Opioid Receptor Gene Research Articles

Micro Opioid Receptor A118G Polymorphism and Post-Operative Pain: Opioids' Effects on Heterozigous Patients

The single-nucleotide-polymorphism (SNP) 118A>G in the micro-1 opioid receptor gene (OPRM1) is associated with a decrease in the analgesic effects of opioids. The aim of this study is to assess whether 118A >G polymorphism could influence the analgesic response to opioid-based postoperative pain (POP) therapy. The study consisted of two parts: section alpha, observational, included 199 subjects undergoing scheduled surgical procedures with pain management standardized on surgery invasiveness and on expected level of postoperative pain; section beta, randomized, included 41 women undergoing scheduled caesarean delivery with continuous intra-operative epidural anesthesia and post-operative analgesia (CEA). In both sections, POP was measured over 48 h (T6h-T24h-T48h) by the visual analogue scale (VAS). In section beta we also tested the responsiveness of hypothalamic-pituitary-adrenal axis (HPA) expressed by cortisol levels. In section alpha, with cluster analysis, subjects were analyzed according to their genotype: a group (no. 1) of 34 patients reporting VAS score >3 at every time lapse was identified and included only A118G carriers, while wild-type (A118A - absence of 118A>G polymorphism) patients were unevenly distributed between those with cluster no. 2 (VAS score <3 at every study steps) and those with cluster no. 3 (VAS score progressively reducing from T6h). In section beta, A118G carriers receiving epidural sufentanil had the lowest VAS scores at T24h; also in these patients, cortisol levels remained more stable, with a mild decrease at T6h. This study shows that the OPRM1 118A>G polymorphism affects postoperative pain response in heterozygous patients: they have a different postoperative pain response than patients with wild-type genes, which may affect the efficacy of the analgesic therapy.

Influence of Endogenous Opioid Systems on T Lymphocytes as Assessed by the Knockout of Mu, Delta and Kappa Opioid Receptors

Here, we evaluated the influence of endogenous opioid activation on immune responses by examining consequences of all three opioid receptor gene (mu, delta and kappa) inactivation. In triple-opioid receptor knockout mice, splenocytes and thymocytes numbers, lymphocyte subsets as well as proliferation and cytokines induced by in vitro stimulation of T lymphocytes were measured. Compared with wild-type mice, similar lymphocyte distribution in thymus and spleen as well as comparable T lymphocyte proliferation were observed, while lower levels of IL-2 and IFNγ as well as higher levels of IL-4 and IL-10 were found in triple-opioid receptor knockout mice. Together, our results indicate a shift from TH1 to TH2 cytokines in triple-opioid receptor knockout animals, suggesting that global endogenous opioid tone drives T lymphocytes toward a TH1 profile under non-pathological conditions.

The Role of the Asn40Asp Polymorphism of the Mu Opioid Receptor Gene (OPRM1) on Alcoholism Etiology and Treatment: A Critical Review

The endogenous opioid system has been implicated in the pathophysiology of alcoholism as it modulates the neurobehavioral effects of alcohol. A variant in the mu opioid receptor gene (OPRM1), the Asn40Asp polymorphism, has received attention as a functional variant that may influence a host of behavioral phenotypes for alcoholism as well as clinical response to opioid antagonists. This paper will review converging lines of evidence on the effect of the Asn40Asp SNP on alcoholism phenotypes, including: (i) genetic association studies; (ii) behavioral studies of alcoholism; (iii) neuroimaging studies; (iv) pharmacogenetic studies and clinical trials; and (v) preclinical animal studies. Together, these lines of research seek to elucidate the effects of this functional polymorphism on alcoholism etiology and treatment response.

Delta opioid receptor analgesia

Delta opioid receptors represent a promising target for the development of novel analgesics. A number of tools have been developed recently that have significantly improved our knowledge of δ receptor function in pain control. These include several novel δ agonists with potent analgesic properties, and genetic mouse models with targeted mutations in the δ opioid receptor gene. Also, recent findings have further documented the regulation of δ receptor function at cellular level, which impacts on the pain-reducing activity of the receptor. These regulatory mechanisms occur at transcriptional and post-translational levels, along agonist-induced receptor activation, signaling and trafficking, or in interaction with other receptors and neuromodulatory systems. All these tools for in-vivo research, and proposed mechanisms at molecular level, have tremendously increased our understanding of δ receptor physiology, and contribute to designing innovative strategies for the treatment of chronic pain and other diseases such as mood disorders.

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.

Parent-Child Interactions Affected by Gene Variant

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessParent-Child Interactions Affected by Gene VariantJoan Arehart-TreichelJoan Arehart-TreichelSearch for more papers by this authorPublished Online:1 Apr 2011https://doi.org/10.1176/pn.46.7.psychnews_46_7_16_2AbstractAn intriguing new study has found that opioid peptides—brain peptides that respond to pain—influence parent-child relationships.The study sample—226 youth aged 9 to 17—was derived from a larger investigation of psychiatric illness and service use in youth in four rural North Carolina counties that have high poverty rates. All participating children and teens were genotyped for the gene that makes the mu-opioid receptor, to which opioid peptides attach. Out of the 226 children, two-thirds had the A variant of the gene (that is, A/A), while the remaining third had the G variant (either G/G or G/A). Each child and one of its parents (usually the mother) were then interviewed using the Child and Adolescent Psychiatric Assessment to obtain information about three aspects of their relationship during the previous three months—separation anxiety, enjoyment of parent-child activities, and parent-child arguments. The researchers then assessed, after taking the youngsters' age and gender into consideration, whether there was a significant link between the mu-opioid receptor gene variant that a child possessed and his or her relationship with the parent during the previous three months. There was such a relationship, they found. Interview findings indicated that children with the G variant indicated that they enjoyed parent-child activities significantly more, had significantly fewer parent-child arguments, and experienced significantly less separation anxiety than did children with the A variant. Furthermore, the findings held even when mental health problems, substance abuse problems, or criminal convictions of the parents were considered. "I think our findings may help explain why some children faced with adversity continue to do well and maintain a good relationship with their parents," William Copeland, Ph.D., an associate professor of psychiatry at Duke University and the lead investigator, told Psychiatric News. "We think this variant may be important for understanding social interactions, which are disrupted in many common psychiatric disorders … . It is probably premature, however, to start thinking about clinical implications. As cautious scientists, we are still interested in seeing this finding replicated in other samples." In addition to benefiting parent-child relationships, the G variant of the opioid receptor gene also seems to confer another advantage—less pain sensitivity—and a disadvantage—an increased risk of substance dependence in adolescence and early adulthood, other researchers have found. The study was funded by the National Institutes of Health.An abstract of "Child Mu-Opioid Receptor Gene Variant Influences Parent-Child Relations" is posted at <www.nature.com/npp/journal/vaop/ncurrent/abs/npp2010251a.html>. ISSUES NewArchived

The Effect of Naltrexone on Alcohol’s Stimulant Properties and Self‐Administration Behavior in Social Drinkers: Influence of Gender and Genotype

Few pharmacological treatments for alcohol dependence are available. Moreover, the best supported treatment, naltrexone hydrochloride, appears to work for only some. To investigate potential predictors of these differential responses, 40 social drinkers (20 women) were administered 6 days of treatment with naltrexone vs. placebo in a double-blind, counterbalanced, crossover design. At the end of each treatment period, participants received a single dose of their preferred alcoholic beverage followed by the opportunity to work for additional alcohol units using a progressive ratio (PR) breakpoint paradigm. All subjects but one were genotyped for the A118G polymorphism of the mu opioid receptor gene (OPRM1). Naltrexone decreased the ethanol-induced 'euphoria' to a priming dose of alcohol in two subgroups: (i) in women, and (ii) in subjects with the A118G polymorphism of the mu opioid receptor gene (OPRM1). Naltrexone did not decrease motivation to work for additional alcoholic beverages on the PR task regardless of gender or genotype. The results add to the evidence that naltrexone decreases positive subjective effects of alcohol, with preferential effects in distinct subgroups. Similar effects in heavier drinkers might decrease alcohol use.

Transcriptional and Epigenetic Regulation of Opioid Receptor Genes: Present and Future

Three opioid receptors (ORs) are known: μ opioid receptors (MORs), δ opioid receptors (DORs), and κ opioid receptors (KORs). Each is encoded by a distinct gene, and the three OR genes share a highly conserved genomic structure and promoter features, including an absence of TATA boxes and sensitivity to extracellular stimuli and epigenetic regulation. However, each of the genes is differentially expressed. Transcriptional regulation engages both basal and regulated transcriptional machineries and employs activating and silencing mechanisms. In retinoic acid-induced neuronal differentiation, the opioid receptor genes undergo drastically different chromatin remodeling processes and display varied patterns of epigenetic marks. Regulation of KOR expression is distinctly complex, and KOR exerts a unique function in neurite extension, indicating that KOR is not simply a pharmacological cousin of MOR and DOR. As the expression of OR proteins is ultimately controlled by extensive posttranscriptional processing, the pharmacological implication of OR gene regulation at the transcriptional level remains to be determined.

Identification and Characterization of Seven New Exon 11-Associated Splice Variants of the Rat Mu Opioid Receptor Gene, OPRM1

BackgroundThe mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat.ResultsThe current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding.ConclusionThe identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various mu opioids seen in rats.

Regulation of opioid gene expression in the rat brainstem by 3,4-methylenedioxymethamphetamine (MDMA): role of serotonin and involvement of CREB and ERK cascade

The amphetamine analogue 3,4-methylendioxymetamphetamine (MDMA, Ecstasy) causes complex adaptations at the molecular and cellular levels altering the activity of different brain neurotransmitters. The present study aims to verify the effects of single and repeated injections of MDMA on dynorphin and nociceptin systems gene regulation in the brainstem, an area rich in neurons containing serotonin. Both acute and chronic (twice a day for 7days) MDMA (8mg/kg) induced a marked increase in prodynorphin mRNA levels as well as in cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation, without causing any effect on kappa opioid receptor or nociceptin system (both pronociceptin and its receptor) genes expression, in this brain region. The blockade of 5HT1/5HT2 receptors by methysergide abolished the acute MDMA-induced increase in prodynorphin. Moreover, the concomitant chronic administration of both methysergide and MDMA (7days) induced a significant increase in all the dynorphin or nociceptin system genes expression and in CREB and ERK phosphorylation. Our data suggest the involvement of dynorphin in the effects evoked by MDMA in the brainstem, possibly via CREB and ERK1/2 cascade activation, since the ERK inhibitor PD98059 prevented the MDMA-induced prodynorphin gene expression, and, acutely, also through the involvement of serotoninergic mechanisms. Chronically, it is also possible to hypothesize a general inhibitor role of serotonin in the effects evoked by MDMA. Moreover, these findings strengthen the hypothesis, already proposed, of a neuroprotective role for both CREB and dynorphin.

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.

Deletion of the δ Opioid Receptor Gene Impairs Place Conditioning But Preserves Morphine Reinforcement

Converging experimental data indicate that δ opioid receptors contribute to mediate drug reinforcement processes. Whether their contribution reflects a role in the modulation of drug reward or an implication in conditioned learning, however, has not been explored. In the present study, we investigated the impact of δ receptor gene knockout on reinforced conditioned learning under several experimental paradigms. We assessed the ability of δ receptor knockout mice to form drug-context associations with either morphine (appetitive)- or lithium (aversive)-induced Pavlovian place conditioning. We also examined the efficiency of morphine to serve as a positive reinforcer in these mice and their motivation to gain drug injections, with operant intravenous self-administration under fixed and progressive ratio schedules and at two different doses. Mutant mice showed impaired place conditioning in both appetitive and aversive conditions, indicating disrupted context-drug association. In contrast, mutant animals displayed intact acquisition of morphine self-administration and reached breaking-points comparable to control subjects. Thus, reinforcing effects of morphine and motivation to obtain the drug were maintained. Collectively, the data suggest that δ receptor activity is not involved in morphine reinforcement but facilitates place conditioning. This study reveals a novel aspect of δ opioid receptor function in addiction-related behaviors.

Haplotype block structure of the genomic region of the mu opioid receptor gene

The opioid system is involved in the action of opiate drugs, opioid addiction, pain experience and analgesia. Individual differences in opioid effect may be attributed in part to genetic variations. Long-range cis regulatory elements and intronic variants are potential sources of functional diversity. Recently, we have detected association of two intronic OPRM1 variants with heroin addiction in European Americans. In the current study, we analyzed the genetic variations in the OPRM1 100 kb 5′ flanking region and intron 1 in the HapMap Caucasian population. Four major linkage disequilibrium (LD) blocks were identified, consisting of 28, 22, 15 and 42 SNPs, respectively. The locations of these blocks are (−100 – −90), (−90 – −67), (−20 - −1) and (+1 – +44) kb, respectively. The two intronic variants, indicated in our recent study, are part of a distinct haplogroup that include SNPs from intron 1, and the proximal 5′ region. The 118G (rs1799971) allele is part of a different haplogroup that includes several variants in the distal 5′ region that may have a regulatory potential. These findings were corroborated by genotyping eight SNPs in a sample of European Americans and suggest an extended OPRM1 locus with potential new regulatory regions.

Differential gene expression activity among species-specific polypyrimidine/polypurine motifs in mu opioid receptor gene promoters

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. An appropriate understanding of MOR gene expression across species is critical for understanding its analgesic functions in humans. Here, we undertake a cross-species analysis of the polymorphic polypyrimidine/polypurine (PPy/u) motif, a key enhancer of MOR gene expression. The mouse PPy/u motif is highly homologous to those of rat (67%) and human (83%), but drives reporter gene expression tenfold and fivefold more effectively than those of rat and human, respectively. Circular dichroism profiles of PPy/u oligonucleotides from different species showed that they are primarily different in structure. Conformational studies of reporter plasmids using confocal Raman spectra, S1 nuclease and restriction enzymes demonstrated that the structural difference is the result of changes in the phosphodiester backbone. Furthermore, these conformational disparities produce differences in torsional stress, as shown by topoisomerase II relaxation and activation of different levels of gene expression under hypertonic conditions. This study demonstrates that homologous PPy/u motifs adopt unique species-specific conformations with different mechanisms and activities for gene expression. We further discuss how structural aspects of transcription regulatory elements, rather than the sequence itself, are significant when studying functional gene expression regulatory elements.

Epigenetic mechanisms involved in the induction of the mu opioid receptor gene in Jurkat T cells in response to interleukin-4

Various immunomodulatory effects of opioids are mediated by mu opioid receptors. While in resting T lymphocytes their expression is repressed, mu opioid receptors are induced by interleukin-4 via the transcription factor STAT6. Here we investigated mechanisms underlying this induction in human Jurkat T cells. Although interleukin-4 induced a rapid activation of STAT6 by phosphorylation within few minutes, chromatin-immune-precipitation analysis revealed that the binding of STAT6 to its regulatory DNA element on the mu opioid receptor promoter occurs later than 2 h after interleukin-4-stimulation. Detectable amounts of the mu opioid receptor mRNA were observed later than 3 h after stimulation. Preceding the binding of STAT6, several epigenetic mechanisms were observed that are known to modify the chromatin architecture of a gene. Thus, we detected by chromatin-immune-precipitation analysis transient association of the mu opioid receptor gene promoter with trimethylated histone H3 at lysine 4, phosphorylated (serine 10) plus acetylated (lysine 14) histone H3, and acetylated histone H4 at lysine 16. In addition, binding of the methyl-cytosine–guanine dinucleotide-binding protein MeCP2 to the mu opioid receptor promoter decreased during the interleukin-4 treatment of Jurkat cells. Furthermore, we detected a transient association of the mu opioid receptor promoter with Brg-1, which is a protein contained in ATP-dependent chromatin remodeling complexes and known to facilitate transcriptional activation of a gene. Together, these data suggest that epigenetic modifications of the chromatin of the mu opioid receptor gene are involved in the transcriptional activation of the gene in response to interleukin-4 in T cells.

In vivo effects of morphine on neuronal fate and opioid receptor expression in zebrafish embryos

Morphine remains one of the most potent analgesic compounds used to control chronic pain despite its known adverse effects. It binds to the opioid receptors mu, delta and kappa, which are involved in aspects of neuronal fate such as cell proliferation, neuroprotection and neuronal differentiation. However, the effect of morphine on these processes is controversial and in vitro studies, as well as in vivo studies on adults and neonates in mammalian models, have not been able to clarify the diverse roles of morphine in the central nervous system. We have used zebrafish embryos to determine in vivo how morphine affects neuronal fate and opioid receptor gene expression and to elucidate if there is a link between these processes. Our results show that at 24 and 48 h post fertilization (hpf) morphine enhances cell proliferation, although it has opposing effects as an inducer of neuronal differentiation at these two stages, increasing the number of certain neuronal populations at 24 hpf and decreasing it at 48 hpf. The present study also demonstrates that in 24-hpf embryos morphine acts as a neuroprotector against glutamate damage in motor neurons and Pax-6-positive neurons. Furthermore, the gene expression of the opioid receptors is altered by embryonic exposition to morphine. In conclusion, our study sheds new light on the in vivo roles of morphine, and it indicates for the first time that its implication in cell proliferation and neuroprotection might be related to changes in the gene expression of opioid receptors.

Pharmacogenetics of Alcohol and Alcohol Dependence Treatment

In this article, we review studies of genetic moderators of the response to medications to treat alcohol dependence, the acute response to alcohol, and the response to the psychotherapeutic treatment of heavy drinking. We consider four neurotransmitter systems: opioidergic, dopaminergic, GABAergic, and glutamatergic and focus on one receptor protein in each: OPRM1 (the &#micro;-opioid receptor gene), DRD4 (the D(4) dopamine receptor gene), GABRA2 (GABA(A) receptor alpha-2 subunit gene), and GRIK1 (the kainite receptor GluR5 subunit gene). We conclude that because parallel developments in alcoholism treatment and the genetics of alcohol dependence are beginning to converge, using genotypic information, it may be possible to match patients with specific treatments. Of greatest clinical relevance is the finding that the presence of an Asp40 allele in OPRM1 modestly predicts a better response to naltrexone treatment. Promising findings include the observations that a polymorphism in GABRA2 predicts the response to specific psychotherapies; a polymorphism in DRD4 predicts the effects of the antipsychotic olanzapine on craving for alcohol and drinking behavior; and a polymorphism in GRIKI predicts adverse events resulting from treatment with the anticonvulsant topiramate. Although variants in other genes have been associated with the risk for alcohol dependence, they have not been studied as moderators of the response either to treatment or acute alcohol administration. We recommend that, whenever possible, treatment trials include the collection of DNA samples to permit pharmacogenetic analyses, and that such analyses look broadly for relevant genetic variation.

Concomitant Duplications of Opioid Peptide and Receptor Genes before the Origin of Jawed Vertebrates

BackgroundThe opioid system is involved in reward and pain mechanisms and consists in mammals of four receptors and several peptides. The peptides are derived from four prepropeptide genes, PENK, PDYN, PNOC and POMC, encoding enkephalins, dynorphins, orphanin/nociceptin and beta-endorphin, respectively. Previously we have described how two rounds of genome doubling (2R) before the origin of jawed vertebrates formed the receptor family.Methodology/Principal FindingsOpioid peptide gene family members were investigated using a combination of sequence-based phylogeny and chromosomal locations of the peptide genes in various vertebrates. Several adjacent gene families were investigated similarly. The results show that the ancestral peptide gene gave rise to two additional copies in the genome doublings. The fourth member was generated by a local gene duplication, as the genes encoding POMC and PNOC are located on the same chromosome in the chicken genome and all three teleost genomes that we have studied. A translocation has disrupted this synteny in mammals. The PDYN gene seems to have been lost in chicken, but not in zebra finch. Duplicates of some peptide genes have arisen in the teleost fishes. Within the prepropeptide precursors, peptides have been lost or gained in different lineages.Conclusions/SignificanceThe ancestral peptide and receptor genes were located on the same chromosome and were thus duplicated concomitantly. However, subsequently genetic linkage has been lost. In conclusion, the system of opioid peptides and receptors was largely formed by the genome doublings that took place early in vertebrate evolution.

966 Localization and Function of Fluorescently-Tagged Delta Opioid Receptor (DOR) Expressed in Enteric Neurons of Knockin Mice

Endogenous opioids and opiate drugs have potent effects on intestinal function through activation of μ, δ and κ opioid receptors. Although the role of the μ receptor in these effects and its distribution within the gut wall has been extensively studied, little is known about the expression and function of the δ opioid receptor (DOR) in the intestine. Since the specificity of available DOR antibodies has been questioned (Cell 137:1-12, 2009), we have used an alternative, genetic strategy to determine the distribution and function of DOR in the enteric nervous system (ENS). Methods and Results. Mice in which enhanced GFP (eGFP) was knocked into the opioid delta receptor gene Oprd1 were studied. Western blotting for GFP revealed a protein of ~80kDa in all regions of the gut, consistent with the expected size of DOReGFP. DOReGFP was localized to myenteric and submucosal ganglia and nerve fibers within the muscularis externa and crypts by immunofluorescence, but not in enterocytes, smooth muscle and ICC. Although DOReGFP-positive neurons were rarely observed in submucosal ganglia, DOReGFP was expressed by ~50% of all PGP9.5-immunoreactive (IR) myenteric neurons throughout the intestine. DOReGFP was detected in both ChATand NOS-IR neurons of the duodenum (41%, 44%, respectively) and ileum (44%, 53%). DOReGFP also colocalized with ChAT in the cecum (30%), and proximal (9%) and distal colon (20%), but most DOReGFP-positive neurons in the large intestine coexpressed NOS-IR (66%, 96%, 65%, respectively). DOReGFP was not coexpressed with NFM in any region. DOReGFP function was investigated by examining agonist-stimulated trafficking of the receptor in myenteric wholemounts. In unstimulated preparations, DOReGFP was detected at the plasma membrane of the soma and neurites. The DOR agonists SNC80 (10, 100 nM) and met-enkephalin (100 nM) induced receptor clustering within 10 min and receptor trafficking to endosomes after 30 min. After 120-240 min, DOReGFP was in lysosomes, identified by LAMP1-IR. Equivalent results were obtained by live imaging of cultured neurons, and after administration of SNC80 to intact mice. SNC80 (100 nM) stimulated contraction of the isolated distal colon, confirming function. Conclusion. By using a novel approach for specific detection, we found that DOR is similarly distributed in cholinergic excitatory neurons and NOS containing inhibitory motor or interneurons in the small intestine, and that DOR is mostly confined to NOS-positive neurons of the large intestine. Activation induces DOR endocytosis and trafficking to lysosomes, where degradation will down-regulate opioid signaling. Supported by DK399857, NHMRC454858.

Dynamic association of p300 with the promoter of the G protein-coupled rat delta opioid receptor gene during NGF-induced neuronal differentiation

The G protein-coupled delta opioid receptor (DOR) plays a critical role in pain control. Emerging evidence shows that DOR also plays a role in neuronal differentiation and survival. Nerve growth factor (NGF) is known to be critical for the development and maintenance of the central and peripheral nervous systems. Our previous studies have shown that sustained activation of NGF/PI3K/Akt/NF-κB signaling is essential for NGF-induced dor gene expression during neuronal differentiation and that the epigenetic modifications at histone 3 lysine 9 temporally correlate with the dor gene transcription. In this study, we cloned the rat dor gene promoter and identified an NGF-responsive region similar to that from the mouse dor gene promoter. We further identified p300, a known NF-κB binding partner with intrinsic histone acetyltransferase activity, to be dynamically associated with the dor gene. We also found that assembling of RNA polymerase II (Pol II) at the promoter took place before NGF stimulation, indicating that p300 could only interact with preassembled Pol II at the promoter after NGF stimulation. Taken together, these results implicate that preassembly of the Pol II preinitiation complex, sustained activation of PI3K/Akt/NF-κB signaling, and dynamic p300 association at the promoters sequentially is one of the mechanisms of induction of the late phase genes during NGF-induced neuronal differentiation.