugan Tiaoshen" group than those in the routine acupuncture group (P<0.01,P<0.05). Results of the second part of this study showed that after injection of antagonist CTOP of OPRM, the therapeutic effect of "Shugan Tiaoshen" acupuncture was weakened in the reduction of behavioral score and serum IL-1ß, IL-6 and TNF-α contents, being minimal, moderate and maximum in the high, medium and low dose of antagonist relevant to PBS in sequence (P<0.05, P<0.01). CONCLUSION: "Shugan Tiaoshen" acupuncture can mitigate pain in migraine rats, which may be associated with its function in up-regulating the expressions of opioid receptors (especially OPRM), and in inhibiting inflammatory reaction in the anterior cingulate cortex.

The Involvement of the Endogenous Opioid System in the Gastrointestinal Aging in Mice and Humans.

Nearly 20% of elderly patients suffer from constipation, but the age-related changes in the gastrointestinal (GI) tract remain insufficiently elucidated. In this study, the alterations within the endogenous opioid system (EOS) as a potential cause of constipation in the elderly were evaluated. The GI functions were assessed in vitro and in vivo and compared between 6-, 12- and 18-month old mice. Moreover, the effect of opioid receptor (MOP, DOP, KOP) agonists on the mouse GI tract functions and the EOS components expression in mouse tissues and colonic biopsies from patients with functional constipation were determined. In the oldest mice, the GI peristalsis was significantly impaired as compared to the younger groups. The tissue response to MOP and DOP, but not KOP, agonists weakened with age in vitro; for DOP, it was confirmed in vivo. In the mouse upper GI tract, Oprm1, Oprd1, Oprk1 expression decreased with age; in the colon, Oprm1 expression increased. There were no differences in the expression of these genes in the colonic biopsies from patients >50 years old as compared to the younger group. In conclusion, the age-related impairment of the GI peristalsis may result from reduced MOP and DOP response to the activation with opioid agonists or the alterations in the EOS expression.

Novel prognostic value and potential utility of opioid receptor gene methylation in liquid biopsy for oral cavity cancer.

Opioids are a class of recreational drugs and prescription medications that bind to a group of G-protein-coupled receptors known as opioid receptors (ORs). ORs are involved in the development of many types of cancer; however, their role in head and neck squamous cell carcinoma (HNSCC) is complex and poorly understood. Here, we analyzed the methylation status of five OR genes in verification (301 HNSCC primary samples) and validation (five circulating tumor DNA [ctDNA] samples) studies using quantitative methylation-specific PCR (Q-MSP). OPRL1 and OPRM1 methylation levels were significantly higher in HNSCC tissues than in corresponding normal tissues from the same individuals (P = 0.001 and P < 0.001, respectively). In Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, two genes (OPRL1 and OPRM1) were significantly associated with increased recurrence in the methylation group with oral cavity cancer. Furthermore, a validation study of ctDNA demonstrated that OPRL1 genes exhibited predictive performance as emerging biomarkers and were each capable of discriminating the plasma from tumor-free individuals. We characterized the relationship between OR gene methylation status and outcomes in oral cavity cancer. Our results highlight the potential utility of ctDNA methylation-based detection in the clinical management of oral cavity cancer.

Peripheral ablation of type III adenylyl cyclase induces hyperalgesia and eliminates KOR-mediated analgesia in mice.

Ca2+/calmodulin-stimulated group I adenylyl cyclase (AC) isoforms AC1 and AC8 have been involved in nociceptive processing and morphine responses. However, whether AC3, another member of group I ACs, is involved in nociceptive transmission and regulates opioid receptor signaling remains elusive. Here, we report that conditional KO of AC3 (AC3 CKO) in L3 and L4 DRGs robustly facilitated the mouse nociceptive responses, decreased voltage-gated potassium (Kv) channel currents, and increased neuronal excitability. Furthermore, we report AC3 CKO eliminated the analgesic effect of κ-opioid receptor (KOR) agonist and its inhibition on Kv channel by classical Gαi/o signaling or nonclassical direct interaction of KOR and AC3 proteins. Interestingly, significantly upregulated AC1 level and cAMP concentration were detected in AC3-deficient DRGs. Inhibition of AC1 completely reversed cAMP upregulation, neuronal excitability enhancement, and nociceptive behavioral hypersensitivity in AC3-CKO mice. Our findings suggest a crucial role of peripheral AC3 in nociceptive modulation and KOR opioid analgesia.

The Expression Pattern of Genes Related to Melanogenesis and Endogenous Opioids in Psoriasis.

The melanocortin system is a major regulator of stress responses in the skin and is responsible for the induction of melanin synthesis through activation of melanogenesis enzymes. The expression of both melanocortin system genes and melanogenesis enzyme genes is altered in psoriasis, and the focus here was on twelve genes related to the signal transduction between them. Additionally, five endogenous opioid system genes that are involved in cutaneous inflammation were examined. Quantitative real-time-PCR was utilized to measure mRNA expression in punch biopsies from lesional and non-lesional skin of psoriasis patients and from the skin of healthy control subjects. Most of the genes related to melanogenesis were down-regulated in patients (CREB1, MITF, LEF1, USF1, MAPK14, ICAM1, PIK3CB, RPS6KB1, KIT, and ATRN). Conversely, an up-regulation occurred in the case of opioids (PENK, PDYN, and PNOC). The suppression of genes related to melanogenesis is in agreement with the reported reduction in pigmentation signaling in psoriatic skin and potentially results from the pro-inflammatory environment. The increase in endogenous opioids can be associated with their involvement in inflammatory dysregulation in psoriasis.

Decrease of morphine-CPP by sinomenine via mediation of tyrosine hydroxylase, NMDA receptor subunit 2B and opioid receptor in the zebrafish brain.

To study the effects of sinomenine on conditioned place preference (CPP) zebrafish induced by morphine and expression levels of intracephalic tyrosine hydroxylase (TH), NMDA receptor subunit 2B (NR2B), µ-opioid receptor (zfmor) and δ-opioid receptors (zfdor1 and zfdor2), morphine (40mg/kg) was administrated to zebrafish and the effect of CPP was detected in these zebrafish treated with sinomenine. The expression of TH and NR2B was detected by immunohistochemistry; and the mRNA expression of opioid receptors zfmor, zfdor1 and zfdor2 in the zebrafish brain was assayed by RT-qPCR. In the CPP test, morphine induced significant behavioral alteration, while pretreatment with sinomenine or methadone, resulted in decreased activity time in the morphine-paired compartment significantly. Morphine also increased the integral optical density value of TH- and NR2B-positive cells in the zebrafish brain, and reduced the amount of opioid receptors. However, the compound sinomenine could attenuate these effects. These findings demonstrate that sinomenine (80mg/kg) decreased the CPP effects of zebrafish induced by morphine significantly, downregulated expression of TH and NR2B, and upregulated µ-opioid (zfmor) and δ-opioid (zfdor1 and zfdor2) receptor expression in the CPP zebrafish brains.

Opioid Addiction and Opioid Receptor Dimerization: Structural Modeling of the OPRD1 and OPRM1 Heterodimer and Its Signaling Pathways.

Opioid addiction is a complex phenomenon with genetic, social, and other components. Due to such complexity, it is difficult to interpret the outcome of clinical studies, and thus, mutations found in individuals with these addictions are still not indisputably classified as opioid addiction-causing variants. Here, we computationally investigated two such mutations, A6V and N40D, found in the mu opioid receptor gene OPRM1. The mutations are located in the extracellular domain of the corresponding protein, which is important to the hetero-dimerization of OPRM1 with the delta opioid receptor protein (OPRD1). The hetero-dimerization of OPRD1-OPRM1 affects the signaling pathways activated by opioids and natural peptides and, thus, could be considered a factor contributing to addiction. In this study, we built four 3D structures of molecular pathways, including the G-protein signaling pathway and the ß-arrestin signaling pathway of the heterodimer of OPRD1-OPRM1. We also analyzed the effect of mutations of A6V and N40D on the stability of individual OPRM1/OPRD1 molecules and the OPRD1-OPRM1 heterodimer with the goal of inferring their plausible linkage with opioid addiction. It was found that both mutations slightly destabilize OPRM1/OPRD1 monomers and weaken their association. Since hetero-dimerization is a key step for signaling processes, it is anticipated that both mutations may be causing increased addiction risk.