MOR mRNA Research Articles

Switching from morphine to fentanyl attenuates the decline of μ-opioid receptor expression in periaqueductal gray of rats with morphine tolerance

Background Opioid switching is a therapeutic maneuver to improve analgesic response and/or reduce adverse side effects although the underlying mechanisms remain unknown. The μ-opioid receptor (MOR) has an important role in mediating the actions of morphine and other analgesic agents. This study is aimed at exploring the changes of MOR in the periaqueductal gray (PAG) in rats when morphine is substituted for equianalgesic fentanyl. Methods Forty rats were randomly assigned to five treatment groups: 7 days normal saline group (N group), 7 days fentanyl group (F group), 7 days morphine group (M group), 7 days morphine and 7 days fentanyl-switching group (MF group), and 14 days morphine group (MM group). Rats repeatedly received subcutaneous injections of morphine sulfate (10 mg/kg) or equianalgesic fentanyl sulfate (0.1 mg/kg) twice daily. Rats' antinociceptive response to thermal pain was evaluated by the tail flick latency assay. MOR mRNA and protein expression in the PAG were measured using RT-PCR and Western blotting analyses respectively. Results This study showed that after morphine was substituted with fentanyl on day 8, the tail flick latency (TFL) increased from (3.9±0.4) seconds to (11.4±0.4) seconds. The results also demonstrated that both MOR mRNA and protein expression in the PAG of rats in the MF group were less than that in the M group (P<0.05) but more than that in MM group (P<0.05). Conclusions Equianalgesic fentanyl was still antinociceptive effective in rats with morphine tolerance, which may be due to the switching from morphine to fentanyl attenuating the decline of MOR expression in the PAG of rats.

Sex differences in μ-opioid receptor expression in trigeminal ganglia under a myositis condition in rats.

Peripheral opioid receptor expression is up-regulated under inflammatory conditions, which leads to the increased efficacy of peripherally administered opioids. Sex differences in the effects of inflammation, cytokines and gonadal hormones on μ-opioid receptor (MOR) expression in trigeminal ganglia (TG) are not well understood. MOR mRNA and protein levels in TG from male and female Sprague Dawley rats following complete Freund's adjuvant (CFA)-induced muscle inflammation were assessed. Cytokine-induced changes in MOR mRNA expression from TG cultures prepared from intact and gonadectomized male and female, and gonadectomized male rats with testosterone replacement were examined. Behavioural experiments were then performed to examine the efficacy of a peripherally administered MOR agonist in male, female and gonadectomized male rats under a myositis condition. CFA and cytokine treatments induced significant up-regulation of MOR expression in TG from male, but not from female, rats. The cytokine-induced up-regulation of MOR mRNA expression was prevented in TG from orchidectomized (GDX) male rats, which was restored with testosterone replacement. Peripherally administered DAMGO, a specific MOR agonist, significantly attenuated CFA-induced masseter mechanical hypersensitivity only in intact male rats. Collectively, these data indicate that testosterone plays a key role in the regulation of MOR in TG under inflammatory conditions, and that sex differences in the anti-hyperalgesic effects of peripherally administered opioids are, in part, mediated by peripheral opioid receptor expression levels.

Reduced Supraspinal Nociceptive Responses and Distinct Gene Expression Profile in CXBH Recombinant Inbred Mice

CXBH mice, known as an “opioid receptor-rich” strain, are a recombinant inbred mouse strain established by crossing the C57BL/6By and BALB/cBy strains. In the present study, we investigated nociceptive and antinociceptive sensitivity in CXBH mice and elucidated the underlying molecular mechanisms. CXBH mice exhibited slightly higher morphine-induced antinociception compared with C57BL/6J and BALB/cBy mice in the hot-plate test but not tail-flick test. CXBH mice exhibited a marked reduction of nociceptive sensitivity, regardless of the type of nociceptive stimulus, with the exception of tail stimulation. Changes in gene expression that corresponded to reduced nociceptive sensitivity in the brains of CXBH mice were observed in 62 transcripts, including pain- and analgesia-related transcripts, in a whole-genome expression assay. The total mRNA expression of opioid receptors was higher in CXBH mice than in C57BL/6J and BALB/cBy mice. However, the expression levels of MOR-1 mRNA, a major transcript of the μ opioid receptor gene, were not different among the C57BL/6J, BALB/cBy, and CXBH strains. In conclusion, supraspinal nociceptive responses were reduced in the CXBH mouse strain, and the expression levels of transcripts were altered in the brain of this strain. PerspectiveThis article presents the nociceptive and antinociceptive properties of CXBH recombinant inbred mice and gene expression differences that may underlie nociceptive tolerance in the strain. The CXBH mouse strain may be a useful animal model to investigate the molecular basis of individual differences in supraspinal pain sensitivity.

Neuroanatomical distribution of μ-opioid receptor mRNA and binding in monogamous prairie voles (Microtus ochrogaster) and non-monogamous meadow voles (Microtus pennsylvanicus)

The opiate system has long been implicated in the rewarding properties of social interactions. In particular, the μ-opioid receptor (MOR) mediates multiple forms of social attachment, including the attachment of offspring to the mother and social bonding between mates. We have previously shown that MOR in the caudate-putamen is involved in partner preference formation in monogamous prairie voles. Here, using in situ hybridization and receptor autoradiography, we mapped in detail the distribution of MOR mRNA and ligand binding in monogamous prairie vole brains and compared MOR binding density with that of promiscuous meadow vole brains. Comparison of MOR binding in these closely related species with distinctly different social behavior revealed that while the distribution of MOR is similar, prairie voles have significantly higher densities of MOR than meadow voles in a majority of regions in the forebrain, including the caudate-putamen, nucleus accumbens shell, lateral septum and several thalamic nuclei, including the anteroventral and anteromedial thalamic nuclei. These differences in MOR expression between prairie and meadow voles could potentially contribute to species differences in behavior, including social attachment.

MicroRNA 339 down‐regulates μ‐opioid receptor at the post‐transcriptional level in response to opioid treatment

μ-Opioid receptor (MOR) level is directly related to the function of opioid drugs, such as morphine and fentanyl. Although agonist treatment generally does not affect transcription of mor, previous studies suggest that morphine can affect the translation efficiency of MOR transcript via microRNAs (miRNAs). On the basis of miRNA microarray analyses of the hippocampal total RNA isolated from mice chronically treated with μ-opioid agonists, we found a miRNA (miR-339-3p) that was consistently and specifically increased by morphine (2-fold) and by fentanyl (3.8-fold). miR-339-3p bound to the MOR 3'-UTR and specifically suppressed reporter activity. Suppression was blunted by adding miR-339-3p inhibitor or mutating the miR-339-3p target site. In cells endogenously expressing MOR, miR-339-3p inhibited the production of MOR protein by destabilizing MOR mRNA. Up-regulation of miR-339-3p by fentanyl (EC(50)=0.75 nM) resulted from an increase in primary miRNA transcript. Mapping of the miR-339-3p primary RNA and its promoter revealed that the primary miR-339-3p was embedded in a noncoding 3'-UTR region of an unknown host gene and was coregulated by the host promoter. The identified promoter was activated by opioid agonist treatment (10 nM fentanyl or 10 μM morphine), a specific effect blocked by the opioid antagonist naloxone (10 μM). Taken together, these results suggest that miR-339-3p may serve as a negative feedback modulator of MOR signals by regulating intracellular MOR biosynthesis.

Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells

BackgroundCocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine.MethodsTo assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin.ResultsBoth cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression.ConclusionsIn the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine’s action on MOR expression in neurons.

Regulation of mammalian MOR-1 gene expression after chronic treatment with morphine

Morphine is an effective analgesic that acts by binding to the μ-opioid receptor (MOR) coded in the human by the OPRM1 gene. In the present study, we investigated the regulation of μ-opioid receptor (MOR-1) mRNA levels in all-trans-retinoic acid-differentiated SH-SY5Y human neuroblastoma cells under in vitro conditions with 10 μM morphine treatment for 24 h. In addition, we measured the MOR-1 levels in recombinant Chinese hamster ovary (CHO) cells, transfected with human μ-opioid receptor gene (hMOR) with 10 μM morphine treatment for 24 h. The isolated mRNA from these cells was subjected to real-time quantitative RT-PCR analysis to determine the regulation of μ-opioid receptor gene expression. It was observed that morphine treatment did not alter MOR-1 levels in undifferentiated SH-SY5Y cells compared to undifferentiated control cells. However, the MOR-1 levels in all-trans-retinoic acid-differentiated cells were significantly higher compared to the undifferentiated cells. Morphine treatment in differentiated SH-SY5Y cells caused significant downregulation of MOR-1 expression compared to the control cells. In the morphine-treated CHO cells, the hMOR-1 mRNA levels remained the same as the untreated control. Finally, pretreatment of SH-SY5Y cells with 10 μM naloxone, the antagonist of μ-opioid receptor, for 1 h significantly blocked the downregulation of MOR-1 mRNA levels with morphine treatment. These findings suggest that regulation of MOR-1 gene expression is cell-type specific after chronic morphine treatment and provide some evidence in the understanding of morphine tolerance.

Propofol increases μ-opioid receptor expression in SH-SY5Y human neuroblastoma cells

The aim of the present study was to explore the effect of propofol, a intravenous sedative-hypnotic agent used widely in inducing and maintaining anesthesia, on µ-opioid receptor (MOR) expression in a human neuronal cell line. SH-SY5Y human neuroblastoma cells were treated with various concentrations of propofol (1, 5, 10 or 20µM) for different lengths of time (6, 12 or 24h). Real-time quantitative RT-PCR showed that at a concentration range of 1-10µM, propofol increased MOR mRNA levels in a statistically significant dose- and time-dependent manner within 12h of treatment. Western blot analyses demonstrated that propofol treatment for 12h dose-dependently increased the MOR protein levels. In the 12-h SH-SY5Y-treated cells, propofol dose-dependently increased MOR density (Bmax) in the cell membranes. In addition, in the presence of the transcription inhibitor actinomycinD (1mg/ml), propofol (10µM) had no significant effect on the MOR mRNA levels over time. The results suggested that propofol dose- and time-dependently enhances MOR expression in SH-SY5Y human neuroblastoma cells at the transcriptional level, leading to an increased density of ligand-binding MORs in the cell membranes. This study demonstrated for the first time a link between propofol and the opioid system, thereby providing new insights into propofol mechanism of action and potential for abuse.

Anxiolytic effects of ethanol are partially related to a reduced expression of adenylyl cyclase 5 but not to μ-opioid receptor activation in rat nucleus accumbens

Anxiolytic effects of alcohol participate in the reinforcing properties of the drug, in which nucleus accumbens (NAcc) is implicated. The opioidergic system in NAcc is considered a main pathway involved in the emotional responses of animals: rats microinjected with morphine in NAcc and the systemic administration of μ-opioid receptors (MOR) agonists yield low anxiety scores in the elevated plus maze (EPM), a behavioral test of anxiety. However, the specific participation of NAcc MOR in the anxiolytic effect of ethanol has not been studied. AC5, a cAMP-synthezising adenylyl-cyclase, is highly expressed in NAcc; it is negatively coupled to MOR and has been implicated in anxiety levels of animals.We evaluated the anxiolytic effects of an intra-gastric administration of ethanol (2.5g/kg) in animals subjected to EPM at 1, 4, and 8h after drug or water exposure. Locomotion was assayed with the open-field test; we also measured accumbal AC5 and MOR mRNA levels by RT-PCR. After 1h, ethanol-exposed animals showed anxiolytic-like behavior, as well as decreased and increased AC5 and MOR expression in NAcc, respectively. Intra-accumbal injection of β-funaltrexamine (FNA), a MOR antagonist, did not block ethanol-induced anxiolysis, rather it induced a tendency to increase anxiety levels in the water-exposed group. FNA partially decreased accumbal AC5 expression in ethanol-treated rats. We concluded that AC5 in NAcc is participating in the emotional effects of ethanol; that MOR was not mediating the drug-induced AC5 reduction in NAcc nor the ethanol-induced anxiolysis. MOR only might be involved in basal levels of anxiety of animals.

Absence of μ opioid receptor mRNA expression in astrocytes and microglia of rat spinal cord

Cumulating evidence has demonstrated that μ opioid receptor (MOR) agonists promote spinal glial activation, lead to synthesis and release of proinflammatory cytokines and chemokines, and contribute to opioid-induced hyperalgesia and development of opioid tolerance and dependence. However, whether these MOR agonists directly or indirectly act on spinal cord astrocytes and microglial cells in vivo is unclear. In the present study, by combining the techniques of in-situ hybridization of MOR mRNA with immunohistochemistry of glial fibrillary acidic protein (GFAP; an astrocyte marker) and Iba1 (a microglial marker), we examined expression and distribution of GFAP, Iba1, and MOR mRNA in the spinal cord of rats under chronic morphine tolerance conditions. Intrathecal injections of morphine twice daily for 7 days reduced morphine analgesic effect and increased both GFAP and Iba1 immunostaining densities in the spinal cord. Surprisingly, neither GFAP nor Iba1 colocalized with MOR mRNA in spinal cord cells. Our findings indicate that MOR expression is absent from spinal cord astrocytes and microglia, suggesting that these cell types are indirectly activated by MOR agonists under chronic opioid tolerance conditions.

Opioid receptor expression in human brain and peripheral tissues using absolute quantitative real-time RT-PCR

BackgroundThe actions of endogenous opioid peptides are mediated by 3 main classes of opioid receptors; mu (MOR), kappa (KOR), and delta (DOR). MethodsWe developed an absolute quantitative real-time reverse transcriptase PCR (AQ-rt-RT-PCR) assay to quantify MOR, DOR, and KOR mRNA in 22 human tissues. ResultsMOR mRNA was greatly enriched (12–20×106copies/μg) in the cerebellum, nucleus accumbens, and caudate nucleus; moderate (6×106copies/μg) in the dorsal root ganglion, spinal cord, and adrenal gland; low (2×104copies/μg) in the pancreas and small intestine; and absent in the lung, spleen, kidney, heart, skeletal muscle, liver, and thymus. High levels (>8.8×106copies/μg) of DOR mRNA were expressed in the brain and dorsal root ganglion; moderate (1.5×106copies/μg) in the adrenal gland and pancreas; low (2×104–6.5×105copies/μg in the cerebellum, spinal cord, small intestine, skeletal muscle, thymus, lung, and kidney); and very low (3.8×103copies/μg) in the heart. DOR mRNA was not detected in the spleen or liver. KOR mRNA was moderate (1×106copies/μg) in brain regions and dorsal root ganglion, but low (1.6–7×105copies/μg) in the cerebellum, temporal lobe and all other peripheral tissues. ConclusionsOur data demonstrate that the AQ-rt-RT-PCR is a highly reproducible and precise method to study the expression of opioid receptors in various tissues and under different disease conditions.

Let-7 microRNAs and Opioid Tolerance.

This chapter will focus on the role of microRNAs (miRs) in regulating the actions of opioid drugs through the opioid receptors. Opioids, such as morphine, are analgesics that are used for treating many forms of acute and chronic pain. However, their chronic use is limited by undesirable effects such as opioid tolerance. The μ opioid receptor (MOR) is the primary receptor responsible for opioids’ analgesia and antinociceptive tolerance. The long 3′-untranslated region (3′-UTR) of MOR mRNA is of great interest since this region may contain elements for the post-transcriptional regulation of receptor expression, such as altering the stability of mRNA, influencing translational efficiency, and controlling mRNA transport. MicroRNAs are small non-coding RNA molecules that exert their functions through base-pairing with partially complementary sequences in the 3′-UTR of target mRNAs, resulting in decreased polypeptide formation from those mRNAs. Since the discovery of the first miR, lin-4 in Caenorhabditis elegans, hundreds of miRs have been identified from humans to viruses, which have provided a crucial and pervasive layer of post-transcriptional gene regulation. The nervous system is a rich source of miR expression, with a diversity of miR functions in fundamental neurobiological processes including neuronal development, plasticity, metabolism, and apoptosis. Recently, the let-7 family of miRs is found to be a critical regulator of MOR function in opioid tolerance. Let-7 is the first identified human miR. Its family members are highly conserved across species in sequence and function. In the review, we will present a brief review of the opioid receptors, their regulation, and opioid tolerance as well as an overview of miRs and a perspective how miRs may interact with MOR and serve as a regulator of opioid tolerance.

Cortical Opioid Markers in Schizophrenia and across Postnatal Development

Certain cognitive deficits in schizophrenia have been linked to dysfunction of prefrontal cortical (PFC) γ-aminobutyric acid (GABA) neurons and appear neurodevelopmental in nature. Since opioids suppress GABA neuron activity, we conducted the first study to determine 1) whether the μ opioid receptor (MOR), δ opioid receptor (DOR), and opioid ligand proenkephalin are altered in the PFC of a large cohort of schizophrenia subjects and 2) the postnatal developmental trajectory in monkey PFC of opioid markers that are altered in schizophrenia. We used quantitative polymerase chain reaction to measure mRNA levels from 42 schizophrenia and 42 matched healthy comparison subjects; 18 monkeys chronically exposed to haloperidol, olanzapine, or placebo; and 49 monkeys aged 1 week-11.5 years. We found higher levels for MOR mRNA (+27%) in schizophrenia but no differences in DOR or proenkephalin mRNAs. Elevated MOR mRNA levels in schizophrenia did not appear to be explained by substance abuse, psychotropic medications, or illness chronicity. Finally, MOR mRNA levels declined through early postnatal development, stabilized shortly before adolescence and increased across adulthood in monkey PFC. In schizophrenia, higher MOR mRNA levels may contribute to suppressed PFC GABA neuron activity and might be attributable to alterations in the postnatal developmental trajectory of MOR signaling.

Chronic high-fat diet drives postnatal epigenetic regulation of μ-opioid receptor in the brain.

Opioid system dysregulation has been observed in both genetic and high-fat diet (HFD)-induced models of obesity. An understanding of the molecular mechanisms of MOR transcriptional regulation, particularly within an in vivo context, is lacking. Using a diet-induced model of obesity (DIO), mice were fed a high-fat diet (60% calories from fat) from weaning to >18 weeks of age. Compared with mice fed the control diet, DIO mice had a decreased preference for sucrose. MOR mRNA expression was decreased in reward-related circuitry (ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC)) but not the hypothalamus, important in the homeostatic regulation of feeding. DNA methylation is an epigenetic modification that links environmental exposures to altered gene expression. We found a significant increase in DNA methylation in the MOR promoter region within the reward-related brain regions. Methyl CpG-binding protein 2 (MeCP2) can bind methylated DNA and repress transcription, and DIO mice showed increased binding of MeCP2 to the MOR promoter in reward-related regions of the brain. Finally, using ChIP assays we examined H3K9 methylation (inactive chromatin) and H3 acetylation (active chromatin) within the MOR promoter region and found increased H3K9 methylation and decreased H3 acetylation. These data are the first to identify DNA methylation, MeCP2 recruitment, and chromatin remodeling as mechanisms leading to transcriptional repression of MOR in the brains of mice fed a high-fat diet.

Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury

Despite using prescribed pain medications, patients with neuropathic pain continue to experience moderate to severe pain. There is a growing recognition of a potent peripheral opioid analgesia in models of inflammatory and neuropathic pain. The goal of this study was to characterize the temporal and spatial expression of mu opioid receptor (mOR) mRNA and protein in primary afferent neurons in a rat L5 spinal nerve ligation model of persistent neuropathic pain. Bilateral L4 and L5 dorsal root ganglia (DRGs), L4 and L5 spinal cord segments, and hind paw plantar skins were collected on days 0 (naïve), 3, 7, 14, and 35 post-spinal nerve ligation or post-sham surgery. We found that expression of mOR mRNA and protein in primary afferent neurons changed dynamically and site-specifically following L5 spinal nerve ligation. Real-time RT–PCR, immunohistochemistry, and Western blot analysis demonstrated a down-regulation of mOR mRNA and protein in the injured L5 DRG. In contrast, in the uninjured L4 DRG, mOR mRNA transiently decreased on day 7 and then increased significantly on day 14. Western blot analysis revealed a persistent increase in mOR protein expression, although immunohistochemistry showed no change in number of mOR-positive neurons in the uninjured L4 DRG. Interestingly, mOR protein expression was reduced in the skin on days 14 and 35 post-nerve injury and in the L4 and L5 spinal cord on day 35 post-nerve injury. These temporal and anatomically specific changes in mOR expression following nerve injury are likely to have functional consequences on pain-associated behaviors and opioid analgesia.

Opioid-receptor gene expression and localization in cancer cells

Abstract This study examined the presence and cellular localization of three types of opioid receptors (MOR, DOR and KOR) in five human cancer cell lines: MCF-7, MDA-MB-231, HT-29, MGH-U1 and SH-SY5Y. Expression levels of opioid receptors were measured quantitatively using real-time PCR, and the localizations of the receptors in the cells were determined by immunocytochemistry. All three types of opioid receptors were present in each of the five cell lines examined. However, three of the cell lines (MCF-7, HT-29 and SH-SY5Y) showed significantly higher levels of MOR mRNA and protein than the other two types of receptors (DOR and KOR). Immunocytochemistry revealed that MOR, DOR and KOR receptors were predominantly present on the surface of cell membranes, although these receptors were also occasionally present in the cell cytoplasm.

Role of u-opioid receptor in attenuation of bone cancer pain by anti-nerve growth factor in rats

Objective To evaluate the role of μ-opioid receptor （MOR） in attenuation of bone cancer pain by anti-nerve growth factor （anti-NGF） in rats. Methods Part Ⅰ Sixty female SD rats weighing 200-220 g were randomly divided into 4 groups （n = 15 each）： sham operation group （group S）, sham operation ＋ anti-NGF group （group SN）, bone cancer pain group （group P） and bone cancer pain＋ anti-NGF group （group PN） . Bonecancer was induced by intra-tibial inoculation of 1 × 105 Walker 256 breast cancer cells in group P and PN. Group S and SN received injection of PBS 10 μl. APE 10 catheter was inserted at L2,3 interspace into the epidural space 13 days after cancer cell inoculation. Three days after the catheter was successfully placed, group SN and PN received intrachecal （IT） injection of anti-NGF 10 μg （in normal saline （NS） 10 μl） and group S and P IT injection of NS 10 μl twice a day for 5 consecutive days. The number of spontaneous flinches （NSF）, paw withdrawal latency （PWL） and paw withdrawal threshold （PWT） were measured before and 13, 16, 18, 21 day after cancer cell inoculation. The animals were sacrificed at 21 day after cancer cell inoculation and the spinal cord dorsal horn and dorsal root ganglion were removed for determination of MOR and MOR mRNA expression. Part Ⅱ Thirty female SD rats weighing 200-220 g were randomly divided into 2 groups （n = 15 each）： bone cancer pain ＋ anti-NGF group （group PN） and bone cancer pain ＋ naloxone ＋ anti-NGF group （group PNN）. Bone cancer was induced by intratibial inoculation of 1 × 105 Walker 256 breast cancer cells. APE 10 catheter was inserted at L2-3 interspace into the epidural space 13 days after cancer cell inoculation. Three days after the catheter was successfully placed,group PN received IT injection of anti-NGF 10 μg （in NS 10 μl） and group PNN IT injection of naloxone 10μg （in NS 25 μl） and 0.5 h later IT injection of anti-NGF 10 μg （in NS 25μl） twice a day for 5 consecutive days. NSF,PWL and PWT were measured before and 13, 16, 18, 21 days after cancer cell inoculation. Results Part ⅠCompared with group S, no significant change was found in NSF, PWL and PWT in group SN, and in MOR and MOR mRNA expression in group SN and PN （P 〉 0.05）, NSF was significantly increased, PWL shortened, PWT decreased at 13-21 days after inoculation in group P and PN, and MOR and MOR mRNA expression was down-regulated in group P （P 〈 0.05 or 0.01）. Compared with group P, NSF was significantly decreased, PWL prolonged, PWT increased, MOR and MOR mRNA expression was up-regulated in group PN at 18-21 days after inoculation （P 〈 0.05 or 0.01）. Part Ⅱ Compared with group PN, NSF was significantly increased, PWL shortened, PWT decreased at 18-21 days after inoculation in group PNN （P 〈 0.05 or 0. 01）. Conclusion The mechanism by which anti-NGF attenuates bone cancer pain in rats is related to the activation of MOR. Key words: Receptors,opioid; Nerve growth factor; Antibodies; Bone neoplasms; Pain

Regulation of Opioid Tolerance by let-7 Family MicroRNA Targeting the μ Opioid Receptor

MicroRNA has emerged as a critical regulator of neuronal functions. This study aimed to test whether let-7 microRNAs can regulate the mu opioid receptor (MOR) and opioid tolerance. Employing bioinformatics, we identified a let-7 binding site in the 3'-untranslated region (UTR) of MOR mRNA, which was experimentally confirmed as a direct target of let-7. The repressive regulation of MOR by let-7 was revealed using a LNA-let-7 inhibitor to knockdown let-7 in SH-SY5Y cells. Conversely, morphine significantly upregulated let-7 expression in SH-SY5Y cells and in a mouse model of opioid tolerance. The LNA-let-7 inhibitor decreased brain let-7 levels and partially attenuated opioid antinociceptive tolerance in mice. Although chronic morphine treatment did not change overall MOR transcript, polysome-associated mRNA declined in a let-7-dependent manner. let-7 was identified as a mediator translocating and sequestering MOR mRNA to P-bodies, leading to translation repression. These results suggest that let-7 plays an integral role in opioid tolerance.

Lack of evidence for the μ‐opioid receptor splice variant MOR1C in rats

We previously reported the existence of MOR1(C) mRNA and MOR1(C)-immunoreactivity (-ir) in rats. However, the sequence that we reported for rat MOR1(C) appears not to be present in the rat genome. We have therefore reexamined whether MOR1(C) mRNA or MOR1(C)-ir exist in rats. We used reverse-transcription polymerase chain reaction (RT-PCR) to attempt to amplify MOR1, MOR1(A), MOR1(B), the rat MOR1(C) sequence we previously reported, and MOR1(C1) and MOR1(C2) (which have recently been reported to exist in rats). In RNA extracted from rats, we were able to demonstrate PCR products representing MOR1, MOR1(A), and MOR1(B) splice variants. All three products were confirmed as related to MOR1 by Southern blot. However, we were unable to detect either the MOR1(C) product reported previously by us or the MOR1(C)-like products reported to exist in rats by others. In RNA extracted from mice we were able to detect MOR1, MOR1(A), MOR1(B), and MOR1(D)-like products. To test the specificity of our MOR1(C) antiserum, we examined MOR1(C)-ir in control and knockout mice. MOR1(C)-ir had a distribution in control mice similar to that previously reported in rats, including coexisting with vGLUT2. However, although MOR1-ir was absent in MOR1 knockout mice, the density and distribution of MOR1(C)-ir were unchanged, suggesting that the antiserum crossreacts with another molecule in tissue. We find no evidence for MOR1(C) mRNA in rats. Furthermore, we conclude that MOR1(C)-ir represents crossreactivity.

M1629 Polaprezinc As a Potent Inducer of Anti-Oxidative Stress Enzyme, Heme Oxygenase 1

G A A b st ra ct s animals that received tap water for 11 days (controls). Rats were observed daily for fluid intake, weight changes, bowel movements, loose stools and rectal bleeding. Samples of distal ileum, and proximal and distal colon were collected for (1) the analysis of myeloperoxidase (MPO) activity, an index of neutrophil infiltration, (2) the histological evaluation of mucosal damage, and (3) the determination of the expression of the proinflammatory cytokine, TNFalpha and MOR mRNA by real time qRT-PCR. Results: Histological analysis showed no surface epithelium lesions in the ileum and colon from control animals as well as in the ileum of treated animals, whereas there were erosions of the epithelium, ulcerations and edema in the distal colon of treated rats. MPO activity was similar in the ileum of control (5.07±0.74 U/mg tissue) and treated group (5.75±0.61 U/mg), whereas it was increased in the proximal colon (11.16±1.67 U/mg vs. controls 4.13±0.55 U/mg) and markedly increased in the distal colon vs. controls (21.96±3.8 U/mg vs. 3.09±0.35 U/mg, p<0.003). There was a 3-fold increase of TNF-alpha mRNA levels in the distal colon in the DSS treated animal (4.47±0.71) compare to the controls (1.42±0.24; p<0.017). By contrast, there was a decrease in the expression of MOR mRNA in the distal colon in DSS treated animals (0.97±0.22) compared to controls (2.0±0.44), whereas the levels of MOR mRNA in the ileum of DSStreated (1.14±0.29), which did not develop detectable inflammation, were comparable to the levels observed in control animals (1.26±0.3). Conclusions: These data show an inverse relationship between the level of inflammation and the expression of MOR mRNA. The lower levels of MOR mRNA in inflamed tissue compared to controls might be due to receptor down regulation induced by increased release of opioids induced by inflammation.