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Delta opioid receptor expression in various models of chronic clinical conditions / Expression du récepteur aux opioïdes delta dans différents modèles de pathologies chroniquesCeredig, Rhian Alice 23 June 2016 (has links)
Les travaux présentés ici visent à déterminer l’implication du récepteur aux opioïdes delta dans des modèles de pathologies chroniques telles que la douleur chronique et l’administration d’opiacés. Nous avons mis en oeuvre des approches génétiques, d’imagerie et comportementales afin de décrire précisément les changements de distribution neuronale du récepteur aux opioïdes delta dans un modèle de douleur neuropathique et dans l’administration chronique de morphine, dans les tissus du système nerveux central et périphérique. Nous avons étudié l’implication des récepteurs aux opioïdes delta périphériques dans l’effet thérapeutique de traitements antiallodyniques dans un modèle de douleur neuropathique, et examiné le rôle des récepteurs aux opioïdes delta dans la sensibilité viscérale et dans les effets thérapeutiques de la Prégabaline. Nos travaux ont permis de décrire précisément les changements et l’implication du récepteur aux opioïdes delta dans plusieurs modèles de pathologies chroniques, dans le but de dégager des pistes thérapeutiques futures. / In this work, we used genetic, imaging and behavioral approaches to describe the changes which the distribution of the delta opioid receptor underwent in models of clinical conditions such as neuropathic pain and chronic opioid exposure, at the peripheral and supraspinal levels. We investigated the role of peripheral delta opioid receptor populations in the antiallodynic effect of chronic treatment by antidepressant and β2 agonist molecules in a model of neuropathic pain. We also described the implication of delta opioid receptors in visceral sensitivity, and their involvement in the pain-relieving effects of Pregabalin in a model of neuropathic pain. Thus, we have brought insight as to the role of delta opioid receptors in these various clinical conditions, and thoroughly described the distribution changes; which may lead the way to therapeutic strategies to treat chronic pain or drug addiction.
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Mechanisms of Channel Arrest and Spike Arrest Underlying Metabolic Depression and the Remarkable Anoxia-tolerance of the Freshwater Western Painted Turtle (Chrysemys picta bellii)Pamenter, Matthew 26 February 2009 (has links)
Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.
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Mechanisms of Channel Arrest and Spike Arrest Underlying Metabolic Depression and the Remarkable Anoxia-tolerance of the Freshwater Western Painted Turtle (Chrysemys picta bellii)Pamenter, Matthew 26 February 2009 (has links)
Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.
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Étude du trafic du récepteur delta-opiacé suite à sa stimulation par différents agonistesCharfi, Iness 06 1900 (has links)
Les opiacés figurent parmi les analgésiques les plus puissants pour le traitement des douleurs sévères. Les agonistes du DOR (récepteur delta opiacé) induisent moins d'effets secondaires que ceux du mu, ce qui les rend une cible d'intérêt pour le traitement des douleurs chroniques. Cependant, ils induisent la tolérance à l'analgésie. Des hypothèses récentes proposent que le potentiel des drogues à induire la tolérance soit la conséquence de la stabilisation de différentes conformations du récepteur induites par la liaison avec différents ligands, chacune ayant différentes propriétés de trafic. Dans ce contexte, nous avons déterminé si différents ligands du DOR différaient dans leur capacité à induire la signalisation et le trafic du récepteur. Nos résultats indiquent que DPDPE et SNC-80 sont les drogues les plus efficaces à inhiber la production d’AMPc, suivis par UFP-512, morphine et TIPP. DPDPE et SNC-80 induisent à eux seuls l’internalisation du DOR dans les cellules HEK-293 de façon dépendante de la β-arrestine mais pas de la GRK2 ni PKC. Ces deux drogues induisent également l’internalisation du DOR dans les neurones corticaux et c’est seulement le DPDPE qui permet au DOR de regagner la membrane des cellules HEK-293 et des neurones après récupération. Cette capacité de recyclage était suggérée comme un mécanisme protégeant contre la survenue de la tolérance. Ces observations indiquent que le DOR peut subir différentes régulations en fonction du ligand lui étant associé. Cette propriété de sélectivité fonctionnelle des ligands pourrait être utile pour le développement de nouveaux opiacés ayant une activité analgésique plus durable. / Opiates are among the most powerful painkillers to treat severe pain. Delta opioid receptor (DOR) agonists induce fewer side effects than mu opioid receptor agonists, which makes them a target of interest for the treatment of chronic pain. However, they induce tolerance to analgesia. Recent hypotheses suggest that drugs tolerance is the result of stabilization of ligand-specific conformations of the receptor, with distinct traffic properties such as internalization and/or recycling. In this context, we determined whether different DOR ligands differed with respect to their ability to induce signaling and receptor trafficking. Our results indicate that DPDPE and SNC-80 are the most effective drugs to inhibit the production of cAMP, followed by UFP-512, morphine and TIPP. Only DPDPE and SNC-80 manage to induce DOR internalization in HEK-293 cells. This effect is dependent on β-arrestin but not on GRK2 or PKC. Of these two internalizing agonists, only DPDPE allows the DOR to recycle back to the membrane of HEK-293 cells after recovery. DPDPE and SNC-80 also trigger similar DOR internalization in cortical neurons, and as observed in HEK293 cells only DPDPE allowed the receptor to recycle back to the membrane. This recycling capacity was suggested as a mechanism to protect against the onset of tolerance. These observations indicate that the DOR can undergo different regulations depending on the ligand bound to it. This property of functional selectivity of DOR ligands could be useful for the development of new opiates with longer lasting analgesic properties.
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The chemistry of Salvia divinorumMunro, Thomas Anthony Unknown Date (has links) (PDF)
Salvia divinorum is a hallucinogenic sage used to treat illness by the Mazatec Indians of Mexico. Salvinorin A (1a), a neoclerodane diterpenoid isolated from the plant, is a potent, selective agonist at the kappa opioid receptor (KOR), and is the first non-nitrogenous opioid. The plant is used recreationally as a hallucinogen, but is unpopular due to its dysphoric effects. 1a has been prohibited in Australia under an invalid systematic name. An early report of psychoactive alkaloids in S. divinorum proved to be irreproducible. Similarly, tests in mice suggesting the presence of psychoactive compounds other than 1a were confounded and therefore unreliable. In this work, an improved isolation method for 1a was developed, using filtration through activated carbon to decolourise the crude extract. Six new diterpenoids were isolated: salvinorins D–F (1d–1f) and divinatorins A–C (28a–28c). Five known terpenoids not previously reported from this species were also isolated. The structure–activity relationships of 1a were evaluated via selective modifications of each functional group. Useful synthetic methods are reviewed, including the first thorough review of furanolactone hydrogenations. Testing of the derivatives at the KOR suggests that the methyl ester and furan ring of 1a are required for activity, but that the lactone and ketone functionalities are not. Other compounds from S. divinorum did not bind to the KOR, suggesting that 1a is the plant’s active principle.
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Mu opioid receptors and neuronal circuits of addiction : genetic approaches in mice / Récepteurs opioïdes mu et circuits neuronaux de l'addiction : approches génétiques chez la sourisCharbogne, Pauline 09 July 2015 (has links)
Le récepteur opioïde mu est responsable des propriétés analgésiques et addictives puissantes de la morphine et de l’héroïne, mais son mode d’action à l’échelle des circuits neuronaux est mal connu et a été peu étudié par des approches génétiques. Le récepteur mu est largement exprimé dans le système nerveux, essentiellement dans des neurones GABAergiques. Le premier objectif de mon projet a été d’inactiver le gène codant pour le récepteur mu dans les neurones GABAergiques du cerveau antérieur et d’en étudier les conséquences comportementales. Notre étude montre que ces récepteurs ne sont pas impliqués dans l’analgésie et la dépendance physique à la morphine, mais qu’ils sont essentiels à l’effet hyperlocomoteur de l’héroïne. De plus, nos résultats indiquent que ces récepteurs limitent la motivation à consommer de l’héroïne et du chocolat, révélant un rôle entièrement nouveau pour cette population particulière de récepteurs (Manuscrit 1 : Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food). Aussi, cette population de récepteurs mu n’est pas responsable du syndrome autistique décrit chez les souris knockout totales (Manuscrit 2 : Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms). Enfin, nous avons développé un nouveau modèle transgénique visant l’inactivation génétique du récepteur mu dans les neurones glutamatergiques, mais qui n’a pas abouti à un knockout conditionnel détectable. Nous avons aussi initié la création d’une lignée transgénique Cre pour l’inactivation de gènes d’intérêt dans l’amygdale étendue, qui permettra notamment d’étudier le rôle du récepteur mu dans ce microcircuit. / Mu opioid receptors mediate the strong analgesic and addictive properties of morphine and heroin;however mu receptor function at circuit levels is not well understood and has been poorly studied by genetic approaches. These receptors are widely expressed throughout the nervous system, essentially in GABAergic neurons. The first aim of my project was to genetically inactivate the mu receptor gene in GABAergic forebrain neurons and study the behavioral consequences. Our study shows that these mu receptors are not implicated in morphine-induced analgesia and physical dependence, but are essential for locomotor effects of heroin. Moreover, our data show that these receptors inhibit motivation to consume heroin and chocolate, revealing an entirely new role for this particular population of mu receptors (Manuscript 1: Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food). Also, mu receptors expressed in forebrain GABAergic neurons are not responsible for the autistic syndrome described in total mu receptor knockout mice (Manuscript 2: Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms). Finally, we developed a new transgenic model targeting the mu receptor gene in glutamatergic neurons, but receptor deletion was not detectable in conditional mice. We also initiated the creation of a transgenic Cre driver line to knockout genes of interest in the extended amygdala, and this tool will enable us to study mu receptor function within this microcircuit.
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Human δ opioid receptor Phe27 and Cys27 variants:the role of heteromerization and pharmacological chaperones in receptor processing and traffickingLeskelä, T. (Tarja) 29 November 2011 (has links)
Abstract
The opioid receptors (δ, κ and μ) are family A G protein-coupled receptors (GPCRs) that have an important role in the regulation of pain. Like all GPCRs they have a common structure that consists of seven transmembrane domains with an extracellular amino (N)-terminus and an intracellular carboxyl-terminus. The human δ opioid receptor (h(δOR) has two polymorphic variants. A single-nucleotide polymorphism causes replacement of Phe with Cys at the amino acid position 27 in the receptor N-terminus. The allelic frequency of hδORCys27, the less common variant, is about 10% in Caucasians.
In this study, the two hδOR variants were expressed in heterologous expression systems and their biosynthesis was characterized in detail using various cell biological and biochemical techniques. In particular, the role of receptor heteromerization and opioid receptor pharmacological chaperones in processing, maturation and trafficking of the variants was assessed.
The hδOR variants showed significant differences in maturation and trafficking. The hδORCys27 had a significantly lower maturation efficiency compared with hδORPhe27. In addition, long-term receptor expression led to the accumulation of hδORCys27 in the endoplasmic reticulum (ER) and also impaired receptor targeting to ER-associated degradation. The hδOR variants also differed at the cell surface, as the hδORCys27 variant was internalized constitutively in a faster and more extensive manner than hδORPhe27. However, the variants had similar pharmacological properties and activated G proteins in an identical manner.
This study also showed that hδORCys27 acted in a dominant negative manner and redirected some hδORPhe27 precursors to degradation. This resulted in impaired plasma membrane expression of hδORPhe27 in co-transfected cells. The hδOR variants were found to form heteromers early in the secretory pathway, which is the most likely reason for the dominant negative behavior of hδORCys27 on hδORPhe27.
The mechanism of action of opioid receptor pharmacological chaperones, membrane-permeable opioid ligands, was investigated in detail using hδORCys27 and its mutant form hδORCys27-(Asp95Ala) as models. Opioid antagonists were found to be able to bind to and stabilize receptor precursors in the ER and enhance their dissociation from the ER molecular chaperone calnexin. This led to an increase in the number of receptors at the plasma membrane. In addition, hδORPhe27, like hδORCys27, was responsive to antagonist treatment whether the variants were expressed together or individually. / Tiivistelmä
Opioidireseptorit kuuluvat G-proteiinikytkentäisiin reseptoreihin, ja niillä on tärkeä rooli kipuaistimuksen säätelyssä. Ne ovat solukalvoproteiineja, joiden aminohappoketju läpäisee kalvon seitsemän kertaa. Reseptorien aminoterminaalipää sijaitsee solun ulkopuolella ja karboksiterminaalipää solun sisällä. Ihmisen δ-opioidireseptori esiintyy kahtena polymorfisena muotona, Phe27:nä ja Cys27:nä, joissa aminohappo 27 on joko fenyylialaniini (Phe) tai kysteiini (Cys). Cys27 on harvinaisempi muoto, ja sen yleisyys on noin 10 % eurooppalaista alkuperää olevalla väestöllä.
Tämän väitöskirjan tavoitteena oli tutkia δ-opioidireseptorin varianttimuotojen biosynteesiä reseptoriproteiinia tuottavissa heterologisissa solumalleissa (HEK293- ja SH-SY5Y-solut) solubiologisilla ja biokemiallisilla menetelmillä..
Väitöskirja osoittaa, että δ-opioidireseptorin varianttimuotojen välillä on eroa prosessoinnissa. Cys27-varianttia kuljetetaan endoplasmakalvostosta solun pinnalle vähemmän kuin Phe27-varianttia, ja pitkäaikainen reseptorituotanto johtaa vastasyntetisoituneiden reseptorien kerääntymiseen solun sisälle. Samalla reseptorien ohjaus proteasomihajotukseen heikkenee. Soluissa, jotka tuottavat molempia varianttimuotoja samanaikaisesti, Cys27-variantin havaittiin ohjaavan myös Phe27-varianttia proteasomihajotukseen vähentäen sen kuljetusta solun pinnalle. Tämä Cys27-variantin dominanttinegatiivinen ominaisuus johtuu todennäköisesti siitä, että variantit muodostavat dimeerisen rakenteen endoplasmakalvostossa. Havaittiin myös, että Cys27-varianttireseptorit ohjataan solun pinnalta lysosomihajotukseen tehokkaammin kuin vastaavat Phe27-varianttimuodot. Prosessointieroista huolimatta variantit eivät poikkea toisistaan farmakologisilta ominaisuuksiltaan, ja ne aktivoivat G proteiineja samalla tavalla.
Väitöskirjassa tutkittiin myös farmakologisten kaperonien toimintamekanismeja käyttämällä mallina δ-opioidireseptorin Cys27-varianttia ja sen pistemutaatiota (Asp95Ala). Farmakologisten kaperonien eli reseptorispesifisten ligandien todettiin sitoutuvan reseptoreihin endoplasmakalvostossa ja stabiloivan niiden rakennetta, mikä vähentää reseptorin ja proteiinien laadunvalvontaan osallistuvan kaperonin, kalneksiinin, välistä vuorovaikutusta. Tämä johtaa reseptorien määrän kasvuun solun pinnalla.
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Characterization of opioid binding sites in spinal cord and other tissuesWood, Malcolm S. January 1988 (has links)
The binding of [³H]opioid ligands to homogenates prepared from the spinal cords of rat and other species has been studied. Similar numbers of sites were seen in all areas of the cord when measured in a rostrocaudal direction. There was found to be approximately 2 x higher density of sites in the dorsal half of the cord compared with the ventral half. Binding studies suggested a similar relative distribution of mu, delta and kappa sites in all areas of the cord. The results are discussed in relation to the reported distribution of opioid peptides. In the above study the kappa binding site was defined as the binding of [³H] unselective opioids in the presence of cold ligands to suppress binding to mu- and delta-sites. Competitive binding assays, however, suggested this site did not have the properties of a single homogeneous group. Approximately 50% of the apparent kappa binding was consistent with a classical kappa site. Saturated binding assays afforded Bmax values which suggested lower 'true' kappa site numbers than previously supposed, values which were confirmed using the kappa peptide' [³H]Dynorphin A-(1-9), and the kappa selective [³H]U-69593. Heterogeneity was also seen in other central nervous system tissues. The heterogeneous nature of the kappa site may be due to different sites, due to interactions at a non-opioid site or may represent different conformations of the same site. The second possibility was discounted since observed binding followed the cellular distribution of the plasma marker Na+/K+-ATPase was stereoselective for levorphanol over dextrorphan, and fully displaceable by naloxone. The third possibility was investigated by studying the role of Na+ and MG2+ ions, which are reported to affect receptor conformation in binding assays employing brain tissues. None of the results obtained suggested that conformational changes were responsible for the observed effects, although the experiments were not exhaustive.
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Studium membránových receptorů pomocí vazby radioligandů / The study of membrane receptors by radioligands bindingRejhová, Alexandra January 2011 (has links)
Drug addiction, opiates respectively, is a social problem which seriousness is currently on the rise. One of key elements causing addiction is tolerance to increasing doses of drug causing abstinence syndrome during withdrawal and craving. Opioid receptors are members of a large group of receptors coupled with heterotrimeric G-proteins (GPCR), whose properties can be investigated using agonist- stimulated binding [35 S] GTPγS. Many extracellular signals are transferred into a cell through GPCR. Opioid receptor agonists inhibit the activity of adenylyl cyclase and are coupled with G-protein group Gi/Go. This work is devoted to the study of changes in isolated plasma membranes of rat forebrain containing opioid receptors of healthy subjects with membranes acquired from morphine addicted subjects. The rats were long-term morphine treated in increasing doses, to develop the dependency. The comparison is done firstly by binding of [3 H]ouabain to Na,K-ATPase, which proves to be a negative standard of changes, secondly by binding [35 S]GTPγS to G-proteins, thereby providing the functional activity of G-protein in stimulating the binding by the agonist of δ-opioid receptors DADLE or agonist of µ-opioid receptors DAMGO. Furthermore, it has been studied the influence of prostaglandin E1 on binding [35...
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Studium molekulárních interakcí opioidních a TRPV1 receptorů / Studies on molecular interactions of the mu-opioid and TRPV1 receptorsMelkes, Barbora January 2020 (has links)
In this work, we investigated the behavior of the -opioid receptor (MOR) and the transient receptor potential vanilloid 1 (TRPV1) ion channel in the plasma membrane and their mutual communication. Both these receptors are implicated in pain perception and analgesia. We observed that the lateral mobility of MOR was strongly affected by different biased opioid agonists. DAMGO and endomorphin-2 display opposite bias towards MOR. According to our results, they also have the opposite effects on the mobility of MOR. Morphine induced only small changes in the mobility of MOR. Moreover, cholesterol depletion and blockage of G protein signaling by pertussis toxin (PTX) affected the ability of different MOR agonists to alter MOR mobility in a unique manner. The effects of DAMGO and endomorphin-2 were compromised under these conditions. On the other hand, we observed increased movement of MOR after the addition of morphine. PTX alone did not affect receptor movement, but it completely disrupted the effect of cholesterol depletion on morphine induced changes the mobility of MOR. Next we studied the mobility of TRPV1. The TRPV1 agonist capsaicin changed the lateral mobility of TRPV1. Surprisingly, after adding the MOR antagonist naloxone, the apparent diffusion coefficient of TRPV1 but to a lower extent than...
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