Opioid and Notch signaling pathways are reciprocally regulated through miR- 29a and miR-212 expression.

BACKGROUND: The abuse of opioids, such as morphine and phentanyl or other drugs as heroin is a social and health problem that affects an increasing number of people each year. The activation of the mu opioid receptor triggers several molecular changes that alter the expression of diverse genes, including miRNAs. The dysregulation of these molecules could explain some of the developmental alterations that are induced after drug intake. In addition, the Notch signaling cascade has also been related to alterations on these processes. METHODS: Zebrafish embryos and SH-SY5Y cells were used to assess the effects of opioid and Notch signaling on the expression on miR-29a and miR-212/132 by qPCR and ChIP-qPCR. Notch1 expression was analyzed using in situ hybridization on 24 hpf zebrafish embryos. In addition, OPRM1 and NICD levels were measured using western blot on the cultured cells to determine the cross-talk between the two pathways. RESULTS: We have observed changes in the levels of miR-212/132 after administrating DAPT to zebrafish embryos indicating that this pathway could be regulating mu opioid receptor expression. In addition, the ISH experiment showed changes in Notch1 expression after morphine and DAPT administration. Moreover, morphine affects the expression of miR-29a through NF-κB, therefore controlling the cleavage and activation of Notch through ADAM12 expression. CONCLUSIONS: This study shows that these two pathways are closely related, and could explain the alterations triggered in the early stages of the development of addiction. GENERAL SIGNIFICANCE: Opioid and Notch pathway are reciprocally regulated by the miRNAs 212/132 and 29a.

Generation and Characterization of Antibodies against Opioid Receptors from Zebrafish.

The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.

Morphine delays neural stem cells differentiation by facilitating Nestin overexpression.

BACKGROUND: Morphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development. METHODS: AB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer. RESULTS: Morphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin. CONCLUSIONS: Morphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells. GENERAL SIGNIFICANCE: The present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog.

Role of the sugar moiety on the opioid receptor binding and conformation of a series of enkephalin neoglycopeptides.

Glycosylation by simple sugars is a drug discovery alternative that has been explored with varying success for enhancing the potency and bioavailability of opioid peptides. Long ago we described two O-glycosides having either ß-Glucose and ß-Galactose of (d-Met2, Pro5)-enkephalinamide showing one of the highest antinociceptive activities known. Here, we report the resynthesis of these two analogs and the preparation of three novel neoglycopeptide derivatives (α-Mannose, ß-Lactose and ß-Cellobiose). Binding studies to cloned zebrafish opioid receptors showed very small differences of affinity between the parent compound and the five glycopeptides thus suggesting that the nature of the carbohydrate moiety plays a minor role in determining the binding mode. Indeed, NMR conformational studies, combined with molecular mechanics calculations, indicated that all glycopeptides present the same major conformation either in solution or membrane-like environment. The evidences provided here highlight the relevance for in vivo activity of the conjugating bond between the peptide and sugar moieties in opioid glycopeptides.

µ Opioid Receptor Expression after Morphine Administration Is Regulated by miR-212/132 Cluster.

Since their discovery, miRNAs have emerged as a promising therapeutical approach in the treatment of several diseases, as demonstrated by miR-212 and its relation to addiction. Here we prove that the miR-212/132 cluster can be regulated by morphine, through the activation of mu opioid receptor (Oprm1). The molecular pathways triggered after morphine administration also induce changes in the levels of expression of oprm1. In addition, miR-212/132 cluster is actively repressing the expression of mu opioid receptor by targeting a sequence in the 3' UTR of its mRNA. These findings suggest that this cluster is closely related to opioid signaling, and function as a post-transcriptional regulator, modulating morphine response in a dose dependent manner. The regulation of miR-212/132 cluster expression is mediated by MAP kinase pathway, CaMKII-CaMKIV and PKA, through the phosphorylation of CREB. Moreover, the regulation of both oprm1 and of the cluster promoter is mediated by MeCP2, acting as a transcriptional repressor on methylated DNA after prolonged morphine administration. This mechanism explains the molecular signaling triggered by morphine as well as the regulation of the expression of the mu opioid receptor mediated by morphine and the implication of miR-212/132 in these processes.

Role of morphine, miR-212/132 and mu opioid receptor in the regulation of Bdnf in zebrafish embryos.

BACKGROUND: Morphine is one of the first-line therapies for the treatment of pain despite its secondary effects. It modifies the expression of epigenetic factors like miRNAs. In the present study, we analyzed miR-212 and miR-132 and their implication in morphine effects in the zebrafish Central Nervous System (CNS) through the regulation of Bdnf expression. METHODS: We used control and knock-down zebrafish embryos to assess the effects of morphine in miRNAs 212/132 and mitotic or apoptotic cells by qPCR, immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB were studied by western blot and through a primary neuron culture. A luciferase assay was performed to confirm the binding of miRNAs 212/132 to mecp2. RESULTS: Morphine exposure decreases miR-212 but upregulates miR-132, as wells as Bdnf and TrkB, and changes the localization of proliferative cells. However, Bdnf expression was downregulated when miRNAs 212/132 and oprm1 were knocked-down. Furthermore, we proved that these miRNAs inhibit mecp2 expression by binding to its mRNA sequence. The described effects were corroborated in a primary neuron culture from zebrafish embryos. CONCLUSIONS: We propose a mechanism in which morphine alters the levels of miRNAs 212/132 increasing Bdnf expression through mecp2 inhibition. oprm1 is also directly involved in this regulation. The present work confirms a relationship between the opioid system and neurotrophins and shows a key role of miR-212 and miR-132 on morphine effects through the regulation of Bdnf pathway. GENERAL SIGNIFICANCE: miRNAs 212/132 are novel regulators of morphine effects on CNS. Oprm1 controls the normal expression of Bdnf.

Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms.

Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide-receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding.

Design, synthesis, pharmacological evaluation and molecular dynamics of ß-amino acids morphan-derivatives as novel ligands for opioid receptors.

Structure-Activity Relationship (SAR) is a current approach in the design of new pharmacological agents. We previously reported the synthesis of a novel analogue of morphine, a 2-azabicyclo[3.3.1]nonane, which contains a ß-amino acid. This bicyclic core exhibits two distinctive chemical handles for further elaboration, which allowed us to create a library of morphan-containing compounds by in silico molecular docking on the µ opioid receptor. Lead candidates were synthesized and biological tests were performed to evaluate their ability to bind to opioid receptors. The four top compounds, three phenyl esters and an N-phenylethyl morphan derivative, were selected for Molecular Dynamics simulations to get topological and thermodynamic information. Aromatic morphan derivatives displayed an interacting domain which fits into a hydrophobic cleft and the effect of the substituents in their affinity was explained by the differences in the calculated binding free energies. Our results indicate that the 3D arrangement of the aromatic ring in the morphine derivatives is not a key issue for a specific ligand - µ receptor interaction. Thus, these morphan derivatives represent a new class of opioid receptor ligands which may be of great use in the clinical practice.

Dysregulation of dopaminergic regulatory mechanisms in the mesolimbic pathway induced by morphine and morphine withdrawal.

Dopamine (DA) is thought to represent a teaching signal and has been implicated in the induction of addictive behaviours. Previously, it has been proposed that the transcription factors Nurr1 and Pitx3, which are critical for transcription of a set of genes involved in DA metabolism in the mesolimbic pathway, are associated with addiction pathology. The aim of our study was to investigate abnormalities in the mesolimbic pathway associated with morphine dependence and withdrawal. Using quantitative real-time PCR, immunofluorescence, HPLC and Western blotting, here we studied the effects of single morphine administration, morphine dependence and morphine withdrawal on Nurr1 and Pitx3 expression as well as on the DA marker tyrosine hydroxylase (TH) and the turnover of DA in the ventral tegmental area (VTA) and/or nucleus accumbens. We showed that the three experimental conditions caused induction of Nurr1 and Pitx3 in the VTA, which correlated with changes in TH expression during chronic morphine administration. Present data also confirmed the colocalization of Nurr1 and Pitx3 with TH-positive neurons in the posterior VTA. Furthermore, during morphine dependence, Nurr1 was detected in the nucleus compartment of VTA TH-positive neurons, whereas Pitx3 was strongly detected in the nucleus of TH-positive neurons after single morphine administration and during morphine withdrawal. The number of TH neurons, number of Nurr1 or Pitx3-positive cells, and the number of TH neurons expressing Nurr1 or Pitx3 were not modified in the subpopulations of DA neurons. Present data provide novel insight into the potential correlation between Nurr1 and Pitx3 and DA neurons plasticity during opiate addiction in the mesolimbic pathway.

Morphine regulates Argonaute 2 and TH expression and activity but not miR-133b in midbrain dopaminergic neurons.

Epigenetic changes such as microRNAs (miRs)/Ago2-induced gene silencing represent complex molecular signature that regulate cellular plasticity. Recent studies showed involvement of miRs and Ago2 in drug addiction. In this study, we show that changes in gene expression induced by morphine and morphine withdrawal occur with concomitant epigenetic modifications in the mesolimbic dopaminergic (DA) pathway [ventral tegmental area (VTA)/nucleus accumbens (NAc) shell], which is critically involved in drug-induced dependence. We found that acute or chronic morphine administration as well as morphine withdrawal did not modify miR-133b messenger RNA (mRNA) expression in the VTA, whereas Ago2 protein levels were decreased and increased in morphine-dependent rats and after morphine withdrawal, respectively. These changes were paralleled with enhanced and decreased NAc tyrosine hydroxylase (TH) protein (an early DA marker) in morphine-dependent rats and after withdrawal, respectively. We also observed changes in TH mRNA expression in the VTA that could be related to Ago2-induced translational repression of TH mRNA during morphine withdrawal. However, the VTA number of TH-positive neurons suffered no alterations after the different treatment. Acute morphine administration produced a marked increase in TH activity and DA turnover in the NAc (shell). In contrast, precipitated morphine withdrawal decreased TH activation and did not change DA turnover. These findings provide new information into the possible correlation between Ago2/miRs complex regulation and DA neurons plasticity during opiate addiction.

Whole-genome expression profile in zebrafish embryos after chronic exposure to morphine: identification of new genes associated with neuronal function and mu opioid receptor expression.

BACKGROUND: A great number of studies have investigated changes induced by morphine exposure in gene expression using several experimental models. In this study, we examined gene expression changes during chronic exposure to morphine during maturation and differentiation of zebrafish CNS. RESULTS: Microarray analysis showed 254 genes whose expression was identified as different by at least 1.3 fold change following chronic morphine exposure as compared to controls. Of these, several novel genes (grb2, copb2, otpb, magi1b, grik-l, bnip4 and sox19b) have been detected for the first time in an experimental animal model treated with morphine. We have also identified a subset of genes (dao.1, wls, bnip4 and camk1γb) differentially expressed by chronic morphine exposure whose expression is related to mu opioid receptor gene expression. Altered expression of copb2, bnip4, sox19b, otpb, dao.1, grik-l and wls is indicative of modified neuronal development, CNS patterning processes, differentiation and dopaminergic neurotransmission, serotonergic signaling pathway, and glutamatergic neurotransmission. The deregulation of camk1γb signaling genes suggests an activation of axonogenesis and dendritogenesis. CONCLUSIONS: Our study identified different functional classes of genes and individual candidates involved in the mechanisms underlying susceptibility to morphine actions related to CNS development. These results open new lines to study the treatment of pain and the molecular mechanisms involved in addiction. We also found a set of zebrafish-specific morphine-induced genes, which may be putative targets in human models for addiction and pain processes.

In vivo regulation of NGF-mediated functions by Nedd4-2 ubiquitination of TrkA.

Trk neurotrophin receptor ubiquitination in response to ligand activation regulates signaling, trafficking, and degradation of the receptors. However, the in vivo consequences of Trk ubiquitination remain to be addressed. We have developed a mouse model with a mutation in the TrkA neurotrophin receptor (P782S) that results in reduced ubiquitination due to a lack of binding to the E3 ubiquitin ligase, Nedd4-2. In vivo analyses of TrkAP782S indicate that defective ubiquitination of the TrkA mutant results in an altered trafficking and degradation of the receptor that affects the survival of sensory neurons. The dorsal root ganglia from the TrkAP782S knock-in mice display an increased number of neurons expressing CGRP and substance P. Moreover, the mutant mice show enhanced sensitivity to thermal and inflammatory pain. Our results indicate that the ubiquitination of the TrkA neurotrophin receptor plays a critical role in NGF-mediated functions, such as neuronal survival and sensitivity to pain.

Morphine modulates cell proliferation through mir133b &mir128 in the neuroblastoma SH-SY5Y cell line.

Neuroblastoma is a childhood cancer with high incidence and high mortality rate. Great efforts are made to find new treatments and molecular markers for diagnosis and prognosis. miRNAs stand for novel strategies to modulate tumor growth, as they can act either as tumor suppressors or as oncogenes. Morphine is an opioid agonist widely used to treat severe and chronic pain, as for example cancer pain. Previous studies have revealed that morphine is able to modify cancer progression, by acting on proliferation or on apoptosis; however, up to date, the available results are contradictory, maybe due to the different doses used, routes of administration and model systems. While some studies show that morphine promotes cell proliferation and metastasis, other authors sustain that morphine effect is mainly antiproliferative and pro-apoptotic. In this study we aim to establish the effect of chronic opiate administration on cell proliferation in the neuroblastoma SH-SY5Y cell line. Low doses of morphine (10nM) promoted cell proliferation in undifferentiated cells and reduced the expression levels of miR133b, while higher doses (1µM) inhibited cell proliferation and correlated with decreased levels of miR133b and miR128 without triggering apoptosis. Naloxone, the classical opioid antagonist, could not fully block the effect of morphine on miR128 expression, so that the observed effect may be mediated by non-opioid mechanisms. Our results represent a further contribution to the hypothesis that a joint regulation of miRNA networks and the specific characteristics of the target tissue may determine the effect of morphine on tumor cell growth.

In vivo regulation of the µ opioid receptor: role of the endogenous opioid agents.

It is well known that genotypic differences can account for the subject-specific responses to opiate administration. In this regard, the basal activity of the endogenous system (either at the receptor or ligand level) can modulate the effects of exogenous agonists as morphine and vice versa. The µ opioid receptor from zebrafish, dre-oprm1, binds endogenous peptides and morphine with similar affinities. Morphine administration during development altered the expression of the endogenous opioid propeptides proenkephalins and proopiomelanocortin. Treatment with opioid peptides (Met-enkephalin [Met-ENK], Met-enkephalin-Gly-Tyr [MEGY] and ß-endorphin [ß-END]) modulated dre-oprm1 expression during development. Knocking down the dre-oprm1 gene significantly modified the mRNA expression of the penk and pomc genes, thus indicating that oprm1 is involved in shaping penk and pomc expression. In addition, the absence of a functional oprm1 clearly disrupted the embryonic development, since proliferation was disorganized in the central nervous system of oprm1-morphant embryos: mitotic cells were found widespread through the optic tectum and were not restricted to the proliferative areas of the mid- and hindbrain. Transferase-mediated dUTP nick-end labeling (TUNEL) staining revealed that the number of apoptotic cells in the central nervous system (CNS) of morphants was clearly increased at 24-h postfertilization. These findings clarify the role of the endogenous opioid system in CNS development. Our results will also help unravel the complex feedback loops that modulate opioid activity and that may be involved in establishing a coordinated expression of both receptors and endogenous ligands. Further knowledge of the complex interactions between the opioid system and analgesic drugs will provide insights that may be relevant for analgesic therapy.

Expression of tachykinin receptors (tacr1a and tacr1b) in zebrafish: influence of cocaine and opioid receptors.

Opioid and tachykinin receptors (TACRs) are closely related in addiction and pain processes. In zebrafish, opioid receptors have been cloned and characterized both biochemically and pharmacologically. However, the tacr1 gene has not yet been described in zebrafish. The aim of this research was to identify the tacr1 gene, study the effects of cocaine on tacr1, and analyze the interaction between tacr1 and opioid receptors. We have identified a duplicate of tacr1 gene in zebrafish, designated as tacr1a and tacr1b. Phylogenetic analyses revealed an alignment of these receptors in the Tacr1 fish cluster, with a clear distinction from other TACR1s of amphibians, birds, and mammals. Our qPCR results showed that tacr1a and tacr1b mRNAs are expressed during embryonic development. Whole-mount in situ hybridization showed tacr1 expression in the CNS and in the peripheral tissues. Cocaine (1.5 µM) induced an upregulation of tacr1a and tacr1b at 24 and 48 h post-fertilization (hpf; except for tacr1a at 48 hpf, which was downregulated). By contrast, HEK-293 cells transfected with tacr1a and tacr1b and exposed to cocaine showed a downregulation of tacr1s. The knockdown of ZfDOR2 and ZfMOR, opioid receptors, induced a down- and upregulation of tacr1a and tacr1b respectively. In conclusion, tacr1a and tacr1b in zebrafish are widely expressed throughout the CNS and peripherally, suggesting a critical role of these tacr1s during embryogenesis. tacr1a and tacr1b mRNA expression is altered by cocaine exposure and by the knockdown of opioid receptors. Thus, zebrafish can provide clues for a better understanding of the relationship between tachykinin and opioid receptors in pain and addiction during embryonic development.

Cocaine modulates the expression of opioid receptors and miR-let-7d in zebrafish embryos.

Prenatal exposure to cocaine, in mammals, has been shown to interfere with the expression of opioid receptors, which can have repercussions in its activity. Likewise, microRNAs, such as let-7, have been shown to regulate the expression of opioid receptors and hence their functions in mammals and in vitro experiments. In light of this, using the zebrafish embryos as a model our aim here was to evaluate the actions of cocaine in the expression of opioid receptors and let-7d miRNA during embryogenesis. In order to determine the effects produced by cocaine on the opioid receptors (zfmor, zfdor1 and zfdor2) and let-7d miRNA (dre-let-7d) and its precursors (dre-let-7d-1 and dre-let-7d-2), embryos were exposed to 1.5 µM cocaine hydrochloride (HCl). Our results revealed that cocaine upregulated dre-let-7d and its precursors, and also increased the expression of zfmor, zfdor1 and zfdor2 during early developmental stages and decreased them in late embryonic stages. The changes observed in the expression of opioid receptors might occur through dre-let-7d, since DNA sequences and the morpholinos of opioid receptors microinjections altered the expression of dre-let-7d and its precursors. Likewise, opioid receptors and dre-let-7d showed similar distributions in the central nervous system (CNS) and at the periphery, pointing to a possible interrelationship between them.In conclusion, the silencing and overexpression of opioid receptors altered the expression of dre-let-7d, which points to the notion that cocaine via dre-let-7 can modulate the expression of opioid receptors. Our study provides new insights into the actions of cocaine during zebrafish embryogenesis, indicating a role of miRNAs, let-7d, in development and its relationship with gene expression of opioid receptors, related to pain and addiction process.

Morphine and microRNA Activity: Is There a Relation with Addiction?

When we talk about drug addiction, we are really dealing with an extremely complex system in which there still remain many unknowns and where many empty spaces or missing links are still present. Recent studies have identified changes in the expression profiles of several specific miRNAs which affect the interactions between these molecules and their targets in various illnesses, including addiction, and which may serve as valuable targets for more efficient therapies. In this review, we summarize results which clearly demonstrate that several morphine-related miRNAs have roles in the mechanisms that define addiction. In this regard, morphine has been shown to have an important role in the regulation of different miRNAs, such as miR-let-7 [which works as a mediator of the movement of the mu opioid receptor (MOR) mRNA into P-bodies, leading to translational repression], miR-23b (involved in linking MOR expression and morphine treatment at the post-transcriptional level), and miR-190 (a key post-transcriptional repressor of neurogenic differentiation, NeuroD). Fentanyl increases NeuroD levels by reducing the amount of miR-190, but morphine does not affect the levels of NeuroD. We also discuss the relationship between morphine, miRNAs, and the immune system, based on the discovery that morphine treatment of monocytes led to a decrease in several anti-HIV miRNAs (mir-28, 125b, 150, and 382). This review is centered on miR-133b and its possible involvement in addiction through the effects of morphine. We establish the importance of miR-133b as a regulatory factor by summarizing its activity in different pathological processes, especially cancer. Using the zebrafish as a research model, we discuss the relationship between mir-133b, the dopaminergic system, and morphine, considering: (1) that morphine modulates the expression of miR-133b and of its target transcript Pitx3, (2) the role of the zebrafish mu opioid receptor (zfMOR) in morphine-induced regulation of miR-133b, which depends on ERK1/2, (3) that morphine regulates miR-133b in hippocampal neurons, and (4) the role of delta opioid receptors in morphine-induced regulation of miR-133b. We conclude that the control of miR-133b levels may be a mechanism for the development of addiction to morphine, or other drugs of abuse that increase dopaminergic levels in the extracellular space. These results show that miR-133b is a possible new target for the design of new treatments against addictive disorders.

Modulation by cocaine of dopamine receptors through miRNA-133b in zebrafish embryos.

The use of cocaine during pregnancy can affect the mother and indirectly might alter the development of the embryo/foetus. Accordingly, in the present work our aim was to study in vivo (in zebrafish embryos) the effects of cocaine on the expression of dopamine receptors and on miR-133b. These embryos were exposed to cocaine hydrochloride (HCl) at 5 hours post-fertilization (hpf) and were then collected at 8, 16, 24, 48 and 72 hpf to study the expression of dopamine receptors, drd1, drd2a, drd2b and drd3, by quantitative real time PCR (qPCR) and in situ hybridization (ISH, only at 24 hpf). Our results indicate that cocaine alters the expression of the genes studied, depending on the stage of the developing embryo and the type of dopamine receptor. We found that cocaine reduced the expression of miR-133b at 24 and 48 hpf in the central nervous system (CNS) and at the periphery by qPCR and also that the spatial distribution of miR-133b was mainly seen in somites, a finding that suggests the involvement of miR-133b in the development of the skeletal muscle. In contrast, at the level of the CNS miR-133b had a weak and moderate expression at 24 and 48 hpf. We also analysed the interaction of miR-133b with the Pitx3 and Pitx3 target genes drd2a and drd2b, tyrosine hydroxylase (th) and dopamine transporter (dat) by microinjection of the Pitx3-3'UTR sequence. Microinjection of Pitx3-3'UTR affected the expression of pitx3, drd2a, drd2b, th and dat. In conclusion, in the present work we describe a possible mechanism to account for cocaine activity by controlling miR-133b transcription in zebrafish. Via miR-133b cocaine would modulate the expression of pitx3 and subsequently of dopamine receptors, dat and th. These results indicate that miRNAs can play an important role during embryogenesis and in drug addiction.

Synthesis, biological evaluation and structural characterization of novel glycopeptide analogues of nociceptin N/OFQ.

To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues, namely: [Thr(5)-O-α-D-GalNAc-N/OFQ] (glycopeptide 1), [Ser(10)-O-α-D-GalNAc]-N/OFQ (glycopeptide 2) and [Ser(10)-O-ß-D-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological activity in competition binding assays using the zebrafish animal model in which glycopeptide 2 exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic environments a certain proportion of the molecules of all these peptides exist as α-helix structures. Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin analogues showing linear helical structures are more complementary and thus interact more efficiently with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly reduced binding affinity for the NOP receptor.

Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).

The nociceptin receptor (NOP) and its endogenous ligand, nociceptin/orphanin FQ (OFQ), are involved in a wide range of biological functions, such as pain, anxiety, learning, and memory. The zebrafish has been proposed as a candidate to study the in vivo effects of several drugs of abuse and to discover new pharmacological targets. We report the cloning, expression, and pharmacological characterization of a NOP receptor from zebrafish (drNOP). The full-length cDNA codes a protein of 363 residues, which shows high sequence similarity to other NOPs. Phylogenetic analysis indicates that NOPs are broadly conserved during vertebrate evolution, and that they stand for the most divergent clade of the opioid/OFQ receptor family. Expression studies have revealed that drNOP mRNA is highly expressed in the central nervous system, and low expression levels are also found in peripheral tissues such as gills, muscle, and liver. Pharmacological analysis indicates that drNOP displays specific and saturable binding for [Leucyl-3,4,5-(3)H]nociceptin, with a K(d)=0.20 ± 0.02 nM and a B(max)=1703 ± 81 fmol/mg protein. [(3)H]Nociceptin binding is displaced by several opioid ligands such as dynorphin A (DYN A), naloxone, bremazocine, or the κ-selective antagonist nor-binaltorphimine. [(35)S]GTPγS stimulation studies showed that drNOP receptor is functional, as nociceptin is able to fully activate the receptor and DYN A behaves as a partial agonist (50% stimulation). Our results indicate that drNOP receptor displays mixed characteristics of both NOP and κ opioid receptors. Hence, drNOP, which has retained more of the likely ancestral features, bridges the gap between nociceptin and opiate pharmacology.