Regulation of the human delta opioid receptor (hDOR) is implicated in the development of tolerance to chronic morphine (Zhu et al., 1999). In addition, DORs are promising analgesic targets for the management of chronic pain states such as inflammatory or neuropathic pain (Cahill et al., 2007). Therefore, in this study, we investigated multiple aspects of hDOR regulation, including receptor phosphorylation, beta-arrestin binding, receptor internalization, down-regulation and desensitization, using recombinant Chinese hamster ovary (CHO) cells expressing the wild-type or various mutant hDOR constructs. We found that structurally diverse delta opioid agonists regulate the hDOR by different mechanisms. We demonstrate that morphine is able to activate the initial step of the regulatory events, phosphorylation of S363, but due to requirements for simultaneous activation of multiple sites, morphine fails to promote beta-arrestin binding, receptor internalization and down-regulation. We also report that peptide delta opioid receptor agonists and a non-peptide agonist SNC80 differ in their ability to down-regulate the hDOR. Further differences in receptor phosphorylation, desensitization and beta-arrestin translocation between these two classes of full DOR agonists are reveled by truncation of the receptor's C-terminus or by mutation of the primary phosphorylation site, S363. Studies using the mutant receptors identify the C-terminus as the important domain for hDOR phosphorylation, beta-arrestin binding and down-regulation by both peptide and non-peptide agonists. S363 within the C-terminus is critically involved in receptor phosphorylation, desensitization and down-regulation, but not in beta-arrestin binding and receptor internalization. In contrast to peptide agonists, SNC80 is able to phosphorylate and activate secondary intracellular domain(s), in addition to the C-terminus, which participate in beta-arrestin recruitment and receptor desensitization and down-regulation. Therefore, agonist-specific differences were detected for multiple regulatory events between morphine, peptide agonists and SNC80. Differential agonist-mediated regulation of the human delta opioid receptor may be used to design pain therapy drugs with improved analgesic properties and minimal side effects.

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA. The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event. With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge. Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.</p>

L’utilisation des opioïdes est à l’origine de phénomènes de tolérance et d’hyperalgésie induite (HIO) aussi bien chez l’animal qu’en utilisation clinique. Ces phénomènes surviennent avec tous les opioïdes de manière dose-dépendante. Les mécanismes qui les sous-tendent sont complexes et imparfaitement connus. Le rémifentanil et le sufentanil sont les opioïdes les plus utilisés en France en anesthésie-réanimation. Leur utilisation s’accompagne d’une HIO qui majore la douleur postopératoire et peut être responsable de la persistance de la douleur à long terme. La perception des stimuli nociceptifs chez un patient sous anesthésie générale n’est pas aisée et repose encore sur des signes cliniques indirects d’activation du système sympathique. Ces signes peu sensibles et peu spécifiques conduisent à sous doser ou sur-doser les patients en opioïdes. Récemment, un nouvel outil de monitorage de la nociception est apparu, l’analgesia nociception index (ANI). L’ANI reflète le tonus parasympathique et de ce fait permettrait aux anesthésistes de mieux adapter le dosage des opioïdes. Dans cette thèse, nous avons d’abord évalué la sensibilité et la spécificité de l’ANI à détecter les stimuli nociceptifs, puis montré qu’elles étaient supérieures à celles des signes cliniques, et supérieures à d’autres indices de monitorage proposés. Ensuite nous avons validé la capacité de l’ANI à guider l’analgésie peropératoire du rémifentanil dans différentes situations.Sur le plan expérimental, nous avons exploré, après une exposition courte ou prolongée à différentes doses de rémifentanil et de sufentanil, les mécanismes associés à l’hyperalgésie thermique in vivo, chez la souris, et ex vivo, sur la voie des MAP kinases ERK1/2 et sur le trafic membranaire des récepteurs opioïdes de type µ (MOR) dans différentes cultures cellulaires. Chez la souris, nous avons mis en évidence une hyperalgésie précoce au saut sur plaque chaude, après exposition aux doses les plus élevées de rémifentanil, mais pas avec le sufentanil. De plus, nous n’avons pas observé d’HIO sur le léchage des pattes.Sur les cultures cellulaires, le rémifentanil comme le sufentanil activent la voie des MAPK ERK1/2 lors d’une exposition courte, avec apparition d’une désensibilisation lorsque l’exposition se prolonge. Le rémifentanil comme le sufentanil induisent une internalisation précoce et progressive des récepteurs MOR. / The use of opioids is associated with tolerance and induced hyperalgesia (OIH). Tolerance and OIH occur with all opioids and have been demonstrated both, in animals and in humans and are likely to be dose-dependent. The underlying mechanisms are complex and partially known. Remifentanil and sufentanil are the most used opioids in France in anesthesia and intensive care. Their use is associated with OIH that increases postoperative pain and may be responsible for persistent pain. In anesthetized patients, nociceptive stimuli are still detected according to clinical signs of sympathetic activation. These signs lack sensitivity and specificity and lead to underdosing or overdosing opioids. Recently, the analgesia nociception index (ANI), has been proposed as surrogate marker of nociception. The ANI reflects the parasympathetic tone and thus may allow anesthetists to better adapt the opioid dosage. In this thesis, we first evaluated the sensitivity and specificity of ANI to detect nociceptive stimuli, and showed that it better detects them than do clinical signs or than other currently available monitoring tools. Subsequently, we validated the ability of the ANI to adequately guide the intraoperative dosing of remifentanil in different clinical setting.After acute and sustained exposure to different doses of remifentanil and sufentanil we investigated, in vivo, the mechanisms associated with thermal hyperalgesia in mice, and ex vivo, the effect on the MAP kinase ERK1/2 pathway and the μ-type opioid receptor (MOR) membrane trafficking in human neuroblastoma and embryonic kidney cell cultures. In mice, high-dose remifentanil induced early hyperalgesia assessed by the jumping latency in a hot-plate test, but not the sufentanil. We did not observe OIH for the hind paw licking test. On cell cultures, after short exposure, both remifentanil and sufentanil produced activation of the MAP kinase ERK1/2 pathway, and rapid desensitization and internalization of the MOR.

Indiana University-Purdue University Indianapolis (IUPUI) / Mu opioid receptors (MORs) mediate the analgesic and rewarding effects of opioids. Most research has focused on MORs in GABAergic neurons; however, MORs are also in glutamatergic neurons and their role in opioid-related behaviors was unclear. Our lab previously showed that MORs inhibit glutamate transmission from vesicular glutamate transporter 2 (vGluT2)-expressing thalamostriatal synapses. The behavioral relevance of MORs in vGluT2-expressing neurons was unknown; therefore, I utilized a conditional MOR knockout mouse with MORs deleted in vGluT2-expressing neurons (MORflox-vGluT2cre). MORflox-vGluT2cre mice have disrupted opioid reward, locomotor stimulation, and withdrawal, compared to cre-recombinase negative littermate controls. However, other MOR-mediated behaviors, including opioid-induced antinociception, alcohol reward, and palatable substance consumption are intact. MORs are expressed in vGluT2 neurons in several reward-related brain regions, including the thalamus and lateral habenula (LHb). To determine whether MORs in these brain regions modulate opioid-related behaviors, an adeno-associated viral (AAV) vector encoding cre-recombinase was stereotaxically injected into the thalamus or LHb of MORflox mice to specifically delete MORs in these brain regions. Opioid reward and locomotor stimulation remained intact in both thalamic and LHb MOR knockout mice; however, basal locomotor activity was increased in LHb MOR knockout mice. Sucrose consumption was also intact in LHb MOR knockout mice. Interestingly, in LHb MOR KO mice opioid withdrawal-induced paw shakes were increased, while withdrawal-induced jumping was completely ablated. Our lab previously showed that MORs inhibit glutamate transmission from the anterior insular cortex (AIC), which is disrupted by in vivo alcohol exposure. To determine the role of AIC MORs, AIC MORs were deleted with AAV vectors. AIC MOR knockout mice had intact opioid, sucrose, and alcohol reward, but had increased basal locomotor activity. MORs in glutamatergic neurons are critical mediators of opioid reward; however, the specific glutamatergic neurons mediating the rewarding effects of opioids remains to be determined.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18859号 / 医博第3970号 / 新制||医||1008(附属図書館) / 31810 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 渡邉 大, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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Kappa opioid receptor (KOPR) is involved in many physiological functions and pharmacological responses such as analgesia, anti-pruritic effect, sedation, motor incoordination and aversion (Simonin et al., 1998; Liu-Chen, 2004). The cellular mechanisms following activation of KOPR involve in part Gi/o protein-dependent pathways (Law et al., 2000). Following KOPR activation, the receptor is phosphorylated and arrestins are recruited. Arrestins mediate agonist-dependent KOPR desensitization, internalization and down-regulation (Liu-Chen, 2004). In recent years, arrestins were found to initiate arrestin-dependent downstream signaling. Thus, agonist-promoted KOPR phosphorylation plays a pivotal role in KOPR regulation and signaling. Previous studies from our lab showed that in Chinese hamster ovary (CHO) cells stably transfected with the human KOPR (hKOPR), U50,488H induced phosphorylation (Li et al., 2002a); however, sites of phosphorylation were not determined. Using LC-MS/MS, our lab recently identified four residues (S356, T357, T363 and S369) to be the sites of U50,488H-promoted phosphorylation in the mouse KOPR (mKOPR) stably expressed in N2A cells (Chen et al., 2016). Antibodies were generated against phosphopeptides and purified and three antibodies were found to have high specificity for the mKOPR phosphorylated at S356/T357, T363 and S369, respectively (Chen et al., 2016). Our lab previously showed that while U50,488H promoted robust hKOPR phosphorylation and internalization, etorphine induced little phosphorylation and internalization, although both were potent full agonists in enhancing [35S]GTPγS (Li et al., 2002a; Zhang et al., 2002; Li et al., 2003). Etorphine caused lower levels of KOPR phosphorylation at all the four residues than U50,488H by immunoblotting with the phospho-specific antibodies (Chen et al., 2016). Using the SILAC (stable isotope labeling by amino acids in cell culture) approach, we have found that compared to etorphine, U50,488H promoted higher levels of single phosphorylation at T363 and S369 and double phosphorylation at T363+S369 and T357+S369 as well as triple phosphorylation at S356+T357+S369 (Chen et al., 2016). These results indicate that an above-threshold phosphorylation is required for KOPR internalization. It has been reported that KOPR is involved in neuronal differentiation and neurogenesis. In the first chapter, I focused on whether there are differences in the mechanisms underlying neurite outgrowth induced by U50,488H and etorphine. In the chapter 2, mechanisms of KOPR phosphorylation were characterized in detail using phospho-specific KOPR antibodies. Protein kinase C was found, for the first time, to be involved in agonist-promoted KOPR phosphorylation. The roles of PKC in behavioral effects induced by KOPR agonists in mice were examined. For the chapter 1, in Neuro2a mouse neuroblastoma cells stably transfected with the hKOPR (N2A-3HA-hKOPR), U50,488H robustly induced neurite outgrowth, but etorphine caused outgrowth to a much lower extent. G protein-dependent pathway was found to be involved in the actions of both agonists, but β-arrestin-dependent pathway was not. Inhibition of ERK1/2 phosphorylation decreased neurite outgrowth promoted by both agonists, indicating the roles of MAP kinase cascades in KOPR agonist-induced neuritogenesis. In contrast, β-arrestin2, 14-3-3ζ, GEC1 and Rap1 are not involved in U50,488H- or etorphine-promoted neurite outgrowth. Thus, the two agonists appear to share the same signaling pathways and the difference between two agonists is likely due to the lower efficacy of etorphine. For the chapter 2, U50,488H caused phosphorylation of the mKOPR at S356, T357, T363 and S369 in N2A cells stably transfected with FmK6H (FmK6H-N2A cells). NorBNI abolished U50,488H-induced KOPR phosphorylation at all four residues. GRKs (GRKs2, 3, 5 and 6) and PKCs were involved in U50,488H-mediated KOPR phosphorylation. In addition, PKC also participated in agonist-independent KOPR phosphorylation. This is the first time that PKC was shown to be involved in agonist-induced KOPR phosphorylation. We found that U50,488H caused KOPR phosphorylation at T363 and S369 in the mouse brain and PKC participated in phosphorylation of S369, but not T363, by using the PKC inhibitor chelerythrine (CHL). Thus, we further characterized effects of PKC inhibition on KOPR-mediated behaviors in CD1 mice. PKC was involved in KOPR-mediated sedation, motor incoordination and conditioned place aversion, but not analgesia and anti-scratching effect in mice. Studies in this thesis revealed the mechanisms of KOPR-mediated neurite outgrowth and KOPR-mediated phosphorylation and the involvement of PKC in KOPR-mediated pharmacological effects in vivo. These studies push the frontier of molecular pharmacology of the KOPR, which may be useful for development of KOPR agonists for therapeutic use. / Pharmacology

Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines. / Molecular Biology and Genetics