Opioidrezeptortypen; Bindungsstudien und selektive Toleranz

Eine langdauernde Vorinkubation des GPI bzw. des MVD mit Morphin-haltiger Nährlösung (unter in vitro-Bedingungen wird anstelle von Morphin häufig Normorphin verwendet) führte zur Entwicklung von Toleranz und im Falle des GPI zu einer zusätzlichen Abhängigkeits-ähnlichen Reaktion. Die Toleranz manifestierte sich als verminderte Ansprechbarkeit gegenüber dem in Anwesenheit des Morphins akut applizierten Normorphin, während die Gewebeabhängigkeit sich als starke Naloxon-induzierte Kontraktion bemerkbar machte. Diese Kontraktion beruhte auf einer massiven Ausschüttung von Acetylcholin aus den postganglionär parasympathischen Nervenendigungen. Als Erklärung wurde hinzugezogen, dass Naloxon das Morphin von seinen Rezeptoren verdrängt und eine entzugsähnliche Reaktion auslöst. Da nicht nur eine in vitro-Vorinkubation mit Morphin in den beiden Präparaten zur Empfindlichkeitsabnahme gegenüber Morphin/Normorphin führte sondern auch die mehrtägige, subkutane Implantation eines Morphin-Pellets oder einer Opioid-Lösung enthaltenden osmotischen Minipumpe, haben wir über den letzteren Weg selektive Toleranz gegenüber μ- (Morphin, Fentanyl), δ- (DADLE) und κ-Agonisten (Ethylketocyclazocin, MR 2034, MRZ) hervorgerufen. Nach in vivo-Behandlung mit den genannten Substanzen wurde das GPI präpariert, in einer Nährlösung, die den jeweiligen Agonisten in der ungefähr 80-fachen Toleranz-induzierenden Kon-zentration enthielt, aufgehängt und mit Feldelektroden elektrisch stimuliert. Die Reizparameter wurden so gewählt (supramaximale Spannung, 0.5 ms Reizbreite, 0.1 Hz Frequenz), dass ausschließlich das neuronale Gewebe stimuliert wurde, nicht aber der Glattmuskel. In vorhergehenden Experimenten konnte die Rezeptorausstattung des GPI nicht eindeutig identifiziert werden. Mit der Erzeugung der selektiven Toleranz an µ-Rezeptoren wurde die akute Wirkung von sowohl μ- als auch δ-Rezeptor-Agonisten wesentlich vermindert. Demgegenüber, übten κ-Rezeptor-Agonisten ihre Wirkung in unveränderter Intensität aus. Die vollständige Kreuz-Toleranz zwischen Morphin und DADLE schloss das Vorhandensein eines δ-Rezeptors aus, während die fehlende Kreuz-Toleranz zwischen Normorphin/DADLE einerseits und Ethylketocyclazocin andererseits das Vorhandensein eines κ-Rezeptors belegte. Es bedarf einer Erklärung, weshalb die in vivo-Behandlung mit Fentanyl nur geringe Toleranz gegenüber Normorphin auslöste und vice versa (wenig Kreuz-Toleranz), obwohl sich eine hochgradige Toleranz gegenüber derselben Substanz entwickelte. Es wurde geschlussfolgert, dass es verschiedene Subtypen von μ-Rezeptoren gibt, eines mit Empfindlichkeit gegenüber Morphin/Normorphin und ein anderes gegenüber Fentanyl.:Inhaltsverzeichnis Inhaltsverzeichnis…………………………………………………………………………… 2 Wissenschaftlicher Anteil der Promovendin an der Publikation……………………….. 3 Bibliographische Beschreibung……………………………………………………………. 4 I. Einleitung………………………………………………………………………………… 5 Grundlegende Erklärungen……………………………………………………………. 5 Multiple Opioidrezeptoren……………………………………………………………… 5 II. Wissenschaftlicher Hintergrund………………………………………………………..8 Eigene Publikationen…………………………………………………………………… 8 Fragestellungen der Dissertation………………………………………………………8 III. Zusammenfassung der Arbeit…………………………………………………………. 9 IV. Originalmitteilungen…………………………………………………………………... 3 V. Derzeitiger Stand der Opioidforschung……………………………………………31 Opioidrezeptortypen…………………………………………………………………… 31 Transduktionsmechanismen der Opioidrezeptoren; Toleranz und Abhängigkeit…………….. 32 Zentrale und periphere Opioidanalgesie…………………………………………….. 38 Schlussfolgerungen……………………………………………………………………. 39 VI. Literaturverzeichnis……………………………………………………………………. 40 Eigenständigkeitserklärung……………………………………………………………….. 44 Lebenslauf und Publikationsverzeichnis…………………………………………… 45 Danksagung………………………………………………………………………………… 4

Beteiligung purinerger Rezeptoren am Schmerzgeschehen und an der neuronalen Entwicklung

Zusammenfassung der Arbeit Beteiligung purinerger Rezeptoren am Schmerzgeschehen und an der neuronalen Entwicklung Patrizia Rubini Illes Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig Diese kumulative Arbeit umfasst neun Publikationen, welche die Beteiligung purinerger P2-Rezeptoren (P2R) am Schmerzgeschehen sowie der neuronalen Entwicklung belegen. Im Fokus stehen zwei ATP-aktivierte Rezeptor-Kanäle (P2X3R, P2X7R) und ein G-Protein-gekoppelter, ATP-empfindlicher Rezeptor (P2Y1R). (1) Da sich humane P2X3R an peripheren und zentralen Endigungen primärer-sensorischer Afferenzen befinden, ist die Entschlüsselung der Agonisten-Bindungsstelle für die Entwicklung potenziell analgetisch wirkender, kompetitiver Antagonisten von großer Bedeutung. Wir identifizierten Gruppen konservierter und nicht-konservierter Aminosäure-Reste, die an der Schnittstelle zweier benachbarter Untereinheiten, die Agonisten-Bindungstasche auskleiden. Abhängig vom Mengenverhältnis der zur Transfektion verwendeten cDNA, bilden P2X2-, P2X3- und P2X6-Untereinheiten heteromere Rezeptoren (P2X2/3R, P2X2/6R) in einer variablen Stöchiometrie. (2) Adulte neurale Progenitorzellen (NPZ) der Subventrikulärzone der Maus besitzen Apoptose- bzw. Nekrose-vermittelnde P2X7R. Diese kommen in Assoziation mit P2X4R vor, bilden jedoch wahrscheinlich keinen heteromeren P2X4/7R. P2X7R könnten zum Absterben der, nach metabolischen Schäden im Überschuss gebildeten, NPZ beitragen. Im Laufe der neuronalen Entwicklung verlieren die NPZ ihre P2X7R. Im Gegensatz hierzu bleiben diese bei ihren astrozytären Nachkommen erhalten. (3) Humane mesencephale embryonale NPZ sind mit P2Y1R ausgestattet, die über eine Ca2+-Wellenaktivität die Proliferation der NPZ beschleunigen und in Differenzierungsvorgänge eingreifen. De-differenzierte, kultivierte Neurone des Ratten-Striatum enthalten ebenfalls P2Y1R. Diese setzen Ca2+ aus seinen intrazellulären Speichern frei und öffnen somit Ca2+-selektive „store-operated channels“ in der Zellmembran. Wir konnten zeigen, dass der P2Y1R-stimulierte Anstieg der intrazellulären Ca2+-Konzentration in den striatalen Neuronen durch die Aktivierung von Dopamin D1- und D2R inhibiert wurde. Die vorliegende Arbeit belegt die Bedeutung einiger P2R für zahlreiche, auch potentiell therapeutisch bedeutsame Funktionen des menschlichen und tierischen Organismus.:Inhaltsverzeichnis Kapitel Seite Abkürzungen …………………………………………………………………………………. 5 1. Einführung in die Thematik.......................................................................... 7 1.1. Freisetzung von Nukleotiden und ihre Metabolisierung im Extrazellulärraum …7………………………………… 1.2. P2X-Rezeptoren…………………………………………………………………….. 9 1.3. P2Y-Rezeptoren……………………………………………………………………. 12 1.4. P2-Rezeptoren und Schmerz……………………………………………………... 14 1.5. P2-Rezeptoren und neuronale Entwicklung; embryonale undadulte neurale Vorläuferzellen……………………………………………………. 17 1.6. Referenzen………………………………………………………………………….. 19 2. Wissenschaftliche Ergebnisse……………………………………………………… 23 2.1. Komplex I. Struktur-Wirkungs-Zusammenhänge bei rekombinanten, humanen P2X3- und P2X2/3-Rezeptoren in Expressionssystemen…………. 24 2.1.1. Amino acid residues constituting the agonist binding site of the human P2X3 receptor……………………………………………………………… 24 2.1.2. ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors……………………………………... 26 2.1.3 Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors……………………………………………………………... 28 2.2. Komplex II. Vorhandensein und Wirkung von P2X7-Rezeptoren an kultivierten adulten, neuralen Vorläuferzellen und Astrozyten………….. 29 2.2.1. P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone………………………………………………………………… 29 2.2.2. Co-expression of functional P2X4 and P2X7 receptors at adult neural precursor cells of the mouse subventricular zone……………………… 31 2.2.3. Functional P2X7 receptors at cultured hippocampal astrocytes but not neurons…………………………………………………………………….. 32 2.3. Komplex III. Vorhandensein und Wirkung von P2Y-Rezeptoren an embryonalen neuralen Vorläuferzellen und dedifferenzierten kultivierten striatalenNeuronen... 33 2.3.1. Increase of intracellular Ca2+ by adenine and uracil nucleotides in human midbrain-derived neuronal precursor cells…………... 33 2.3.2. Regulation of intracellular Ca2+ by P2Y1 receptors may depend on the developmental stage of cultured rat striatal neurons…………………... 35 2.3.3. Modulation by D1 and D2 dopamine receptors of ATP-induced release of intracellular Ca2+ in cultured rat striatal neurons 36 3. Zusammenfassung und Ausblick………………………………………… ………... 37 4. Erklärungen, Danksagung…………………………………………… 38 Erklärung über die eigenständige Anfertigung der Arbeit………… 38 Erklärung über den eigenen Anteil an den einzelnen Arbeiten…………….. 39 Danksagung………………………………………………………………… 4

Increasing Efficiency of Repetitive Electroacupuncture on Purine- and Acid-Induced Pain During a Three-Week Treatment Schedule

Acupuncture (AP) is an important constituent of the therapeutic repertoire of traditional Chinese medicine and has been widely used to alleviate chronic painful conditions all over the world. We studied in rats the efficiency of electroacupuncture (EAP) applied to the Zusanli acupoint (ST36) as an analgesic treatment over a 3-week period of time on purine (α,β-methylene ATP, dibenzoyl-ATP)- and acid (pH 6.0 medium)-induced pain in the rat paw. The two ATP derivatives stimulated P2X3 and P2X7 receptors, respectively, while the slightly acidic medium stimulated the “acid-sensitive ion channel 3” (ASIC3). It was found that the P2X7 receptor and ASIC-mediated pain was counteracted by EAP with greater efficiency at the end than at the beginning of the treatment schedule, while the P2X3 receptor–mediated pain was not. Our findings have important clinical and theoretical consequences, among others, because they are difficult to reconcile with the assumption that AP is primarily due to the release of peripheral and central opioid peptides causing the well-known tolerance to their effects. In consequence, AP is a convenient therapeutic instrument to treat subacute and chronic pain

Inherent P2X7 Receptors Regulate Macrophage Functions during Inflammatory Diseases

Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophagelocalized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1 (pro-IL-1)-degrading caspase-1 to lead to IL-1 release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences

Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNS

Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood–brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5′-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation

Takayasu arteritis: a cohort of Italian patients and recent pathogenetic and therapeutic advances

Takayasu arteritis (TAK) is a rare granulomatous vasculitis of unknown etiology that mainly affects the aorta and its major branches. The aim is to describe the clinical features, diagnostic procedures, pathogenesis, and management of TAK in a longitudinal cohort of patients recruited within a single region of southern Italy. The cohort included 43 patients who were diagnosed with TAK and followed up according to a standard protocol, in a collaboration between four university tertiary referral centers and a regional hospital. Clinical and imaging classification criteria were those established by the American College of Rheumatology. Thirty-five patients (81.4%) were female, and the mean age at disease onset was 32.6 (range 16-54) years. Angiographic assessment of the vascular involvement allowed disease classification in five different types. Clinical features ranged from constitutional symptoms in the early inflammatory stage of the disease to cardiovascular ischemic symptoms in the late, chronic stage. Noninvasive imaging techniques were employed to assess the extent and severity of the arterial wall damage and to monitor the clinical course and response to therapy. Medical treatment, based on pathogenetic insights into the roles of humoral and cell-mediated immune mechanisms, included glucocorticoids mostly combined with steroid-sparing immunosuppressive agents and, in patients with relapsing/refractory disease, biologic drugs. Significant clinical and angiographic differences have been detected in TAK patients from different geographic areas. Patients with life-threatening cardiovascular and neurologic manifestations as well as sight-threatening ophthalmologic signs and symptoms should be promptly diagnosed, properly treated, and closely followed up to avoid potentially severe consequences

High, in Contrast to Low Levels of Acute Stress Induce Depressive-like Behavior by Involving Astrocytic, in Addition to Microglial P2X7 Receptors in the Rodent Hippocampus

Extracellular adenosine 50-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans

Narrative review of multiparametric ultrasound in parotid gland evaluation

Disorders affecting parotid gland represent a heterogeneous group comprising congenital, inflammatory and neoplastic diseases which show a focal or diffuse pattern of appearance. The differentiation of neoplastic from non-neoplastic conditions of parotid glands is pivotal for the diagnostic imaging. Frequently there is evidence of overlapping between the clinical and the imaging appearance of the various pathologies. The parotid gland is also often object of study with the combination of different techniques [ultrasound-computed tomography-magnetic resonance imaging (US-CT-MRI), ex.]. Compared to other dominant methods of medical imaging, US has several advantages providing images in real-time at lower cost, and without harmful use of ionizing radiation and of contrast enhancement. B-mode US, and the microvascular pattern color Doppler are usually used as first step evaluation of parotid lesions. Elastography and contrast-enhanced US (CEUS) has opened further possible perspectives to improve the differentiation between benign and malignant parotid lesions. The characterization of the parotid tumors plays a crucial role for their treatment planning and for the prediction of possible surgical complications. We present, here an updated review of the most recurrent pathologies of parotid gland focusing on the diagnostic power of multiparametric US including CEUS and ultrasound elastography (USE); limitations, advantages and the main key-points will be presented

Reliability of Lupus Anticoagulant and Anti-phosphatidylserine/prothrombin Autoantibodies in Antiphospholipid Syndrome: A Multicenter Study

Background: Is it well-known that one of the major drawbacks of Lupus Anticoagulant (LA) test is their sensitivity to anticoagulant therapy, due to the coagulation based principle. In this study we aimed to assess the reproducibility of LA testing and to evaluate the performance of solid assay phosphatidylserine/prothrombin (aPS/PT) antibodies.Methods: We included 60 patients that fulfilled the following inclusion criteria: (I) diagnosis of thrombotic antiphospholipid syndrome (APS); (II) patients with thrombosis and (a) inconstant previous LA positivity and/or (b) positivity for antiphospholipid antibodies (aPL) at low-medium titers [defined as levels of anti-β2Glycoprotein-I or anticardiolipin (IgG/IgM) 10–30 GPL/MPL] with no previous evidence of LA positivity. aPL testing was performed blindly in 4 centers undertaking periodic external quality assessment.Results: The 60 patients enrolled were distributed as follows: 43 (71.7%) with thrombotic APS, 7 (11.7%) with thrombosis and inconstant LA positivity and 10 (16.7%) with low-medium aPL titers. Categorical agreement for LA among the centers ranged from 0.41 to 0.60 (Cohen's kappa coefficient; moderate agreement). The correlation determined at the 4 sites for aPS/PT was strong, both quantitatively (Spearman rho 0.84) and when dichotomized (Cohen's kappa coefficients = 0.81 to 1.0). Discordant (as defined by lack of agreement in ≥3 laboratories) or inconclusive LA results were observed in 27/60 (45%) cases; when limiting the analysis to those receiving vitamin K antagonist (VKA), the level of discordant LA results was as high as 75%(15/20). Conversely, aPS/PT testing showed an overall agreement of 83% (up to 90% in patients receiving VKA), providing an overall increase in test reproducibility of +28% when compared to LA, becoming even more evident (+65%) when analyzing patients on VKA. In patients treated with VKA, we observed a good correlation for aPS/PT IgG testing (Cohen's kappa coefficients = 0.81–1; Spearman rho 0.86).Conclusion: Despite the progress in the standardization of aPL testing, we observed up to 45% of overall discrepant results for LA, even higher in patients on VKA. The introduction of aPS/PT testing might represent a further diagnostic tool, especially when LA testing is not available or the results are uncertain

State of the art of 18F-FDG PET/CT application in inflammation and infection: a guide for image acquisition and interpretation

Aim The diagnosis, severity and extent of a sterile inflammation or a septic infection could be challenging since there is not one single test able to achieve an accurate diagnosis. The clinical use of 18F-fluorodeoxyglucose ([F-18]FDG) positron emission tomography/computed tomography (PET/CT) imaging in the assessment of inflammation and infection is increasing worldwide. The purpose of this paper is to achieve an Italian consensus document on [F-18]FDG PET/CT or PET/MRI in inflammatory and infectious diseases, such as osteomyelitis (OM), prosthetic joint infections (PJI), infective endocarditis (IE), prosthetic valve endocarditis (PVE), cardiac implantable electronic device infections (CIEDI), systemic and cardiac sarcoidosis (SS/CS), diabetic foot (DF), fungal infections (FI), tuberculosis (TBC), fever and inflammation of unknown origin (FUO/IUO), pediatric infections (PI), inflammatory bowel diseases (IBD), spine infections (SI), vascular graft infections (VGI), large vessel vasculitis (LVV), retroperitoneal fibrosis (RF) and COVID-19 infections. Methods In September 2020, the inflammatory and infectious diseases focus group (IIFG) of the Italian Association of Nuclear Medicine (AIMN) proposed to realize a procedural paper about the clinical applications of [F-18]FDG PET/CT or PET/MRI in inflammatory and infectious diseases. The project was carried out thanks to the collaboration of 13 Italian nuclear medicine centers, with a consolidate experience in this field. With the endorsement of AIMN, IIFG contacted each center, and the pediatric diseases focus group (PDFC). IIFG provided for each team involved, a draft with essential information regarding the execution of [F-18]FDG PET/CT or PET/MRI scan (i.e., indications, patient preparation, standard or specific acquisition modalities, interpretation criteria, reporting methods, pitfalls and artifacts), by limiting the literature research to the last 20 years. Moreover, some clinical cases were required from each center, to underline the teaching points. Time for the collection of each report was from October to December 2020. Results Overall, we summarized 291 scientific papers and guidelines published between 1998 and 2021. Papers were divided in several sub-topics and summarized in the following paragraphs: clinical indications, image interpretation criteria, future perspectivess and new trends (for each single disease), while patient preparation, image acquisition, possible pitfalls and reporting modalities were described afterwards. Moreover, a specific section was dedicated to pediatric and PET/MRI indications. A collection of images was described for each indication. Conclusions Currently, [F-18]FDG PET/CT in oncology is globally accepted and standardized in main diagnostic algorithms for neoplasms. In recent years, the ever-closer collaboration among different European associations has tried to overcome the absence of a standardization also in the field of inflammation and infections. The collaboration of several nuclear medicine centers with a long experience in this field, as well as among different AIMN focus groups represents a further attempt in this direction. We hope that this document will be the basis for a "common nuclear physicians' language" throughout all the country