Mu-opioid Receptor mRNA Levels Research Articles

Acupuncture alleviates inflammatory pain by activating CXCL1/CXCR2 signaling in the primary somatosensory cortex in adjuvant induced arthritis rats.

To observe whether acupuncture up-regulates chemokine CXC ligand 1 (CXCL1) in the brain to play an analgesic role through CXCL1/chemokine CXC receptor 2 (CXCR2) signaling in adjuvant induced arthritis (AIA) rats, so as to reveal its neuro-immunological mechanism underlying improvement of AIA. BALB/c mice with relatively stable thermal pain reaction were subjected to planta injection of complete Freund adjuvant (CFA) for establishing AIA model, followed by dividing the AIA mice into simple AF750 (fluorochrome) and AF750+CXCL1 groups (n=2 in each group). AF750 labeled CXCL1 recombinant protein was then injected into the mouse's tail vein to induce elevation of CXCL1 level in blood for simulating the effect of acupuncture stimulation which has been demonstrated by our past study. In vivo small animal imaging technology was used to observe the AF750 and AF750+CXCL1-labelled target regions. After thermal pain screening, the Wistar rats with stable pain reaction were subjected to AIA modeling by injecting CFA into the rat's right planta, then were randomized into model and manual acupuncture groups (n=12 in each group). Other 12 rats that received planta injection of saline were used as the control group. Manual acupuncture (uniform reinforcing and reducing manipulations) was applied to bilateral "Zusanli" (ST36) for 4×2 min, with an interval of 5 min between every 2 min, once daily for 7 days. The thermal pain threshold was assessed by detecting the paw withdrawal latency (PWL) using a thermal pain detector. The contents of CXCL1 in the primary somatosensory cortex (S1), medial prefrontal cortex, nucleus accumbens, amygdala, periaqueductal gray and rostroventromedial medulla regions were assayed by using ELISA, and the expression levels of CXCL1, CXCR2 and mu-opioid receptor (MOR) mRNA in the S1 region were detected using real time-quantitative polymerase chain reaction. The immune-fluorescence positive cellular rate of CXCL1 and CXCR2 in S1 region was observed after immunofluorescence stain. The immunofluorescence double-stain of CXCR2 and astrocyte marker glial fibrillary acidic protein (GFAP) or neuron marker NeuN or MOR was used to determine whether there is a co-expression between them. In AIA mice, results of in vivo experiments showed no obvious enrichment signal of AF750 or AF750+CXCL1 in any organ of the body, while in vitro experiments showed that there was a stronger fluorescence signal of CXCL1 recombinant protein in the brain. In rats, compared with the control group, the PWL from day 0 to day 7 was significantly decreased (P<0.01) and the expression of CXCR2 mRNA in the S1 region significantly increased in the model group (P<0.05), while in comparison with the model group, the PWL from day 2 to day 7, CXCL1 content, CXCR2 mRNA expression and CXCR2 content, and MOR mRNA expression in the S1 region were significantly increased in the manual acupuncture group (P<0.05, P<0.01). Immunofluorescence stain showed that CXCR2 co-stained with NeuN and MOR in the S1 region, indicating that CXCR2 exists in neurons and MOR-positive neurons but not in GFAP positive astrocytes. Acupuncture can increase the content of CXCL1 in S1 region, up-regulate CXCR2 on neurons in the S1 region and improve MOR expression in S1 region of AIA rats, which may contribute to its effect in alleviating inflammatory pain.

Ethnicity-dependent effect of rs1799971 polymorphism on OPRM1 expression in the postmortem brain and responsiveness to antipsychotics

Schizophrenia is associated with aberration of inhibitory neurons. Although the mu-opioid receptor (MOR) is an essential modulator of inhibitory neurons, the effect of rs1799971 polymorphism in the MOR gene on risk of schizophrenia is controversial. Moreover, the disturbance of opioids systems in patients with schizophrenia has not been fully examined. We firstly conducted preliminary meta-analyses integrating Asian and European populations separately over 12,000 subjects to assess the effect of rs1799971 on risk of schizophrenia. Based on the above result, we also investigated the effect on the expression levels of MOR mRNA in the prefrontal cortex (PFC) and caudate nucleus of 41 postmortem brains. In addition, we determined whether these levels were related to antemortem schizophrenia symptoms and pharmacotherapeutic effects. The rs1799971 G-allele reduced the risk of schizophrenia in Asian populations (OR: 0.56, 95%CI: 0.32–0.98, p = 0.042) but increased it in European populations (OR: 1.66, 95%CI: 1.08–2.56, p = 0.022). It decreased MOR mRNA levels in PFC in the Japanese population (p = 0.031). Increased MOR mRNA level in PFC correlated with higher total score of antemortem schizophrenia symptoms (p = 0.017). Furthermore, the pharmacotherapeutic effect of first-generation antipsychotics was higher for genotype AA than AG/GG of rs1799971 (p = 0.036). The rs1799971 affects risk of schizophrenia and MOR mRNA expression and the effect varies according to ethnicity. Overexpression of MOR might induce severe schizophrenia symptoms. Therefore, MOR modulation may be the key clue for treating antipsychotics-resistant schizophrenia, and genotyping rs1799971 may provide a better pharmacotherapeutic strategy.

The role of testosterone in mu-opioid receptor expression in the trigeminal ganglia of opioid-tolerant rats

Although tolerance serves as a major limitation in the long-term clinical use of opioids in patients with chronic severe pain, mechanisms of opioid tolerance are poorly understood. In this study, a morphine tolerance model was established by subcutaneously injecting male rats with morphine (10 mg/kg) twice a day for 10 consecutive days. In addition, a subset of morphine-tolerant rats underwent testosterone replacement therapy. The levels of mu-opioid receptor (MOR) mRNA and protein in the trigeminal ganglia (TGs) of morphine-tolerant versus control rats and of morphine-tolerant rats with vs. without testosterone replacement therapy were measured. We found that testosterone levels were significantly lower in morphine-tolerant rats than in the controls (1.248 ± 0.231 ng/ml vs. 2.223 ± 0.153 ng/ ml, respectively; p = 0.008). Furthermore, chronic morphine exposure led to a downregulation in the levels of MOR mRNA to 79.3%, and of MOR protein to 68.9%. Testosterone replacement therapy restored MOR mRNA and protein levels specifically in rats who had developed a tolerance to morphine, thereby suggesting a potential role of testosterone in the opioid-receptor response to chronic morphine exposure. In summary, our study provides evidence for the involvement of testosterone in the proper regulation of the peripheral MOR system in rats following prolonged morphine exposure. We also suggest that analgesic therapeutic measures should take into account the testosterone levels of patients who have built up a tolerance to morphine.

Role of Mu-Opioid Receptor in Nociceptive Modulation in the Anterior Cingulate Cortex of Rats with Inflammatory Pain

Objective: The present study was performed to explore the role of mu opioid receptor (MOR) in nociceptive modulation in anterior cingulate cortex (ACC) of rats with inflammatory pain. Methods: To set up an inflammatory pain model, rats received a subcutaneous injection of 0.1 ml of 2% carrageenan into the left hindpaw. The hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation, by hot plate and Randall Selitto Test respectively, was used to evaluate the rat's responses to noxious stimulation. The effects induced by intra-ACC administration of morphine and opioid receptor antagonists were observed. The influence of inflammatory pain on MOR mRNA level and MOR expression in ACC were assayed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot. Results: We found that intra-ACC administration of morphine induced significant antinociceptive effects in a dose-dependent manner in rats with inflammatory pain. Furthermore, the antinociceptive effects induced by morphine were attenuated by intra-ACC injection of the opioid receptor antagonist naloxone, indicating an involvement of opioid receptor in nociceptive modulation in ACC in rats with inflammatory pain. Moreover, intra-ACC administration of the MOR antagonist β-funaltrexamine (β-FNA) attenuated the morphine-induced antinociception significantly in rats with inflammatory pain. The results demonstrated that blockade MOR by β-FNA inhibited the morphine-induced antinociception, indicating that MOR plays an important role in nociceptive modulation in ACC in rats with inflammatory pain. We further found that the morphine-induced antinociception was lower in rats with inflammatory pain than that in normal rats. Interestingly, we found that there were significant decreases in MOR mRNA level and MOR expression in ACC in rats with inflammatory pain compared with intact rats tested by RT-PCR and western blot, indicating that there is a down regulation in MOR expression in rats with inflammatory pain, which support our above results that morphine-induced antinociception was lower in rats with inflammatory pain than that in normal rats. Conclusion: These findings suggest that MOR plays an important role in nociceptive modulation in ACC in rats with inflammatory pain and there is a down-regulation in MOR expression in rats with inflammatory pain.

DNMT3a methylation in neuropathic pain.

BackgroundMu opioid receptor (MOR) plays a crucial role in mediating analgesic effects of opioids and is closely associated with the pathologies of neuropathic pain. Previous studies have reported that peripheral nerve injury downregulates MOR expression, but the epigenetic mechanisms remain unknown.ObjectiveTherefore, we investigated DNA methyltransferase3a (DNMT3a) expression or methylation changes within MOR promoter in the spinal cord in a neuropathic pain induced by a chronic constriction injury (CCI) mouse model and further determined whether these injury-associated changes are reversible by pharmacological interventions.MethodsA CCI mouse model was established and tissue specimens of lumbar spinal cords were collected. The nociception threshold was evaluated by a Model Heated 400 Base. DNMT3a and MOR mRNA and protein level were detected by real-time-polymerase chain reaction and Western blot, respectively. Methylation of DNMT3a gene was measured by methylation-specific PCR.ResultsOur data showed that chronic nerve injury led to a significant upregulation of DNMT3a expression that was associated with increased methylation of MOR gene promoter and decreased MOR protein expression in the spinal cord. Inhibition of DNMT3a catalytic activity with DNMT inhibitor RG108 significantly blocked the increase in methylation of the MOR promoter, and then upregulated MOR expression and attenuated thermal hyperalgesia in neuropathic pain mice.ConclusionThis study demonstrates that an increase of DNMT3a expression and MOR methylation epigenetically play an important role in neuropathic pain. Targeting DNMT3a to the promoter of MOR gene by DNMT inhibitor may be a promising approach to the development of new neuropathic pain therapy.

Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1.

Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5'-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). J. Cell. Physiol. 232: 576-584, 2017. © 2016 Wiley Periodicals, Inc.

Identification and Characterization of Gamma‐Synuclein as a PCBP1‐Interacting Protein in Various Cancer Cell Lines

PolyC binding protein1 (PCBP1), a transcriptional regulator of mu‐opioid receptor (MOR) gene, has multiple functions, which can be regulated by various protein‐protein interactions. Using PCBP1 as bait, a human brain cDNA library was screened via two‐hybrid system. One candidate clone was identified as g‐synuclein with glutamic acid (E) at the 110 position rather than valine (V). It was called g‐synuclein110E in here to differentiate from the 110V form. The interaction between PCBP1 and g‐synuclein110E was confirmed by in vivo validation and GST‐pull down assay. Confocal analysis showed g‐synuclein110E mainly expressed in the cytosol of neuronal NMB cells. Ectopic expression of g‐synuclein110E or 110V did not alter MOR mRNA level or hMOR promoter activity, suggesting no involvement of g‐synuclein in modulating hMOR expression. Co‐immunoprecipitation using anti‐PCBP1 antibody and lysates of NMB cells overexpressing g‐synuclein110E or 110V revealed a stronger band intensity of co‐immunoprecipitated 110E form than 110V form, indicating that the change of amino acid at the 110 position affected the interaction with PCPB1. Synuclein110E form was found in H292 (lung), HT29 (colon) and T47D (breast) cells, and co‐immunoprecipitation demonstrated that this physical interaction with PCPB1 also occurred in these cells. To further examine if different endogenous g‐synuclein forms were also present in various cancer cells, RT‐PCR using RNAs from different cell lines and a specific primer set for g‐synuclein were used. The full length of g‐synuclein PCR products were cloned into a cloning vector and then several clones were subjected to DNA sequencing. Different sequences were detected in certain cell lines. Overall, this study reports g‐synuclein110E as a new PCBP1‐interacting protein and provides some insight into its complex role.Support or Funding InformationConfocal microscope was acquired via the NSF grant. This research was supported by NIH research grant DA016673 and BioResearch Fund from Seton Hall University.

Sex differences in peripheral mu-opioid receptor mediated analgesia in rat orofacial persistent pain model.

Unilateral ligation of the tendon of anterior superficial part of rat masseter muscle (TASM) leads to long-lasting allodynia. Sex differences in peripheral mu-opioid receptor (MOR)-mediated analgesia under persistent myogenic pain are not well understood. In this study, we examined (1) whether locally applied MOR agonists attenuate persistent pain following TASM ligation in a sex dependent manner, (2) whether there are sex differences of MOR expression changes in rat trigeminal ganglia (TG). The effects of MOR agonist, D-Ala2, N–Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO), were assessed 14 days after TASM ligation in male, female and orchidectomized (GDX) male rats. MOR mRNA and protein levels in TG 14 days following tendon ligation were also determined. The mechanical thresholds of the injured side were significantly decreased in both male and female rats, from 3 days to 28 days after TASM ligation. A10 μg DAMGO significantly attenuated allodynia in male rats. A 10-fold higher dose of DAMGO was required in female and GDX male rats to produce the level of anti- allodynia achieved in male rats. The level of MOR mRNA in TG from male rats was significantly greater 14 days after TASM ligation compared with the sham-operated male rats, but not from female and GDX male rats. After TASM ligation, males had significantly more MOR immunoreactivity in TG compared to sham-operated males. The MOR levels increased to 181.8% of the sham level in male rats receiving tendon injury. But there was no significant change in female rats receiving tendon injury compared to the sham female rats. Taken together, our data suggest that there were sex differences in the effects of peripheral MOR agonists between male and female rats under TASM ligation developing long-lasting pain condition, which is partly mediated by sex differences in the changes of MOR expressions and testosterone is an important factor in the regulation of MOR.

Identify PCBP1‐interacting protein and its novel functional role on human MOR gene expression

PCBP1 is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein‐protein interaction with PCBP1. A human brain cDNA library was screed using the two‐hybrid system with PCBP1 as the bait. RACK1 protein, containing WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation, and by co‐immunoprecipitation with anti‐PCBP1 antibody using human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 overexpression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in NMB cells.*This research was supported by NIH research grant, NSF MRI grant and SHU Biological Research.

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu-opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co-regulator modifying human MOR gene expression by protein-protein interaction with PCBP1. A human brain cDNA library was screened using the two-hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over-expression resulted in a dose-dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock-down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT-PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by (3) H-diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.

Voluntary Alcohol Drinking Enhances Proopiomelanocortin Gene Expression in Nucleus Accumbens Shell and Hypothalamus of Sardinian Alcohol‐Preferring Rats

Evidence obtained in humans and rodents indicates that beta-endorphin (encoded by the proopiomelanocortin [POMC] gene) is critical in the regulation of alcohol drinking behavior. However, the alcohol effect on POMC gene expression has not been studied in rodent mesolimbic regions, such as the nucleus accumbens (NAc). In this study, we first utilized POMC-enhanced green fluorescent protein (EGFP) transgenic mice to visualize POMC neurons and found that POMC-EGFP cells were modestly distributed throughout the NAc shell and core, in addition to the hypothalamic arcuate nucleus. POMC mRNA expression in the NAc of mice and rats was confirmed using reverse transcriptase-polymerase chain reaction and solution hybridization assays. We then investigated whether there are genetically determined differences in basal mRNA levels of POMC and mu opioid receptor (MOP-r) between selectively bred Sardinian alcohol-preferring (sP) and nonpreferring (sNP) rats, and whether these mRNA levels are altered in sP rats after alcohol drinking (10%, unlimited access) for 17 days. Alcohol-naïve sP rats had higher basal POMC mRNA levels than sNP rats only in hypothalamus. Alcohol drinking increased POMC mRNA levels in both the NAc shell (by 100%) and the hypothalamus (by 50%) of sP rats. Although sP rats had lower basal levels of MOP-r mRNA and GTPγS binding in NAc shell than sNP rats, voluntary alcohol consumption had no effect on MOP-r mRNA levels in the NAc shell. Our results define the distribution of POMC-expressing neurons in the NAc of mice and rats. Higher POMC expression at basal levels in sP rats (genetically determined), along with increases after drinking (alcohol-induced) in the NAc shell and hypothalamus, suggests that the POMC systems play a role in high alcohol preference and consumption.

Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP‐ribose) polymerase‐1

The pharmacological actions of morphine and morphine-like drugs such as heroin mediate primarily through the mu opioid receptor (MOR). It represents the target of the most valuable painkiller in contemporary medicine. Here we report that poly(ADP-ribose) polymerase 1 (PARP-1) binds to the double-stranded poly(C) element essential for the MOR promoter and represses promoter activity at the transcriptional level. We identified PARP-1 by affinity column chromatography using the double-stranded poly(C) element, followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. PARP-1 binding to the poly(C) sequence of the MOR gene was sequence-specific as confirmed by the supershift assay. In cotransfection studies, PARP-1 repressed the MOR promoter only when the poly(C) sequence was intact. When PARP-1 was disrupted in NS20Y cells using siRNA, transcription of the endogenous target MOR gene increased significantly. Chromatin immunoprecipitation assays showed specific binding of PARP-1 to the double-stranded poly(C) element essential for the MOR promoter. Inhibition of PARP-1's catalytic domain with 3-aminobenzamide increased endogenous MOR mRNA levels in cultured NS20Y cells, suggesting that automodification of PARP-1 regulates MOR transcription. Our data suggest that PARP-1 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of PARP-1 as a transcriptional repressor in MOR gene regulation.

Identification of Endomorphin‐1 and Endomorphin‐2 Binding Sites in Human μ‐Opioid Receptor by Antisense Oligonucleotide Strategy

The effects of phosphorothioate antisense oligodeoxynucleotides against exons-1, -2, -3 and -4 of the human mu-opioid receptor were studied in the CHO-mu-opioid receptor cells using aequorin luminescence-based calcium assay. All four antisense oligodeoxynucleotides significantly decreased the level of mu-opioid receptor mRNA in comparison with the non-treated cells, used as control. However, no statistically significant differences between antisense oligodeoxynucleotides were observed. antisense oligodeoxynucleotides against exon-2 attenuated endomorphin-1-induced intracellular calcium response in a concentration-dependent manner. antisense oligodeoxynucleotides against exons-1, -2, -3 and -4 inhibited endomorphin-2-induced intracellular calcium response in a concentration-dependent manner and the effect of antisense oligodeoxynucleotides against exons-3 and -4 was most pronounced. The mismatch oligodeoxynucleotides against respective exons failed to exert any effect. The selective actions of antisense probes directed against different exons of the human mu-opioid receptor gene, that resulted, at the protein level, in attenuation of calcium responses induced by endomorphin-1 and endomorphin-2, suggest that the binding sites for endomorphins are structurally and functionally different. The presence of functionally distinct binding sites might play a crucial role in the modulation of pain and may be important clinically.

Effects of trichostatin A on neuronal mu-opioid receptor gene expression

In this study, we determined the effects of a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on neuronal mu-opioid receptor (MOR) gene expression using human neuronal NMB cells, endogenously expressing MOR. Recruitment of two classes of HDAC, HDAC1 and HDAC2, to MOR promoter region in situ was detected via chromatin immunoprecipitation (ChIP) analysis with NMB cells. Functional analysis using the luciferase reporter gene system showed that TSA induced an approximately 3-fold increase of the promoter activity as compared to the vehicle treated group. Mutation analysis demonstrated that TSA response was mediated by both dsDNA (Sp1/Sp3 binding site) and ssDNA (PolyC binding protein1, PCBP, binding site) elements located in mouse MOR proximal core promoter region, further suggesting the functional importance of this cis-element, which shows high sequence homology between human and mouse MOR genes. ChIP analysis further suggested that TSA enhanced the recruitment of Sp1/Sp3 and PCBP to the promoter region, whereas no significant changes of total proteins were observed in response to TSA using Western blot analysis. Moreover, confocal images showed TSA-induced nuclear hot spots of endogenous PCBP in neuronal cells, whereas no obvious nuclear PCBP hotspot was observed in vehicle treated cells. Taken together, these results suggested that TSA enhanced neuronal MOR gene expression at the transcriptional level. RT-PCR analysis further revealed that TSA also decreased the steady-state level of MOR mRNA in a time-dependent manner by enhancing its instability. Thus, data suggest that TSA, an epigenetic regulator, affects neuronal MOR gene expression at both transcriptional and post-transcriptional levels.

Transcriptional activation of human mu‐opioid receptor gene by insulin‐like growth factor‐I in neuronal cells is modulated by the transcription factor REST

The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I up-regulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription-1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells.

Expression of the Mu Opioid Receptor in the Human Immunodeficiency Virus Type 1 Transgenic Rat Model

Opioids, via the mu opioid receptor (MOR), can exacerbate bacterial infections and the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. Recently, an HIV-1 transgenic (HIV-1Tg) rat model containing circulating HIV-1 gp120 was created. Using real-time reverse transcription-PCR, we found that MOR mRNA levels were significantly higher in the peritoneal macrophages of the HIV-1Tg rat than those in control animals. Lipopolysaccharide, a bacterial endotoxin, induced secretion of the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-beta (IL-beta), and IL-10 in the HIV-1Tg rat and further increased MOR expression. Ex vivo studies showed that MOR expression was up-regulated in the peritoneal macrophages of F344 control rats by exposure to serum from HIV-1Tg rats and that MOR up-regulation was abolished by addition of gp120 antibody to the serum. In human TPA-differentiated HL-60 cells, which are macrophage-like cells, LPS-induced MOR mRNA up-regulation was greater in gp120-pretreated cells than in vehicle-pretreated cells. Our data suggest that in individuals infected with HIV-1, the MOR is up-regulated, possibly by circulating HIV-1 proteins such as gp120, and HIV-1 proteins may play a significant role in modulating the response to bacterial infection in opioid-using HIV-infected individuals. Furthermore, our results demonstrate that the new HIV-1Tg rat model can be a valuable tool with which to study MOR gene expression and its effects in the continuous presence of HIV viral proteins.

Tolerance to the Antinociceptive and Antiexudative Effects of Morphine in a Murine Model of Peripheral Inflammation

Opioids are used in humans in the management of chronic osteoarticular pains, but the development of tolerance to the analgesic effects after continuous administration is still not well understood. Our aim was to characterize morphine tolerance in a murine model of arthritis that mimics the sequence of events occurring in humans. Inflammation was induced by the intraplantar injection of complete Freund's adjuvant (CFA) and tolerance by the implantation of a 75-mg morphine pellet. We assessed the antihyperalgesic (plantar and Randall-Selitto tests), antiallodynic (Von Frey test), and antiexudative (Evans blue) effects of morphine, the mu-opioid receptor (MOR) mRNA levels in dorsal root ganglia (DRG), and MOR protein levels in DRG and plantar tissue. Inflammation induced plasma extravasation, and it significantly increased the antihyperalgesic effects of morphine (p < 0.05). Morphine pellet implantation decreased morphine potency in all tests. ED(50) values decreased 4.4 and 7.3 times in the absence and presence of inflammation in the plantar test and 2.7 and 5.3 times in the Randall-Selitto test, whereas plasma extravasation decreased 4.2 times. MOR mRNA levels in the DRG were not affected 7 days after inflammation, whereas chronic morphine administration induced a discrete increase (p < 0.05). MOR protein in the DRG or the paw was unchanged. The results show that inflammation enhances the development of tolerance to the antihyperalgesic and antiexudative effects of morphine. At the molecular level, our results suggest that these effects are not mediated by changes in MOR expression but by other changes in receptor activation/internalization.

A vitamin A deficient diet enhances proinflammatory cytokine, Mu opioid receptor, and HIV-1 expression in the HIV-1 transgenic rat

The HIV-1 (HIV) transgenic (Tg) rat develops several immune abnormalities in association with clinical impairments that are similar to what are seen with HIV infection in humans. In HIV infection, retinoids and opioids can have separate and potentially combined effects on the clinical course of HIV disease. In these studies, the effects of a vitamin A deficient diet on T cell proinflammatory cytokine and mu opioid receptor (MOR) expression were examined in the Tg and in wild-type (WT) rats. The effects of the diet on HIV gene expression were also analyzed in the Tg rats. Phytohemagglutinin-stimulated T cells from WT rats on the vitamin A diet and from Tg rats on either diet were more likely to either produce increased percentages of T cells expressing intracytoplasmic IFN-γ, secrete higher levels of TNF-α, and express higher levels of MOR mRNA and surface MOR. Mitogen stimulation also increased Tg rat HIV env, tat, and nef mRNA expression with even higher env and nef mRNA produced in association with the vitamin A deficient diet. All together, these data suggest that a vitamin A deficient diet can result in cellular effects that increase T cell proinflammatory responses and HIV expression, which may alter the course of disease in the HIV Tg rat model.

Effects of acute “binge” cocaine on mRNA levels of μ opioid receptor and neuropeptides in dopamine D1 or D3 receptor knockout mice

In humans, elevations of mu opioid receptor (MOP-r) binding potential (BP) in the frontal cortex (FC) are associated with cocaine craving during early abstinence. In rats, decreases in dopaminergic (DAergic) transmission in the medial FC are associated with increased cocaine-seeking behavior. DA D1 or D3 receptor homozygous knockout (D1-/- or D3-/-) mice offer the opportunity to test the roles of these specific receptors in regulating MOP-r gene expression in response to cocaine. In the present studies, we found an increase in basal MOP-r mRNA levels in the FC of both D1-/- and D3-/- mice compared to wild type controls, with no change in the nucleus accumbens (NAc) core or caudate-putamen (CPu). Acute "binge" cocaine (3 x 15 mg/kg for 2.5 h) returned FC MOP-r mRNA levels in D1-/- or D3-/- mice to those in wild type controls. In the NAc core, the MOP-r mRNA levels after acute "binge" cocaine were decreased in D1-/- mice while increased in D3-/- mice. In the CPu, however, the MOP-r mRNA levels after acute "binge" cocaine were increased in D1-/- mice while decreased in D3-/- mice. We also found a decrease in basal orexin mRNA levels in the lateral hypothalamus of the D3-/- mice, which was unaltered by acute "binge" cocaine. Together, our findings suggest that: (1) both D1 and D3 receptors are involved in FC MOP-r gene regulation; and (2) D1 and D3 receptors play opposite roles in the effects of cocaine on MOP-r gene regulation differentially in the NAc core or CPu.

Both GnRH agonist and continuous oral progestin treatments reduce the expression of the tyrosine kinase receptor B and mu-opioid receptor in deep infiltrating endometriosis

Deep infiltrating endometriosis (DIE) is commonly associated with severe pain. The pain can be managed successfully with GnRH agonists or continuous progestins. The precise molecular mechanism by which DIE causes pain or why hormonal treatment is effective, however, remains unclear. We recently identified three potential candidate genes that might be involved in DIE pain pathways: tyrosine kinase receptor B (TrKB), mu-opioid receptor (MOR) and serotonin transporter (5HTT). We hypothesized that if these three genes were involved in DIE-associated pain, their expression levels would probably be modulated by GnRH agonist or progestin. In this study, we compared mRNA expression levels of TrKB, MOR and 5HTT in DIE among patients pre-operatively treated with GnRH agonist, progestin or without pre-operative medical treatments. The expression levels of TrKB, MOR and 5HTT mRNA in DIE were determined using laser capture microdissection and real-time RT-PCR techniques. The expression levels of TrKB in epithelial cells and MOR in stromal cells from DIE were significantly decreased in patients with pre-operative GnRH agonist or progestin. There was no significant difference in 5HTT expression levels among untreated, GnRH agonist- and progestin-treated patients. The expression levels of TrKB and MOR genes in DIE appeared to be modulated by GnRH agonist or progestin. However, the functional roles of TrKB and MOR in DIE remain to be clarified.