Chronic postoperative pain impacts millions of individuals worldwide that undergo a variety of surgical procedures. Opioids remain the mainstay analgesics of acute and perioperative pain; however, prolonged opioid therapy may lead to life-threating adverse effects, tolerance, dependence, and addiction. Therefore, unraveling the cellular mechanisms that drive persistent pain states and opposing endogenous analgesia provided by opioid receptor signaling, may lead to novel analgesics. Evidence suggests that tissue injury leads to increased sensitization of the spinal cord nociceptive neurons which increases susceptibility to chronic pain via an N-methyl-D-aspartate (NMDA) receptor activation of calcium-sensitive adenylyl cyclase isoform 1 (AC1). This phenomenon, named latent pain sensitization (LS), is mediated by a compensatory response of endogenous inhibitory systems. In this dissertation, we test the hypothesis that surgical insult promotes prolonged activation of kappa opioid receptors (KOR) which mask LS via attenuation of pro-nociceptive AC1 signaling pathways in both male and female animals. We employed a murine model of chronic postoperative pain that promotes LS in the spinal cord and closely resembles the phenotypic features of postoperative pain in human subjects. When behavioral signs of hyperalgesia resolved, we targeted spinal opioid receptor systems and pronociceptive modulators with intrathecal delivery of selective pharmacological antagonists and assessed behavioral signs of hyperalgesia and spinal nociceptive sensitization. We propose that LS is kept in remission by a long-lasting compensatory response of tonic endogenous KOR signaling that hinders a pronociceptive LS pathway that includes not only AC1 but also two downstream targets: protein kinase A (PKA) and exchange protein activated by cAMP (Epac1/2) - in a sex-dependent manner. Our results propose new therapeutic targets for the management of persistent postoperative pain and underscore the importance of tailoring sex-specific pain management strategies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22333号 / 医博第4574号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 萩原 正敏, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

It has recently been discovered that the effect of morphine can significantly reduce the tissue damage that occurs during myocardial ischemia. The molecular mechanisms by which morphine acts on the heart are still little understood. The aim of this thesis was to monitor the effect of chronic 27-day and 10-day administration of low (1 mg/kg/day) and high (10 mg/kg/ day) doses of morphine on the expression of selected G-protein-coupled receptors (GPCR) and on the expression and activity of adenylyl cyclase (AC). Chronic (27 days) morphine treatment reduced the expression of к-opioids receptors, but 10-day morphine exposure did not influence the expression of these receptors. Assessment of β1- and β2-AR by immunoblot technique did not show any significant change in the expression, but the more accurate determination of β-AR expression using the saturation binding studies revealed that 27-day treatment with high doses of morphine appreciable increased the total number of these receptors. Administration of high doses of morphine led to marked up-regulation of adenylyl cyclase (AC) isoforms V/VI, and the amount of AC decreased proportionally with the time of discontinuation of morphine administration. Low doses of morphine up- regulated AC only during 27-day administration. Chronic morphine exposure did...

Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...

The D3 dopamine receptor (D3R) belongs to D2-class of dopamine receptors (DARs) and is involved in emotion, movement, and reward. D3R dysfunction has been reported in some neuropsychiatric disorders such as addiction, cognitive deficits, depression, schizophrenia, and Parkinson’s disease. Genetic studies have shown two polymorphic variants of the D3R gene resulting from substitution of serine to glycine at position nine of the amino terminus. Isoform 5 of adenylyl cyclase (AC5) is one of the nine transmembrane bound ACs in the brain and myocardium. Previous studies in rats have shown that AC5 is expressed in the striatum, nucleus accumbens and olfactory tubercle and at lower levels in islands of Calleja, where the D3R is also expressed. Previous studies showed that although D2R and D4R inhibit ACs activity in different cell types, inhibition of ACs by D3R is weak and often undetectable. It has been shown that D3R selectively inhibits AC5 activity in human embryonic kidney 293 (HEK293) cells co-transfected with D3R and AC5. Co-expression of D3R and AC5 in brain regions which are major coordinators of normal and pathological movement, and the selective inhibition of AC5 activity by D3R raise the possibility of a functional link between AC5 and D3R in the modulation of signal transduction and trafficking. I hypothesized that AC5 plays a unique role in modulation of D3R trafficking and signaling pathways through interaction between D3R and AC5. Herein, I demonstrated an interaction between D3R and AC5 in vivo and in vitro using reciprocal co-immunoprecipitation/immunoblotting (co-IP/IB) assays. Interestingly, DA may facilitate the formation of protein complex between D3R and AC5 in vitro. Radio ligand binding assays revealed that heterodimerization of D3R polymorphic variants with AC5 does not change ligand binding affinity and expression of the D3R. Furthermore, taking advantages of GloSensor assays, selective inhibition of AC5 activity by D3Ser9 and D3Gly9 has been shown following activation by DA and quinpirole. Using ELISA studies showed that AC5 promotes cell surface expression and total expression of D3Ser9 and D3Gly9. Moreover, ELISA results suggested that AC5 facilitates DA-induced D3Ser9 endocytosis in dynamin and β-arrestin 2 dependent process, while having no effect on D3Gly9 polymorphic variant. The results also revealed that AC5 attenuates heterologous (PKC-induced) internalization of D3Ser9, while it does not have any effect on D3Gly9 heterologous internalization. My results also displayed a complex formation between D3R, AC5 and, β-arrestin 2 under basal and DA stimulation conditions, which emphasize the role of β-arrestin 2 in D3R signal transduction. Overall, a new regulatory mechanism for D3R has been suggested. My results suggested that complex formation between both D3R polymorphic variants with AC5 can regulate signaling and trafficking properties of D3R without changing the binding affinity of the receptor. These data will be meaningful for understanding of diseases and developing treatment strategies.

No description available.

Le récepteur aux cannabinoïdes de type 1 (CB1) est un récepteur couplé aux protéines G, abondamment exprimé dans le cerveau et régulant plusieurs processus physiologiques. Cependant, les mécanismes cellulaires par lesquels les CB1 régulent ces processus n’ont été que peu analysés. Bien que les CB1 localisés dans les membranes plasmiques sont connus pour induire la transduction de signal; une partie de ces récepteurs sont aussi fonctionnels au niveau des mitochondries (mtCB1), où leur stimulation réduit la respiration mitochondriale. L’objectif de cette thèse fut d’évaluer l’impact de l’activation des récepteurs mtCB1 du cerveau sur les effets connus des cannabinoïdes. Afin de distinguer la fonction des mtCB1 de celle des autres populations de récepteurs, nous avons développé des outils basés sur la signalisation induite par les mtCB1. Dans les mitochondries isolées de cerveau, l’activation des protéines Gαi/o, dépendante des mtCB1 diminue l’activité de l’adénylyl cyclase soluble (sAC). L'inhibition locale de l’activité de sAC prévient l’amnésie, la catalepsie et partiellement l’hypolocomotion induite par les cannabinoïdes. De plus, nous avons généré une protéine fonctionnelle mutante CB1 (DN22-CB1) dépourvue des 22 premiers acides aminés des CB1 ainsi que de sa localisation mitochondriale. Contrairement aux CB1, l'activation des DN22-CB1 n’affecte pas l'activité mitochondriale. Enfin, l’expression des DN22-CB1 dans l’hippocampe bloque à la fois la diminution de la transmission synaptique et l’amnésie induites par les cannabinoïdes. Ces travaux démontrent l’implication des mtCB1 dans certains effets des cannabinoïdes et le rôle clé des processus bioénergétiques contrôlant les fonctions cérébrales. / Type-1 cannabinoid receptor CB1 is a G protein-coupled receptor (GPCR), widely expressed in the brain, which regulates numerous physiological processes. However, the cellular mechanisms of CB1-mediated control of these functions are poorly understood. Although CB1 are known to signal at the plasma membrane, a portion of these receptors are also present in mitochondria (mtCB1), where mtCB1 activation decreases mitochondrial activity. The goal of this thesis was to dissect the impact of brain mtCB1 signaling in known behavioral effects induced by cannabinoids. To distinguish the functions of mtCB1 from other receptor pools, we developed tools based on the characterization of the intra-mitochondrial molecular cascade induced by mtCB1 receptors. In isolated brain mitochondria, we found that intra-mitochondrial decrease of soluble-adenylyl cyclase (sAC) activity links mtCB1- dependent activation of Gαi/o proteins to decrease cellular respiration. Local brain inhibition of sAC activity blocks cannabinoid-induced amnesia, catalepsy and contributes to the hypolocomotor effect of cannabinoids. In addition, we generated a functional mutant CB1 protein (DN22-CB1) lacking the first 22 amino acid of CB1 and its mitochondrial localization. Differently from CB1, activation of DN22-CB1 does not affect mitochondrial activity. Hippocampal in vivo expression of DN22-CB1 abolished both cannabinoid-induced impairment of synaptic transmission and amnesia in mice. Together, these studies couple mitochondrial activity to behavioral performances. The involvement of mtCB1 in the effects of cannabinoids on memory and motor control highlights the key role of bioenergetic processes as regulators of brain functions.

Dopamine (DA) receptor supersensitivity refers to the phenomenon of an enhanced physiological, behavioral or biochemical response to a DA agonist. Literature related to ontogenetic aspects of this process was reviewed. Neonatal 6-hydroxydopamine (6-OHDA) destruction of rat brain DA neurons produces overt sensitization to D1 agonist-induced oral activity, overt sensitization of some D2 agonist-induced stereotyped behaviors and latent sensitization of D1 agonist-induced locomotor and some stereotyped behaviors. This last process is unmasked by repeated treatments with D1 (homologous "priming") or D2 (heterologous "priming") agonists. A serotonin (5-HT) neurotoxin (5,7-dihydroxytryptamine) and 5-HT2C receptor antagonist (mianserin) attenuate some enhanced behavioral effects of D1 agonists, indicating that 5-HT neurochemical systems influence D1 receptor sensitization. Unlike the relative absence of change in brain D1 receptor number, DA D2 receptor proliferation accompanies D2 sensitization in neonatal 6-OHDA-lesioned rats. Robust D2 receptor supersensitization can also be induced in intact rats by repeated treatments in ontogeny with the D2 agonist quinpirole. In these rats quinpirole treatments produce vertical jumping at 3-5 wk after birth and subsequent enhanced quinpirole-induced antinociception and yawning. The latter is thought to represent D3 receptor sensitization. Except for enhanced D1 agonist-induced expression of c-fos, there are no changes in the receptor or receptor-mediated processes which account for receptor sensitization. Adaptive mechanisms by multiple "in series" neurons with different neurotransmitters may account for the phenomenon known as receptor supersensitivity.

La douleur chronique est une maladie fréquente et grave qui, pour de nombreuses personnes, ne peut pas être entièrement traitée avec les choix thérapeutiques actuels. Des agonistes des récepteurs opioïdes delta (DORs) ont été proposés comme interventions thérapeutiques pour ces maladies. Des recherches précliniques ont montré que l'activation des DOR produit des effets anti-hyperalgiques et antidépresseurs avec moins d'effets secondaires associés aux analgésiques opioïdes disponibles sur le plan clinique. Cependant, de nombreux agonistes DOR induisent une tolérance analgésique, entravant ainsi leur développement en tant que médicaments. Les travaux de cette thèse visent à mieux comprendre les causes cellulaires et moléculaires de la tolérance ainsi que ce qui rend certains agonistes plus résistants à la tolérance que d'autres. Dans le premier projet, nous nous sommes concentrés sur la superactivation de l'adénylyl cyclase induite par un ligand, un modèle de réponse adaptative médiée par les isoformes de l'adénylyl cyclase (AC). La superactivation de l'adénylyl cyclase (SA) a été associée à l’hyperalgésie, la tolérance analgésique et à des symptômes de sevrage. Ainsi, nous étions curieux de voir si les profils de signalisation cellulaire créés pour la découverte de médicaments pouvaient nous fournir des informations sur la capacité d'un ligand à induire la SA. Pour répondre à cette question, nous avons généré des profils de signalisation complets pour six agonistes différents du DORs (Met-enképhaline, deltorphine II, DPDPE, SNC-80, ARM390 et TIPP) tout en surveillant 12 différents résultats de signalisation avec des biocapteurs à base de BRET. L'analyse des profils de signalisation a montré une sélectivité fonctionnelle remarquable parmi les ligands étudiés. Ensuite, nous avons pu classer les agonistes DOR en fonction de la similarité de leurs profils en utilisant l'approche que nous avons adaptée de notre laboratoire. Nous avons par la suite démontré que, à l'exception de TIPP, dont la réponse SA était Ca2+-indépendante, les catégories de médicaments résultant du regroupement sont corrélées avec la capacité du ligand à provoquer une SA. Une investigation plus approfondie des mécanismes a révélé que Gαi/o était essentiel tant pour la SA déclenchée par TIPP que par Met-Enkepkaline, mais les mécanismes en aval étaient assez distincts pour ces ligands. Ensemble, nos résultats indiquent que les mécanismes sous-jacents à la tolérance cellulaire induite par les agonistes DOR sont spécifiques au ligand. Dans le deuxième projet, nous nous sommes principalement intéressés aux mécanismes de tolérance aux agonistes DOR qui peuvent être en partie expliqués par la désensibilisation et la régulation négative des récepteurs. Il a été établi que, les ligands qui induisent le recyclage du récepteur après l'internalisation ont été trouvés pour fournir une analgésie de longue durée. Par conséquent, les expériences menées dans cette étude ont été menées pour révéler davantage les déterminants moléculaires sous-jacents au recyclage du récepteur et sur la manière dont l'interaction agoniste-récepteur pourrait produire des modèles distincts de régulation des récepteurs. Nous avons évalué l'activation de l'agoniste et la désensibilisation du signal DOR-Gαi1. Nos données ont rapporté que le DPDPE était pratiquement sans effet sur la désensibilisation de l'activation de Gαi1, tandis que la désensibilisation par la deltorphine II était plus importante que celle induite par le DPDPE mais moins que celle induite par l'ARM390 et le SNC-80. Ensuite, nous avons établi que les DORs stimulés par le DPDPE se recyclaient de manière plus efficace que ceux activés par la deltorphine II. De plus, nous fournissons des preuves phénoménologiques que des interventions similaires ont des effets distincts sur le recyclage évoqué par chaque ligand. En particulier, la truncation du DOR ou la surexpression de βarr2 avaient des effets différentiels sur le recyclage par le DPDPE et la deltorphine II. Il est admis que les mécanismes sous-jacents à ces différences restent à être pleinement décrits, mais la phénoménologie de nos observations soutient l'idée que le DPDPE et la deltorphine II mettent en œuvre des processus de recyclage distincts. / Chronic pain is a common and severe disease that, for many people, cannot be fully treated with current therapeutic choices. Agonists of the delta opioid receptor (DOR) have been proposed as therapeutic interventions for this illness. Preclinical research has shown that DORs produce antihyperalgesic and antidepressant-like effects with fewer side effects than the ones associated with clinically available opioid analgesics. However, numerous DOR agonists induce analgesic tolerance, hampering their development as medications. Thus, further investigations are needed to understand the mechanisms underlying the tolerance associated with chronic opioid use. This thesis aimed to further understand the cellular and molecular mechanisms that causes tolerance as well as what makes some agonists more resistant to tolerance than others. In the first project, we focused on ligand-induced cyclase superactivation (SA), a pattern of adaptive response mediated by adenylyl cyclase (AC) isoforms. Cyclase SA has been associated with hyperalgesia, analgesic tolerance, and withdrawal symptoms. Therefore, we were curious to assess weather cell-based signalling profiles created for drug discovery could provide us with information on the ability of a ligand to induce cyclase SA. To address this question, we generated comprehensive signalling profiles for six different DOR agonists (Met-enkephalin, deltorphin II, DPDPE, SNC-80 and ARM390) while monitoring 12 different signalling outcomes with BRET-based biosensors. Analysis of the signalling profiles showed remarkable functional selectivity among the investigated ligands. Next, we were able to classify DOR agonists based on the similarity of their profiles using the approach we adapted from our lab. We subsequently demonstrated that except for TIPP, whose SA response was Ca2+-independent, the drug categories resulting from clustering are correlated with ligand capacity to cause SA. Further investigation of the mechanisms revealed that Gαi/o was essential for both TIPP and Met-Enkepkalin-driven cyclase SA. However, downstream mechanisms were quite distinct for these two ligands. Altogether, our findings indicate that mechanisms underlying cellular tolerance induced by DOR agonists are ligand-specific. In the second project, we were primarily concerned with the mechanisms of tolerance to DOR agonists that may be, in part, explained the receptor desensitization and downregulation. Obviously, ligands that induce receptor recycling after internalization have been found to provide long-lasting analgesia. Therefore, the objectives of the experiments in this project were to assess the molecular determinants affecting receptor recycling and how agonist-receptor interaction can result in different patterns of receptor regulation. We assessed agonist inducing activation and desensitization of DOR-Gαi1 signal. Our data showed that DPDPE was efficient in activating the receptor without noticeable desensitization effect. On the other hand, deltorphin II exerted a significant desensitization effect. However, this effect was low when compared to ARM390 and SNC-80. Then, we established that DORs stimulated by DPDPE recycle more efficiently than those activated by deltorphin II. We also provided phenomenological evidence on receptor recycling elicited by each ligand. In particular, DOR truncation or the overexpression of βarr2 had differential effects on receptor recycling by DPDPE and deltorphin II. While our data shed light on the mechanism underlying these differences, further investigation is needed for the mechanism to be fully elucidated. Admittedly, our observations support the notion that DPDPE and deltorphin II engage distinct recycling processes.

The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....